Patent Application
20240287596
This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Aug. 30, 2022, is named VOSSP0127US_ST25_2.txt and is 5,658 bytes in size.
The invention relates to a method for sequencing oligonucleotides comprising RNA, the method comprising the steps of (a) providing permeabilized cells and/or nuclei comprising a first oligonucleotide comprising RNA; (b) combining said cells and/or nuclei of (a) with a second oligonucleotide comprising DNA in a first reaction compartment, wherein the second oligonucleotide comprises at least a first sequence at least partially complementary to a sequence of the first oligonucleotide, a second sequence comprising an indexing sequence, and a third sequence comprising a primer binding site, under conditions to allow annealing of the first sequence of the second oligonucleotide to the first oligonucleotide; (c) reversely transcribing the first oligonucleotide in said cells/nuclei to obtain an elongated second oligonucleotide; (d) combining said cells and/or nuclei obtained in step (c) with a microbead-bound third oligonucleotide in a second reaction compartment, wherein the third oligonucleotide comprises (i) a first sequence corresponding to a fourth sequence comprised in the second oligonucleotide used in step (b); or (ii) a first sequence complementary to a first sequence of a fourth oligonucleotide, wherein the fourth oligonucleotide further comprises a second sequence at least partially complementary to the third sequence of the second oligonucleotide; wherein for (i), the method further comprises a step of second strand DNA synthesis subsequent to step (c) and prior to step (d) and wherein for (ii) the method further comprises a step of DNA ligation; and wherein the third oligonucleotide further comprises a second sequence comprising an indexing sequence and a third sequence comprising a primer binding site; (e) amplifying the DNA oligonucleotides obtained in step (d); and (f) sequencing of amplified DNA oligonucleotides. The invention furthermore relates to uses of such methods and devices used for such methods. Further provided are kits comprising one or more components used in the methods of the invention.
Cell atlas projects (e.g., the Human Cell Atlas (Rozenblatt-Rosen et al. (2017) Nature 550, 451-3) and single-cell CRISPR screens (e.g. using CROP-seq (Datlinger et al. (2017) Nat Methods 14, 297-301)) hit the limits of current technology, as they require profiling of millions of single cells. Most single-cell RNA-seq studies that reach beyond the scale of what is feasible using standard microtiter (96-well or 384-well) plates are currently based on either sub-nanoliter well plates or on microfluidic droplet generators. Both technologies build on a micro-manufacturing method called soft lithography.
In sub-nanoliter well-based scRNA-seq (Cyto-Seq (Chen et al. (2015) Science 348, aaa6090), Seq-Well (Gierahn et al. (2017) Nat Methods 14, 395-8), Microwell-Seq (Han et al. (2018) Cell 172, 1091-1107), sci-RNA-seq (Cao et al. (2017) Science 357, 661-7)), a plate with miniaturized reaction compartments in the sub-nanoliter range is cast from a material such as PDMS or agarose. Beads and cells are loaded by gravity. While beads are typically loaded to near saturation, cells are loaded at a limiting dilution (i.e., very low concentration) to avoid cells entering the same reaction compartment. If two cells did enter the same well on the plate, they would end up with the exact same cell barcode and would be indistinguishable in the downstream analysis. On the plate, cells are lysed and their transcriptome anneals to complementary oligonucleotides on the microbeads. Typically, beads are then collected, and the reverse transcription is performed in bulk. Currently, there is a lack of well-validated and readily available protocols and commercial solutions, so that most labs prefer microfluidic droplet generators (described next).
Soft lithography is not limited to open designs such as sub-nanoliter well plates. When using PDMS as the material, the open side can be sealed by bonding it to a glass slide to realize complex channel designs. This has allowed the manufacturing of microfluidic droplet generators for scRNA-seq (Drop-seq (Macosko et al. (2015) Cell 161, 1202-14), inDrop (Klein et al. (2015) Cell 161, 1187-1201), 10× Genomics Chromium (Zheng et al. (2017) Nat. Commun. 8, 14049)). A typical microfluidic device for scRNA-seq has four inputs (for cells, barcoded microbeads, reverse transcription reagents, and carrier oil) and one output (for the droplet emulsion). The reverse transcription reaction is typically performed inside the droplets. While deformable beads can be loaded to near saturation, cells are supplied at a limiting dilution to make it unlikely that two cells enter the same droplet. If two cells did enter the same droplet, they would receive the exact same cell barcode and would be indistinguishable in the downstream analysis. As a consequence, while most droplets contain both reagents and beads and are thus fully functional, they are ultimately not used because they do not contain a cell.
The throughput of sub-nanoliter well plates and microfluidic droplet generators is limited by the requirement to load cells at a limiting dilution to avoid cell doublets. These platforms typically reach a throughput of about 10,000 cells per experiment (e.g. per sub-nanoliter well plate or per channel on the 10× Genomics Chromium chip) but this can be increased by parallelization (multiple plates, multiple channels on the microfluidic device). However, this often comes at high cost and is labour-intensive.
In combinatorial indexing, the number of cells profiled can scale exponentially with the number of barcoding rounds. Two rounds of barcoding allow the profiling of roughly 10,000 cells (when using 384×384 barcodes), which generates a lot of manual work, but does not provide any advantage over sub-nanoliter well plates or droplet generators. Only when a third round of indexing is introduced, the processing of over one million cells becomes possible. The currently largest dataset generated with sci-RNA-seq v3 comprises 2 million single-cell transcriptomes from the developing mouse embryo (Cao et al. (2019) Nature 566, 496-502). However, this comes with several drawbacks: (1) most NGS library preparation protocols are not immediately compatible with three rounds of combinatorial indexing (e.g. assays such as ATAC-seq, DNA methylation profiling, Hi-C). (2) In each barcoding step, nuclei or cells have to remain intact despite aggressive reaction buffers and high temperature incubations. With three barcoding rounds, the loss of material is typically >90%. (3) It is challenging to design an elegant library read structure to sequence the combination of three barcodes cost effectively (this is particularly problematic when ligation overhangs have to be sequenced along with the barcodes such as in SPLIT-seq or sci-RNA-seq v3. (4) Synthesis and sequencing errors in the barcodes accumulate, so that a larger percentage of reads cannot be assigned with confidence. (5) Running reactions on intact cells or nuclei is only partially efficient. The more reactions have to be run this way, the lower the overall efficiency of the library preparation and quality of the resulting single-cell transcriptomes. (6) To achieve high cell numbers, a large number of indices have to be used for each barcoding round. As an example, to generate the 2 million cell dataset a combination of 384×384×768 barcodes was used. This is both labor-intensive and wasteful in terms of the reagent volumes required. Given these disadvantages, it is hard to imagine that published methods for combinatorial indexing scRNA-seq will be universally adopted by research labs or become a commercial success.
In a typical experiment, the cell suspension is loaded onto a microfluidic chip, along with a population of microbeads with unique DNA barcodes, reverse transcription reagents, and carrier oil (
Importantly, if two cells enter the same droplet or the same well, e.g. on a sub-nanoliter well plate, their transcriptomes are labelled with the exact same cell barcode, resulting in a cell doublet that confounds the analysis. To avoid this issue, state-of-the-art droplet generators are supplied with the cell suspension at a limiting dilution, with most droplets carrying 0 or 1 cells. This makes microfluidic scRNA-seq highly inefficient. While most emulsion droplets are fully functional (they contain both barcoded microbeads and reverse transcription reagents), they do not receive a cell and thus do not result in a productive library preparation event.
Accordingly, there is a need for improved methods for analyzing RNA oligonucleotides, in particular methods allowing high throughput analysis.
The technical problem is solved by the embodiments provided herein and in particular as provided in the claims.
The present invention relates to, inter alia, the following items:
1. A method for sequencing oligonucleotides comprising RNA, the method comprising the steps of:
2. The method of item 1, wherein in step (c) untemplated nucleotides are added to the 3′-end of the second oligonucleotide.
3. The method of item 2, wherein second strand DNA synthesis comprises the use of primers comprising a sequence complementary to the added untemplated nucleotides.
4. The method of item 2, wherein a primer comprising RNA nucleotides complementary to the added untemplated nucleotides is added for extension.
5. The method of item 1, wherein second strand DNA synthesis comprises
6. The method of item 1 or 5, further comprising subsequent to or concurrently with second strand DNA synthesis a step of introducing untemplated nucleotides at the 5′-end of the synthesized second strand DNA.
7. The method of item 6, wherein untemplated nucleotides are introduced using a transposase enzyme, in particular Tn5 transposase.
8. The method of item 1, wherein the method further comprises a step of linear extension subsequent to DNA ligation, wherein linear extension comprises adding a primer comprising RNA nucleotides and adding a reverse transcriptase enzyme.
9. The method of item 1, wherein the method further comprises a step of linear extension comprising adding a primer comprising random nucleotides.
10. The method of any one of items 1 to 9, wherein the sequence of the first oligonucleotide bound by the first sequence of the second oligonucleotide is located at the 3′-end of the first oligonucleotide.
11. The method of any one of items 1 to 10, wherein the first sequence of the second oligonucleotide is complementary to the 3′ poly-A tail of the first oligonucleotide.
12. The method of any one of items 1 to 11, wherein the first reaction compartment comprises permeabilized intact cells and/or nuclei.
13. The method of any one of items 1 to 12, wherein the first reaction compartment comprises 5000 to 10000 cells.
14. The method of any one of items 1 to 13, wherein the second reaction compartment comprises lysed cells and/or nuclei.
15. The method of any one of items 1 to 14, wherein the second reaction compartment comprises more than one cell and/or nuclei per microbead, preferably 10 cells/nuclei per microbead.
16. The method of any one of items 1 to 15, wherein the second reaction compartment is a microfluidic droplet or a well on a microtiter plate, in particular a sub-nanoliter well plate.
17. The method of item 16, wherein the second reaction compartment is a microfluidic droplet and the third oligonucleotide is released from the microbead upon formation of the droplets.
18. The method of any one of items 1 to 17, wherein the second oligonucleotide further comprises a unique molecular identifier (UMI).
19. The method of any one of items 1 to 18, wherein the cells and/or nuclei are obtained from in vitro cultures or fresh or frozen samples.
20. The method of any one of items 1 to 19, wherein the cells/nuclei are
21. The method of any one of items 1 to 20, wherein DNA ligation uses a thermostable DNA ligase.
22. Use of a microfluidic system, in particular to generate microfluidic droplets or to deliver material into a microfluidic well-based device, in the method of any one of items 1 to 21.
23. The use of item 22, wherein the microfluidic system is a droplet generator.
24. The use of item 22, wherein the microfluidic system comprises a sub-nanoliter well plate.
25. A kit comprising a second oligonucleotide as defined in item 1, preferably together with instructions regarding the use of the method of any one of items 1 to 21.
26. The kit of item 25 further comprising a transposase enzyme.
27. The kit of item 25 further comprising second strand synthesis reagents and/or a thermostabe ligase.
28. The kit of any one of items 25 to 27 further comprising the fourth oligonucleotide.
The present invention relates to a method for sequencing oligonucleotides comprising RNA, the method comprising the steps of (a) providing permeabilized cells and/or nuclei comprising a first oligonucleotide comprising RNA; (b) combining said cells and/or nuclei of (a) with a second oligonucleotide comprising DNA in a first reaction compartment, wherein the second oligonucleotide comprises at least a first sequence at least partially complementary to a sequence of the first oligonucleotide, a second sequence comprising an indexing sequence, and a third sequence comprising a primer binding site, under conditions to allow annealing of the first sequence of the second oligonucleotide to the first oligonucleotide; (c) reversely transcribing the first oligonucleotide in said cells/nuclei to obtain an elongated second oligonucleotide; (d) combining said cells and/or nuclei obtained in step (c) with a microbead-bound third oligonucleotide in a second reaction compartment, wherein the third oligonucleotide comprises (i) a first sequence corresponding to a fourth sequence comprised in the second oligonucleotide used in step (b); or (ii) a first sequence complementary to a first sequence of a fourth oligonucleotide, wherein the fourth oligonucleotide further comprises a second sequence at least partially complementary to the third sequence of the second oligonucleotide; wherein for (i), the method further comprises a step of second strand DNA synthesis subsequent to step (c) and prior to step (d) and wherein for (ii) the method further comprises a step of DNA ligation; and wherein the third oligonucleotide further comprises a second sequence comprising an indexing sequence and a third sequence comprising a primer binding site; (e) amplifying the DNA oligonucleotides obtained in step (d); and (f) sequencing of amplified DNA oligonucleotides. The present method(s) as provided herein may also comprise an additional step of fixation of the permeabilized cells and/or nuclei comprising said first oligonucleotide comprising RNA. Corresponding embodiments are also provided herein below.
The present inventors have surprisingly found that microfluidic scRNA-seq could be used at full capacity when entire transcriptomes are pre-indexed with a first barcode prior to the microfluidic run (
The herein provided method for single-cell RNA sequencing at ultra-high throughput is named scifi-RNA-seq (for: single-cell combinatorial indexing with fluidic indexing RNA sequencing). The method of the invention extends state-of-the-art droplet-based scRNA-seq by single-round combinatorial pre-indexing and thereby increases the throughput by at least 15-fold, at least 20-fold, at least 25-fold or more. This is mainly achieved due to possible loading of multiple cells into one droplet without creating indistinguishably labelled readouts.
