Patent Application
20210214721
The disclosure relates to tools for understanding gene expression and biology.
In living organisms, genetic information is stored in DNA. Genes in the DNA are transcribed into messenger RNA (mRNA), which is translated into proteins. Proteins play critical functional and structural roles in living organisms. For example, most enzymes are made of proteins, and those enzymes catalyze the metabolic reactions that keep us alive. It is also enzymes that copy DNA into mRNA. Proteins are also structural, and constitute the essential fibers of muscles, the predominant material of hair, as well as basic structural linkages within the cytoskeleton. Essentially, all such proteins are made by translating an mRNA into the protein. In fact, one mRNA can serve as the template for synthesizing multiple copies of a protein.
Because living cells change in response to different environmental conditions, nutrient availability, and even intra-cellular signaling, the cells need different proteins at different times. It is beneficial to the cell's ability to change that any given mRNA is short-lived. It is thought that most mRNA molecules have a lifetime measured in seconds or minutes. The essential and ephemeral nature of mRNA presents a challenge to biological understanding. On the one hand, the mRNAs that are present in a cell at a given moment could reveal much about how the cell is responding to a pathogen, or a drug, or to age-specific developmental changes. On the other hand, in any attempt to remove a cell from its natural environment and study the mRNAs present, time is of the essence. Those mRNAs begin to degrade within seconds or minutes. As time is spent in the laboratory to set up a clinical or research assay, the very molecular ingredients of the cell to be studied begin to decay and the information they represent is lost.
The disclosure provides methods for reverse transcribing mRNA into complementary DNA (cDNA) while simultaneously isolating cells into aqueous partitions. Methods of the disclosure provide for the very rapid capture of the information in mRNA into cDNA, which is more stable than mRNA. The cDNAs are made immediately as the sample is emulsified into droplets. Methods of the disclosure make use of particles that serve as templates for making a large number of monodisperse emulsion droplets simultaneously in a single tube or vessel. By adding cells into an aqueous mixture that includes a plurality of hydrogel template particles, layering oil over the aqueous phase, and vortexing or pipetting the tube, the particles serve as templates while the shear force of the vortexing or pipetting causes the formation of water-in-oil monodisperse droplets with on particle in each droplet. Reverse transcription reagents can be included in the initial mixture, allowing reverse transcriptase to begin simultaneously with shearing the water/oil mixture to form the emulsions. Making cDNAs from the RNAs immediately during the first stage of the droplet-making process preserves the information present as mRNA in the original cells. The disclosure provides suitable reagents and conditions for successfully reverse transcribing mRNA into cDNA while isolating a plurality of cells into monodisperse droplets in a single tube.
Because the cDNA is made simultaneously with mixing the emulsions in the tube, important biological information is not lost due to the short lifetime of RNA molecules in living cells. Because the information of mRNA is preserved as cDNA, methods of the disclosure provide an additional useful tool for understanding the phenotype and gene expression of a given cell at any time. In fact, the cDNA can be amplified by, e.g., polymerase chain reaction, into a plurality of stable DNA amplicons that can be stored or studied under a variety of conditions or methods. Methods of the disclosure are well-suited to making DNA libraries suitable for sequencing on a next-generation sequencing (NGS) instrument.
An insight of the disclosure is that a plurality of droplets can be made in a single tube at a temperature and/or at a mixing speed that promote cDNA synthesis. For example, by mixing at about 50 degrees C. and/or at about 500 rpm, methods can successfully initiate cDNA synthesis while, in the single tube, forming the droplets that contain the cells thereby isolated into individual aqueous partitions. Thus, methods of the disclosure provide important tools for basic biology, clinical research, and patient testing.
