In situ combinatorial labeling of cellular molecules

Abstract

To provide methods of uniquely labeling complementary DNA molecules within a plurality of cells.SOLUTION: A method comprises: fixing and permeabilizing a plurality of cells; reverse-transcribing RNA molecules within the cells to form cDNA molecules; dividing the cells comprising the cDNA molecules into at least two primary aliquots; providing primary nucleic acid tags to the primary aliquots; coupling the cDNA molecules within the primary aliquots with the provided primary nucleic acid tags; combining the primary aliquots; dividing the combined primary aliquots into at least two secondary aliquots; providing secondary nucleic acid tags to the secondary aliquots; coupling the cDNA molecules within the secondary aliquots with the provided secondary nucleic acid tags; combining final aliquots; forming a cell lysate; and adding a protease inhibitor and a binding agent to the lysate.SELECTED DRAWING: Figure 20

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 561,806, filed Sep. 22, 2017, which is hereby incorporated by reference in its entirety.

[0002] Technical Field The present disclosure generally relates to methods for uniquely labeling or barcoding molecules within nuclei, multiple nuclei, cells, multiple cells, and / or tissues. The present disclosure also relates to kits for uniquely labeling molecules within nuclei, multiple nuclei, cells, multiple cells, and / or tissues. In particular, the methods and kits can relate to the labeling of RNA and / or cDNA.

Background Art

[0003] Background Next - generation sequencing (NGS) can be used to identify and / or quantify individual transcripts derived from a sample of cells. However, such techniques can be too complex to perform on individual cells within a large - scale sample. In such methods, generally, RNA transcripts are purified from lysed cells (i.e., cells that have been broken apart), and subsequently, reverse transcription is used to convert the RNA transcripts into complementary DNA (cDNA). Thereafter, the cDNA sequences can be sequenced using NGS. In such procedures, since all the cDNA sequences are mixed together before sequencing, the RNA expression of the entire sample is measured, and individual sequences cannot be associated with the original individual cells.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Methods for uniquely labeling or barcoding transcripts derived from individual cells may involve manually separating individual cells into separate reaction vessels and may require specialized equipment. Alternative techniques for sequencing individual cell transcripts are to use microscopy to identify individual fluorescent bases. However, this technique is difficult to implement and may be limited to sequencing a small number of cells.

[0005] The embodiments disclosed herein will become more fully apparent from the following description and the appended claims, when considered in conjunction with the accompanying drawings.

Means for Solving the Problems

[0006] Detailed Description The present disclosure generally relates to methods for uniquely labeling or barcoding molecules in nuclei, multiple nuclei, cells, multiple cells, and / or tissues. The present disclosure also relates to kits for uniquely labeling or barcoding molecules in nuclei, multiple nuclei, cells, multiple cells, and / or tissues. Molecules to be labeled can include, but are not limited to, RNA, cDNA, DNA, proteins, peptides, and / or antigens.

Brief Description of the Drawings

[0007]

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DETAILED DESCRIPTION OF THE INVENTION

[0008] It will be readily understood that the embodiments generally described herein are exemplary. The following more detailed description of the various embodiments is not intended to limit the scope of the present disclosure and merely represents the various embodiments. Furthermore, those skilled in the art can change the order of the steps or operations of the methods disclosed herein without departing from the scope of the present disclosure. In other words, the specific order of steps or operations can be modified unless a particular order of steps or operations is necessary for the proper operation of the embodiments.

[0009] The term "binding" is widely used throughout the present disclosure to refer to any form for attaching or coupling two or more components, entities, or objects. For example, two or more components may be bound to each other by chemical bonds, covalent bonds, ionic bonds, hydrogen bonds, electrostatic forces, Watson-Crick type hybridization, etc.