In scifi-RNA-seq (
The herein provided means and methods can, inter alia, be used on the Chromium platform commercialized by 10× Genomics (“Chromium™”), which is currently the most popular scRNA-seq platform. However, the method(s) of the invention can be adopted to boost the throughput of any microfluidic or plate-based platforms, in particular nano and/or sub-nanoliter microplate-based platforms, and/or any protocols involving barcoding, like combinatorial indexing protocols. For example, the methods of the invention can be used to improve results obtained using the BectonDickinson Rhapsody system (see e.g. Shum et al. (2019) Adv Exp Med Biol, 1129:63-79/“BD Rhapsody™”). Such an improvement can, inter alia, be seen in a substantially higher cell/nuclei input and/or the potential multiplexing of hundreds or thousands of samples since with the present method no individual channels for assessment are needed. The present invention also provides for cleaner data, like a high single-cell purity. Moreover, the inventors have shown that the method(s) of the invention solve various drawbacks of the standard method(s) used on prior art systems, like the above mentioned Chromium™ platform of 10× Genomics. These surprising ameliorations over the prior art, like Chromium™, comprise for example, reduced “backgrounds” (which are often due to free-floating RNA or cell preparation artefacts) and/or improved (single-)cell purity (as inter alia, illustrated in
As such, the scifi-RNA-seq method as provided herein and variations thereof, i.e. the methods of the present invention can be used, inter alia, in organ-scale and/or organism-scale single-cell sequencing projects (e.g. Human Cell Atlas) and/or developmental studies at the organ and/or organism level. The methods of the present invention can also be used for the identification of extremely rare and/or transient cell types, developmental stages and/or cellular phenotypes. Such applications may include the identification of extremely rare reprogramming and/or transdifferentiation events that are so far difficult to capture with selectable marker proteins. In a further application of the methods of the present invention, CRISPR single-cell sequencing (e.g. by CROP-seq, Perturb-seq, CRISP-seq, Mosaic-seq) with combined whole transcriptome and/or CRISPR gRNA readout may be envisaged. As a further example, CRISPR single-cell sequencing (e.g. by CROP-seq, Perturb-seq, CRISP-seq, Mosaic-seq) with combined single transcript and CRISPR gRNA readout, or transcript panel and CRISPR gRNA readout may be done using the methods of the present invention. Furthermore, a combination of scifi-RNA-seq and CRISPR single-cell sequencing with CRISPR activation, to profile the response of the whole transcriptome, or a subset of the transcriptome to a perturbation is envisaged. The scifi-RNA-seq method as provided herein and variations thereof, i.e. the methods of the present invention, may also be employed in the drug screenings and/or the testing of compounds, for example the testing of (a) compound(s) for its/their capacity to elucidate a chance in the cellular expression profile and the like. Accordingly, the present invention also provides for screening methods. The means and methods provided herein are also useful in biological/biochemical research approaches, like, inter alia, in the elucidation of ligand-receptor relationships and/or of signal-cascades and their (cellular) consequences.
The methods of the present invention, scifi-RNA-seq, may serve as a readout for CRISPR single-cell sequencing with multiple perturbations per cell, where ultra-high throughput is required to capture all possible combinations.
The methods of the present invention may be combined with single-cell ATAC-seq for integrated transcriptome/epigenome readout. The methods of the present invention may also be combined with lineage tracing methods, for an integrated readout of lineage information and/or transcriptome.
Further provided is the use of scifi-RNA-seq, the methods of the present invention, for immune repertoire sequencing at ultra-high throughput, by specific enrichment of transcripts encoding for the B cell receptor, T cell receptor, or other relevant proteins (
Also provided is the use of the methods of the present invention, scifi-RNA-seq, for integrated transcriptome and immune repertoire sequencing.
Further provided is the use of the methods of the present invention, scifi-RNA-seq and variations thereof, for the identification of antigen-specific, reactive T-cells, B-cells and/or other immune cells, for example, by means of their activation signature. Also provided is the use for the detection of barcoded antibodies or other biomolecules interacting with extracellular and/or intracellular partners such as targets and/or antigens.
Also provided is the combination of the methods of the present invention with the enrichment of transcripts of interest (single transcripts, panels of transcripts, CRISPR gRNAs, feature barcodes obtained inter alia from barcoded antibodies or other biomolecules), for instance by specific PCR or transcript capture. This includes diagnostic applications.
The means and methods of the present invention are also useful in the assessment of cell-cell interactions and/or in cell-cell interaction profiling. In accordance with this embodiment of the invention the cells are not separated but allowed to physically interact. Cell-cell interactions will allow cells to pass through the same first reaction compartment. Interactions between cells can be stabilized by fixation methods.
Specifically, in a first experiment, the loading capacity of the microfluidic system was tested by substituting the lysis reagents for standard EB buffer. Thus, the number of nuclei contained in the microfluidic droplets could be counted under a light microscope. As shown in
In a second experiment, a first barcode index was introduced using a specialized library preparation method depicted in
The next step in this exemplary protocol of the method of the invention was to introduce a second defined end for the ensuing enrichment PCR reaction. This was achieved using a custom Tn5 transposase loaded with an Illumina-compatible i7-only adapter. Alternative means in the methods of the invention to achieve the same outcome are, inter alia, template switching by the reverse transcriptase when provided with an appropriate oligonucleotide; random priming with Klenow Exo- or a similar enzyme; single-stranded ligation with or without RNA base tailing.
Importantly and advantageously over methods of the prior art, throughout the process, nuclei and/or cells remain intact, and are loaded onto the microfluidic device at an unusually high concentration to promote loading of multiple cells per droplet. In the methods of the invention, one microbead is co-encapsulated with multiple barcoded cells/nuclei. Due to the buffer composition, nuclei are lysed and annealing of the transcriptomes to the microbead-tethered oligos is allowed. The microfluidic droplets were then subjected to multiple rounds of linear extension to introduce the second (microfluidic) barcode into the transcriptomes. After this reaction, the droplet emulsion was broken and the sequencing library was PCR-enriched, which allowed the introduction of an additional, channel-specific barcode. While both the first and second barcodes can be shared by multiple cells, the combination of the two barcodes is unique for an individual cell. During the bioinformatic analysis, cells were identified by their cell barcode comprising both the plate-based first and the microfluidic second barcodes. The combination of both led to the surprising results provided herein. Specifically, the results of a typical library preparation experiment are depicted in
For several reasons, it was believed in the art that combinatorial indexing RNA-seq could not be combined with droplet microfluidics. Most importantly, it was believed that subjecting cells or nuclei to reverse transcription, second strand synthesis, and tagmentation is inevitably damaging. It was thus surprising and unexpected that the methods of the invention lead to a significant improvement over the prior art methods.
In the appended examples, it is shown that the 10× Genomics Chromium assay can be overloaded with 100-fold higher nuclei amounts as maximally recommended. Surprisingly, stable droplet emulsions were achieved without clogging of the microfluidic channels even at the highest loading concentration. Detailed metrics on the nuclei fill rate over a range of high loading concentrations are provided, and it is demonstrated that it can be tightly controlled even at unusually high loading concentrations. For instance, a stable mean fill rate of 9.6 cells per droplet was achieved when loading 1.53 million nuclei per channel (100× the maximum recommended amount). It is also shown that there is no physical limit to filling droplets with nuclei. For instance, loading 1.53 million nuclei per channel resulted in a fill rate of 95.5%.
Moreover, it is shown in the appended examples that nuclei subjected to a combinatorial pre-indexing round are sufficiently stable to withstand the pressure and shear stress inside a microfluidic device. This was unexpected, as they are in some instances of the present invention subjected to three enzymatic reactions: reverse transcription, second strand synthesis, and tagmentation. These steps involve high-temperature incubations and aggressive buffers that were expected to compromise the integrity of nuclei. It was therefore not obvious to combine a pre-indexing step with microfluidics. Surprisingly, the optimized workflow for scifi-RNA-seq as provided herein recovers pre-indexed cells/nuclei at a rate comparable to standard microfluidic scRNA-seq.
The methods of the invention constitute the first use of linear barcoding for single-cell transcriptome sequencing. In some instances, the present invention also provides the first use of a thermostable ligase for next-generation sequencing library preparation. Linear barcoding refers to the introduction of a cell barcode by annealing to a bead-tethered oligonucleotide followed by linear extension with a suitable DNA polymerase. While linear barcoding has been recently described for single-cell ATAC-seq, it has not been suggested for scRNA-seq. There is no other scRNA-seq method using linear barcoding prior to the present invention. Through the invention as described herein, it was demonstrated that linear barcoding is effective for preparing single-cell transcriptome libraries. The resulting data is of high quality and complexity, with minimal technical noise or sequencing artefacts. Similarly, there is no other scRNA-seq method using a thermostable ligase prior to the present invention. For the relevant methods provided herein, it was demonstrated that use of a thermostable ligase is effective for preparing single-cell transcriptome libraries. The resulting data is of high quality and complexity, with minimal technical noise or sequencing artefacts.
By employing droplet microfluidics for the second index, about 750,000 sequences can be used for the second combinatorial barcoding round in the methods of the invention. This results in roughly 288 million barcode possibilities when using a 384-well plate for the first indexing round (384×750,000). Two rounds of state-of-the-art combinatorial indexing in 384-well plates only results in 147,456 combinations. The combination of combinatorial indexing and microfluidic droplet generators also enables scaling of NGS protocols that-due to their design—are not immediately compatible with three rounds of indexing.
In summary, in the methods of the present invention, a pre-indexing step is used to barcode entire single-cell transcriptomes prior to the microfluidic run. The methods of the invention are not subject to the aforementioned limitation because cells can be distinguished even if they enter the same droplet. Thus, microfluidic droplet generators (but also sub-nanoliter well plates) can be loaded with a much higher number of cells than in existing protocols.
As such, the methods of the present invention can be used, inter alia, as a high content readout for saturation mutagenesis, for instance for the experimental annotation of genetic variants in cells. The methods of the present invention can also be used as a high content readout for synthetic biology, e.g. when a large number of synthesized DNA modules are introduced into cells, both natural and artificial.
Accordingly, the present invention, in a first embodiment, relates to a method for sequencing oligonucleotides comprising RNA, the method comprising the steps of (a) providing permeabilized cells and/or nuclei comprising a first oligonucleotide comprising RNA; (b) combining said cells and/or nuclei of (a) with a second oligonucleotide comprising DNA in a first reaction compartment, wherein the second oligonucleotide comprises at least a first sequence at least partially complementary to a sequence of the first oligonucleotide, a second sequence comprising an indexing sequence, and a third sequence comprising a primer binding site, under conditions to allow annealing of the first sequence of the second oligonucleotide to the first oligonucleotide; (c) reversely transcribing the first oligonucleotide in said cells/nuclei to obtain an elongated second oligonucleotide; (d) combining said cells and/or nuclei obtained in step (c) with a microbead-bound third oligonucleotide in a second reaction compartment, wherein the third oligonucleotide comprises (i) a first sequence corresponding to a fourth sequence comprised in the second oligonucleotide used in step (b); or (ii) a first sequence complementary to a first sequence of a fourth oligonucleotide, wherein the fourth oligonucleotide further comprises a second sequence at least partially complementary to the third sequence of the second oligonucleotide; wherein for (i), the method further comprises a step of second strand DNA synthesis subsequent to step (c) and prior to step (d) and wherein for (ii) the method further comprises a step of DNA ligation; and wherein the third oligonucleotide further comprises a second sequence comprising an indexing sequence and a third sequence comprising a primer binding site; (e) amplifying the DNA oligonucleotides obtained in step (d); and (f) sequencing of amplified DNA oligonucleotides. As discussed herein the permeabilized cells and/or nuclei comprising a first oligonucleotide comprising RNA may also be fixed, for axmaple via chemical cross linking of the RNA to be analyzed on or to cellular structures or on or to structures of the nuclei. Details of this embodiment of an additional fixing step are also provided herein below. The fixation step may in particular of interest when fresh samples, like, non-preserved cells/nuclei (e.g. material that is previously not formalin-fixed) is to be analyzed in accordance with means and methods of the present invention.
Thus, in general, the invention relates to a method for sequencing oligonucleotides comprising RNA. The term “sequence” refers to sequence information about an oligonucleotide or any portion of the oligonucleotide that is two or more units (nucleotides) long. The term can also be used as a reference to the oligonucleotide itself or a relevant portion thereof.
Oligonucleotide sequence information relates to the succession of nucleotide bases in the oligonucleotide, in particular RNA, in particular RNA of the first oligonucleotide as in the methods of the present invention. For example, if the oligonucleotide contains bases Adenine, Guanine, Cytosine, and/or Uracil, or chemical analogs thereof, the oligonucleotide sequence can be represented by a corresponding succession of letters A, G, C, or U, respectively. Such oligonucleotides may be sequenced using the methods of the present invention.
Accordingly, in a first step, the methods of the invention comprise a step of providing permeabilized cells and/or nuclei comprising a first oligonucleotide comprising RNA. The first oligonucleotide comprises RNA. However, the methods of the present invention are not limited by the type of RNA of the first oligonucleotide or as comprised in the cells/nuclei used in the methods of the invention. Thus, the RNA may of any type known to the person skilled in the art. The RNA may preferably be messenger RNA. It may preferably represent parts or the entirety of the transcriptome as comprised in the cells/nuclei used in the methods of the present invention, preferably the transcriptome in its entirety. As such, the RNA comprised in the first oligonucleotide is preferably in the form of messenger RNA (mRNA). As the skilled person will appreciate, mRNA generally comprises a polyadenylated tail at its 3′ end. Accordingly, it is preferred that the first sequence of the second oligonucleotide is at least partially complementary to the 3′ end of the first oligonucleotide, i.e. the poly-A-tail. However, the methods of the present invention are not limited to binding to the 3′ end. Rather, the first sequence of the second oligonucleotide can be at least partially complementary to a sequence of the first oligonucleotide, wherein said sequence is located in 5′ direction from the 3′ end of the first oligonucleotide. This can, inter alia, be used in cases where the target sequence is known or at least partially known.
The cells/nuclei may be present in various states and may be obtained from samples of various states or origins.
For example, in one embodiment, the cells and/or nuclei are obtained from in vitro cultures or fresh or frozen samples. Cells/nuclei might be obtained from preserved tissue samples, such as formalin-fixed paraffin-embedded (FFPE) material.
Within the present invention, the cells/nuclei may be of any origin as long as the cells/nuclei comprise oligonucleotides comprising RNA. For example, the cells may be cell lines, primary cells, blood cells, somatic cells, derived from organoids or xenografts. Furthermore, cells might be obtained from cell preparations used in immune oncology such as, for example, CAR-T cells, CAR-NK cells, modified T cells, B cells, NK cells or other immune cells, or isolated from patients treated with such products. Moreover, cells might be induced pluripotent stem cells (iPS) or embryonic stem cells undergoing natural differentiation or artificially induced reprogramming or transdifferentiation. Accordingly, the nuclei may be derived from any of the above cells, including e.g. blood cells, somatic cells, induced pluripotent stem cells (iPS) or embryonic stem cells. As such, the methods of the present invention can, inter alia, be used in immune oncology (CAR-T cells, CAR-NK cells, bispecific engagers, BiTEs, immune checkpoint blockade, cancer vaccines delivered as mRNA), molecularly targeted cancer therapy, the dissection of drug resistance and toxicity mechanisms and/or target discovery and/or validation.