In certain aspects, the disclosure provides a library preparation method. The method includes preparing a mixture that includes cells and reagents for reverse transcription and vortexing or optionally pipetting the mixture. During the vortexing (or pipetting), the mixture partitions into aqueous droplets that each essentially include zero or one cell, the cells are lysed to release mRNA into the droplets, and reverse transcriptase copies the mRNA into cDNAs. The method preferably further includes amplifying the cDNAs into a library of amplicons. Preferably the mixture includes particles such that, during vortexing, the particles template the formation of the droplets. The particles may be gels that include the reagents therein. The mixture may be aqueous and the method may include adding an oil onto the mixture prior to the vortexing/pipetting. The method may include, during the vortexing, heating the mixture to a temperature that promotes activity of the reverse transcriptase (e.g., between about forty and about fifty degrees C.). The mixture is preferably sheared by any suitable mechanism or device, such as a benchtop vortexer or shaker, a pipette (e.g., micropipette), a magnetic or other stirrer or similar.
In certain embodiments, the particles are linked to capture oligos that have a free, 3′ poly-T region. The particles may also include cDNA capture oligos that have 3′ portions that hybridize to cDNA copies of the mRNA. The 3′ portions of the cDNA capture oligos may include gene-specific sequences or oligomers. The oligomers may be random or “not-so-random” (NSR) oligomers (NSROs), such as random hexamers or NSR hexamers. The particles may be linked to capture oligos that include one or more handles such as primer binding sequences cognate to PCR primers that are used in the amplifying step or the sequences of NGS sequencing adaptors. The cDNA capture oligos may include template switching oligos (TSOs), which may include poly-G sequences that hybridize to and capture poly-C segments added during reverse transcription.
In some embodiments, the vortexing is performed on a vortexing instrument, e.g., which vortexes the mixture at a suitable rate such as between about two hundred and about seven hundred rpm (preferably about 500 rpm). The vortexing instrument may include a heater that heats the mixture during vortexing.
The mixture may be pre-prepared with a plurality of template particles at a number to capture a suitable target number of cells. For example, the mixture may initially include thousands, tens of thousands, hundreds of thousands, millions, or at least about 10 million template particles. Methods may be used to capture and partition any number of cells such as thousands, tens of thousands, hundreds of thousands, millions, or at least about 10 million cells.
Each of the particles may contain some of the reagents for reverse transcription. The particles may be used to template the formation of monodisperse droplets. Preferably, each of the particles serves as a template to initiate formation of aqueous monodisperse droplets in oil, in which each droplet comprises one particle. The particles may be hydrogel particles and may include, for example, polyacrylamide (PAA) or polyethylene glycol (PEG).
Aspects of the disclosure provide a sample preparation method. The method includes preparing, in a sample vessel, an aqueous mixture that includes nucleic acids and polymerase enzymes. An oil is added to the sample vessel, and the method includes shaking or vortexing the sample vessel to simultaneously: (i) partition the aqueous mixture into droplets surrounded by the oil and (ii) synthesize a DNA copy of at least one of the nucleic acids with the polymerase during the shaking. The nucleic acids may initially be in cells and the shaking step may cause droplets to form that contain the cells. The method may include lysing the cells within the droplets to release the nucleic acids into the droplets. Lysing may be done by adding a lytic agent to the vessel (such as a detergent like sodium dodecyl sulfate (SDS)). In some embodiments, the vessel is heated to a temperature that promotes reverse transcription. It may be found that detergent, heat, and shaking work in combination to lyse the cells. In preferred embodiments, the nucleic acids include mRNA and the polymerase enzymes include reverse transcriptase enzymes.
Preferably the aqueous mixture includes a plurality of template particles, and shaking the sample vessel causes each template particle to serve as a template in the formation of one of the droplets. The nucleic acids may initially be in cells and the shaking step may cause droplets to form such that each of the droplet contains one template particle and one or zero cells. The method may include lysing the cells within the droplets to release the nucleic acids into the droplets and the method may include, during the shaking step, heating the aqueous mixture to a temperature that promotes reverse transcription.