[0010] One aspect of the present disclosure relates to a method of labeling nucleic acids. In some embodiments, the method may include the step of labeling nucleic acids in a first cell. The method includes: (a) generating complementary DNA (cDNA) in a plurality of cells including the first cell by reverse transcribing RNA using a reverse transcription primer comprising a 5' overhang sequence; (b) dividing the plurality of cells into (n) aliquots; (c) providing a plurality of nucleic acid tags to each of the n aliquots, wherein each labeling sequence of the plurality of nucleic acid tags provided to a given aliquot is the same, and different labeling sequences are provided to each of the n aliquots; (d) binding at least one of the cDNA of each of the n aliquots to the nucleic acid tag; (e) combining the n aliquots; and (f) repeating steps (b), (c), (d), and (e) using the combined aliquots. In various embodiments, the plurality of cells can be selected from eukaryotic cells and prokaryotic cells. In various other embodiments, the plurality of cells can be selected from at least one of, but not limited to, mammalian cells, yeast cells, and / or bacterial cells.

[0011] In certain embodiments, each nucleic acid tag may comprise a first strand that includes a 3' hybridization sequence extending from the 3' end of the labeled sequence and a 5' hybridization sequence extending from the 5' end of the labeled sequence. Each nucleic acid tag may also comprise a second strand that includes a protruding sequence. The protruding sequence may include (i) a first portion complementary to at least one of the 5' hybridization sequence and the 5' protruding sequence, and (ii) a second portion complementary to the 3' hybridization sequence. In some embodiments, the nucleic acid tag (e.g., the final nucleic acid tag) may include a capture agent such as, but not limited to, 5'-biotin. cDNA labeled with a nucleic acid tag containing 5'-biotin can enable or permit the attachment or coupling of streptavidin-coated magnetic beads to the cDNA. In some other embodiments, a plurality of beads may be coated with a capture strand (i.e., a nucleic acid sequence) configured to hybridize to the final sequence overhang of the barcode. In yet some other embodiments, the cDNA may be purified or isolated by using a commercially available kit (e.g., the RNEASY™ kit). 。

[0012] In various embodiments, step (f) (i.e., steps (b), (c), (d), and (e)) can be repeated a sufficient number of times to generate a unique set of labeled sequences for the cDNA of the first cell. Stated in another manner, step (f) may be repeated a number of times such that the cDNA of the first cell has a first unique set of labeled sequences, the cDNA of the second cell has a second unique set of labeled sequences, the cDNA of the third cell has a third unique set of labeled sequences, and so on. The methods of the present disclosure provide for the labeling of cDNA sequences derived from single cells with unique barcodes, which can identify or assist in the identification of the cell from which the cDNA is derived. In other words, a portion, majority, or substantially all of the cDNA derived from a single cell may have the same barcode, which need not be repeated in cDNA derived from one or more other cells in the sample (e.g., derived from a second cell, a third cell, a fourth cell, etc.).

[0013] In some embodiments, the barcoded cDNAs can be mixed together and sequenced (e.g., using NGS) to collect data regarding RNA expression at the single cell level. For example, certain embodiments of the methods of the present disclosure may be useful for the evaluation, analysis, or study of the transcriptome (the various RNA species transcribed from the genome of a given cell) of one or more individual cells.

[0014] As discussed above, aliquots or groups of cells may be separated into different reaction vessels or containers, and a first set of nucleic acid tags may be added to multiple cDNA transcripts. The vessels or containers may herein be referred to interchangeably as vessels, samples, and wells. Thus, the terms vessel, container, vessel, sample, and well may be used interchangeably herein. Thereafter, the aliquots of cells may be regrouped, mixed, and separated again, and a second set of nucleic acid tags may be added to the first set of nucleic acid tags. In various embodiments, the same nucleic acid tag may be added to more than one aliquot of cells in a single or given round of labeling. However, after repeated rounds of separation, tagging, and repooling, the cDNA of each cell can bind to a unique combination or sequence of nucleic acid tags that form a barcode. In some embodiments, the cells in a single sample may be separated into a number of different reaction vessels. For example, the number of reaction vessels may include four 1.5 ml microcentrifuge tubes, multiple wells of a 96-well plate, or another suitable number and type of reaction vessel.

[0015] In certain embodiments, step (f) (i.e., steps (b), (c), (d), and (e)) may be repeated a number of times, and the number of times may be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, etc. In certain other embodiments, step (f) may be repeated a sufficient number of times such that it is likely that the cDNA of each cell is bound to a unique barcode. This number can be selected to provide that there is a greater than 50% likelihood, a greater than 90% likelihood, a greater than 95% likelihood, a greater than 99% likelihood, or some other probability that the cDNA of each cell is bound to a unique barcode. In yet other embodiments, step (f) may be repeated some other appropriate number of times.

