Patent Application
20220064717
Single cell analysis is important. for understanding how cells respond to drugs and other perturbations because phenotypic responses are asynchronous and cells are often both genetically and epigenetically heterogeneous. In the absence of artificial or external perturbations, the natural processes of cellular differentiation during development and malignant transformation in cancer also occur asynchronously and give rise to phenotypic heterogeneity. While single cell RNA-Seq is emerging as a high-dimensional and increasingly scalable tool for assessing phenotypic heterogeneity, many aspects of cellular phenotype cannot be inferred from the transcriptome alone. However, high-content imaging by microscopy could greatly expand the space of observables in a single cell analysis, particularly if it can be integrated with single cell RNA-Seq. High-content imaging assays using optical microscopy provide access to numerous phenotypic observables for cellular metabolism, protein localization, protein synthesis, cell cycle states, and cell signaling all with single cell resolution. However, existing tools for merging single cell imaging and sequencing are expensive, low-throughput, and incompatible with short-term cell culture and stimulation. The instant disclosure addresses this issue by combining a highly scalable microfluidic platform for single cell RNA-Seq and imaging with associated methodologies that allow for the association of sequence barcodes for inexpensive pooled library preparation with optical barcodes in devices described herein.
The disclosure allows for devices, systems, and methods relating to genome-wide profiling of RNA from hundreds to thousands of individual cells in parallel for only a few cents per cell. The devices, systems, and methods described herein address the scalability problem of parallel preparation of low-input single cell libraries for RNA sequencing. Devices, systems, and methods described herein allow for parallel RNA profiling of individual cells in a device that is compatible with short term cell culture, drug stimulation experiments, and high-content fluorescence imaging. In an aspect, this is because the cells are physically segregated into microwells but still in sufficiently close proximity to communicate via diffusible factors.
In an aspect, the disclosure provides for a device or system comprising
In an aspect, the disclosure provides for a method of drug discovery, drug profiling, and/or drug testing comprising
In an aspect, the one or more optical barcodes include one or more optical barcode sequences. In another aspect, the one or more optical barcodes include one or more fluorescent dyes including different colors and/or intensities.
The disclosure further provides for method of drug discovery, drug profiling, and/or drug testing described herein comprising (f) adding a buffer to the plurality of chambers, microchambers, or microwells configured for reversible sealing. In an aspect, the buffer includes a lysis buffer.
The disclosure further provides for a method of drug discovery, drug profiling, and/or drug testing comprising analyzing said cell-identifying optical barcode sequences by fluorescence.
In another aspect, the device or system of described herein comprise a plurality of chambers, microchambers, or microwells attached to a polysiloxane substrate. In yet another aspect, said polysiloxane substrate comprises polydimethylsiloxane.
In another aspect, the device or system described herein comprise cell-identifying optical barcode sequences are adapted for florescence detection.
In yet another aspect, the device, system, or methods comprise cells and said cells are not arranged in a droplet configuration.
In another aspect, the disclosure provides for a method for single cell RNA capture and sequencing, wherein said method comprises
In yet another aspect, the disclosure provides for a device for single cell RNA capture and sequencing, wherein said device comprises a plurality of microwells; wherein said plurality of microwells are attached to a first polysiloxane substrate; a second substrate comprising glass that faces said first substrate, wherein oligo printers are grafted onto to the glass surface of said second substrate.
The disclosure further provides for a rnicrowell comprising a composition comprising one or more mRNA capture beads; one or more cell-identifying optical barcode sequences; one or more cells, one of more buffers.
In an aspect, the disclosure provides for a microwell array system, devices, and methods for pairing individual cells with mRNA capture beads. In another aspect, the disclosure provides for a microwell array system, device, and methods for pairing individual cells with mRNA capture beads that introduce, cell-identifying barcode sequences into cDNA generated after mRNA capture. In an aspect, the system is compatible with high-content cellular imaging and drug stimulation experiments.
The disclosure further provides for capture beads, for example a mRNA capture beads, or probes comprising one or more optical barcodes described herein. In an aspect, the capture beads, for example a mRNA capture beads, or probes are used to identify drug and/or to be used in drug simulation experiments.
In an aspect, the disclosure provides for devices, systems, and/or methods wherein cells are arranged in a grid and/or a chamber. In yet another aspect, the disclosure provides for devices, systems, and/or methods wherein cells are arranged in a grid and/or chamber as compared to devices, systems, and/or methods wherein cells are arranged in droplets or a configuration where cells are physically segregated from one another. In an aspect, a microfluidic device, system, or method described herein comprises a flow cell with an array of microwells embedded in either the top or bottom of the device. In another aspect, the device described herein is a solid state device that allows for single cell isolation, imaging, and/or uniform parallel introduction of reagents to a plurality of cells.
The disclosure provides for devices, systems, and/or methods wherein cells are arranged in a reversible chamber or microchamber wherein the chamber and/or microchamber can be open and/or closed. The disclosure also provides for devices, systems, and/or methods wherein cells are arranged in reversible chamber or microchamber where the chamber and/or microchamber can be opened or closed as compared to devices, systems, and/or methods wherein cells are arranged in non-reversible droplets or a non-reversible configuration wherein cells are physically segregated from one another. In such a non-reversible configuration, the droplets and/non-reversible configuration wherein cells are physically segregated cannot be readily toggled back and forth, that is, may not be opened or closed. As a result, in a non-reversible configuration, fluidics cannot be uniformly distributed to the cells and then uniformly removed and replaced by a second fluid.
The disclosure further provides for devices or system comprising
In an aspect, the disclosure provides for devices or system comprising
The disclosure further provides for probes comprising one or more mRNA capture beads and one or more cell-identifying barcode sequences associated or coupled with the one or more mRNA capture beads.
The disclosure further provides for compositions comprising one or more mRNA capture beads and one or more cell-identifying barcode sequences associated or coupled with the one or more mRNA capture beads and one or more cells.
The disclosure further provides for methods of drug discovery, drug profiling, and/or drug testing comprising
The disclosure further provides for a probe comprising
The disclosure further provides for methods for single cell RNA capture and sequencing, wherein said method comprises
In an aspect, the device, system, and/or microarray is imaged using florescence.
In another aspect, the disclosure provides for a device for single cell RNA capture and sequencing, wherein said device comprises a plurality of microwells; wherein said plurality of microwells are attached to a first polysiloxane substrate; a second substrate comprising glass that faces said first substrate, wherein oligo primers are grafted onto to the glass surface of said second substrate.
In an aspect, the substrate or surface comprises a polysiloxane substrate. In an aspect, the polysiloxane substrate comprises PDMS. In another aspect, the substrate comprises polymethylmethyacrylate (PMMA). In another aspect, the substrate comprises a thermoplastic for fabrication by, for example, hot embossing. In another aspect, the substrate comprises glass for fabrication by, for example laser machining. In another aspect, the substrate comprises silicon or silicon-on-glass or photoresist-on-silicon.
In another aspect, a system or device described herein further comprises one or more drugs, buffers, active agents, and/or a plurality of cells.