In certain embodiments, the template particles are linked to capture oligos, which are linked to the template particles at their 5′ ends, and in which 3′ ends of the capture oligos include a poly-T sequence. Each of the template particles may contain some of the reverse transcriptase enzymes. The method may include, after the adding step, loading the sample vessel into an instrument that performs the shaking step. In some embodiments, during the shaking: the droplets form, cells are lysed within the droplets to release the nucleic acids, template particles capture the nucleic acids, and the polymerase enzymes synthesize the DNA copies.
The aqueous mixture may include a plurality of template particles (e.g., hydrogel particles), and the method may include, after the adding step, loading the sample vessel into an instrument that performs the shaking step and wherein shaking the sample vessel causes each template particle to serve as a template in the formation of one of the droplets. The nucleic acids may initially be in cells and the shaking step may cause droplets to form in which each of the droplets contains one template particle and one or zero cells. In some embodiments: the nucleic acids are mRNAs in cells in the aqueous mixture; the droplets contain the cells; the polymerase enzymes are provided in template particles within the aqueous mixture; and the template particles serve as template to cause formation of the droplets during the shaking. The method may include—after partitioning the aqueous mixture into the droplets—lysing the cells to release the mRNAs into the droplets. In certain embodiments the template particles are bound to capture oligos that capture the mRNAs and prime extension reactions by which the polymerase enzymes copy the mRNAs.
The disclosure generally relates to single-tube “direct to sequencing library” methods that can be used to isolate cells into fluid partitions (e.g., droplets) while also reverse transcribing RNA into cDNA while isolating the cells into the partitions. In some embodiments, premade particles, such as hydrogel particles, serve as templates that cause water-in-oil emulsion droplets to form when mixed in water with oil and vortexed or sheared. For example, an aqueous mixture can be prepared in a reaction tube that includes template particles and target cells in aqueous media (e.g., water, saline, buffer, nutrient broth, etc.). An oil is added to the tube, and the tube is agitated (e.g., on a vortexer aka vortex mixer). The particles act as template in the formation of monodisperse droplets that each contain one particle in an aqueous droplet, surrounded by the oil.
The droplets all form at moment of vortexing—essentially instantly as compared to the formation of droplets by flowing two fluids through a junction on a microfluidic chip. Each droplet thus provides an aqueous partition, surrounded by oil. An important insight of the disclosure is that the particles can be provided with reagents that promote useful biological reactions in the partitions and even that reverse transcription can be initiated during the mixing process that causes the formation of the partitions around the template droplets. Moreover, the pre-templated instant partitions may be formed while the reaction mixture is being heated to a temperature that promotes activity of reverse transcriptase. In fact, data show mixing conditions and particle compositions that promote successful copying of mRNA into cDNA during mixing of the mixture to form the pre-templated instant partitions.
Methods of the disclosure are useful in making a cDNA library. A cDNA library may be a useful way to capture and preserve information from RNAs present in a sample. For example, a sample that includes one or more intact cells may be mixed with template particles to form a partition (e.g., droplet) that includes the cell. The cell can be lysed and mRNAs can be reverse transcribed into cDNAs in the droplet during the mixing stage that forms the partitions. Similarly, a sample that includes cell-free RNA can be mixed with oligo-linked template particles and mixed (e.g., shaken, vortexed, or sheared) to form droplets while simultaneously beginning the transcribe the RNA to cDNA. Whether starting with whole cells or cell-free RNA, the result is the formation of droplets that include cDNA copies of the starting RNA. Because the cDNA is more stable than RNA (e.g., cDNA does not include 2′ hydroxyl groups that autocatalyze the molecule's own hydrolysis), the droplets provide a stable cDNA library that may be used in downstream assays to study the RNA content of the starting sample.
Forming the cDNAs while initially forming the droplets avoids problems caused by the ephemeral nature of mRNA. Sample preparation and library preparation methods of the disclosure improve the ability of laboratory techniques to study RNA compositions of a sample. In fact, cells can be sequestered into aqueous partitions while also, simultaneously copying the mRNAs into stable cDNA that may be stored and studied downstream.