In further embodiments, the cells and/or nuclei may be obtained from biological material used in forensics, reproductive medicine, regenerative medicine or immune oncology. Accordingly, the cells and/or nuclei may be cells/nuclei derived from a tumor, blood, bone marrow aspirates, lymph nodes and/or cells/nuclei obtained from a microdissected tissue, a blastomere or blastocyst of an embryo, a sperm cell, cells/nuclei obtained from amniotic fluid, or cells/nuclei obtained from buccal swabs. It is preferred that the tumor cells/nuclei are disseminated tumor cells/nuclei, circulating tumor cells/nuclei or cells/nuclei from tumor biopsies. It is furthermore preferred that the blood cells/nuclei are peripheral blood cells/nuclei or cells/nuclei obtained from umbilical cord blood. It is particularly preferred that the RNA oligonucleotides comprised in the cells/nuclei represent the transcriptome of the cells/nuclei.
Within the methods of the present invention, the cells/nuclei are provided in a permeabilized state. The skilled person is well-aware of methods suitable to provide cells/nuclei in said state. For example, methanol permeabilization may be used for whole cells, whereas incomplete lysis with detergents such as Igepal CA-630, Digitonin or Tween-20 may be used. As such, the first reaction compartment may comprise permeabilized intact cells and/or nuclei.
The number of cells in the first reaction compartment is not particularly limited. However, the total number of cells will depend on the lengths chosen for first and second indexing sequences and the number of unique first and second indices in order to ensure proper sample attribution. Typically, in the methods of the present invention, the first reaction compartment comprises 5000 to 10000 cells.
In a second step of the methods of the invention, the cells and/or nuclei comprising the first oligonucleotide comprising RNA are combined with a second oligonucleotide comprising DNA in a first reaction compartment, wherein the second oligonucleotide comprises at least a first sequence at least partially complementary to a sequence of the first oligonucleotide, a second sequence comprising an indexing sequence, and a third sequence comprising a primer binding site, under conditions to allow annealing of the first sequence of the second oligonucleotide to the first oligonucleotide.
In a preferred embodiment of the invention, the cells and/or nuclei comprising the first oligonucleotide comprising RNA are combined with a second oligonucleotide comprising DNA in a first reaction compartment, wherein the second oligonucleotide comprises at least a first sequence at least partially complementary to the 3′-end of the first oligonucleotide, a second sequence comprising an indexing sequence, and a third sequence comprising a primer binding site, under conditions to allow annealing of the first sequence of the second oligonucleotide to the 3′-end of the first oligonucleotide.
As detailed further above, the methods of the invention allow a surprisingly high throughput of cells/nuclei to be analyzed/sequenced. This is at least partially due to the introduction of at least two indexing sequences into the oligonucleotide comprising RNA that is to be analyzed/sequenced. The first of said at least two indexing sequences is introduced by combining the cells/nuclei comprising the first oligonucleotide with a second oligonucleotide comprising DNA in a first reaction compartment, wherein the second oligonucleotide comprises at least a first sequence at least partially complementary to a sequence of the first oligonucleotide, a second sequence comprising an indexing sequence, and a third sequence comprising a primer binding site, under conditions to allow annealing of the first sequence of the second oligonucleotide to the first oligonucleotide. In a particular embodiment, the first of at least two indexing sequences is introduced by combining the cells/nuclei comprising the first oligonucleotide comprising RNA with a second oligonucleotide comprising DNA in a first reaction compartment, wherein the second oligonucleotide comprises at least a first sequence at least partially complementary to the 3′-end of the first oligonucleotide, a second sequence comprising an indexing sequence, and a third sequence comprising a primer binding site, under conditions to allow annealing of the first sequence of the second oligonucleotide to the 3′-end of the first oligonucleotide.
Accordingly, a second oligonucleotide is employed in the methods of the present invention. The second oligonucleotide comprises DNA and at least three functional sequences/parts. A first sequence of the second oligonucleotide is at least partially complementary to a sequence of the first oligonucleotide, preferably to the 3′end of the first oligonucleotide. As described above, it is preferred within the present invention that the first oligonucleotide comprising RNA comprises a polyadenylated 3′ end, for example as generally comprised in mRNA. Thus, it is preferred that the first sequence of the second oligonucleotide employed in the methods of the present invention comprises a sequence at least partially complementary to the 3′-end of the first oligonucleotide, in particular a sequence predominantly comprising thymine residues or consisting of thymine residues. As such, the first sequence of the second oligonucleotide may partially or completely anneal to the 3′ end of the first oligonucleotide. Provided is thus a method, wherein the first sequence of the second oligonucleotide is complementary to the 3′ poly-A tail of the first oligonucleotide. However, as also provided herein, the methods of the invention are not limited to the first sequence of the second oligonucleotide being at least partially complementary to the poly-A-tail of the first oligonucleotide. The first sequence of the second oligonucleotide can be at least partially complementary to a sequence lying 5′ from the 3′ end of the first oligonucleotide.
The second sequence/part of the second oligonucleotide comprises or consists of an indexing sequence. The term “indexing sequence” is known to the person skilled in the art, although it is surprising that an indexing sequence is used as part of the second oligonucleotide employed in the methods of the invention.
The term “indexing sequence” in accordance with the invention is to be understood as a sequence of nucleotides that is known or may not be known, wherein each position has an independent and equal probability of being any nucleotide. In a preferred embodiment of the methods of the present invention, the first indexing sequence is known and the second indexing sequence may be known or unknown. The nucleotides of the indexing sequence can be any of the nucleotides, for example G, A, C, T, U, or chemical analogs thereof, in any order, wherein: G is understood to represent guanylic nucleotides, A adenylic nucleotides, T thymidylic nucleotides, C cytidylic nucleotides and U uracylic nucleotides. The skilled person will appreciate that known oligonucleotide synthesis methods may inherently lead to unequal representation of nucleotides G, A, C, T or U. For example, synthesis may lead to an overrepresentation of nucleotides, such as G in randomized DNA sequences. This may lead to a reduced number of unique sequences as expected based on an equal representation of nucleotides. However, the skilled person is well aware that the overall number of unique sequences comprised in the second oligonucleotide used in the methods of the invention will generally be sufficient to clearly identify each target RNA comprising oligonucleotide. This is because the skilled person will also be aware of the fact that the length of the indexing sequence may be varied depending on the number of expected first oligonucleotides. The expected number of first oligonucleotides may be derived from the number of genes expected to be expressed and/or the number of cells/nuclei expected to be analyzed/sequenced. Accordingly, the potential unequal representation of nucleotides in the indexing sequence of the second oligonucleotide used in the methods of the invention, which is due to unequal coupling efficiencies of nucleotides in known standard oligonucleotide synthesis methods, can easily be taken into account by the skilled person based on the general knowledge in the art. In particular, the skilled person is well aware that the length of the indexing sequence may be increased in order to obtain an increased number of unique sequences.
The third sequence comprised in the second oligonucleotide used in the methods of the present invention comprises a primer binding site. The skilled person is well aware of suitable sequences. As such, any sequence can be employed as long as a primer employed in the methods of the present invention is allowed to bind to the third sequence of the second oligonucleotide used in the methods of the present invention.
Within the methods of the present invention, the first sequence of the second oligonucleotide is allowed to anneal to a sequence comprised in the first oligonucleotide, preferably to the 3′ end of the first oligonucleotide. The skilled person is well aware of conditions allowing the annealing of these sequences to each other. Within the present invention, the constitution of the first sequence of the second oligonucleotide favours the annealing. Namely, the first sequence of the second oligonucleotide predominantly comprises nucleotides complementary to nucleotides comprised in the target sequence of the first oligonucleotide, preferably constituting the 3′-end of the first oligonucleotide. In a preferred embodiment, the 3′ end of the first oligonucleotide comprises adenine nucleotides and as such will anneal to thymine nucleotides comprised in the first sequence of the second oligonucleotide.
In certain embodiments of the invention, the second oligonucleotide further comprises a unique molecular identifier (UMI).
Subsequent to annealing of the first sequence of the second oligonucleotide to the first oligonucleotide, preferably to the 3′ end of the first oligonucleotide, the methods of the present invention comprise a step of reversely transcribing the first oligonucleotide in said cells/nuclei to obtain an elongated second oligonucleotide. The skilled person is well-aware of means and methods that can be employed to reversely transcribe the first oligonucleotide within the methods of the present invention. More specifically, the reaction will generally involve the use of a reverse transcriptase enzyme. In certain embodiments of this invention a reverse transcriptase with the ability to add untemplated nucleotides might be preferred.
Reverse transcriptases are enzymes composed of distinct domains that exhibit different biochemical activities. RNA-dependent DNA polymerase activity and RNase H activity are the predominant functions of reverse transcriptases, although depending on the source organisms there are variations in functions, including, for example, DNA-dependent DNA polymerase activity. The reverse transcription process typically involves a number of steps:
In the presence of an annealed primer, reverse transcriptase binds to an RNA template and initiates the reaction. RNA-dependent DNA polymerase activity synthesizes the complementary DNA (cDNA) strand, incorporating dNTPs. Optional RNase H activity degrades the RNA template of the DNA:RNA complex. DNA-dependent DNA polymerase activity (if present) recognizes the single-stranded cDNA as a template, uses an RNA fragment as a primer, and synthesizes the second-strand cDNA to form double-stranded cDNA. In the methods of the present invention, various types of reverse transcriptase enzymes can be used, in particular enzymes having RNA-dependent DNA polymerase activity only or enzymes having RNA-dependent DNA polymerase activity combined with RNase H activity. Enzymes having all of the above three activities may also be used.
For example, the method may be carried out by incubating the first reaction compartment, for example a multi-well plate, for a given time at an elevated temperature, for example for 5 or more minutes at about 55° C., such that RNA secondary structures are resolved. Subsequent to resolving secondary structures, the first reaction compartment may be placed on ice to prevent their re-formation. Then, a reaction mix comprising buffer, dNTPs and a reverse transcription enzyme may be added to initiate the reverse transcription reaction. Additives such as RNase inhibitors or DTT might be added to the reaction. Preferably, the reaction is carried out at increasing temperatures starting with about 4° C. and gradually increasing the temperature to about 55° C.
Certain reverse transcriptases may also display terminal nucleotidyl transferase (TdT) activity, which results in non-template-directed addition of nucleotides to the 3′ end of the synthesized DNA. TdT activity occurs only when the reverse transcriptase reaches the 5′ end of the RNA template, adds extra nucleotides to the cDNA end, and exhibits specificity towards double-stranded nucleic acid substrates (e.g., DNA:RNA in the first-strand cDNA synthesis and DNA:DNA in the second-strand cDNA synthesis). An exemplary reverse transcriptase enzyme having such activity is Maxima H Minus RT. While this activity is oftentimes undesirable because the added nucleotides do not correspond to the template, the methods of the invention may comprise the use of such enzymes. As such, in a particular embodiment, the methods of the invention comprise a step (c), wherein untemplated nucleotides are added to the 3′-end of the second oligonucleotide. In a more particular embodiment of the invention, second strand DNA synthesis may then comprises the use of primers comprising a sequence complementary to the added untemplated nucleotides.
Accordingly, subsequently to reverse transcription, the methods of the invention may comprise a step of second strand DNA synthesis to obtain double-stranded cDNA.
Subsequent to reverse transcription and/or second strand DNA synthesis, the methods of the invention comprise the transfer of the permeabilized cells/nuclei to a second reaction compartment. At this stage, the cells/nuclei are permeabilized but preferably still intact, that is non-lysed. As such, the methods of the present invention allow using permeabilized intact cells/nuclei during the first indexing reaction, whereas methods of the prior art comprise a lysis step prior to the first indexing reaction.
The second reaction compartment may be a microfluidic droplet or a microtiter plate. The microtiter plate may be a miniaturized microtiter plate. In another embodiment of the invention, both the first and second reaction compartment may be generated by a microfluidic droplet generator or may be a miniaturized plate. Within the present invention, both reaction compartments may also be standard microwell plates. Exemplary plates include Seq-Well (Gierahn et al. (2017) Nature Methods 14, 395-8) or Microwell-seq (Han et al. (2018) Cell 172(5), 1091-1107).
In the second reaction compartment, the cells and/or nuclei obtained in step (c) are combined with a microbead-bound third oligonucleotide, wherein the third oligonucleotide comprises
The cells/nuclei may be lysed subsequent to transfer to the second reaction compartment. As such, the second reaction compartment may comprise lysed cells/nuclei.
The third oligonucleotide used in the methods of the present invention comprises at least three functional parts/sequences and is initially bound to a microbead. In the second reaction compartment, the microbead may be dissolved and the third oligonucleotide released. A first sequence comprised in the third oligonucleotide is used to either directly or indirectly direct the cDNA comprised in the cells/nuclei obtained in the previous method steps to the microbead-bound third oligonucleotide.
Whether the first sequence of the third oligonucleotide binds the cDNA directly or indirectly depends on the presence of a second strand DNA synthesis step prior to combining the cDNA with the microbead-bound third oligonucleotide. In one embodiment, the first sequence of the third oligonucleotide may correspond to a fourth sequence part of the second oligonucleotide. As the skilled person will appreciate, a sequence corresponding to a part of the second oligonucleotide will be complementary to the synthesized second strand DNA. As such, this embodiment of the invention comprises a step of second strand DNA synthesis subsequent to step (c) and prior to step (d).
In a preferred embodiment of the invention, second strand DNA synthesis comprises introducing nicks in the first oligonucleotide; extending nicked oligonucleotides; and ligating extended oligonucleotides. The nicks may be introduced by addition of a further enzyme, for example RNase H. As detailed above, the reverse transcriptase enzyme may have RNase H activity and may thus also be used to introduce nicks in the first oligonucleotide. The nicked oligonucleotides are then extended by the reverse transcriptase enzyme and/or a further enzyme such as a DNA polymerase and are subsequently ligated to form cDNA oligonucleotides for further processing.
The methods of the present invention may further comprise subsequent to or concurrently with second strand DNA synthesis a step of introducing untemplated nucleotides at the 5′-end of the synthesized second strand DNA. Preferably, untemplated nucleotides are introduced using a transposase enzyme, in particular Tn5 transposase.