In an aspect, the disclosure provides for devices, systems, methods, and/or probes capable of profiling as hundreds or thousands of cells from an organ or tumor. In art aspect, the cells analyzed are healthy human cells, abnormal cells, cancer cells, neural cells, immune cells, epithelial cells, mesenchymal cells, or stem cells. Cells could also originate from microorganisms including parasites, fungi, or bacteria. Other units containing nucleic acids such as viruses both in isolation or inside of art infected host cell could be analyzed.
In yet another aspect, different drugs or chemical constituents or compositions, for example, lysis buffer, may be introduced to a device or system described herein, for example, in a chamber or microchamber, at any time given the reversible nature of the chambers or microchambers. This provides for additional flexibility and advantages over other devices or systems, such as those employing, for example, droplet. microfluidic technology, nanoliter droplet technology, and/or droplet sequence, “Drop-seq,” “Drop-seq single cell analysis” technology, and/or technology where cells are captured in “droplets.”
In art aspect, the device or system described herein, for example, the microfluidic device does not involve any on-chip valves and/or moving parts, which can result in a high feature density. In another aspect, the device or system described herein is not a droplet microfluidics device or does not include droplet microfluidic technology, nonoliter droplet technology, and/or droplet sequence, “Drop-seq,” “Drop-seq single cell analysis” technology, and/or technology where cells are captured in “droplets.” In an aspect, the device or system described herein is more compatible with cell culture and cell perturbation assays than a droplet microfluidic device because fluids can be readily exchanged in a uniform fashion and cells can communicate with each other via diffusible factors. Hence, cell viability will be higher and cells will exhibit normal physiology in comparison to cells that are sequestered in droplets. In another aspect, in the device or system described herein, cell are arranged in regular array and are in contact with a flat, optically transparent surface as opposed to a droplet microfluidic device in which cells are isolated inside of a spherical enclosure. Therefore, in yet another aspect, the device or system described herein is intrinsically more compatible with imaging, for example optical imaging or microscopy, than droplet devices.
In are aspect, devices, systems, and methods described herein have a higher throughput than devices, systems, or methods employing droplet microfluidic technology, nonoliter droplet technology, and/or droplet sequence, “Drop-seq,” “Drop-seq single cell analysis” technology, and/or technology where cells are captured in “droplets.” In yet another aspect, analysis of cells, probes, and beads using devices, systems, and methods described herein have a throughput that is 5, 10, 15, 20, 25, or 100 or more times less expensive than devices, systems, or methods employing droplet microfluidic technology, nonoliter droplet technology, and/or droplet sequence, “Drop-seq,” “Drop-seq single cell analysis” technology, and/or technology were cells are captured in “droplets.”
In another aspect, the device described herein is compatible with automation, for example, a computer-controlled system that is capable of facilitating automated introduction of any of various fluids to the device, temperature control, and reversible sealing of the microwells. Accordingly, in a preferred aspect the device described herein can capture mRNA from thousands of individual cells in parallel and produce pooled, single cell RNA-Seq libraries for ˜$0.10/cell.
In an aspect, barcoded capture beads are introduced to the microwell array in either a random process or ordered process. In yet another aspect, barcoded capture beads are introduced into the microwell array in a random process with little or no control over which barcoded capture bead enter each microwell. Under such a scenario, in an aspect, imaging information acquired from the cells in the device is not directly associated with the single cell transcriptomes that ultimately are quantified by deep sequencing. In an aspect, this issue is addressed by introducing barcodes, for example, optical barcodes, to the mRNA capture heads that indicate the sequence barcode attached to each bead. In another aspect, the optical barcodes can be read out using the same or different fluorescence microscope used for live cell imaging on the device. In yet another aspect, the disclosure provides for a reagent that allows for the capture RNA from individual cells, allows for association of a unique sequence barcode with each single cell cDNA library, and/or reveals the identity of the attached sequence barcode by optical methods.
In an aspect, the disclosure provides for an optical barcode and associated methods described herein. In an aspect, the optical barcode is a sequence. In another aspect, the optical barcode is not a sequence. In another aspect, optical barcodes described herein are not DNA sequences.
In an aspect, an optical barcode described herein comprises a combination of fluorescent dyes comprising at least one different color and optionally more than one different intensity that make the bead uniquely identifiable on art optical microscope. In another aspect, an optical barcode described herein comprises a combination of fluorescent dyes comprising two, three, four, five or more different colors and optionally more, than one different intensity that make a respective bead uniquely identifiable on an optical microscope
The disclosure further provides for proteomic methods and systems utilizing a device, probe, or bead described herein. In an aspect, an oligonucleotide-based optical barcode is loaded onto beads that also harbor reactive groups for capturing protein (e.g. amine-, carboxyl-, or thiol-reactive groups). In yet another aspect, optical barcodes are loaded onto beads along with one or more antibodies capable of capturing specific proteins. Proteins can be quantified by fluorescence methods (e.g. fluorescently labeled antibodies) or mass spectrometry. In a further aspect, the disclosure provides for the use of DNA-labeled antibodies that are amplified, identified, and quantified by deep sequencing.
The devices, systems, and methods described herein provide for key distinctions and advantages over other devices, systems, and methods. For example, the microwell array device is constructed in such a way that it can be reversibly sealed during cell lysis and RNA capture. Significant loss of RNA occurs in our arrays when cells are lysed in unsealed or even imperfectly sealed arrays due to rapid diffusion of RNA molecules. In the context of the bead capture and RNA-Seq experiments, this could result not only in reduced RNA capture, but also significant cross-talk. Herein, advantage is taken of the physical properties of PDMS, namely its flexibility and hydrophobicity, for high-fidelity, reversible sealing which is difficult to achieve using the agarose hydrogel device reported previously. In addition, the devices, systems, and methods described herein demonstrate genome-wide single cell RNA-Seq. Additionally, the single cell capture and pooled library preparation scheme described herein costs $0.10-$0.20/cell even at a relatively modest scale of several hundred cells per run (see, for example, Table 7), compared to the <$1/cell estimated at the 10,000-cell scale for alternative approach, for example, one described in Fan HC, Fu GK, Fodor SPA: Combinatorial labeling of single cells for gene expression cytometry. Science 2015, 347:628-636, which is herein incorporated by reference in its entirety.
In an aspect, the disclosure provides for optical demultiplexing or optical barcoding of sequence-barcoded capture beads by, for example, attaching a unique combination of short oligonucleotides to each bead. In an aspect, while the sequence barcode associated with the RNA capture primer can be read out by DNA sequencing, the optical barcode can be read out on a standard fluorescence microscope. In another aspect, the oligonucleotide combination is unique to each sequence barcode and can be identified by hybridizing fluorescently labeled complementary oligonucleotides to beads. The presence or absence of a series of fluorescently labeled oligonucleotides on each bead after hybridization indicates the unique combination of short oligonucleotides attached to each bead and therefore the sequence barcode attached to each bead. Once the sequence barcode attached to each bead is identified, single cell transcriptome captured on each bead can be identified and read out using a sequencer with phenotypic information obtained from imaging the cells associated with each bead in each microwell of our device.