[0016] In some embodiments, the method of labeling the nucleic acid of the first cell may include a step of fixing a plurality of cells before step (a). For example, the components of the cells may be fixed or cross-linked so that the components are immobilized or retained in place. The plurality of cells may be fixed using formaldehyde in phosphate buffered saline (PBS). For example, the plurality of cells may be fixed with about 1-4% formaldehyde in PBS. In various embodiments, the plurality of cells may be fixed using methanol (e.g., 100% methanol) at about -20°C or about 25°C. In various other embodiments, the plurality of cells may be fixed using methanol (e.g., 100% methanol) at about -20°C to about 25°C. In yet various other embodiments, the plurality of cells may be fixed using ethanol (e.g., about 70-100% ethanol) at about -20°C or room temperature. In yet various other embodiments, the plurality of cells may be fixed using ethanol (e.g., about 70-100% ethanol) at about -20°C to room temperature. In yet various other embodiments, the plurality of cells may be fixed using acetic acid at, for example, about -20°C. In yet various other embodiments, the plurality of cells may be fixed using acetone at, for example, about -20°C. Other suitable methods for fixing a plurality of cells are also within the scope of the present disclosure.

[0017] In certain embodiments, the method of labeling the nucleic acid of the first cell may include a step of permeabilizing a plurality of cells before step (a). For example, holes or openings may be formed in the outer membranes of the plurality of cells. TRITON (trademark) X-100 may be added to the plurality of cells, followed optionally by the addition of HCl to form one or more holes. Approximately 0.2% of TRITON (trademark) X-100 may be added to the plurality of cells, followed, for example, by the addition of approximately 0.1 N HCl. In certain other embodiments, ethanol (e.g., approximately 70% ethanol), methanol (e.g., approximately 100% methanol), Tween20 (e.g., approximately 0.2% Tween20), and / or NP-40 (e.g., approximately 0.1% NP-40) may be used to permeabilize the plurality of cells. In various embodiments, the method of labeling the nucleic acid of the first cell may include a step of fixing and permeabilizing a plurality of cells before step (a).

[0018] In some embodiments, the cells may be adherent cells (e.g., adherent mammalian cells). Fixation, permeabilization, and / or reverse transcription may be performed or carried out on the adherent cells (e.g., on cells adhered to a plate). For example, the adherent cells may be fixed, permeabilized, and / or reverse transcribed, followed by trypsin treatment to detach the cells from the surface. Alternatively, the adherent cells may be detached before the separation and / or tagging step. In some other embodiments, the adherent cells may be trypsinized before the fixation and / or permeabilization step.

[0019] In some embodiments, a method of labeling nucleic acids of a first cell may include ligating at least two nucleic acid tags that are bound to cDNA. The ligation may be performed before or after the lysis and / or cDNA purification step. The ligation may include covalently linking the 5' phosphate sequence of a nucleic acid tag to an adjacent strand or the 3' end of an adjacent nucleic acid tag such that individual tags are formed into a contiguous or substantially contiguous barcode sequence that is bound to the 3' end of the cDNA sequence. In various embodiments, a double-stranded DNA or RNA ligase may be used with an additional linker strand configured to hold the nucleic acid tag with an adjacent nucleic acid in a "nicked" double-stranded conformation. Thereafter, a double-stranded DNA or RNA ligase may be used to fill in the "nick". In various other embodiments, a single-stranded DNA or RNA ligase may be used without an additional linker. In certain embodiments, the ligation may be performed in multiple cells.

[0020] Figure 1 shows ligating multiple nucleic acid tags to form a substantially contiguous label or barcode. For example, when multiple nucleic acid tags are added, each cDNA transcript can be bound or ligated to a series of nucleic acid tags. By using a ligase, portions of the nucleic acid tags can be ligated or covalently linked to form a substantially contiguous label or barcode that is bound or attached to the cDNA transcript.