Transposase is an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. Transposases are classified under EC number EC 2.7.7. Genes encoding transposases are widespread in the genomes of most organisms and are the most abundant genes known. A preferred transposase within the context of the present invention is Transposase (Tnp) Tn5, in particular a customized transposase. Tn5 is a member of the RNase superfamily of proteins which includes retroviral integrases. Tn5 can be found in Shewanella and Escherichia bacteria. The transposon codes for antibiotic resistance to kanamycin and other aminoglycoside antibiotics. Tn5 and other transposases are notably inactive. Because DNA transposition events are inherently mutagenic, the low activity of transposases is necessary to reduce the risk of causing a fatal mutation in the host, and thus eliminating the transposable element. One of the reasons Tn5 is so unreactive is because the N- and C-termini are located in relatively close proximity to one another and tend to inhibit each other. This was elucidated by the characterization of several mutations which resulted in hyperactive forms of transposases. One such mutation, L372P, is a mutation of amino acid 372 in the Tn5 transposase. This amino acid is generally a leucine residue in the middle of an alpha helix. When this leucine is replaced with a proline residue the alpha helix is broken, introducing a conformational change to the C-Terminal domain, separating it from the N-Terminal domain enough to promote higher activity of the protein. Accordingly, it is preferred that such a modified transposase be used, which has a higher activity than the naturally occurring Tn5 transposase. In addition, it is particularly preferred that the transposase employed in the methods of the invention is loaded with oligonucleotides, which are inserted into the target double-stranded oligonucleotide, preferably loaded with untemplated nucleotides.
Accordingly, it is preferred to use a hyperactive Tn5 transposase and a Tn5-type transposase recognition site (Goryshin and Reznikoff, J. Biol. Chem., 273:7367 (1998)), or MuA transposase and a Mu transposase recognition site comprising RI and R2 end sequences (Mizuuchi, K., Cell, 35: 785, 1983; Savilahti, H, et al, EMBO J., 14: 4893, 1995). More examples of transposition systems that can be used in the methods of the present invention include Staphylococcus aureus Tn552 (Colegio et al, J. Bacteriol, 183: 2384-8, 2001; Kirby C et al, Mol. Microbiol, 43: 173-86, 2002), Tyl (Devine & Boeke, Nucleic Acids Res., 22: 3765-72, 1994 and International Publication WO 95/23875), Transposon Tn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, Review in: Curr Top Microbiol Immunol, 204:27-48, 1996), Tn/O and IS 10 (Kleckner N, et al, Curr Top Microbiol Immunol, 204:49-82, 1996), Mariner transposase (Lampe D J, et al, EMBO J., 15: 5470-9, 1996), Tel (Plasterk R H, Curr. Topics Microbiol. Immunol, 204: 125-43, 1996), P Element (Gloor, G B, Methods Mol. Biol, 260: 97-1 14, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. 265: 18829-32, 1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol. 204: 1-26, 1996), retroviruses (Brown, et al, Proc Natl Acad Sci USA, 86:2525-9, 1989), and retrotransposon of yeast (Boeke & Corces, Annu Rev Microbiol. 43:403-34, 1989). More examples include IS5, TnIO, Tn903, IS91 1, and engineered versions of transposase family enzymes (Zhang et al, (2009) PLOS Genet. 5:el000689. Epub 2009 Oct. 16; Wilson C. et al (2007) J. Microbiol. Methods 71:332-5) and those described in U.S. Pat. Nos. 5,925,545; 5,965,443; 6,437,109; 6,159,736; 6,406,896; 7,083,980; 7,316,903; 7,608,434; 6,294,385; 7,067,644, 7,527,966; and International Patent Publication No. WO2012103545, all of which are specifically incorporated herein by reference in their entirety.
While any buffer suitable for the used transposase may be used in the methods of the present invention, it is preferred to use a buffer particularly suitable for efficient enzymatic reaction of the used transposase. In this regard, a buffer comprising dimethylformamide is particularly preferred for use in the methods of the present invention, in particular during the transposase reaction. In addition, buffers comprising alternative buffering systems including TAPS, Tris-acetate or similar systems can be used. Moreover, crowding reagents as polyethylenglycol (PEG) are particularly useful to increase tagmentation efficiency of very low amounts of DNA. Particularly useful conditions for the tagmentation reaction are described by Picelli et al. (2014) Genome Res. 24:2033-2040.
The transposase enzyme catalyzes the insertion of a nucleic acid, in particular a DNA in a target nucleic acid, in particular target DNA. The transposase used in the methods of the present invention is loaded with oligonucleotides, which are inserted into the target nucleic acid, in particular the target DNA. The complex of transposase and oligonucleotide is also referred to as transposome. Preferably, the transposome is a heterodimer comprising two different oligonucleotides for integration. In this regard, the oligonucleotides that are loaded onto the transposase comprise multiple sequences. In particular, the oligonucleotides comprise, at least, a first sequence and a second sequence. The first sequence is necessary for loading the oligonucleotide onto the transposase. Exemplary sequences for loading the oligonucleotide onto the transposase are given in US 2010/0120098. The second sequence comprises a linker sequence necessary for primer binding during amplification, in particular during PCR amplification, optionally further comprising untemplated nucleotides. Accordingly, the oligonucleotide comprising the first and second sequence is inserted in the target nucleic acid, in particular the target DNA, by the transposase enzyme. The oligonucleotide may further comprise sequences comprising barcode sequences. Barcode sequences may be random sequences or defined sequences. In this regard, the term “random sequence” in accordance with the invention is to be understood as a sequence of nucleotides, wherein each position has an independent and equal probability of being any nucleotide. The random nucleotides can be any of the nucleotides, for example G, A, C, T, U, or chemical analogs thereof, in any order, wherein: G is understood to represent guanylic nucleotides, A adenylic nucleotides, T thymidylic nucleotides, C cytidylic nucleotides and U uracylic nucleotides. The skilled person will appreciate that known oligonucleotide synthesis methods may inherently lead to unequal representation of nucleotides G, A, C, T or U. For example, synthesis may lead to an overrepresentation of nucleotides, such as G in randomized DNA sequences. This may lead to a reduced number of unique random sequences as expected based on an equal representation of nucleotides. The oligonucleotide for insertion into the target nucleic acid, in particular DNA, may further comprise sequencing adaptors.
The person skilled in the art is well-aware that the time required for the used transposase to efficiently integrate a nucleic acid, in particular a DNA, in a target nucleic acid, in particular target DNA, can vary depending on various parameters, like buffer components, temperature and the like. Accordingly, the person skilled in the art is well-aware that various incubation times may be tested/applied before an optimal incubation time is found. Other factors may be the ratio of transposomes to tagmented DNA. Optimal in this regard refers to the optimal time taking into account integration efficiency and/or required time for performing the methods of the invention.
The first sequence of the third oligonucleotide may alternatively be complementary to a first sequence of a fourth oligonucleotide present in the second reaction compartment. Accordingly, the third oligonucleotide may comprise a first sequence complementary to a first sequence of a fourth oligonucleotide, wherein the fourth oligonucleotide further s a second sequence at least partially complementary to the third sequence of the second oligonucleotide. The presence of the fourth oligonucleotide directs the second oligonucleotide to the third oligonucleotide. In this embodiment, the second oligonucleotide is then ligated to the third oligonucleotide. As the skilled will appreciate, in this embodiment, the second oligonucleotide comprises a 5′-phosphorylation for ligation. In this embodiment, the fourth oligonucleotide is preferably blocked on its 3′-end to prevent extension by DNA polymerases. Thus, in this embodiment, the method further comprises a step of DNA ligation to obtain an oligonucleotide comprising the second and third oligonucleotide. In a preferred embodiment of this invention, the ligase is thermostable. Exemplary thermostable ligases include, but are not limited to, Ampligase (Lucigen) or Taq HiFi DNA Ligase (New England Biolabs). This allows the use of heat denaturation and cooling, i.e. temperature cycles, to anneal the second, third and fourth oligonucleotides without compromising the activity of the ligase. Specifically, emulsion droplets containing said oligonucleotides and the ligase enzyme can be subjected to multiple rounds of thermal cycling between heat denaturation and annealing, which allows efficient annealing and ligation.
In the methods of the present invention, the third oligonucleotide further comprises a second sequence comprising an indexing sequence and a third sequence comprising a primer binding site. As such, a second indexing sequence is introduced in the methods of the present invention. The combined use of the first and second indexing sequences enables the surprisingly high throughput of cells/nuclei achieved in the methods of the present invention. This is because due to the presence of two independent indexing sequences, the second reaction compartment in the methods of the present invention may comprise more than one cell/nuclei per microbead, preferably 10 cells/nuclei per microbead. Methods of the prior art allow much lower throughput, because the number of cells/nuclei is limited in theory to 1 cell/nuclei per microbead in order to ensure that RNA of a cell/nuclei receives a unique indexing sequence. In practice, methods of the prior art are even further limited due to practical reasons to 0.1-0.2 cells/nuclei per microbead.
The methods of the present invention further comprise a step of amplifying the DNA oligonucleotides obtained by combining the second and third oligonucleotides, optionally together with the fourth oligonucleotide. This step comprises linear extension for incorporation of the second indexing sequence comprised in the third oligonucleotide and amplification for sequencing.
The methods of the invention then comprise a step of sequencing of amplified DNA oligonucleotides.
The skilled person is well-aware of methods suitable to sequence DNA oligonucleotides. Exemplary, non-limiting methods to be used in order to determine the sequence of an oligonucleotide are e.g. methods for sequencing of nucleic acids (e.g. Sanger di-deoxy sequencing), massive parallel sequencing methods such as pyrosequencing, reverse dye terminator, proton detection, phospholinked fluorescent nucleotides or nanopore sequencing.
In particular, the resulting amplified oligonucleotides may be subjected to either conventional Sanger-based dideoxy nucleotide sequencing methods or employing novel massive parallel sequencing methods (“next generation sequencing”) such as those marketed by Roche (454 technology), Illumina (e.g. Solexa technology, sequencing-by-synthesis technology), ABI (Solid technology), Oxford Nanopore (e.g. nanopore sequencing) or Pacific Biosciences (SMRT technology). It is preferred to use the Illumina NextSeq 500/550 platform, the Illumina NovaSeq 6000 platform, or the NextSeq 1000/2000 platform for sequencing.
Various steps of the methods of the invention involve oligonucleotide generation and/or amplification. Such reactions, as well as the sequencing reaction, may comprise the use of primer sequences.
Accordingly, the present invention relates to an oligonucleotide capable of specifically amplifying the oligonucleotides of the present invention. Accordingly, oligonucleotides within the meaning of the invention may be capable of serving as a starting point for amplification, i.e. may be capable of serving as primers. Such oligonucleotide may comprise oligoribo- or desoxyribonucleotides which are complementary to a region of one of the strands of an oligonucleotide. According to the present invention, a person skilled in the art would readily understand that the term “primer” may also refer to a pair of primers that are with respect to a complementary region of an oligonucleotide directed in the opposite direction towards each other to enable, for example, amplification by polymerase chain reaction (PCR). Purification of the primer(s) is generally envisaged, prior to its/their use in the method of the present invention. Such purification steps can comprise HPLC (high performance liquid chromatography) or PAGE (polyacrylamide gel-electrophoresis), and are known to the person skilled in the art.
When used in the context of primers, the term “specifically” means that preferably or exclusively the desired oligonucleotides as described herein are amplified. Thus, a primer according to the invention is preferably a primer, which binds to a region of an oligonucleotide which is unique for this molecule. In connection with a pair of primers, according to the invention, it is possible that one of the primers of the pair is specific in the above described meaning or both of the primers of the pair are specific.
The 3′-OH end of a primer is used by a polymerase to be extended by successive incorporation of nucleotides. Preferably, the primer or pair of primers of the present invention are used for amplification reactions on template oligonucleotides. The term “template” refers to oligonucleotides or fragments thereof of any source or composition, that comprise a target oligonucleotide sequence. It is known that the length of a primer results from different parameters (Gillam, Gene 8 (1979), 81-97; Innis, PCR Protocols: A guide to methods and applications, Academic Press, San Diego, USA (1990)). Preferably, the primer should only hybridize or bind to a specific region of a target oligonucleotide. The length of a primer that statistically hybridizes only to one region of a target nucleotide sequence can be calculated by the following formula: (¼)x(whereby x is the length of the primer). However, it is known that a primer exactly matching to a complementary template strand must be at least 9 base pairs in length, otherwise no stable-double strand can be generated (Goulian, Biochemistry 12 (1973), 2893-2901). It is also envisaged that computer-based algorithms can be used to design primers capable of amplifying DNA. It is also envisaged that the primer or pair of primers is labeled. The label may, for example, be a radioactive label, such as 32P, 33P or 35S. In a preferred embodiment of the invention, the label is a non-radioactive label, for example, digoxigenin, biotin and fluorescence dye or dyes.
The invention furthermore relates to the use of a microfluidic system, in particular a microfluidic droplet generator, in the methods of the invention. The microfluidic system may be in particular used to generate (microfluidic) droplets or to deliver material into a well- or chamber-based device, like into microfluidic well-based device Such devices are known in the art and are, inter alia, based on integrated fluidic circuit technologies. An example of such a provider for such devices is Fluidigm Corporation/U.S.A. Accordingly, the generation of (microfluidic) droplets or the delivery of material into a well- or chamber-based device may also be part of the methods of the present invention. An exemplary droplet generator is the Chromium™ Controller provided by 10× Genomics (Pleasanton, CA). Further examples include Drop-seq and inDrop platforms. Moreover, the invention can be used to boost the throughput of sub-nanoliter well based platforms such as CytoSeq (Fan et al., 2015), Seq-Well (Gierahn et al., 2017), Microwell-Seq (Han et al, 2018) or microfluidic systems with built-in reaction chambers. A compatible commercial version is the above mentioned BD Rhapsody™ system on which the methods of the invention can be shown to provide surprising results.
The methods of the invention may further comprise an additional layer of multiplexing by cell hashing.