In an aspect, optical barcoding technology described herein is combined with applications beyond single cell RNA-Seq. In an aspect, an extension of the invention is bulk RNA-Seq or RNA-Seq of small numbers of cells. These can be seeded in the microwells or propagated from a single cell seeded in each microwell. The disclosure further provides for single cell DNA-Seq. The disclosure further provides for barcoded capture oligonucleotides that contain oligo(dT) for capturing mRNA from individual eukaryotic cells. Alternatively, the beads are functionalized with primers that are specific to targeted DNA loci or RNA transcripts. In yet another aspect, the beads are functionalized with primers that have a random sequence and can therefore capture any DNA or RNA sequence from an individual cell. In an aspect, in each case, a corresponding optical barcode is used to link imaging data acquired for a target cell or group of cells and sequencing data acquired from whatever nucleic acids are captured on the bead.
The disclosure further provides for proteomic methods and systems utilizing a device described herein. In an aspect, an oligonucleotide-based optical barcode is loaded onto beads that also harbor reactive groups for capturing protein (e.g. amine-, carboxyl-, or thiol-reactive groups). In yet another aspect, optical barcodes are loaded onto beads along with one or more antibodies capable of capturing specific proteins. Proteins can be quantified by fluorescence methods (e.g. fluorescently labeled antibodies) or mass spectrometry. In a further aspect, the disclosure provides for the use of DNA-labeled antibodies that are amplified, identified, and quantified by deep sequencing.
Single cell analysis is important for understanding how cells respond to drugs and other perturbations because phenotypic responses are inherently asynchronous and cells
In an aspect, the microfluidic platform described herein comprises a flow cell with an array of microwells embedded in either the top or bottom of the device. In an aspect, the device or system described herein comprises a high-throughput DNA sequencing and digital PCR device, as described in, for example, White A K, VanInsberghe M, Petriv O I, Hamidi M, Sikorski D, Marra M A, Piret J, Aparicio S, Hansen C L: High-throughput microfluidic single cell RT-qPCR. Proceedings of the National Academy of Sciences of the United Stares of America 2011, 108(34):13999-14004 or Men Y, Fu Y, Chen Z, Sims P A, Greenleaf W J, Huang Y: Digital polymerase chain reaction in an array of femtoliter polydimethylsiloxane microreactors. Anal Chem 2012, 84(10):4262-4266, the contents of which are incorporated by reference herein in their entirety.
In an aspect, fluids can be driven through the flow cell manually by, for example, laminar flow using a syringe or pipette. Fluid exchange in the microwells occurs by diffusion, while cells and beads can be loaded by gravity. The microwell arrays may be fabricated from, a polysiloxane substrate, for example, polydimethylsiloxane (PDMS), a silicone rubber commonly used in soft lithography. PDMS allows inexpensive, rapid, and repeatable fabrication from molds produced on silicon in photoresist using standard photolithography. See, for example, McDonald J C, Whitesides G M: Poly(dimethylsiloxane) as a material for fabricating microtluidic devices. Accounts of chemical research 2002, 35(7):491-499 and First Single Cell Expression Analysis with PDMS, Marcus et al., Analytical Chemistry, 2006. (Quake Lab), which are herein incorporated by reference in their entirety. In addition, the material properties of PDMS, including its hydrophobicity and flexibility, facilitate reversible sealing of the microwells against a flat surface using mechanical deformation and negative pressure (see, for example,
In an aspect, the device or system described herein is capable of solid-phase capture of RNA from individual cells via two modes of operation—RNA “printing” on glass and RNA capture on beads. For example, in an aspect, in RNA printing mode, individual cells are loaded in the microwells, which are fabricated in a PDMS slab that faces a glass coverslip. Oligo(dT) primers are covalently grafted to the glass surface so that mature mRNA molecules can be immobilized by hybridization of their poly(A) tails. Following the introduction of lysis buffer, the microwells can be sealed by mechanically placing them in conformal contact with the functionalized glass surface. Cell lysis releases mRNA into the solution confined by the microwells, resulting in hybridization to the oligo(dT)-coated glass coverslip. By placing the flow channel under negative pressure, the seal can be maintained in the absence of mechanical force, making the device transportable and readily accessible to an optical microscope. See, for example, White A K, VanInsberghe M, Petriv O I, Hamidi M, Sikorski D, Marra M A, Piret J, Aparicio S, Hansen C L: High-throughput microfluidic single cell RT-qPCR. Proceedings of the National Academy of Sciences of the United States of America 2011, 108(34):13999-14004, which is herein incorporated by reference in its entirety.
Because the mRNA is immobilized on a glass surface, enzymatic processing steps can take place on-chip, simply by sequential flow of reagents through the device. After incubating the trapped, single cell lysates with the glass capture surface, the seal may be released and the flow cell can be rinsed with a detergent-containing buffer followed by a reaction mixture containing DNase. Because the oligo(dT) primers are comprised of locked nucleic acid (LNA), they are resistant to nuclease digestion. See, for example, Koshkin A A, Nielsen P, Meldgaard M, Rajwanshi V K, Singh S K, Wengel J: LNA (locked nucleic acid): an RNA mimic forming exceedingly stable LNA: LNA duplexes. Journal of the American Chemical Society 1998, 120(50):13252-13253, the content of which is incorporated by reference in its entirety.
The immobilized single cell mRNA libraries can then be reverse transcribed in parallel, and the resulting mRNA/cDNA hybrids can be visualized by fluorescence microscopy after staining with a fluorogenic intercalator dye. For example,
A scalable platform for single cell RNA-Seq based on the bead capture modality is described herein. The low reagent volumes required for microfluidic processing result in a significant cost reduction relative to some conventional methods, for example, as described in Wu A R, Neff N E, Kalisky T, Dalerba P, Treutlein B, Rothenberg M E, Mburu F M, Mantalas G L, Sim S, Clarke M F et al: Quantitative assessment of single-cell RNA-sequencing methods. Nat Methods 2014, 11(1):41-46, which is herein incorporated by reference in its entirety.
A further reduction in cost can be realized by, for example, using microfluidics in combination with scheme for cDNA barcoding. A representative example of this, such as the CEL-Seq strategy, can be found in Hashimshony T, Wagner F, Sher N, Yanai I: CEL-Seq: single, cell RNA-Seq by multiplexed linear amplification. Cell Rep 2012, 2(3):666-673, which is herein incorporated by reference in its entirety. By introducing a cell-specific barcode to the cDNA during reverse transcription, all subsequent sequencing library preparation steps can be accomplished on pooled cDNA from multiple cells, further reducing hands-on time and reagent consumption.
A pool of mRNA capture beads was generated in which each bead is attached to ˜1 billion copies of a primer terminated on the 3′-end with one of 960 possible barcode sequences followed by oligo(dT) using a combinatorial synthesis technique (
A PDMS microwell device containing five flow channel lanes for physical multiplexing of samples and >10,000 microwells was constructed (
After loading the cells and barcoded beads, procedures described above and herein can be employed to trap single cell lysates in sealed microwells, captured mRNA on beads, and reverse transcribe (
In an aspect, the microfluidic device described herein is used to obtain RNA-Seq profiles from ˜600 cells across five lanes from two commonly used human cancer cell lines (described herein as “Experiment 1” throughout the text). One of the five lanes contained U87 human glioma cells, one contained MCF10a human breast cancer cells, and the other three contained a mixture of both cell lines. These two cell lines are highly mesenchymal, have been cultured for numerous passages, and have relatively similar expression profiles. Nonetheless, they are distinguishable by a few key genes and can be readily separated the data set. In addition, in an aspect, a slightly different protocol with less expensive reagents are used to obtain profiles of ˜500 cells across five lanes for a different cell pair (U87 cells and the diploid cell line WI-38, which has not undergone malignant transformation) in Experiment 2.