During the vortexing, several things are accomplished. The mixture partitions 109 into aqueous droplets that each include zero or one cell. When the sample includes whole cells such as PBMCs, the cells are lysed 115 to release mRNA into the droplets. The lysing 115 is an optional step, as the method 101 may be used where the original sample includes cell-free RNA. Additionally, reverse transcriptase copies 123 the mRNA into cDNAs. Lysis may be performed chemically (e.g., using micelles to deliver lysis agents), by activated chemistry (e.g., thermal, light, etc), and/or enzymatically (heat activated). A mix of micelle/chemical plus heat-activated enzymes has been tested.
Embodiments of the disclosure employ chemical lysis methods including, for example, micelle-based methods. Methods may include using micelles to deliver suitable lysis agents. Suitable lysis agents include Sarkosyl, SDS, Triton X-100. One or more surfactants is used to micellize the lysis agent into the oil phase. Suitable surfactants for creating micelles may include, for example Ran or ionic Krytox. It may be useful to use a super-concentrated co-solvent to aid dissolution of the lysis agent. Some embodiments use a combination of fluoro-phase surfactant Krytox 157-FSH (acidic form) or neutralized form (ammonium counter-ion, potassium counter-ion or sodium counter-ion) in 0.05%-5% in Novec 7500 or 7300 or 7100 or Fuorinert to form micelles that include a sysis agent such as Sarkosyl or SDS at 0.05%-5%. In certain embodiments, a fluoro-phase surfactant such as Perfluorpolyether PEG-conjugates is used with a non-ionic lsysis agent such as Triton-X100 or IGEPAL at 0.05%-2%. Fluorocarbon based oil system may be used, e.g., 3M Novec HFE (e.g. HFE7000, 7100, 7200, 7300, 7500, 7800, 8200) or 3M Fluorinert (e.g. FC-40, -43, -70, -72, -770-3283.-3284). Embodiments may use surfactant for fluorocarbon based oil, e.g., commercially available compounds such as Chemour Krytox 157FSH, Chemour Capstone etc. Ionic type fluorophase surfactants may include Perfluoroalkyl carboxylates, Perfluoroalkyl sulfonates, Perfluoroalkyl sulfates, Perfluoroalkyl phosphates, Perfluoropolyether carboxylates, Perfluoropolyether sulfonates, or Perfluoropolyether phosphates. Non-ionic type fluorophase surfactant may include Perfluoropolyether ethoxylates or Perfluoroalkyl ethoxylates. A silicone based oil system may be used such as polydimethylsiloxane (PDMS) with viscosity range between 0.5-1000 cst. Suitable surfactant for silicone based oil may be used such as Gelest Reactive Silicones, Evonik ABIL surfactant, etc. An ionic type silicone phase surfactant may be carboxylate terminated PDMS or Amine terminated PDMS. A non-ionic type silicone phase surfactant may be hydroxyl terminated PDMS or PEG/PPG functionalized PDMS. A hydrocarbon based oil system may use heavy alkane hydrocarbons with carbon atoms number greater than 9. The oil could include a single compound or a mixture from multiple compounds. For example, tetradecane, hexadecane, mineral oil with viscosity range between 3 to 1000 cst. Suitable surfactant for hydrocarbon based oil (ionic) may include Alkyl carboxylates, Alkyl sulfates, Alkyl sulfonates, Alkyl phosphates or (non-ionic) PEG-PPG copolymers (e.g. Pluronic F68, Pluronic F127, Pluronic L121, Pluronic P123), PEG-alkyl ethers (e.g. Brij L4, Brij 58, Brij C10), PEG/PPG functionalized PDMS (e.g. Evonik ABIL EM90, EM180), Sorbitan derivatives (e.g. Span-60, Span-80, etc.), or Polysorbate derivatives (e.g. Tween-20, Tween 60, Tween 80). To achieve best micellization/co-dissolution performance and minimum disruption of water-in-oil droplet interface, the general rule of thumb for lysis agent/oil phase surfactant combination is as follow: (i) an ionic type lysis agent is preferred for combination with ionic oil phase surfactant, such lysis agent may include but not limited to: SDS, Sarkosyl, sodium deoxycholate, Capstone FS-61, CTAB; (ii) a non-ionic type lysis agent is preferred for combination with non-ionic oil phase surfactant, such lysis agent may include but not limited to: Triton X-100, Triton X-114, NP-40, Tween-80, Brij 35, Octyl glucoside, octyl thioglucoside; and/or (iii) a zwitterionic type lysis agent may be used in combination with either ionic or non-ionic oil phase surfactant, such lysis agent may include but not limited to: CHAPS, CHAPSO, ASB-14, ASB-16, SB-3-10, SB-3-12.