As provided herein, the methods of the present invention may be used in synthetic biology. For example, the methods of the present invention may be used with a gene panel readout (e.g. a few 10s to 100s of specifically assayed genes instead of a whole-transcriptome readout). As such, provided is a device that uses single-cell RNA-seq, the methods of the present invention, to replace flow cytometry as a key diagnostic assay (especially when combined with barcoded antibodies and/or TCR/BCR immune repertoire profiling) in cancer, immune disorders, and many other diseases. In a further envisaged embodiment, the methods of the present invention are combined with guide-RNA enrichment for massive-scale CRISPR single-cell sequencing (CROP-seq, Perturb-seq, etc. —using CRISPR knockout, CRISPR activation, CRISPR knockdown, CRISPR knock-in of natural or synthetic sequences, CRISPR epigenome editing, saturation mutagenesis or similar assays for the perturbation step) with hypothesis-driven gene set/pathway readout.
Further provided are the methods of the present invention combined with ChIPmentation as described in WO 2017/025594 as a separate assay based on the same technology (e.g. for single-cell epigenome profiling) or combined with guide-RNA enrichment (e.g. for epigenome-based CROP-seq screens).
The methods of the invention also provided for use in drug discovery, drug screening, testing of compounds and/or target validation. As such, the methods of the invention are able to derive, inter alia, relevant screening signatures directly from the transcriptome of control cells, so that no prior knowledge about the mechanism of action of a drug and/or test compound is required. Moreover, the single-cell resolution of the methods of the invention allows to assess the effect of a drug/test compound to be screened on different cell types in a complex mixture (for instance, but not limited to, PBMCs), or on a mixture of cells from distinct donors.
Accordingly, provided herein, is a method for identifying and/or screening a test compound able to alter the transcriptome of a cell, the method comprising the steps of:
In the above method, said “first oligonucleotide comprising RNA” as comprised in said cells and/or nuclei may be a naturally occurring RNA but may also be a synthetically synthesized, chimeric and/or artificial RNA construct, like an guide RNA and/or shRNAs as employed in the CRISPR technology, a viral or viral derived nucleic acid as, inter alia, used for gene transfers, etc. Non-limiting examples of such “first oligonucleotides comprising RNA” include: the cell's naturally occurring transcriptome, other naturally occurring or artificial small RNAs, such as tRNA, snRNA, snoRNA, micro-RNA, rRNA, synthetic biology tools such as riboswitches and RNA aptamers, combinations of RNAs as employed in CRISPR technologies, like combinations of guide RNAs or shRNAs in the same cell e.g. (co-essentiality, combined action), synthetic genes and synthetic mutagenized gene libraries, RNA barcodes, e.g. to mark sample of origin, spatial location, treatment, transgenes, RNA barcodes from lineage tracing experiments, RNA barcodes connected to antibodies expressed in a given cell, RNA barcodes marking the location on a tissue slice, RNA barcodes marking cell-cell interactions, RNA barcodes that label (cell surface) proteins (intracellular proteins or modified amino acid residues (for example via antibodies), RNA barcodes used as synthetic readers of biological processes, viral RNA, for example to assess the infection state of a cell, immune receptors such as chimeric antigen receptors or T cell receptors, (synthetic) transcription factors, (synthetic) homing receptors, etc.
As with all means and methods as provided in the present invention, also this method for identifying and/or screening a test compound able to alter the transcriptome of a cell as provided herein and comprising the step “permeabilized cells and/or nuclei comprising a first oligonucleotide comprising RNA” (herein above in step (b)), may comprise an additional, optional step wherein said cells/nuclei are fixed. Fixation of cells/nuclei are known in the art and comprise, inter alia but preferably, chemical cross-linking (like, e.g, with formaldehyde or alcohols, like methanol). This fixing step may comprise the fixing of the RNAs to be analyzed in context of the herein provided methods in and on their cellular context, for example, on structural components of the cells/nuclei etc. Such an optional fixing step has also the advantage that said cells/nuclei may be preserved/conserved and/or that these fixed cells/nuclei may be employed/analyzed at a later point of time. Such a preservation/conservation may comprise freezing of said permeabilized and fixed cells/nuclei.
Said one or more test compound(s) to be screened/validated/identified and/or used in the method as recited above may be selected from the group of small molecules, large molecules, RNA, DNA and other compounds, including chemical compounds and/or pharmaceuticals. But also biological material and/or pathogens may be the “test compound” to be screened/identified and/or used in the methods of the present invention. Such biological material and/or pathogens may comprise bacteria, viruses, fungi and/or other biological material, like multicellular pathogens, like nematodes, jellyfish, etc. The term “biological material and/or pathogens” also comprises parts of said materials/pathogens, like, inter alia, proteins, peptides, nucleic acids, mixtures of such materials/pathogens, extracts etc. Said test compound(s) may also be a compound or group of compounds resulting in genetic perturbations, such as CRISPR modifications and/or edits in the genome of the cell and/or nuclei.
Further examples of the “test compound” to be employed in the methods of the present invention comprise, but are not limited to, compounds that lead to a status modification and/or change in a given cell, like a change in differentiation status or leading to apoptosis. The “test compound” may also be an mRNA to be introduced in the cells/nuclei, a plasmid, a viral vector etc. Such compounds may also be used, inter alia, for gene transfer. Such “coding” nucleic acids and/or gene transfer shuttles may encode, without being limiting for transcription factors, epigenetic regulators, kinases, homing receptors to control the localization of cells within an organism or tissue, immune co-stimulatory domains (such as 41BB, CD27, CD28, OX40, CD2, or CD40L), or immune co-inhibitory domains (such as BTLA, CTLA4, LAG3, LAIR1, PD-1, TIGIT or TIM3). Also constituents of receptor/ligand systems (or isolated parts thereof, like extracellular domains and/or soluble parts) may be employed as “test compounds”. Non-limiting examples of such receptor/ligand systems include, inter alia, molecules of signaling pathways and/or immunomodulation pathways, like the PD-1/PD-L1/PD-L2 system(s), or CD40/CD40L system(s), B7-1, B7-2, etc.
As is evident from the current description and in context of this invention, the examples for “test compounds” as provided herein above are not limited to the above discussed “method for identifying and/or screening a test compound able to alter the transcriptome of a cell”. These “test compounds” may be also employed in the general method for sequencing oligonucleotides provided herein, i.e. in the inventive scifi-RNA-seq method and variations thereof.
The methods of the invention may also combine various steps as also illustrated herein and in the appended examples. Particularly preferred are versions of the invention, like EXT-TN5 (Example 3), LIG-TS (Example 4), EXT-RP (Example 5), LIG-RP (Example 6) and/or EXT-TS (Example 7). Each of these versions of the inventive mean and method are particularly useful to increase the number of uniquely labeled cells and thus the throughput as compared to existing methods.
Thus, in a particular embodiment, the present invention relates to a method for sequencing oligonucleotides comprising RNA (EXT-TN5), the method comprising the steps of:
In a particular embodiment, the present invention relates to a method for sequencing oligonucleotides comprising RNA (LIG-TS), the method comprising the steps of:
In a particular embodiment, the present invention relates to a method for sequencing oligonucleotides comprising RNA (EXT-RP), the method comprising the steps of:
In a particular embodiment, the present invention relates to a method for sequencing oligonucleotides comprising RNA (LIG-RP), the method comprising the steps of:
In a particular embodiment, the present invention relates to a method for sequencing oligonucleotides comprising RNA (EXT-TS), the method comprising the steps of:
The above recited versions of the present invention, like EXT-TN5 (also illustrated in appended Example 3), LIG-TS (also illustrated in appended Example 4), EXT-RP (also illustrated in appended Example 5), LIG-RP (also illustrated in appended Example 6) and EXT-TS (also illustrated in appended Example 7) may also, optionally, comprise an additional step wherein the permeabilized cells and/or nuclei comprising a first oligonucleotide comprising RNA are fixed before the following steps are carried out. Accordingly, if desired, an optional fixation step may be carried out after step (a) as recited for the scifi-RNA-seq method and its variants as provided herein above.
The present invention also relates to kits, in particular research kits. The kits of the present invention comprise the second oligonucleotide of the present invention, preferably together with instructions regarding the use of the methods of the invention. The kits of the invention may further comprise a hyperactive, preferably also oligonucleotide loaded, tranposase and/or reagents for second strand synthesis. The kits of the invention may also comprise the transposase enzyme in a ready-to-use form. Further comprised may be one or more of the other oligonucleotides used in the present invention, for example the fourth oligonucleotide and/or the thermostable ligase. The kits of the invention may be used inter alia in research applications such as the sequencing of RNA molecules.
In a particularly preferred embodiment of the present invention, the kits (to be prepared in context) of this invention or the methods and uses of the invention may further comprise or be provided with (an) instruction manual(s). For example, said instruction manual(s) may guide the skilled person (how) to employ the kit of the invention in the diagnostic uses provided herein and in accordance with the present invention. Particularly, said instruction manual(s) may comprise guidance to use or apply the herein provided methods or uses.
The kit (to be prepared in context) of this invention may further comprise substances/chemicals and/or equipment suitable/required for carrying out the methods and uses of this invention. For example, such substances/chemicals and/or equipment are solvents, diluents and/or buffers for stabilizing and/or storing and/or enabling enzymatic reactions or terminating enzymatic reactions, (a) compound(s) required for the uses provided herein, like stabilizing and/or storing the chemical agent(s) and/or transposase comprised in the kits of the present invention.
Further embodiments are exemplified in the scientific part. The appended figures provide for illustrations of the present invention. Whereas the experimental data in the examples and as illustrated in the appended figures are not considered to be limiting. The technical information comprised therein forms part of this invention.
The invention thus also covers all further features shown in the figures individually, although they may not have been described in the previous or following description. Also, single alternatives of the embodiments described in the figures and the description and single alternatives of features thereof can be disclaimed from the subject matter of the other aspect of the invention.
a) Standard droplet-based scRNA-seq using a microfluidic droplet generator is highly inefficient in its use of the droplets. Most droplets contain both a barcoded microbead and the reverse transcription reagents (and are thus fully functional), but never receive a cell; furthermore, the reagents within a droplet are sufficient to barcode more than one cell. b) scifi-RNA-seq unlocks the full potential of microfluidic droplet generators. Prior to the microfluidic run, entire transcriptomes are pre-indexed by reverse transcription inside permeabilized cells or nuclei (round1 barcodes indicated by letters A to F). The differentially barcoded pool of cells/nuclei is loaded at fill rates e.g. around 10 per droplet. Cells inside the same emulsion droplet are labelled with an identical microfluidic (round2) barcode, but can still be distinguished via their transcriptome (round1) index.
a) Fraction of exact matches for round1 and round2 barcodes. b) Experiment performance of a typical scifi-RNA-seq experiment based on thermoligation and template switching. Left: Reads per cell plotted against unique UMIs per cell reveal that single-cell transcriptomes are highly complex. Right: The rate of unique reads per cell averages around 90% over a wide range of reads sequenced. c) Ranked barcodes plotted against reads reveal a characteristic inflection point that separates cells from background noise. In this particular experiment 15,300 nuclei were loaded into the microfluidic device. d) Species-mixing plot for a 1:1 mixture of human (Jurkat-Cas9-TCR) and mouse (3T3) nuclei.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).
While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. It will be understood that changes and modifications may be made by those of ordinary skill within the scope and spirit of the following claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above and below.
The invention also covers all further features shown in the figures individually, although they may not have been described in the afore or following description. Also, single alternatives of the embodiments described in the figures and the description and single alternatives of features thereof can be disclaimed from the subject matter of the other aspect of the invention.
Furthermore, in the claims the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single unit may fulfil the functions of several features recited in the claims. The terms “essentially”, “about”, “approximately” and the like in connection with an attribute or a value particularly also define exactly the attribute or exactly the value, respectively. Any reference signs in the claims should not be construed as limiting the scope.
1.1 Preparation of Permeabilized Whole Cells from Human and Mouse Cell Lines
5 million cells were washed with 10 ml of ice-cold 1×PBS (Gibco cat. no. 14190-094, centrifugation: 300 rcf, 5 min, 4° C.) and fixed in 5 ml of ice-cold methanol (Fisher Scientific cat. no. M/4000/17) at −20° C. for 10 min. After two additional washes (centrifugation: 300 rcf, 5 min, 4° C.) with 5 ml of ice-cold PBS-BSA-SUPERase (1×PBS supplemented with 1% w/v BSA (Sigma cat. no. A8806-5) and 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific cat. no. AM2696)) permeabilized cells were resuspended in 200 μl of ice-cold PBS-BSA-SUPERase, and filtered through a cell strainer (40 UM or 70 UM depending on the cell size). 10 μl of the sample were used for cell counting on a CASY device (Schärfe System), and diluted to 5,000 cells per μl with ice-cold PBS-BSA-SUPERase. It was immediately proceeded with the reverse transcription step.
1.2 Preparation of Fresh Nuclei from Human and Mouse Cell Lines
5 million cells were washed with 10 ml of ice-cold 1×PBS (Gibco cat. no. 14190-094, 300 rcf, 5 min, 4° C.). Nuclei were prepared by resuspending cells in 500 μl of ice-cold Nuclei Preparation Buffer (10 mM Tris-HCl PH 7.5 (Sigma cat. no. T2944-100ML), 10 mM NaCl (Sigma cat. no. S5150-1L), 3 mM MgCl2 (Ambion cat. no. AM9530G), 1% w/v BSA (Sigma cat. no. A8806-5), 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific cat. no. AM2696), 0.1% v/v Tween-20 (Sigma cat. no. P7949-500ML), 0.1% v/v IGEPAL CA-630 (Sigma cat. no. 18896-50ML), 0.01% v/v Digitonin (Promega cat. no. G944A)), followed by 5 min of incubation on ice. Lysis of the plasma membrane was stopped by adding 5 ml of ice-cold Nuclei Wash Buffer (10 mM Tris-HCl PH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In Rnase Inhibitor, 0.1% v/v Tween-20). Nuclei were collected by centrifugation (500 rcf, 5 min, 4° C.), resuspended in 200 μl of ice-cold PBS-BSA-SUPERase (1×PBS supplemented with 1% w/v BSA and 1% v/v SUPERase-In Rnase Inhibitor (20 U/μl, cat. no.)) and filtered through a cell strainer (40 μM or 70 μM depending on the cell size). 10 μl of the sample were used for cell counting on a CASY device (Schärfe System), and diluted to 5,000 cells per μl with ice-cold PBS-BSA-SUPERase. It was immediately proceeded with the reverse transcription step.