A factor with any pooled library scheme is cross-talk between cell-identifying barcodes. As described herein, this issue is addressed by quantitative analysis of Experiment 1. For example, the disclosure provides for methodology wherein cross-talk is quantified by using both sequencing and imaging data. In an aspect, because the device or system described herein is compatible with fluorescence microscopy, a fraction of the streptavidin molecules on each bead can be labelled with red-fluorescent AlexaFluor 647 and pre-stained the cells with a blue-excitable live stain. This allows quantification of the number of cells paired with a barcoded capture bead and also allows for the estimation of the number of barcodes that are expected to be observed in the sequencing data for each lane. The sequencing data reveals that more barcodes are present in the library for each lane than expected based on the imaging data. Analysis reveals that the number of molecules associated with a given barcode placed the barcodes in two distinct populations. The size of the population of barcodes associated with a larger number of molecules was highly consistent with the imaging data (within ˜8%), which we take to demarcate our single cell RNA-Seq profiles. The second, larger population of barcodes with relatively few associated molecules likely results from multiple potential sources including sequencing error, actual cross-talk or spurious capture within oar microfluidic device, and PCR jumping as observed in other implementations of multiplex single cell RNA-Seq. Across all five lanes the cell-identifying barcodes that were not associated with actual cells in the device had 200-300× fewer molecules per barcode than those associated with cells (based on the ratio of median unique molecules in the two populations).
As described in
To assess the similarity of our single cell expression profiles to conventional, population-level RNA-Seq, the Pearson correlation between bulk RNA-Seq and single-cell medians constructed from different numbers of individual cells after normalizing by the total number of molecules detected in each cell were calculated (
To further demonstrate the robustness of our data set, classifier for U87 and MCF10a cells in Experiment 1 were built. Single cell profiles from lanes that contained either exclusively U87 cells of MCF10a cells were used to identify 189 differentially expressed genes (p<0.05, Wilcoxin rank-sum test).
In an aspect, the microfluidic platforms described throughout are compatible with transcriptome-wide analysis of individual cells by RNA-Seq. In an another aspect, either could be combined with a sequence-based barcoding scheme to generate a pooled cDNA library from hundreds or thousands of individual cells. For example, in the bead capture device, barcoding is not strictly necessary because physical means could be used to extract the beads from the microwells for downstream processing with conventional labware. Alternatively, fluorescently labeled oligonucleotide probes could be used to image captured RNA molecules similar to RNA-FISH. Probes could be introduced sequentially, imaged, and removed in cycles or combined with previously reported multiplexing schemes. Similarly, sequential rounds of qRT-PCR in sealed microwells could allow targeted detection of specific genes or mutations in captured RNA.
In another aspect, the disclosure provides for a kit comprising, consisting essentially of, or consisting of any of probes, beads, and regents disclosed herein. In art aspect, the kit includes any of the combination of probes, beads, and regents disclosed herein in
In an aspect, the kit includes instructions describing the methodology described herein.
The following examples serve to illustrate certain aspects of the disclosure and are not intended to limit the disclosure.
The invention is illustrated herein by the experiments described by the following examples, which should not be construed as limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. Those skilled in the art will understand that this invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will fully convey the invention to those skilled in the art. Many modifications and other embodiments of the invention will come to mind in one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description. Although specific terms are employed, they are used as in the art unless otherwise indicated.
Generation of Optically Threaded mRNA Capture Beads for Optical Demultiplexing by Multi-Color Fluorescence Imaging for Single Cell RNA-Seq
N-succinimide-coated Sepharose beads with a mean diameter of ˜30 μm were obtained from GE Healthcare in isopropanol. The heads were washed three times with water by centrifugation and split into 125 different tubes. The beads were then re-suspended in a reaction mixture with a final concentration of 100 mM sodium borate (pH 8.5) along with three differently-labeled streptavidin proteins (streptavidin-AlexaFluor 488, streptavidin-AlexaFluor 546, and Streptavidin-AlexFluor 647 from Life Technologies). For each of the 125 reaction mixtures, the three proteins were added at one of 5 concentrations such that each of the 125 reaction mixtures had one of 5{circumflex over ( )}3=125 unique combinations of labels and label intensities. The reaction was incubated at room temperature for one hour on a rotisserie to allow the labeled streptavidins to covalently react with the beads, form amide bonds. The beads were then washed five times in 50 mM Tris-HCl pH 8, 50 mM NaCl, and 0.1% Tween-20 before using to completely quench any remaining reactive groups on the beads. Each of the 125 uniquely labeled sets of beads were then incubated with a uniquely barcoded, 5′-biotinylated oligonucleotide. The oligonucleotides had a universal adapter sequence on the 5′-end to facilitate amplification, a barcode sequence, and oligo(dT) on the 3′-end to facilitate mRNA capture. The oligonucleotides became bound to the beads through the biotin-streptavidin interaction, resulting in beads that could be identified by fluorescence microscopy based on their unique configuration of fluorescent labels to harbor a specific nucleic acid barcode sequence.
Generation of Optically Barcoded mRNA Capture Beads for Optical Demultiplexing by Sequential Fluorescence Hybridization for Single Cell RNA-Seq
2.4 million beads aminated polystyrene beads (Custom Primer Support 200 Amino, GE) were reacted with an 8% solution of glutaraldehyde in Cyanoborohydride Coupling Buffer (Sigma) for six hours a room temperature. After washing the beads in phosphate buffered saline (PBS), the beads were divided evenly into each of 32 tubes. To each tube, 62.5 pmol of a unique 5′-aminated oligonucleotide were added containing an adapter sequence, a unique capture barcode sequence, and a linker sequence. In addition, zero or more 5′-aminated oligonucleotides were added from a set of five, each of which contains a unique optical barcode sequence and a linker sequence. The constellation of oligonucleotides on the head surface forms a binary code. For example, if there are five oligonucleotides in a set, and the first, third, and fourth oligonucleotides are present on the bead, then the code could be in an aspect, 10110. However, there will also be a bead in the set with the code 00000, where none of the oligonucleotides are present. This also provides a signal, a bead that does not fluoresce when exposed to any of the probes
2.5 pmol of each optical barcode oligonucleotide was further added. Each capture barcode sequence is associated with a unique set of optical barcode sequences. In this embodiment, there are 32 capture barcode sequences and 25=32 possible combinations of optical barcode configurations added in the first round of synthesis. The 5′-aminated oligonucleotides were reacted with the aldehyde-conjugated beads in Cyanoborohydride Coupling buffer for 12 hours at room temperature. The reaction was then quenched by the reaction by adding 1 M Tris-HCI to each well. The beads were then washed in PBS.