[0021] In certain other embodiments, the method may include lysing (i.e., breaking down the cell structure of) multiple cells to release cDNA from within the multiple cells, for example, after step (f). In some embodiments, the multiple cells are lysed in a lysis solution (e.g., 10 mM Tris-HCl (pH 7.9), 50 mM EDTA (pH 7.9), 0.2 M In NaCl, 2.2% SDS, 0.5 mg / ml ANTI-RNase (protein ribonuclease inhibitor; AMBION (registered trademark)), and 1000 mg / ml proteinase K (AMBION (registered trademark)), it may be dissolved, for example, with shaking (e.g., vigorously shaking) at about 55 °C for about 1 to 3 hours. In some other embodiments, the plurality of cells may be lysed by sonication and / or by passing through an 18-25 gauge injection needle at least once. In yet some other embodiments, the plurality of cells may be lysed by heating to about 70-90 °C. For example, the plurality of cells may be lysed by heating to about 70-90 °C for about 1 hour or longer. Thereafter, the cDNA may be isolated from the lysed cells. In some embodiments, RNase H may be added to the cDNA to remove the RNA. The method may further include the step of ligating at least two of the nucleic acid tags bound to the released cDNA. In some other embodiments, the method of labeling the nucleic acid of the first cell may include the step of ligating at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, etc. of the nucleic acid tags bound to the cDNA.

[0022] In various embodiments, a method of labeling nucleic acids of a first cell may include the step of removing one or more unbound nucleic acid tags (e.g., the step of washing a plurality of cells). For example, the method may include the step of removing a portion, a majority, or substantially all of the unbound nucleic acid tags. The unbound nucleic acid tags may be removed such that further rounds of the methods of the present disclosure are not contaminated with one or more unbound nucleic acid tags from a previous round of a given method. In some embodiments, the unbound nucleic acid tags may be removed by centrifugation. For example, a plurality of cells can be centrifuged such that a pellet of the cells forms at the bottom of a centrifuge tube. The supernatant (i.e., the liquid containing the unbound nucleic acid tags) can be removed from the centrifuged cells. The cells may then be resuspended in a buffer (e.g., a fresh buffer that does not contain or substantially does not contain unbound nucleic acid tags). In another example, a plurality of cells may be coupled or linked to magnetic beads coated with an antibody configured to bind to a cell membrane or nuclear membrane. A magnet can then be used to attract the plurality of cells to one side of a reaction vessel to pellet the plurality of cells. In some other embodiments, a plurality of cells may be placed in a cell strainer (e.g., a PLURISTRAINER® cell strainer) and washed with a wash buffer. For example, the plurality of cells may be left in the cell strainer and the wash buffer may pass through the cell strainer. The wash buffer may include a surfactant, a detergent, and / or about 5-60% formamide.

[0023] As discussed above, multiple cells may be re-pooled, and this method may be repeated any number of times to add more tags to the cDNA and generate a set of nucleic acid tags that can act as barcodes. Adding even more rounds increases the number of paths the cells can take, and consequently, the number of possible barcodes that can be generated. With sufficient rounds and splits, the number of possible barcodes will be much larger than the number of cells, increasing the likelihood that each cell will have a unique barcode. For example, when splitting in a 96-well plate, after 4 splits, there will be 4 = 84,934,656 possible barcodes.

[0024] In some embodiments, the reverse transcription primer may be configured to reverse transcribe all or substantially all of the RNA in the cell (e.g., a random hexamer with a 5' overhang). In some other embodiments, the reverse transcription primer may be configured to reverse transcribe RNA having a poly(A) tail (e.g., a poly(dT) primer such as a dT(15) primer with a 5' overhang). In yet some other embodiments, the reverse transcription primer may be configured to reverse transcribe a predetermined RNA (e.g., a transcript-specific primer). For example, the reverse transcription primer may be configured to barcode specific transcripts such that fewer transcripts per cell may be profiled, but each of the transcripts can be profiled across a larger number of cells.

[0025] Figure 2 shows the formation of cDNA by in situ reverse transcription. Panel A shows cells that have been fixed and permeabilized. Panel B shows the addition of a poly(T) primer that can prime the reverse transcription of polyadenylated transcripts, as discussed above. Panel C shows the addition of random hexamers that can prime the reverse transcription of substantially any transcript, as discussed above. Panel D shows the addition of primers designed to target specific transcripts such that only a subset of transcripts can be amplified, as discussed above. Panel E shows the cells of Panel A after reverse transcription, showing that the cDNA has hybridized to the RNA.