As shown, two important phenomena are accomplished during the vortexing 107 step: aqueous partitions form 109 and reverse transcription 123 occurs.
Importantly, a plurality (e.g., thousands, tens of thousands, hundreds of thousands, millions, or tens of millions or more) of aqueous partitions are formed 109 essentially simultaneously. Results have shown that this consistently works. It may be preferable to use template particles (e.g., a corresponding number of hydrogel particles that serve as templates to the formation of droplets). Reagents may be provided to promote cell lysis or initiate reverse transcription. Once the vortexing 107 step has been performed, at least one of the droplets will have at least one cDNA copy of an RNA from the starting sample. For background overview, see generally Gubler, 1983, A simple and very efficient method for generating cDNA libraries, Gene 25(2-3):263-9 and Figueiredo, 2007, Cost effective method for construction of high quality cDNA libraries, Biomolecular Eng 24:419-421, both incorporated by reference. Preferably, one or a plurality of the droplets will each have a plurality of cDNAs that include droplet-specific oligonucleotide barcodes for a plurality of corresponding RNAs that were partitioned into the droplets by the partitioning 109. Forming the cDNA(s) may include attaching amplification primer-binding sites (such as first and second universal priming sequences at the ends of the cDNAs), and the method 101 optionally includes amplifying 127 the cDNA(s) into amplicons, which may be stored or analyzed. For example, the amplicons may be sequenced using a sequencer such as a next-generation sequencing (NGS) instrument.
To prepare 103 the mixture that includes cells and reagents, template particles may be provided. Template particles may be made of any suitable material such as, for example, polyacrylamide, poly (lactic-co-glycolic acid), polyethylene glycol, agarose, or other such material. In some embodiments, hydrogel particles are prepared. In some embodiments, 6.2% acrylamide (Sigma-Aldrich), 0.18% N,N′-methylene-bis-acrylamide (Sigma-Aldrich), and 0.3% ammonium persulfate (Sigma-Aldrich) are used for PAA particle generation. A total of 14% (w/v) 8-arm PEGSH (Creative PEGworks) in 100 mM NaHCO3 and PEGDA (6 kDa, Creative PEGworks) in 100 mM NaHCO3 may be used for PEG particle generation. A 1% low melting temperature agarose (Sigma-Aldrich) may be used for agarose particle generation. The agarose solution is warmed to prevent solidification. Agarose and PEG solutions are injected into a droplet generation device with the oil (HFE-7500 fluorinated oil supplemented with 5% (w/w) deprotonated Krytox 157 FSH) using syringe pumps (New Era, NE-501). The PAA solution is injected into the droplet generation device with the fluorinated oil supplemented with 1% TEMED. The hydrogel solution and oil are loaded into separate 1 mL syringes (BD) and injected at 300 and 500 μL, respectively, into the droplet generation device using syringe pumps. The PAA and PEG droplets are collected and incubated for 1 h at room temperature for gelation. The agarose droplets are incubated on ice for gelation. After gelation, the gelled droplets are transferred to an aqueous carrier by destabilizing them in oil with the addition of an equal volume of 20% (v/v) perfluoro-1-octanol in HFE-7500. The particles are washed twice with hexane containing 2% Span-80 (Sigma-Aldrich) to remove residual oil. Following the hexane wash, the particles are washed with sterile water until all oil is removed.