In a second barcode synthesis, the 32 reactions from the first round were pooled and then re-distributed into each of 128 tubes. To each tube, 150 pmol of a unique oligonucleotide was added containing a linker sequence complementary to that in the capture barcode oligonucleotides already attached to the beads, a second capture barcode sequence, a random sequence (for unique molecular identification), and a poly(A) sequence. In addition, zero or more oligonucleotides from a set of seven were added, each of which contains a unique optical barcode sequence and a linker sequence complementary to that in the optical barcode oligonucleotides already attached to the heads. 20 pmol of each optical barcode oligonucleotide. Each capture barcode sequence is associated with a unique set of optical barcode sequences. There are 128 capture barcode sequences and 27=128 possible combinations of optical barcode configurations added in the second round of synthesis. In combination with the first round of synthesis, the second round of synthesis results in a set of 32×128=4,096 uniquely barcoded beads with both unique capture and optical barcodes attached. The second round oligonucleotides was hybridized to the oligonucleotides attached in the first round in Buffer 2 (New England BioLabs) with 1% tween-20 (Sigma) at 50C for 20 minutes and at room temperature for 12 hours. The beads were then washed and then copied the second round oligonucleotides onto the 3′-end of the first round oligonucleotides by primer extension with Klenow Large Fragment DNA Polymerase (New England BioLabs) at room temperature for 2 hours. The primer extension reaction was quenched with EDTA followed by sodium hydroxide. The beads from all 128 reactions were then pooled and washed them extensively in sodium hydroxide and Wash Buffer (10 mM Tris pH 8, 1 mM EDTA, 0.01% tween-20).
Microfabricated arrays of cylindrical pillars in photoresist on a silicon wafer were obtained from FlowJEM. The pillars were 50 microns in diameter and 50 microns tall. A second wafer contained a relief pattern of a flow channel. A 1:10 mixture of liquid polydimethylsiloxane (PDMS) base and curing agent (Sylgard 184, Dow-Corning) was poured on each wafer. A glass microscope slide was pressed onto the uncured liquid PDMS that had been poured on the wafer containing cylindrical pillars, forming a thin layer of PDMS between the water and the glass slide. Both wafers with liquid PDMS were then placed in an oven to cure at. 90° C. overnight. The cured, solidified PDMS were then peeled from both wafers resulting in a thin microwell array on the glass slide and a PDMS flow channel. After using a punch to insert inlet and outlet holes on either end of the flow channel, air plasma was used to bond the flow channel over the microwell array. The resulting microwell array flow cell device was then used for single cell RNA-Seq and optical demultiplexing.
Prior to the experiment, the device was flushed with 0.1% Tween-20 and incubated for several hours to hydrate the microwells, which were subsequently washed with PBS. Cell suspensions were counted using the Countess automated cell counter (Life Technologies). A suspension of cells in PBS mixed with Calcein AM (live stain) was flowed into the device and incubated for five minutes. Cells were deposited in the microwells by gravity. After thoroughly removing any excess cells with PBS, a suspension of barcoded capture beads that had been pre-counted by microscopy was introduced in PBS and allowed to load under gravity for five minutes. Excess beads were thoroughly removed with PBS and the flow cell was incubated on ice. A lysis buffer containing 0.08% Triton X- 100 supplemented with SUPERaseIN (Life Technologies) was flowed under ice-cold conditions immediately followed by fluorinert oil (Sigma) to seal the device. After two cycles of freeze-thaw at −80C to enhance cell lysis, the device was incubated at room temperature for 60 minutes for mRNA capture. After one hour of incubation for mRNA capture, the device was unsealed by rapid removal of the fluorinert oil with 20 mM Tris-HCl pH 8 containing 1% Triton X-100 and SUPERaseIN followed by thorough washing of the device with 20 mM Tris-HCl pH 8, 50 mM NaCl, and 0.1% Tween-20.
The mRNA captured on the beads was reverse transcribed using ProtoScript II Reverse Transcriptase (New England BioLabs) for two hours at 42C in 1× ProtoScript Reverse Transcriptase Buffer supplemented with 10 mM DTT, 0.5 mM dNTPs, 0.1% Tween-20, and SUPERaseIN. The reaction mixture was then removed with 20 mM Tris-HCl pH 8, 50 mM NaCl, and 0.1% Tween-20. Second strand synthesis was carried out using the MessageAmp II aRNA amplification kit (Ambion) according to the manufacturer's instructions. This involves a reaction mixture with DNA polymerase and RNaseH that is incubated for two hours at 16C. After removing the second strand reaction mixture with 20 mM Tris-HCl pH 8, 50 mM NaCl, and 0.1% Tween-20, in vitro transcription (IVT) was carried out using reagents in the MessageAmp II aRNA amplification kit (Ambion) including T7 RNA polymerase according to the manufacturer's instructions for 13 hours at 37C. The reaction linearly amplified the double-stranded cDNA libraries on the beads, eluting pools of barcoded aRNA into the flow channel which was subsequently removed using a pipette and purified using an RNA Clean & Concentrator Column (Zymo). aRNA was then reverse transcribed using uniquely barcoded random hexamer primers using PrimeScript Reverse Transcriptase (Clontech) supplemented with 0.5 mM dNTPs, 10 mM DTT, 1× PrimeScript Buffer, and 0.1 U/uL SUPERaseIN. The reaction proceeded for 10 minutes at 25C and two hours at 42C. The RNA-cDNA hybrid product was purified twice using 0.65× Ampure XP beads (Beckman). The resulting cDNA libraries were then enriched by PCR using Phusion High Fidelity DNA polymerase (New England BioLabs) using the Illumina RPI and RPI primers. The resulting PCR product was then purified on a 1.5% agarose gel, extracted using a Gel Extraction Kit (Qiagen), purified using 0.65× Ampure XP beads (Beckman), and sequenced on a NextSeq 500 sequencer (Illumina).
Cells and mRNA capture beads were loaded into the microwell array device as described above. The device was then placed on a computerized temperature and fluidic control system for automated single cell library production. The system consists of a manually controlled pressurization system, software, an electronic six-port rotary selector valve (Rheodyne), and a thermoelectric heater-cooler module (TE Technology). After cooling the system to 4C, a lysis solution comprised of TCL Buffer (Qiagen) and 1% 2-mercaptoethanol was introduced to the device, which was rapidly and automatically sealed with fluorinert (Sigma). After removing the oil and unsealing the microwells, a reverse transcription reaction mixture comprised of 1× Maxima RT Buffer, 1 mM dNTPs, 1 U/uL SUPERaseIN, 2.5 uM template-switching oligonucleotide, 0.1% Tween-20, and 10 U/uL Maxima H-Reverse Transcriptase (ThermoFisher) was introduced and incubated for 30 minutes at room temperature followed by 90 minutes at 42C.
After removing the reverse transcription reaction mixture, the beads were removed from the microwells by sonication, concentrated by centrifugation and incubated for 30 minutes at 37C with Exonuclease I in 1× Exonuclease I Buffer (New England BioLabs). The beads were then washed in 10 mM Tris-HCl pH 8, 1 mM EDTA, and 0.5% sodium dodecyl sulfate (SDS) followed by 10 mM Tris-HCl pH 8, 1 mM EDTA, and 0.01% Tween-20. The beads were then re-suspended in a PCR reaction mixture containing 1× HiFi Hot Start Ready Mix (Kappa) and 1 uM PCR primer and thermocycled to amplify a full-length, double-stranded, pooled cDNA library. The PCR product was purified with 0.6× Ampure XP beads (Beckman). The library was then fragmented and a sequencing adapter introduced using the Nextera XT library preparation kit (IIlumina) according to the manufacturer's instructions. Finally, the sequencing library was enriched by PCR using a reverse PCR primer from the Nextera XT kit and a custom forward PCR primer along with amplification reagents from the Nextera XT kit used according to the manufacturer's instructions. The resulting PCR product was purified with 0.6× Ampure XP beads (Beckman) and sequenced on a NextSeq 500 sequencer (Illumina).