[0026] Reverse transcription may be performed or carried out on multiple cells. In certain embodiments, reverse transcription may be performed on multiple fixed and / or permeabilized cells. In some embodiments, a mutant of the M-MuLV reverse transcriptase may be used for reverse transcription. Any suitable method of reverse transcription is within the scope of the present disclosure. For example, the reverse transcription mix may include a reverse transcription primer that includes a 5' overhang, and the reverse transcription primer may be configured to initiate reverse transcription and / or act as a binding sequence for a nucleic acid tag. In some other embodiments, the portion of the reverse transcription primer that is configured to bind to RNA and / or initiate reverse transcription may be any of the following: a random hexamer of nucleotides, a septamer, an octomer, a nonamer, a decamer, a poly(T) stretch, and / or may include one or more of one or more gene-specific primers.

[0027] Another aspect of the present disclosure relates to a method for uniquely labeling molecules within a cell or within a plurality of cells. In some embodiments, the method comprises: (a) binding an adapter sequence or a universal adapter to molecules within a plurality of cells; (b) dividing the plurality of cells into at least two primary aliquots, wherein the at least two primary aliquots include at least a first primary aliquot and a second primary aliquot; (c) providing a primary nucleic acid tag to the at least two primary aliquots, wherein the primary nucleic acid tag provided to the first primary aliquot is different from the primary nucleic acid tag provided to the second primary aliquot; (d) binding the adapter sequence within each of the at least two primary aliquots to the provided primary nucleic acid tag; (e) combining the at least two primary aliquots; (f) dividing the combined primary aliquots into at least two secondary aliquots, wherein the at least two secondary aliquots include at least a first secondary aliquot and a second secondary aliquot; (g) providing a secondary nucleic acid tag to the at least two secondary aliquots, wherein the secondary nucleic acid tag provided to the first secondary aliquot is different from the secondary nucleic acid tag provided to the second secondary aliquot; and (h) binding the molecules within each of the at least two secondary aliquots to the provided secondary nucleic acid tag.

[0028] In certain embodiments, the method further comprises step (i), namely using subsequent aliquots It may further include steps of repeating steps (e), (f), (g), and (h). Step (i) can be repeated a sufficient number of times to generate a series of nucleic acid tags specific to the molecule of a single cell. In various embodiments, the number of times can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, etc. In certain other embodiments, step (i) can be repeated another suitable number of times.

[0029] In some embodiments, the molecule may be located inside a cell or multiple cells. In some other embodiments, the molecule may be coupled to a cell or multiple cells. For example, the molecule may be a cell surface molecule. In still some other embodiments, the molecule may be located inside a cell or multiple cells and / or coupled to a cell or multiple cells.

[0030] As discussed above, the method may include a step of fixing and / or permeabilizing a plurality of cells before step (a). In various embodiments, each of the nucleic acid tags may include a first strand. The first strand may include a barcode sequence including a 3' end and a 5' end. The first strand may further include a 3' hybridization sequence and a 5' hybridization sequence adjacent to the 3' end and the 5' end of the barcode sequence, respectively. In some embodiments, each of the nucleic acid tags may include a second strand. The second strand may include a first portion complementary to at least one of the 5' hybridization sequence and the adapter sequence, and a second portion complementary to the 3' hybridization sequence.

[0031] In certain embodiments, the molecule is a macromolecule. In various embodiments, the molecule is selected from at least one of RNA, cDNA, DNA, protein, peptide, and / or antigen.

[0032] In some embodiments, the molecule is RNA and the adapter sequence may be single-stranded. Further, step (a) may include one of ligating the 5' end of the single-stranded adapter sequence to the 3' end of the RNA and / or ligating the 3' end of the single-stranded adapter sequence to the 5' end of the RNA. In some other embodiments, the molecule is RNA and step (a) may include the step of hybridizing the adapter sequence to the RNA.