In some embodiments, the template particles are provided in some form of tube or sample vessel for steps of the method 101. Any suitable vessel may be used. For example, a sample vessel may be an, e.g., 50 or 150 mL, microcentrifuge tube such as those sold under the trademark EPPENDORF. The sample vessel may be a blood collection tube such as the collection tube sold under the trademark VACUTAINER. The tube may be a conical centrifuge tube sold under the trademark FALCON by Corning Life Science. In preferred embodiments of the method, the template particles are provided in a tube within an aqueous media such as a buffer, nutrient broth, saline, or water.
A sample that contains RNA is obtained, to be added to the particles. Any suitable sample may be used. Suitable samples include environmental, clinical, library specimen, or other samples with known or unknown RNA present as cell-free RNA or present in tissue or cells (living or preserved) containing the RNA. Suitable samples may include whole or parts of blood, plasma, cerebrospinal fluid, saliva, tissue aspirate, microbial culture, uncultured microorganisms, swabs, or any other suitable sample, For example, in some embodiments, a blood sample is obtained (e.g., by phlebotomy) in a clinical setting. Whole blood may be used, or the blood may be spun down to isolate a component of interest from the blood, such as peripheral blood monocytes (PBMCs). The sample is then preferably added to a mixture such as the particles in the tube. For the method 101 it is preferable that the mixture include reagents for reverse transcription such as reverse transcriptase.
The mixture may be vortexed by any suitable method or mechanism. The mixture may be contained in a tube such as a microcentrifuge tube. The tube may be manually flicked, or pressed down on a benchtop vortexer. The mixture may be in a well in a plate, such as a 96-well plate, and the plate may be loaded onto a benchtop mixer or shaker. The mixture may be in one tube of an 8-tube strip of microcentrifuge tubes such as the 8-tube strip sold under the trademark EPPENDORF. In a preferred embodiment, the tube is loaded into a vortexing instrument.
For example, in some embodiments, certain molecular reagents such as polymerase enzymes are packaged in the particles, some reagents such as oligonucleotides are linked (e.g., covalently) to the particles, and some reagents such as lysis agents (e.g., detergent), dNTPs, and metal ions are added independently.
In preferred embodiments, the target RNAs are mRNAs. For example, methods of the disclosure may be used to make a cDNA library useful for showing an expression profile of a cell. Where the target RNAs are mRNAs, the particles may include mRNA capture oligos 701 useful to at least synthesize a first cDNA copy of an mRNA. The particles 213 may further include cDNA capture oligos 709 with 3′ portions that hybridize to cDNA copies of the mRNA. For the cDNA capture oligos, the 3′ portions may include gene-specific sequences or hexamers. As shown, the mRNA capture oligos 701 include, from 5′ to 3′, a binding site sequence P5, an index, and a poly-T segment. The cDNA capture oligos include, from 5′ to 3′, a binding sequence P7 and a hexamer. Any suitable sequence may be used for the P5 and P7 binding sequences. For example, either or both of those may be arbitrary universal priming sequence (universal meaning that the sequence information is not specific to the naturally occurring genomic sequence being studied, but is instead suited to being amplified using a pair of cognate universal primers, by design). The index segment may be any suitable barcode or index such as may be useful in downstream information processing. It is contemplated that the P5 sequences, the P7 sequence, and the index segment may be the sequences use in NGS indexed sequences such as performed on an NGS instrument sold under the trademark ILLUMINA, and as described in Bowman, 2013, Multiplexed Illumina sequencing libraries from picogram quantities of DNA, BMC Genomics 14:466 (esp. in
As shown, a capture oligo 701 hybridizes to an mRNA 715. A reverse transcriptase 725 binds and initiates synthesis of a cDNA copy of the mRNA 715. Note that the mRNA 715 is connected to the particle 213 non-covalently, by Watson-Crick base-pairing. The cDNA that is synthesized will be covalent linked to the particle 213 by virtue of the phosphodiester bonds formed by the reverse transcriptase 725.