1.3 Preparation of Nuclei from Primary Cells with Formaldehyde Fixation and Permeabilization
5 million primary cells were washed with 10 ml of ice-cold 1×PBS (Gibco cat. no. 14190-094, centrifugation: 300 rcf, 5 min, 4° C.). Nuclei were prepared by resuspending cells in 500 μl of ice-cold Nuclei Preparation Buffer without Digitonin and without Tween-20 (10 mM Tris-HCl PH 7.5 (Sigma cat. no. T2944-100ML), 10 mM NaCl (Sigma cat. no. S5150-1L), 3 mM MgCl2 (Ambion cat. no. AM9530G), 1% w/v BSA (Sigma cat. no. A8806-5), 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific cat. no. AM2696), 0.1% v/v IGEPAL CA-630 (Sigma cat. no. 18896-50ML)), followed by 5 min of incubation on ice. Lysis of the plasma membrane was stopped by addition of 5 ml of Nuclei Wash Buffer without Tween-20 (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In Rnase Inhibitor). Nuclei were collected by centrifugation (500 rcf, 5 min, 4° C.), and fixed in 5 ml of ice-cold 1×PBS containing 4% Formaldehyde (Thermo Fisher Scientific cat. no. 28908) for 15 min on ice. Fixed nuclei were collected (500 rcf, 5 min, 4° C.), the pellet was resuspended in 1.5 ml of ice-cold Nuclei Wash Buffer without Tween-20 and transferred to a 1.5 ml tube. After one more wash with 1.5 ml of ice-cold Nuclei Wash Buffer without Tween-20 (500 rcf, 5 min, 4° C.), fixed nuclei were resuspended in 200 μl of Nuclei Wash Buffer without Tween-20, snap-frozen in liquid nitrogen and stored at −80° C.
For processing with scifi-RNA-seq, frozen samples were thawed in a 37° C. water bath for exactly 1 min, and immediately placed on ice. Following centrifugation (500 rcf, 5 min, 4° C.), fixed nuclei were resuspended in 250 μl of ice-cold Permeabilization Buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In Rnase Inhibitor, 0.01% v/v Digitonin (Promega cat. no. G944A), 0.1% v/v Tween-20 (Sigma cat. no P7949-500ML)). After 5 min of incubation in ice, 250 μl of Nuclei Wash Buffer without Tween-20 were added per sample, and nuclei were collected (500 rcf, 5 min, 4° C.). After one more wash with 250 μl of Nuclei Wash Buffer without Tween-20, nuclei were taken up in 100 μl of 1×PBS containing 1% w/v BSA and 1% v/v SUPERase-In Rnase Inhibitor. 5 μl of the sample were used for cell counting on a CASY device (Schärfe Systems), and diluted to 5,000 cells per μl with PBS-BSA-SUPERase. It was immediately proceeded with the reverse transcription step.
Human Jurkat cells (clone E6-1) were cultured in RPMI medium (Gibco cat. no. 21875-034) supplemented with 10% FCS (Sigma) and penicillin-streptomycin (Gibco cat. no. 15140122). Fresh nuclei were isolated as described above. Next, samples of 15.3 k, 191 k, 383 k, 765 k and 1.53M nuclei were prepared, 1.5 μl of Reducing Agent B (10× Genomics cat. no. 2000087) and 1× Nuclei Buffer (10× Genomics cat. no. 2000153) were added to a total volume of 80 μl. This buffer does not contain detergents, hence the nuclei remain intact during the microfluidic run and can be visualized inside the emulsion droplets with a standard light microscope. At the same time, Reducing Agent B dissolves the Gel Beads, which might otherwise obstruct the view. The microfluidic chip (Single Cell E Chip, 10× Genomics 2000121) was loaded as follows: 75 μl of nuclei sample at the indicated loading concentrations into inlet 1, 40 μl of Single Cell ATAC Gel Beads (10× Genomics cat. no. 2000132) into inlet 2, and 240 μl of Partitioning Oil (10× Genomics cat. no. 220088) into inlet 3. To image the resulting droplets, 15 μl of Partitioning Oil were pipetted onto a glass slide, followed by 5 μl of emulsion droplets, and images were taken at 10× magnification. An average of 653 droplets per condition were counted.
To measure the bead fill rate, the Single Cell E Chip (10× Genomics 2000121) was loaded with 80 μl of 1× Nuclei Buffer (10× Genomics cat. no. 2000153) into inlet 1, 40 μl of Single Cell ATAC Gel Beads (10× Genomics cat. no. 2000132) into inlet 2, and 240 μl of Partitioning Oil (10× Genomics cat. no. 220088) into inlet 3. By leaving out Reducing Agent B, it was ensured that Gel Beads remain intact throughout the microfluidic run, such that they can be visualized inside the emulsion droplets using a standard light microscope. The fill rate calculations are based on a total of 1,265 droplets.
Reverse Transcription: Sets of 96 and 384 indexed reverse transcription primers were synthesized by Sigma Aldrich and shipped at 100 μM in EB Buffer in 96-well plates. Primers had the sequence (5′-TCGTCGGCAGCGTCGGATGCTGAGTGATTGCTTGTGACGCCTTCNNNNNNNNN XXXXXXXXXXXVTTTTTTTTTTTTTTTTTTTVN-3′), where N indicates a random base, the underlined bases are known for a given primer, and X is an 11-base-long primer-specific index sequence. 96-well plates with barcoded oligo-dT primers were prepared prior to the experiment and stored at −20° C. (1 μl of 25 μM per well). 10,000 permeabilized cells or nuclei (2 μl of a 5,000/μl suspension) were added to the pre-dispensed primers and well assignments were recorded. The plate was incubated for 5 min at 55° C. (to resolve RNA secondary structures), then placed immediately on ice (to prevent their re-formation). Per well, a mix of 3 μl nuclease-free water, 2 μl 5× Superscript IV Buffer, 0.5 μl of 100 mM DTT, 0.5 μl of 10 mM dNTPs (Invitrogen cat. no. 18427-088), 0.5 μl of RNaseOUT RNase inhibitor (40 U/ml, Invitrogen cat. no. 10777019), and 0.5 μl of Superscript IV Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific cat. no. 18090200) was added. The reverse transcription was incubated as follows: (heated lid set to 60° C.), 4° C. for 2 min, 10° C. for 2 min, 20° C. for 2 min, 30° C. for 2 min, 40° C. for 2 min, 50° C. for 2 min, 55° C. for 15 min, storage at 4° C.
Second Strand Synthesis and Cell/Nuclei Recovery: For the second strand synthesis, a mix of 1.33 μl Second Strand Synthesis Reaction Buffer and 0.67 μl Second Strand Synthesis Enzyme Mix (NEB cat. no. E6111L) was added per well, followed by 2 hours of incubation at 16° C. Processed nuclei were recovered from the plates and pooled in one 15 ml tube per plate. Wells were washed with 1×PBS-1% BSA, which was transferred to the same tube for maximum recovery. The volume was topped up to 10 ml with 1×PBS-1% BSA, and nuclei were collected (500 rcf, 5 min, 4° C.). We used two additional wash steps with 1×PBS-1% BSA to remove cellular debris. The resulting pellet was resuspended in 1.5 ml of 1× Nuclei Buffer (10× Genomics cat. no. 2000153), transferred to a 1.5 ml tube and centrifuged (500 rcf, 5 min, 4° C.). The supernatant was removed completely, and the tube was centrifuged briefly (500 rcf, 30 s, 4° C.) to collect the remaining liquid at the bottom of the tube. Typically, this resulted in less than 10 μl of a highly concentrated suspension, which was diluted 1:50 and counted in a Fuchs Rosenthal counting chamber (Incyto cat. no. DHC-F01). Tagmentation: For the tagmentation, processed nuclei were combined with 1× Nuclei Buffer for a total volume of 5 μl, and mixed with 7 μl of ATAC Buffer (10× Genomics cat. no. 2000122) and 6 μl of custom i7-only transposome (prepared as described below). Double-stranded cDNA inside the processed nuclei was tagmented at 37° C. for 1 hour, followed by storage at 4° C.
Linear barcoding: Unused channels in the Chromium Chip E (10× Genomics cat. no. 2000121) were filled with 75 μl (inlet 1), 40 μl (inlet 2) or 240 μl (inlet 3) of 50% glycerol solution (Sigma cat. no. G5516-100ML). Right before loading the chip, a mix of 61.5 μl Barcoding Reagent, 1.5 μl Reducing Agent B and 2.0 μl of Barcoding Enzyme (all from 10× Genomics cat. no. 1000110) was added per tagmentation reaction. The microfluidic chip was loaded with 75 μl of tagmented nuclei in barcoding mix (inlet 1), 40 μl of Single Cell ATAC Gel Beads (inlet 2, 10× Genomics cat. no. 2000132) and 240 μl of Partitioning Oil (inlet 3, 10× Genomics cat. no. 220088) and run on the 10× Genomics Chromium controller. The linear barcoding reaction was incubated as follows: (heated lid set to 105° C., volume set to 125 μl), 72° C. for 5 min, 98° C. for 30 s, 12× (98° C. for 10 s, 59° C. for 30 s, 72° C. for 1 min), storage at 15° C. The emulsion was broken by addition of 125 μl of Recovery Agent (10× Genomics cat. no. 220016) and 125 μl of the pink oil phase were removed by pipetting. The remaining sample was mixed with 200 μl of Dynabead Cleanup Master Mix (per reaction: 182 μl Cleanup Buffer (10× Genomics cat. no. 2000088), 8 μl Dynabeads MyOne Silane (Thermo Fisher Scientific cat. no. 37002D), 5 μl Reducing Agent B (10× Genomics cat. no. 2000087), 5 μl of nuclease-free water). After 10 min of incubation at room temperature, samples were washed twice with 200 μl of freshly prepared 80% ethanol (Merck cat. no. 603-002-00-5) and eluted in 40.5 μl of EB Buffer (Qiagen cat. no. 19086) containing 0.1% Tween (Sigma cat. no. P7949-500ML) and 1% v/v Reducing Agent B. Bead clumps were sheared with a 10 μl pipette or needle. 40 μl of the sample were transferred to a fresh tube strip and subjected to a 1.2× cleanup with SPRIselect beads (Beckman Coulter cat. no. B23318), eluting in 40.5 μl of EB Buffer.
Enrichment PCR: Each sample was enriched in eight separate PCR reactions containing 50 μl of NEBNext High Fidelity 2× Master Mix (NEB cat. no. M0541S), 5 μl of primer 06-11_Partial-P5 (10 μM, 5′-AATGATACGGCGACCACCGAGA-3′), 1 μl of 100×SYBR Green in DMSO (Life Technologies cat. no. S7563), 34 μl of water, 5 μl of indexed 06-11_P7-Read2N-00X primer (10 μM, 5′-CAAGCAGAAGACGGCATACGAGAT[indexi7] GTCTCGTGGGCTCGG-3′) and 5 μl of sample from the previous step. Reactions were incubated in a qPCR machine: 98° C. for 45 s, 40× (98° C. for 20 s, 67° C. for 30 s, 72° C. for 30 s followed by the plate read). During the run, the fluorescence signal was monitored and samples were removed from the thermocycler when they reached saturation. To complete unfinished PCR products, the sample was incubated for 2 min at 72° C. in another thermocycler.
Size selection and quality control: PCR reactions were cleaned with a 0.7× standard SPRI cleanup, followed by a double-sided 0.5×/0.7×SPRI cleanup. The library size distribution was checked on a Bioanalyzer HS chip (Agilent cat. no. 5067-4626 and 5067-4627) and the concentration of dsDNA was measured in a Qubit dsDNA HS assay (Thermo Fisher Scientific cat. no. Q32854).
Reverse Transcription: Sets of 96 and 384 indexed reverse transcription primers were synthesized by Sigma Aldrich and shipped at 100 μM in EB Buffer in 96-well plates. Primers had the sequence (5′-[phos]ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNXXXXXXXXX XXVTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′), where N indicates a random base, the underlined bases are known for a given primer, X is an 11-base-long primer-specific index sequence and a 5′ phosphate group allows the ligation of this oligo. 96-well plates with barcoded oligo-dT primers were prepared prior to the experiment and stored at −20° C. (1 μl of 25 μM per well). 10,000 permeabilized cells or nuclei (2 μl of a 5,000/μl suspension) were added to the pre-dispensed primers and well assignments were recorded. The plate was incubated for 5 min at 55° C. (to resolve RNA secondary structures), then placed immediately on ice (to prevent their re-formation). Per well, a mix of 3 μl nuclease-free water, 2 μl 5× Reverse Transcription Buffer, 0.5 μl of 100 mM DTT, 0.5 μl of 10 mM dNTPs (Invitrogen cat. no. 18427-088), 0.5 μl of RNaseOUT RNase inhibitor (40 U/ml, Invitrogen cat. no. 10777019), and 0.5 μl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific cat. no. EP0753) was added. The reverse transcription was incubated as follows: (heated lid set to 60° C.), 50° C. for 10 min, 3 Cycles of {8° C. for 12 sec, 15° C. for 45 sec, 20° C. for 45 sec, 30° C. for 30 sec, 42° C. for 2 min, 50° C. for 3 min}, 50° C. for 5 min, store at 4° C.
Cell/Nuclei recovery and pooling: Processed cells/nuclei were recovered from the plates and pooled in one 15 ml tube per plate. Wells were washed with 1×PBS-1% BSA, which was transferred to the same tube for maximum recovery. The volume was topped up to 15 ml with 1×PBS-1% BSA, and nuclei were collected (500 rcf, 5 min, 4° C.). The resulting pellet was resuspended in 1.0 ml of 1× HiFi Taq DNA Ligase Buffer (NEB #M0647S) or 1× Ampligase Reaction Buffer (Lucigen #A0102K), filtered through a cell strainer (40 μm or 70 μm depending on the cell/nuclei size) into a 1.5 ml tube and centrifuged (500 rcf, 5 min, 4° C.). The supernatant was removed completely, and the tube was centrifuged briefly (500 rcf, 30 s, 4° C.) to collect the remaining liquid at the bottom of the tube. Typically, this resulted in less than 10 μl of a highly concentrated suspension, which was diluted 1:50 and counted in a Fuchs Rosenthal counting chamber (Incyto cat. no. DHC-F01). The desired number of cells/nuclei was brought to a volume of 15 μl with 1× HiFi Taq DNA Ligase Buffer (NEB #M0647S) or 1× Ampligase Reaction Buffer (Lucigen #A0102K).