Optical Demultiplexing of Barcoded mRNA Capture Beads
Following cDNA synthesis on the surface of optically barcoded capture beads, the microwell array device was transferred to an epifluorescence microscope system. The microscope system contains an inverted microscope frame (Ti-U, Nikon), an electron-multiplied charge coupled device (EM-CCD) camera (iXon, Andor), a 20×0.75 NA air objective lens (Nikon), a three-axis motorized stage (ASI), a 140 mW 637 nm optically pumped semiconductor laser (OBIS, Coherent), and custom optics for coupling the laser beam into the microscope. The custom optical path includes an engineered diffusor (Thorlabs) and electronic phase scrambling device for flattening the illumination field. An automated, computer-controlled fluidics system is attached the microscope system and contains two, ten-channel rotary selector valves (Rheodyne). A single computer program controls the fluidics system, laser shutter, camera, and microscope stage. Each of twelve, Cy5-labeled optical barcode probes was loaded into the channels of the fluidics system. The optical barcode probes were each complementary to one of the optical barcode oligonucleotides attached the beads as described above. In addition, a wash buffer (20 mI Tris-HCl pH 8, 50 mM NaCl, and 0.1% Tween-20) and a denaturing solution (100 mM NaOH) were loaded into independent channels of the fluidics system. The first of twelve optical barcode probes was flowed into the flow cell, incubated at room temperature for ten minutes, and washed out with wash buffer. After washing, the microscope automatically scanned and imaged the entire microwell array and identifies the beads in each microwell that are fluorescently labeled with the first optical barcode probe by hybridization, indicating the presence of the first optical barcode sequence. The denaturing solution was then introduced to the flow cell which causes the first optical barcode probe to dissociate from the beads and exit the flow cell. After washing with wash buffer, the process was repeated for all of the remaining optical barcode probes, At conclusion, the configuration of optical barcode sequences attached to each bead can be. interred from the series of twelve fluorescence images. Because each unique configuration of optical barcode sequences corresponds to a specific capture barcode sequence associated with the cDNA that is also attached to each bead, the capture barcode sequence for each individual cell can be identified (see, for example,
Silicon wafer masters (˜4 in) with cylindrical pillars (diameter 50 micron; height 30 micron) for photolithography were obtained from Stanford Microfluidics Foundry and were subsequently exposed to 1H,1H-2H-2H-perfluorooctyltrichlorosilane (Alfa Aesar) vapor under vacuum for ˜30 minutes to avoid curing of the PDMS on the wafer. PDMS (Sylgard 184, Dow Corning) was thoroughly mixed 10:1 (base:curing agent) and degassed under house vacuum for 2 hours. ˜15 g of degassed PDMS was poured onto the 4 in silicon water master and allowed to cure overnight at 90° C. This slab with microwells was then gently peeled. off from the master and used to construct PDMS microreactor flow cells.
VistaVision Microscope cover glass (22×50×0.16 mm) was plasma sterilized (Harrick Plasma) for ˜5 mins, and immediately immersed in 10% acetic acid (pH 3.5) ethanol solution containing 0.5% trimethoxysilanealdehyde (United Chemical Technologies), and incubated for 15 mins. The cover glass was then washed with ethanol, air-dried and heat cured at 90° C. for 10 mins. A 2.5 μM solution of 5′-aminated-LNA-oligo(dT) (Exiqon) in cyanoborohyride coupling buffer (Sigma) supplemented with 1M NaCl was added on the aldehyde surface of the cover glass. The cover glass was incubated for 3 hours at room temperature inside a humid chamber, and then washed with DI water. The aldehyde surface was then incubated in 10% ethanolamine in cyanoborohydride coupling buffer for 30 mins to quench the unreacted aldehydes.
A rectangular slab (3.5×1.5×0.1 cm) of PDMS containing the microwell array in the center was cut and a double-sided adhesive tape (˜120 micron thickness, Grace BioLabs) was adhered to the flat side of the PDMS slab that contained the microwells. The tape was cut in an elongated hexagonal shape, which formed the microchannel in the flowcell. The other side of the tape was pasted on the LNA coated cover glass to build the microfluidic device. Two holes were punched at the two end of the microchannel with a biopsy punch, which acted as the inlet and outlet of the device and tubing were attached to allow liquid flow. The periphery of the PDMS slab was sealed on to the cover glass using epoxy glue.
Experimental Procedure for Single Cell mRNA Printing on Glass
A suspension of U87 cells in PBS was flowed in to the device and loaded into the microwells by gravity (kept upside down) for 5 mins at room temperature. After washing with PBS buffer supplemented with SUPERaseIN (Ambion), the microwells were sealed using an automated mechanical device by placing the flow cell upside down on a screw mounted on a motorized z-stage (ASI) so that the top PDMS slab containing the microwells was pressed against the glass bottom. After sealing the wells mechanically, the seal was retained by hermetic sealing to trap the single cell lysate within a single microwell. The cells were lysed by freeze-thaw. Once the cells lysed, the mRNAs were captured on the LNA surface by hybridization of the 3′-polyA tail of the mRNA to the LNA-oligo(dT) during a 60 minute incubation. The microwells were then unsealed and the flow cell was immediately and vigorously washed with the Wash Buffer (20 mM Tris pH 8.0, 50 mM NaCl, 0.1% Tween-20), supplemented with SUPERaseIN (
The epifluorescence imaging system was constructed on an inverted Nikon Eclipse Ti-U microscope with 20×, 0.75 NA air objective (Plan Apo λ, Nikon). SYTOX Orange was excited using a 532 nm diode-pumped solid state laser (Dragon Lasers), and the fluorescence was collected and imaged onto an electron multiplying charge coupled device (EMCCD) camera (iXON3, Andor Technologies). The images were acquired with. 0.5 s exposure time (controlled by external shutter) at 1 MHz digitization (with no EM gain). Automated scanning of the surface (motorized X-Y stage, ASI), image acquisition, and illumination were controlled with custom software written in C/C++. The images were analyzed using ImageJ software.