It will be observed that copying the first sense copy 915 of the cDNA 814 using the free forward primer 901 (to produce the) is the first depicted step producing a molecular product not-covalently linked to the particle 213. Copying the sense copy 915 produces an antisense copy 914 that is not covalently linked to the particle 213. Of the sense copies 915, only the first sense copy 915 was covalently linked to the particle 213. After copying the first sense copy, every template has a barcode (“index”). This allows droplets 401 to be broken, after which multiplexing can proceed in bulk aqueous phase. In fact, where multiple droplets were formed and used to perform reverse transcription, each template strand may be barcoded by droplet. After “breaking the emulsion” (releasing contents from droplets into bulk aqueous phase), the same free forward primer 901 and free reverse primer 909 may be used to amplify, in parallel and together, any number of sense copies 915 and antisense copies 914 (each barcoded back to original droplet and optionally to individual strand).
Other variants and equivalents are within the scope of the disclosure. A feature that is preferably in common among embodiments of the disclosure is that some form of vortexing, shaking, shearing, agitating, or mixing is performed to encapsulate a plurality of particles simultaneously into droplets while some reverse transcription occurs at least partially during the vortexing, shaking, shearing, agitating, or mixing stage. Preferably, either wholly or at least in part, shaking/vortexing to form droplets is contemporaneous with synthesizing a cDNA copy of an mRNA resulting in the cDNA copy being contained within the droplet, once formed. Because methods of the disclosure are useful for making cDNAs that may serve well as samples for sequencing or quantification assays (e.g., digital PCR, for example), methods of the disclosure are useful for preparing samples where the input includes RNA.
Preferably in the method 1201, the nucleic acids (e.g., mRNA 715) are initially in cells 209 and the shaking step 1213 forms droplets wherein each of the droplet 401 contains one template particle 213 and one or zero cells. The method 1201 may also include lysing the cells 209 in the droplets 401 to release the nucleic acids into the droplets. Lysing may be done by introducing a detergent such as SDS. Beneficially, the combination of shaking at about 500 rpm, the addition of SDS, and heating to about 40 to about 50 degrees C. may be sufficient to lyse the cells 209. Preferably, during the shaking step, the aqueous mixture is heated to a temperature that promotes reverse transcription (e.g., about 40 to about 50 degrees C.).
In some embodiments of the method 1201, the template particles are linked to capture oligos 701, linked to the template particles at their 5′ ends, wherein the 3′ ends of the capture oligos include a poly-T sequence. Each of the template particles 213 may contain some of the reverse transcriptase enzymes. During the shaking: the droplets 401 form, cells 209 are lysed within the droplets 401 to release the nucleic acids, template particles 213 capture the nucleic acids, and the polymerase enzymes synthesize the DNA copies 814. The method 1201 is suitable for the production of a plurality of monodisperse droplets where the aqueous mixture includes a plurality of template particles, and the method comprises, after the adding step, loading the sample vessel into an instrument that performs the shaking step and wherein shaking the sample vessel causes each template particle to serve as a template in the formation of one of the droplets.
The nucleic acids may initially be in cells and the shaking step forms droplets such that each of the droplets contains one template particle and one or zero cells. Preferably the nucleic acids are mRNAs in cells in the aqueous mixture, and the droplets contain the cells; and the polymerase enzymes are provided in template particles within the aqueous mixture. The method 1201 may include—after partitioning the aqueous mixture into the droplets—lysing the cells to release the mRNAs into the droplets. The template particles 2013 may be bound to capture oligos 701 that capture the mRNAs 715 and prime extension reactions by which the polymerase enzymes 725 copy the mRNAs 715.
| Number | Date | Country | |
|---|---|---|---|
| 62960283 | Jan 2020 | US |