Microfluidic thermoligation barcoding: Unused channels in the Chromium Chip E (10× Genomics cat. no. 2000121) were filled with 75 μl (inlet 1), 40 μl (inlet 2) or 240 μl (inlet 3) of 50% glycerol solution (Sigma cat. no. G5516-100ML). Right before loading the chip, a mix of 47.4 μl nuclease-free water, 11.5 μl of either HiFi Taq DNA Ligase Buffer (10×, NEB #M0647S) or Ampligase Reaction Buffer (10×, Lucigen #A0102K), 2.3 μl of either HiFi Taq DNA Ligase (NEB #M0647S) or Ampligase (Lucigen #A0102K), 1.5 μl of Reducing Agent B (10× Genomics cat. no. 2000087) and 2.3 μl of Bridge Oligo (100 HM, 5′-CGTCGTGTAGGGAAAGAGTGTGACGCTGCCGACGA[ddC]-3′) was added per sample. The microfluidic chip was loaded with 75 μl of cells/nuclei in thermoligation mix (inlet 1), 40 μl of Single Cell ATAC Gel Beads (inlet 2, 10× Genomics cat. no. 2000132) and 240 μl of Partitioning Oil (inlet 3, 10× Genomics cat. no. 220088) and run on the 10× Genomics Chromium controller. The thermoligation barcoding reaction was incubated as follows: (heated lid set to 105° C., volume set to 100 μl), 12× (98° C. for 30 s, 59° C. for 2 min), storage at 15° C. The emulsion was broken by addition of 125 μl of Recovery Agent (10× Genomics cat. no. 220016) and 125 μl of the pink oil phase were removed by pipetting. The remaining sample was mixed with 200 μl of Dynabead Cleanup Master Mix (per reaction: 182 μl Cleanup Buffer (10× Genomics cat. no. 2000088), 8 μl Dynabeads MyOne Silane (Thermo Fisher Scientific cat. no. 37002D), 5 μl Reducing Agent B (10× Genomics cat. no. 2000087), 5 μl of nuclease-free water). After 10 min of incubation at room temperature, samples were washed twice with 200 μl of freshly prepared 80% ethanol (Merck cat. no. 603-002-00-5) and eluted in 40.5 μl of EB Buffer (Qiagen cat. no. 19086) containing 0.1% Tween (Sigma cat. no. P7949-500ML) and 1% v/v Reducing Agent B. Bead clumps were sheared with a 10 μl pipette or needle. 40 μl of the sample were transferred to a fresh tube strip and subjected to a 1.0×cleanup with SPRIselect beads (Beckman Coulter cat. no. B23318), eluting in 22 μl of EB Buffer.
Template switching: 20 μl of sample from the previous step were mixed with 10 μl of 5× Reverse Transcription Buffer, 10 μl of Ficoll PM-400 (20%, Sigma #F5415-50ML), 5 μl of 10 mM dNTPs (Invitrogen cat. no. 18427-088), 1.25 μl of Recombinant Ribonuclease Inhibitor (Takara #2313A), 1.25 μl of Template Switching Oligo (100 HM, 5′-AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG-3′, where r indicates RNA bases) and 2.5 μl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific cat. no. EP0753). The template switching reaction was incubated for 30 min at 25° C., 90 min at 42° C., storage at 4° C. and cleaned with a 1.0×SPRI cleanup, eluting in 17 μl of EB buffer.
cDNA enrichment: 15 μl of the above sample were mixed with 33 μl of nuclease-free water, 50 μl of NEBNext High Fidelity 2× Master Mix (NEB #M0541S), 0.5 μl of Partial P5 primer (100 μM, 5′-AATGATACGGCGACCACCGAGA-3′), 0.5 μl of TSO Enrichment Primer (100 μM, 5′-AAGCAGTGGTATCAACGCAGAGT-3′) and 1 μl of SYBR Green (100× in DMSO). cDNA was amplified in a thermocycler: 98° C. for 30 sec, Cycle until fluorescent signal >2000 RFU {98° C. for 20 sec, 65° C. for 30 sec, 72 C for 3 min}, 72° C. for 5 min in another thermocycler, storage at 4° C. cDNA was cleaned by one 0.8×SPRI cleanup followed by a 0.6×SPRI cleanup, quantified with a Qubit HS assay (ThermoFisher Scientific #Q32854) and 1.5 ng were checked on a Bioanalyzer High-Sensitivity DNA chip (Agilent #5067-4626 and #5067-4627).
Library preparation: cDNA can be converted into NGS-ready libraries by various established methods: (i) tagmentation of double-stranded cDNA with a commercially available (e.g. Illumina Nextera) or custom-made Tn5 transposase (instructions on how to prepare the transposome are included below) followed by PCR enrichment. (ii) fragmentation of double-stranded cDNA by mechanical (e.g. sonication) or enzymatic (e.g. NEB dsDNA fragmentase) means followed by end repair, A-tailing, adapter ligation and PCR enrichment. (iii) linear extension by random priming with a high-processivity polymerase (e.g. Klenow fragment) followed by PCR enrichment.
Random priming (RP) provides an alternative means to introduce a defined sequence at the end of the library fragment distal to the sequence captured during the reverse transcription (e.g. the poly-A tail). It is compatible with version TN5 (where it replaces the tagmentation step) and version LIG (where it replaces the template switching step). Reverse transcription, second strand synthesis and cell/nuclei recovery and counting were performed as described above for version EXT-TN5 (Example 3). However, the tagmentation is no longer required. Instead, processed cells/nuclei in a total volume of 11 μl 1× Nuclei Buffer were mixed with 7 μl of ATAC Buffer (10× Genomics cat. no. 2000122), 61.5 μl Barcoding Reagent, 1.5 μl Reducing Agent B and 2.0 μl of Barcoding Enzyme (all from 10× Genomics cat. no. 1000110) and the microfluidic chip was loaded and run as described previously. The sample was cleaned by silane and SPRI bead cleanups as described above for version EXT-TN5, eluting in a volume of 43 μl nuclease-free water. 41.75 μl of the cleaned sample were mixed with 5 μl of Blue Buffer (10×, Enzymatics #P7010-HC-L), 1.25 μl 10 mM dNTPs (Invitrogen cat. no. 18427-088) and 1 μl of Random Primer (100 μM, 5′-[Btn]GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNN, where the underlined part corresponds to a stretch of random bases ideally four to eight bases in length and the biotin modification is optional). The sample was then denatured for 5 min at 95° C., and immediately cooled on ice to prevent the re-formation of secondary structures and allow the annealing of the random primer. Next, 1 μl of Klenow Exo-Polymerase (50 U/μl, Enzymatics #P7010-HC-L) was added, the reaction was mixed by pipetting and incubated in a thermocycler: 4° C. for 15 min, then ramp to 37° C. at 1° C./min, 37° C. for 1 hour, then 70° C. for 10 min (enzyme inactivation), storage at 4° C. Excess random primer was removed by addition of 2.5 μl Exonuclease I (20 U/μl, NEB #M0293S) and 1.25 μl of rSAP (1 U/μl, NEB #M0371S) followed by incubation for 1 hour at 37° C. and heat inactivation for 20 min at 80° C., then store at 4° C. After performing a 0.8×SPRI cleanup or a Streptavidin-Bead cleanup, the library was enriched by PCR as described above for version EXT-TN5.
Reverse transcription, cell/nuclei recovery and counting, thermoligation barcoding on the microfluidic device and the silane cleanup are performed as described above for version LIG (Example 4). At the end of the SPRI cleanup, the sample is eluted in 43 μl of nuclease-free water. Random priming replaces the Template Switching step and is performed as follows. 41.75 μl of the cleaned sample are mixed with 5 μl of Blue Buffer (10×, Enzymatics #P7010-HC-L), 1.25 μl 10 mM dNTPs (Invitrogen cat. no. 18427-088) and 1 μl of Random of Primer (100 μM, 5′-[Btn]GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNN, where the underlined part corresponds to a stretch of random bases ideally four to eight bases in length and the biotin modification is optional). The sample is then denatured for 5 min at 95° C., and immediately cooled on ice to prevent the re-formation of secondary structures and allow the annealing of the random primer. Next, 1 μl of Klenow Exo-Polymerase (50 U/μl, Enzymatics #P7010-HC-L) are added, the reaction is mixed by pipetting and incubated in a thermocycler: 4° C. for 15 min, then ramp to 37° C. at 1° C./min, 37° C. for 1 hour, then 70° C. for 10 min (enzyme inactivation), storage at 4° C. Excess random primer is removed by addition of 2.5 μl Exonuclease I (20 U/μl, NEB #M0293S) and 1.25 μl of rSAP (1 U/μl, NEB #M0371S) followed by incubation for 1 hour at 37° C. and heat inactivation for 20 min at 80° C., then store at 4° C. After performing a 0.8×SPRI cleanup or a Streptavidin-Bead cleanup, the library is enriched by PCR as described above for version EXT-TN5.
Template Switching (TS) provides an alternative means to introduce a defined sequence at the end of the library fragment distal to the sequence captured during the reverse transcription (e.g. the poly-A tail). TS is already used in version LIG-TS, but is also compatible with version EXT-TN5, as described below. Reverse transcription is performed with Maxima H Minus Reverse Transcriptase or an alternative reverse transcriptase that adds untemplated C bases to the cDNA upon reaching the transcript end. Reverse transcription primers have the sequence (5′-TCGTCGGCAGCGTCGGATGCTGAGTGATTGCTTGTGACGCCTTCNNNNNNNNN XXXXXXXXXXXVTTTTTTTTTTTTTTTTTTTTTTTVN-3′), where N indicates a random base, the underlined bases are known for a given primer, and X is an 11-base-long primer-specific index sequence. 96-well plates with barcoded oligo-dT primers are prepared prior to the experiment and stored at −20° C. (1 μl of 25 μM per well). 10,000 permeabilized cells or nuclei (2 μl of a 5,000/μl suspension) are added to the pre-dispensed primers and well assignments are recorded. The plate is incubated for 5 min at 55° C. (to resolve RNA secondary structures), then placed immediately on ice (to prevent their re-formation).
Per well, a mix of 1 μl 5× Reverse Transcription Buffer, 1 μl of Ficoll PM-400 (20%, Sigma #F5415-50ML), 0.5 μl of 10 mM dNTPs (Invitrogen cat. no. 18427-088), 0.125 μl of Recombinant Ribonuclease Inhibitor (Takara #2313A), 0.125 μl of Template Switching Oligo (100 μM, 5′-AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG-3′, where r indicates RNA bases) and 0.25 μl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific cat. no. EP0753) is added. The combined reverse transcription and template switching reaction is incubated as follows: (heated lid set to 60° C.), 25° C. for 30 min, 42° C. for 90 min, storage at 4° C. Cell/nuclei recovery and counting are performed as described above for version EXT-TN5. However, the tagmentation is no longer required. Instead, processed cells/nuclei in a total volume of 9.7 μl 1× Nuclei Buffer are mixed with 7 μl of ATAC Buffer (10× Genomics cat. no. 2000122), 61.5 μl Barcoding Reagent, 1.5 μl Reducing Agent B, 2.0 μl of Barcoding Enzyme (all from 10× Genomics cat. no. 1000110) and 1.3 μl of TSO Enrichment Primer (100 μM, 5′-AAGCAGTGGTATCAACGCAGAGT-3′). The microfluidic chip is loaded and run and the droplet emulsion is incubated as described previously. The sample is cleaned by silane and SPRI bead cleanups as described above for version EXT-TN5. The cDNA is amplified and libraries are prepared as described above for version LIG-TS.
Oligonucleotides Tn5-top_ME (5′-[Phos]CTGTCTCTTATACACATCT-3′) and Tn5-bottom_Read2N (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′) were synthesized by Sigma Aldrich and reconstituted in EB buffer (Qiagen cat. no. 19086) at 100 μM. 22.5 μl of each oligonucleotide and 5 μl of 10× Oligonucleotide Annealing Buffer (10 mM Tris-HCl (Sigma cat. no. T2944-100ML), 50 mM NaCl (Sigma cat. no. S5150-1L), 1 mM EDTA (Invitrogen cat. no. AM9260G)) were mixed and annealed in a thermocycler: 95° C. for 3 min, 70° C. for 3 min, ramp to 25° C. at 2° C. per minute. The annealing reaction was then diluted by addition of 180 μl of water. At this point, the diluted oligonucleotide cassette can be aliquoted and frozen for future transposome assemblies. To load the Tn5 transposase, we mixed 20 μl of diluted oligonucleotide cassette from the previous step with 20 μl of 100% glycerol (Sigma cat. no. G5516-100ML) and 10 μl of EZ-Tn5 Transposase (Lucigen cat. no. TNP92110), and incubated for 30 min at 25° C. in a thermocycler. The resulting 50 μl of assembled transposome are sufficient for eight scifi-RNA-seq reactions with the EXT-TN5 protocol (6 μl per reaction) or over 200 library preparations for scifi-RNA-seq implementations with cDNA enrichment. The transposome can be stored at −20° C. for at least one month.
Tagmented DNA flanked by two Illumina i7 adapters is suppressed in PCR reactions due to competition between intramolecular annealing and primer binding. The custom i7-only transposome is therefore tested in a negative qPCR assay as described previously (Rykalina et al., 2017). Briefly, a defined PCR product is subjected to one tagmentation reaction and one no-enzyme control reaction. Both samples are then re-amplified with the same primers in a qPCR reaction. Since the tagmentation fragments the PCR product, the corresponding reaction should yield higher Ct values. The tagmentation efficiency can then be calculated from the shift of Ct values:
Generation of the PCR product: Oligonucleotides pUC19-FWD (5′-AAGTGCCACCTGACGTCTAAG-3′) and pUC19-REV (5′-CAACAATTAATAGACTGGATGGAGGCGG-3′) were synthesized by Sigma Aldrich and reconstituted in EB buffer (Qiagen cat. no. 19086) at 100 μM. Next, a 1,961 bp PCR product was generated by mixing 128.7 μl of water, 33 μl of 50 pg/μl pUC19 plasmid (NEB cat. no. N3041S), 1.65 μl each of primers pUC19-FWD and pUC19-REV (100 μM) combined with 165 μl of 2× Q5 HotStart High-Fidelity Master Mix (NEB cat. no. M0494L). The resulting 6.6× master mix was distributed into a tube strip (six reactions of 50 μl) and amplified in a thermocycler: 98° C. for 30 s; 31× (98° C. for 10 s, 68° C. for 30 s, 72° C. for 1 min), 72° C. for 2 min, storage at 12° C. To each 50 μl PCR reaction, we added 6.25 μl of 10× CutSmart Buffer and 6.25 μl of Dpnl (NEB cat. no. R0176L) and incubated at 37° C. for 1 hour to digest the PCR template plasmid. The six PCR reactions were pooled and cleaned with the QiaQuick PCR Purification Kit (Qiagen cat. no. 28106) using two columns and eluting with 30 μl of EB buffer per column. Eluates were pooled, and the purity of the PCR fragment was checked on a 1% agarose gel containing ethidium bromide. We then measured the concentration of dsDNA with a Qubit HS assay (Thermo Fisher Scientific cat. no. Q32854), and diluted the PCR product to 25 ng/μl with EB buffer.