For the single cell RNA-Seq experiment a monolithic PDMS was designated based multi-channel device, by fabricating each channel with a microwell array. Two key soft lithography techniques were used to fabricate this device. First, instead of using silicon wafer master directly for fabricating the microwell array as done in the case of RNA printing device, we generated a secondary master made out of PDMS. This was done because the aspect ratio of the micropillars results in a relatively fragile silicon master. It was found that the PDMS master to be more durable. Second, instead of using a double-sided adhesive tape for the device assembly, the bottom and the top of the device were bonded together by partial curing. This provided us with more durable and reliable partitions between the individual channels of our device than could be generated using tape. For the multi-lane microfluidic device, two different silicon wafer masters were fabricated, one for the top and other for the bottom containing the array of microwells. Masters for soft lithography were generated from 4-inch silicon test wafers (University Wafer) coated with SU-8 2005 (MicroChem) photoresist. The wafer master for the bottom of the device contained five arrays of cylindrical pillars (diameter 50 micron; height 50 micron). The wafer was then fluorosilanized as described above. To avoid repeated use of the silicon wafer, we fabricated secondary masters in PDMS as follows. 40 g of degassed PDMS 10:1 (base:curing agent) was poured and cured on the wafer, and then peeled off and cut into a rectangular slab. The surface containing an array of microwells was oxidized in plasma chamber (Harrick Plasma) for ˜2 mins and immediately fluorosilanized. Using this microwell-containing slab as a master, ˜10 g of degassed PDMS was cured on it and peeled off. This new PDMS slab containing array of pillars is an exact replica of the silicon wafer, and is fluorosilanized and served as a secondary master for soft lithography for microfabrication of the bottom part of the microfluidic flowcell device. ˜2 g of degassed PDMS 10:1 (base:curing agent) was poured on a plasma cleaned glass slide and the secondary master with pillar array was placed gently with pillars immersed into the liquid PDMS. The slide, PDMS and master (on top) was degassed for ˜5 mins and then cured hard at 90° C. for 2 hours. After curing, the master is peeled off and a thin layer of PDMS is bonded to the glass slide with 5 lane arrays of microwells.
A second silicon wafer master was constructed containing five longitudinal ridges (with a height of 100 microns) with rounded ends on which ˜30 g of degassed PDMS 15:1 (base:curing agent) was poured and allowed to cure partially at 60° C. for 90 mins. The partially cured PDMS was cut into a slab, holes were punched at either end of each channel, and the slab was placed gently on the top of the glass slide containing the microwell array in such a way that the longitudinal grooves were aligned over each of the five microwell arrays. The slide assembly was then incubated at 90° C. overnight to form a single monolithic PDMS structure as shown In
Synthesis of Uniquely Barcoded Beads for mRNA Capture
N-succinimide-coated Sepharose beads with a mean diameter of ˜30 μm were obtained from GE Healthcare in isopropanol. The beads were washed three times with water by centrifugation and re-suspended in a reaction mixture with a final concentration of 100 mM sodium borate (pH 8.5) and ˜0.8 mg/mL streptavidin (streptayidin from New England BioLabs was spiked with ˜2% AlexaFluor 647-labeled streptavidin from Life Technologies). The reaction was incubated at room temperature for one hour on a rotisserie to allow the streptavidin to covalently attach to the beads. The beads were then washed five times in Wash Buffer and incubated in 1 Wash Buffer for 30 minutes before using to completely quench any remaining reactive groups on the beads.
A dual-biotinylated oligonucleotide containing both the T7 promoter sequence and a partial Illumina adapter sequence (Table 1) were annealed to each of 96 oligonucleotides (Table 2) that are complementary to the partial Illumina adapter sequence on the 3′-end and contain a unique barcode and universal anchor sequence on the 5′-end (
Table 2 describes a list of oligonucleotide sequences used to generate the first set of barcoded beads (FBC) for combinatorial synthesis in Experiment 1.
The dual-biotinylated oligonucleotide was annealed at a final concentration of 2 μM in the presence of a four-fold molar excess of the barcoded oligonucleotide in a 96-well plate by stepwise cooling from 85C to 30C over 30 minutes. A DNA polymerase master mix was then added to each well such that the final concentration of the reaction components was 1× NEB Buffer 2 (New England BioLabs), 0.25 U/μL Klenow Fragment (exo-) (New England BioLabs), and 0.5 mM dNTPs. The reaction was incubated in each well at 37 C. for 30 minutes before heat inactivating the polymerase at 75° C. for 20 minutes.
An equal volume of beads was then added to each reaction mixture so that the extended, dual-biotinylated oligonucleotide could conjugate to the streptavidin coated beads at a final density of ˜1 billion oligonucleotide primers per bead. The conjugation reaction was incubated at room temperature overnight on a rotisserie and quenched with biotin at a final concentration of 2 mM and sodium hydroxide at a final concentration of 125 mM to melt the template strand off of the beads. The beads were then pooled and washed five times in 125 mM sodium hydroxide supplemented with 0.1 mM biotin and then washed an additional three times with Wash Buffer and 0.1 mM biotin. The beads were then re-suspended in Hybridization Buffer (20 mM Tris pH 8.0, 1 M NaCI, 0.1% Tween-20) supplemented with 0.1 mM biotin.
The pooled heads were split into ten reactions to which one of ten partially complementary oligonucleotides (Table 3) each containing a specific second barcode was added at a final concentration of 5 μM. The second barcode-containing oligonucleotides were allowed to hybridize to the beads at room temperature overnight on a rotisserie.
Table 3 describes oligonucleotide sequences used to generate the second set of barcoded beads (SBC) for combinatorial synthesis in Experiment 1.
The beads were then washed five times in Wash Buffer supplemented with 0.1 mM biotin and then re-suspended in a reaction mixture with final concentrations of 0.5 mM dNTPs, 1× NEB Buffer 2 (New England BioLabs), and 0.1 mM biotin. We included biotin in the wash and storage buffers in order to saturate any remaining streptavidin sites on the beads so that, in the even that a barcoded capture primer dissociates form a beads, it cannot re-associate with a different bead. The reactions were cooled to 16° C. on a thermocycler and Klenow Fragment (exo-) (New England BioLabs) was added at a final concentration of 0.25 U/μL. The reaction was incubated for one hour at 16° C. with mixing every 10 minutes with a pipette followed by heat inactivation at 75° C. for 20 minutes.
The ten reaction mixtures were then quenched and the hybridized strand was denatured by addition of sodium hydroxide to a final concentration of 125 mM. The reaction mixtures were then washed five times in 125 mM sodium hydroxide with 0.1 mM biotin, pooled, and then further washed three times with Wash Buffer supplemented with 0.1 mM biotin.
Prior to the experiment, each lane of the device was flushed with 0.1% Tween-20 solution and incubated for several hours to hydrate the microwells, which were subsequently washed with 2 mL of phosphate-buffered saline (PBS). Cell suspensions were counted using Countess automated cell counter (Life Technologies). A suspension of cells in PBS mixed with Calcein AM (live stain) dye was flowed in to each lane and incubated for ˜5 mins, so that the cells load in to the microwells under gravity. After thoroughly washing out the excess cells with PBS, a suspension of barcoded capture beads that had been pre-counted by microscopy was introduced in PBS and allowed to load under gravity for ˜5 mins. In an aspect, ˜3,000 cells to each lane of the device was introduced. It is also noted that only 25% of the lower surface of each channel contains a microwell array, and so by expanding this area, the number of cells captured without incurring increased reagent costs for on-chip library generation were significantly increased (for example, as long as the size of the barcode pool was pooled). Excess beads were washed out thoroughly with PBS and the flow cell was incubated on ice. 20 μL 0.08% TritonX-100 (Sigma) supplemented with SUPERaseIN in PBS was flowed under ice-cold conditions immediately followed by fluorinert oil (Sigma) to seal the device, After two cycles of freeze-thaw at ˜80° C. to enhance cell lysis, the device was incubated at mom temperature for 60 mins for mRNA capture (
Two of the lanes contained pure U87 and MCF10a cells, respectively, and other lanes were loaded with a mixture of both the cell types. All lanes were imaged twice, first with blue laser (λex=473 nm, Dragon Lasers) for imaging the cells and secondly with a red laser (λex=637 nm, Obis, Coherent) for imaging the beads labeled with AlexaFluor 647 tagged streptavidin. We used the two-color images to determine number of bead-cell pairs in the array. After an hour of incubation for mRNA capture, all the lanes were unsealed by rapid washing of the oil with 20 mM Tris, containing 1% TritonX-100 and SUPERaseIN, followed by Wash Buffer supplemented with SUPERaseIN. After this point the microwells stay open and subsequent enzymatic steps occur simultaneously in separate lanes of the open device.
The single cell library preparation protocol is adopted from the recently reported CEL-Seq protocol with few modifications as described below. The mRNA captured on the beads was reverse transcribed using ProtoScript II Reverse Transcriptase (New England Biolabs) for 2 hours at 42° C. in 1× ProtoScript Reverse Transcriptase, buffer, supplemented with 10 mM DTT, 0.5 mM dNTPs, 0.1% Tween-20 and SUPERaseIN. The reaction mixture was washed out with Wash Buffer. The second strand synthesis was carried out using reagents front the MessageAmp II aRNA amplification kit (Ambion), where a mixture of DNA polymerase and RNaseH in second strand buffer was used along with dNTPs by incubating the device at 16° C. for 2 hours. After flushing out the second strand reaction mixture with Wash Buffer, an in vitro transcription mixture from the MessageAmp II kit containing four nucleotides and T7 RNA polymerase enzyme mix in T7 buffer was introduced to all lanes and incubated for 13 hours at 37° C. (
Experiment 2 was identical to Experiment 1 with a few exceptions. First, the two cell types under study were U87 human glioma cells and WI-38 human fibroblast cells (a diploid, limited-passage, non-cancer cell line). Second, reagents from the HiScribe In Vitro Transcription kit (New England BioLabs) were substituted for the MessageAmp II kit for the IVT portion of the protocol. Third, some of the oligonucleotides used were different from in Experiment and are tabulated as detailed in Tables 4-6.
Table 4 describes a group of preferred oligonucleotides used for barcoding and library preparation for Experiment 2.
Table 5 describes oligonucleotide sequences used to generate the first set of barcoded beads (FBC) for combinatorial synthesis in Experiment 2.
Table 6 provides for oligonucleotide sequences used to generate the second set of barcoded beads (SBC) for combinatorial synthesis in Experiment 2.
Read 1 of the single cell RNA-Seq data contains a cell-identifying barcode sequence followed by poly(dT), and read 2 contains a 8-base UMI followed by a 6-base lane-identifying barcode and a transcript sequence. The reads are first demultiplex based on the lane-identifying barcode while recording the corresponding UMI using a custom Python script. We then map the remainder of read 2 to the human genome and transcriptome (hg19, Ensembl annotation from Illumina iGenomes) using the STAR aligner. Mapped reads for each lane are then demultiplexed based on the cell-identifying barcodes in read 1 and assigned to a gene using HTSeq. Both the lane- and cell-identifying barcodes were allowed to have a single-base mismatch during demultiplexing.
The set of reads that uniquely mapped to the transcriptome were collected and assigned an address comprised of its cell-identifying barcode, gene, UMI, and mapping position. In addition, the reads that mapped to both the genome and transcriptome were kept, but that mapped to only one position on the transcriptome and mapped to that position with the appropriate strand-specificity. The reads to identify unique molecules were filtered. Reads with identical addresses were collapsed to a single molecule. In addition, reads with identical cell-identifying barcodes, genes, mapping positions, and with UMIs having a Hamming distance less than or equal to two were collapsed to a single molecule. All reads considered identical molecules by the above definition (UMIs with a Hamming distance less than or equal to two and mapping position within six bases) were removed but that also occurred with different cell-identifying barcodes within the same lane. This approach likely underestimates of the true number of molecules associated with each cell and gene and results in some loss of gene detection. However, it also removes molecules that may become spuriously associated with the incorrect cell via PCR recombination, as observed and similarly filtered in previous studies that used very similar library construction protocols.
To identify barcodes that correspond to actual individual cells in our device in Experiment 1, the observed cell-identifying barcodes were filtered by progressively downsampling the corresponding gene profiles to the same number of total reads and assessing the number of unique molecules detected from each cell-identifying barcode. After excluding cell-identifying barcodes having zero associated molecules, it was found that the distribution of associated unique molecules to be bimodal, with one small subpopulation having nearly as many unique molecules as reads at low read totals. It was found the size of this subpopulation to be in excellent agreement with our device imaging data. These 598 profiles were taken to represent the actual individual cells captured in the device with a barcoded head. We used the same approach to assess the cell-identifying barcodes in Experiment 2.
The 396 single-cell profiles were kept with the highest coverage in the data set (all five lanes represented). The U87 and MCF10a single cell profiles were compared to bulk RNA-Seq profiles of U87 and MCF10a cells. Bulk RNA-Seq library from ˜107 U87 cells were prepared using the, TruSeq RNA-Seq library preparation kit (Illumina) and sequenced the library to a depth of ˜30M, 100-base single-end reads on an Illumina HiSeq 2500. Publically available bulk RNA-Seq profile of MCF10a cells was obtained from the Gene Expression Omnibus (entry GSE45258). Reads were mapped to the transcriptome as described above and expression values (FPKM) were computed using Cufflinks. Pearson correlation coefficients between single cell and hulk profiles were computed between log-transformed single-cell expression profiles (unique molecules per million reads plus one) and log-transformed hulk values (EPKM plus one). Single cell median profiles were generated from different numbers of randomly selected single cell profiles and repeated this random sampling ten times without replacement for each data point in
Differential expression analysis was conducted by comparing each detected gene in the two cell type-exclusive lanes using Wilcoxin's rank-sum test. Genes with p<0.05 were used for clustering analysis. Regardless of differential expression +/−(1−p) was used (which is positive for expression biased in one cell type and negative for expression biased towards a second cell type) for each gene as input to iPAGE, a mutual information-based algorithm that can associate gene ontologies with genes based on an assigned numerical value[35]. A matrix of pairwise Spearman correlation coefficients was generated based on unique molecules detected across 396 single cell profiles in Experiment 1 (247 profiles in Experiment 2) using only the differentially expressed genes. The data was then clustered with the MATLAB implementation t-SNE using the correlation matrix as input. The single cell profiles were color coated in the t-SNE clusters using a simple classifier score given by the log-ratio of the number of cell type-specific genes for each of the two cell types in a given cell with an above-average rank in expression level (
Table 7 describes estimated costs per reagents and associated costs in an aspect of the disclosure.
This application claims priority to U.S. Application Ser. No. 62/166,565, filed May 26, 2015, the content of which is herein incorporated by reference in its entirety.
This invention was made with Government support under Contract No. K01EB016071 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62166565 | May 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15576925 | Nov 2017 | US |
| Child | 17480839 | US |