Tagmentation: Tagmentation reactions were set up by mixing 2 μl of 25 ng/μl pUC19 PCR product from the previous step, 7 μl of ATAC Buffer (10× Genomics cat. no. 2000122), and either 6 μl of custom i7-only transposome (tagmentation reaction) or 6 μl of water (no-enzyme control reaction). After 60 min of incubation at 37° C., the Tn5 enzyme was stripped from the DNA by addition of 1.75 μl of 1% SDS solution (Sigma cat. no. 71736-100ML) followed by incubation at 70° C. for 10 min. The two reactions were diluted 1/100 with EB buffer, and qPCR reactions were set up in triplicates: 2 μl of 1/100-diluted reaction, 10 μl of 2× GoTaq qPCR Master Mix (Promega cat. no. A600A), 0.1 μl each of 100 μM pUC19-FWD and pUC19-REV primers and 7.8 μl of water. qPCR reactions were incubated as follows: 95° C. for 2 min, 40× (95° C. for 30 s, 68° C. for 30 s, 72° C. for 2 min and plate read).
Resulting scifi-RNA-seq libraries were sequenced on the Illumina NextSeq 500 platform, using High Output v2.5 reagents (75 Cycles, Illumina cat. no. 20024906). We used custom sequencing primers 18-12_scifi_SEQ_inDrop_read1 (5′-GGATGCTGAGTGATTGCTTGTGACGCC*T*T*C, where * denotes phosphorothioate bonds) for Read1 and 18-12_scifi_SEQ_inDrop_index2 (5′-GCATCCGACGCTGCCGA*C*G*A-3′) for Index2. The machine was set to read lengths of 21 bases (Read1), 47 bases (Read2), 8 bases (Index1, i7) and 16 bases (Index2, i5).
Large single-cell libraries were sequenced on the Illumina NovaSeq 6000 platform, using NovaSeq 6000 SP (100 Cycles, Illumina cat. no. 20027464) or S2 (100 Cycles, Illumina cat. no. 20012862) reagents. Custom sequencing primer 18-12_scifi_SEQ_inDrop_read1 (5′-GGATGCTGAGTGATTGCTTGTGACGCC*T*T*C, where * denotes phosphorothioate bonds) was applied for Read1. Due to a different sequencing chemistry, Index2 can be read with standard NovaSeq primers. The sequencer was set to a read structure of 21 bases (Read1), 55 bases (Read2), 8 bases (Index1, i7) and 16 bases (Index2, i5).
In some implementations of scifi-RNA-seq, a primer binding site compatible with standard Illumina sequencing primers was used, so that custom primers were no longer required.
Cell culture: Human Jurkat-Cas9-TCRlib cells were cultured in RPMI medium (Gibco #21875-034) containing 10% FCS (Sigma) and penicillin-streptomycin and were continuously selected with 25 μg/ml blasticidin (Invivogen #ant-bl-5) and 2 μg/ml puromycin (Fisher Scientific #A1113803). Mouse 3T3 cells were cultured in DMEM medium (Gibco #10569010) containing 10% FCS (Sigma) and penicillin-streptomycin.
Single-cell RNA-seq: A nuclei suspension from human Jurkat-Cas9-TCRlib cells and mouse 3T3 cells was freshly prepared, as described in Example 1.2, supra. To evaluate the performance of scifi-RNA-seq as a function of droplet overloading, 15,300, 383,000, or 765,000 pre-indexed nuclei were loaded into a single channel of the Chromium system. Both the number of single-cell transcriptomes and the average number of nuclei inside each droplet scaled linearly with the loading amount (
Finally, the dataset allowed to conclusively resolve a third feasibility concern for scifi-RNA-seq-whether the reagents in each droplet would be sufficient for effective barcoding of the transcriptomes from multiple nuclei. When plotting UMI counts and fractions of unique reads per cell against the number of nuclei per droplet (
Cell culture: Jurkat-Cas9-TCRlib, K562 and NALM-6 cell lines were cultured in RPMI medium (Gibco #21875-034) containing 10% FCS (Sigma) and penicillin-streptomycin. Jurkat-Cas9-TCRlib cells were continuously selected with 25 μg/ml blasticidin (Invivogen #ant-bl-5) and 2 μg/ml puromycin (Fisher Scientific #A1113803). HEK293T cells were cultured in DMEM medium (Gibco #10569010) containing 10% FCS (Sigma) and penicillin-streptomycin.
Single-cell RNA-seq: A nuclei suspension from four human cell lines with unique characteristics (Jurkat, K562, NALM-6, HEK293T) was freshly prepared, as described in Example 1.2, supra. Next, these nuclei were subjected to scifi-RNA-seq as described in Example 4, supra, according to the protocol based on thermocycling ligation and template switching (LIG-TS). During the reverse transcription step on a 384-well plate, each cell line was assigned a specific set of pre-indexing (round1) barcodes. After the pre-indexing samples were pooled and 383,000 nuclei were loaded into a single microfluidic channel of the Chromium system. 151,788 single-cell transcriptomes passed quality control (
Isolation of primary human T cells: Peripheral blood from healthy donors was obtained from as blood packs with buffered sodium citrate as anti-coagulant. For each donor, we prepared T cells from 3×15 ml of peripheral blood, according to the following protocol. 15 ml of peripheral blood were mixed with 750 μl of RosetteSep Human T Cell Enrichment Cock-tail (Stemcell #15061). After 10 min of incubation at room temperature, the sample was diluted by addition of 15 ml 1×PBS (Gibco #14190-094) containing 2% v/v FCS (Sigma). SepMate tubes (Stemcell #86450) were loaded with 15 ml of Lymphoprep density gradient medium (Stemcell #07851) and the blood sample was poured on top. After centrifugation (1,200 rcf, 10 min, room temperature, brake set to 9), the supernatant was transferred to a fresh 50 ml tube, topped up to 50 ml with 1×PBS containing 2% FCS, and centrifuged (1200 rcf, 10 min, room temperature, brake set to 3). After one additional wash with 50 ml of 1×PBS containing 2% FCS (1200 rcf, 10 min, room temperature, brake set to 3), T cells were resuspended in 10 ml of 1×PBS containing 2% FCS, filtered through a 40 μM cell strainer, and counted using a CASY device (Schärfe Systems). For accurate cell counting, it was important to exclude contaminating erythrocytes, which will be lysed during the subsequent nuclei preparation.
Anti-CD3/CD28 stimulation of human T cells: Freshly isolated primary human T cells were resuspended at a density of 1 million cells per ml in Human T Cell Medium (OpTmizer medium (Thermo Fisher #A1048501) containing 1/38.5 volumes of OpTmizer supplement, 1× GlutaMax (Thermo Fisher #35050061), 1× Penicillin/Streptomycin (Thermo Fisher #15140122), 2% heat-inactivated human AB serum (Fisher Scientific #MT35060CI), 10 ng/ml of recombinant human IL-2 (PeproTech #200-02)). The culture was split into two flasks, and one was treated with Human T-Activator CD3/CD28 Dynabeads (25 μl beads per 1 million cells, Thermo Fisher #11131D). After 16 hours, we prepared formaldehyde-fixed nuclei and snap-froze the nuclei suspension as described herein.
Flow cytometry analysis of T cell populations: A total of 1 million primary human T cells were washed twice with 1×PBS containing 0.1% BSA and 5 mM EDTA (PBS-BSA-EDTA). Single-cell suspension was incubated with anti-CD16/CD32 (clone 93, 1:200, Biolegend #101301) to prevent nonspecific binding and stained with combinations of antibodies against CD4 (PE-TxRed, clone OKT4, 1:200, Biolegend #317448), CD8 (APC-Cy7, clone SK1, 1:150, Biolegend #344746), CD25 (PE-Cy7, clone BC96, 1:100, Biolegend #302612), CD45RA (PerCp-Cy5.5, clone HI100, 1:100, Biolegend #304122), CD45RO (AF700, clone UCHL1, 1:100, Biolegend #304218), CD69 (AF488, clone FN50, 1:100, Biolegend #310916), CD127 (APC, clone A019D5, 1:100, Biolegend #351342), CD197 (CCR7, PE, clone G043H7, 1:100, Biolegend #353204), and DAPI viability dye (Biolegend #422801) for 30 min at 4° C. After two washes with PBS-BSA-EDTA, cells were acquired with an LSRFortessa Cell Analyzer (BD). CD4+ and CD8+ T cells were subdivided into naive T cells (CD45RA+CCR7+), effector memory T cells (CD45RA-CCR7−), central memory T cells (CD45RA-CCR7+) and TEMRA cells (CD45RA+CCR7−). T cell receptor-mediated activation of CD4+ and CD8+ T cells was assessed based on CD25 and CD69 expression.
Single-cell RNA-seq: scifi-RNA-seq was performed as described in Example 4, following the protocol based on thermocycling ligation and template switching (LIG-TS). During the reverse transcription step on a 384-well plate, donor identity and TCR stimulation status were barcoded with a set of unique round1 pre-indices. After the pre-indexing samples were pooled and 765,000 nuclei were loaded into a single microfluidic channel of the Chromium system. Results are shown in
In this experiment the performance of the methods of the invention was compared to existing multi-round combinatorial indexing technologies. Publicly available data was obtained for sci-RNA-seq v1 (Cao, Packer et al., 2017), SPLIT-seq (Rosenberg, Roco et al, 2018), sci-RNA-seq v3 (Cao, Spielmann et al., 2019) and sci-Plex (Srivatsan, McFaline-Figueroa, Ramani et al., 2020). Using mouse 3T3 cells as a common point of reference, it was demonstrated that the library quality of scifi-RNA-seq was consistently superior to sci-RNA-seq v1, sci-RNA-seq v3 and sci-Plex (
In addition, the library design and sequencing read structure was compared between the methods, in order to assess their cost-effectiveness. Because scifi-RNA-seq does not read uninformative ligation overhangs, all sequencing cycles spent on cell barcodes are informative, in contrast to sci-RNA-seq v1 (58% informative), sci-RNA-seq v3 and sci-Plex (87% informative), and SPLIT-seq (33% informative). As a result, scifi-RNA-seq greatly reduces the bottleneck of sequencing cost for ultra-high throughput single-cell RNA-seq. (
For a comparison to microfluidic single-cell RNA-seq, scifi-RNA-seq was benchmarked against the widely used 10× Genomics technology, using the latest v3 chemistry. In a series of new wet-lab experiments, test samples were split and processed side-by-side with both assays, loading the same number of 7,500 nuclei/cells per microfluidic channel and comparing the results between permeabilized nuclei, methanol-fixed cells, and intact cells. An equal mixture of four human cell lines with variable transcript content was used (K562, HEK293T, Jurkat, NALM-6) as well as a cross-species mixture of human (Jurkat) and mouse (3T3) cells. This setup allowed separation of the effects of permeabilization methods, technology platform, cell type, species and transcript content.
In summary, these experiments showed that: (i) Pre-indexed cells/nuclei in scifi-RNA-seq are recovered at almost the same rate as native cells/nuclei on the 10× Genomics system. Due to the minimal sequencing coverage spent on background this can be compensated for by increased loading concentrations (
It was shown that droplet overloading according to the methods of the present invention is compatible with the Chromium Single Cell ATAC v.1.1 (NextGEM) kit (
The advantages of the whole transcriptome pre-indexing step in scifi-RNA-seq are two-fold. First, barcoded cells/nuclei can be loaded into the second compartment at a rate of multiple cells/nuclei per compartment, allowing the ultra-high throughput processing of the sample. Secondly, the round1 pre-index can label hundreds to thousands of experimental conditions, thereby enabling large-scale perturbation studies such as drug screens or genetic perturbation screens at the single-cell level.
To demonstrate the multiplexing capabilities of the invention, and the benefits of profiling very high numbers of single-cells for drug development and target discovery, the following experiment was performed. The human Jurkat cell line was transduced with a lentiviral vector to express the Cas9 nuclease. These cells were further modified with a second lentiviral vector expressing 48 distinct CRISPR guide RNAs (gRNAs), targeting 20 genes with 2 gRNAs each plus 8 non-targeting control gRNAs. We allowed 10 days for efficient genome editing under antibiotic selection. Afterwards, the 48 single knockout cell lines were split into two parts, which received stimulation of the T cell receptor with anti-CD3/CD28 antibodies or were left untreated. For the resulting 96 samples methanol-fixed cells were prepared and scifi-RNA-seq according to the described methods of the invention was performed (
The above highlights the potential of the methods of the invention for drug discovery and target validation. The methods of the invention derive relevant screening signatures directly from the transcriptome of control cells, so that no prior knowledge about the mechanism of action of a drug is required. This can save valuable time in prioritizing lead candidates and in bringing a drug product to the market. Moreover, the single-cell resolution of the methods of the invention can assess the effect of drug treatments on different cell types in a complex mixture (for instance PBMCs), or on a mixture of cells from distinct donors.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19196008.7 | Sep 2019 | EP | regional |
| 19216696.5 | Dec 2019 | EP | regional |
The present application is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2020/074985, filed Sep. 7, 2020, which claims the priority benefit of European Application No. 19216696.5, filed Dec. 16, 2019, and European Application No. 19196008.7, filed Sep. 6, 2019, the entire contents of each of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/074985 | 9/7/2020 | WO |