[0033] Methods for binding or coupling an adapter sequence to RNA can be used, for example, in RNA transcriptome sequencing, ribosome profiling, small RNA sequencing, non-coding RNA sequencing, and / or RNA structure profiling. In some embodiments, a plurality of cells may be fixed and / or permeabilized. The 5' end of the single-stranded adapter sequence may be ligated to the 3' end of the RNA (see FIGS. 3A and 3B). In certain embodiments, the ligation may be performed or carried out by T4 RNA ligase 1. In certain other embodiments, the ligation may be performed by T4 RNA ligase 1 using a single-stranded adapter sequence containing a 5' phosphate. In various embodiments, the ligation may be performed by THERMOSTABLE 5' APPDNA / RNA LIGASE (TM) (NEW ENGLAND BIOLABS (R)). In various other embodiments, the ligation may be performed by THERMOSTABLE 5' APPDNA / RNA LIGASE (TM) using a 5'-preadenylated single-stranded adapter sequence. Other suitable ligases and adapter sequences are also within the scope of the present disclosure.

[0034] In some embodiments, for example, hybridization by Watson-Crick base pairing can be used to label RNA with an adapter sequence (see Figure 4). The adapter sequence may be configured to initiate reverse transcription to form or generate cDNA after the labeling step and / or cell lysis, as discussed above (see Figure 5).

[0035] The 3’ end of the single-stranded adapter sequence may be ligated to the 5’ end of the RNA. In certain embodiments, the ligation may be performed or carried out by T4 RNA ligase 1. In certain other embodiments, the ligation may be performed by T4 RNA ligase 1 using RNA containing a 5’ phosphate. In various embodiments, the ligation may be performed by THERMOSTABLE 5’ APPDNA / RNA LIGASE (trademark) (NEW ENGLAND BIOLABS (registered trademark)). In various other embodiments, the ligation may be performed by THERMOSTABLE 5’ APPDNA / RNA LIGASE (trademark) using 5’ preadenylated RNA. As described above, other suitable ligases and adapter sequences are also within the scope of the present disclosure.

[0036] In some embodiments, the molecule may be cDNA. Methods for binding or coupling an adapter sequence to cDNA can be used, for example, for RNA transcriptome sequencing. In certain embodiments, a plurality of cells may be fixed and / or permeabilized. Reverse transcription may be performed on the plurality of fixed and / or permeabilized cells using a primer containing an adapter sequence at its 5’ end. As discussed above, the 3’ end of the primer may be a gene-specific random hexamer or a poly(T) sequence. The resulting cDNA may contain an adapter sequence at its 5’ end (see Figure 5).

[0037] In some embodiments, when the molecule is DNA (e.g., genomic DNA), the method may further include, before step (a), digesting the DNA with a restriction enzyme. Further, step (a) may include ligating an adapter sequence to the digested DNA.

[0038] Methods for binding or coupling an adapter sequence to DNA can be used, for example, in whole genome sequencing, targeted genome sequencing, DNase-Seq, ChIP sequencing, and / or ATAC-seq. In certain embodiments, one or more restriction enzymes may be used to digest the DNA to produce at least one of blunt-ended fragments and / or fragments having overhangs. A single-stranded universal adapter or a partial double-stranded sequence with an adapter sequence having an overhang at one end may be ligated to the digested genomic DNA. For example, a partial double-stranded sequence with a single-stranded adapter sequence having an overhang that is compatible with the overhang generated by one or more restriction enzymes may be ligated to the digested genomic DNA.

[0039] In various embodiments, Tn5 transposase can be used to incorporate (e.g., directly incorporate) an adapter sequence into genomic DNA, and the transposase can be released by adding sodium dodecyl sulfate (SDS) to expose the adapter sequence. Other transposases and methods for incorporating adapter sequences into genomic DNA are also within the scope of the present disclosure.

[0040] In certain embodiments, the molecule is a protein, peptide, and / or antigen, and the adapter sequence is a unique identifier sequence (e.g., including a nucleic acid) coupled to an antibody. It can be coupled to (mu). The unique identifier array may be configured to uniquely identify the antibody to which the unique identifier array is coupled. Further, step (a) may include the step of binding an antibody containing each of the adapter array and the unique identifier array to a protein, a peptide, and / or an antigen. In certain other embodiments, the molecule is a protein, a peptide, and / or an antigen, and the adapter array may be incorporated into an aptamer. Further, step (a) may include the step of binding the aptamer to a protein, a peptide, and / or an antigen.

[0041] Methods for binding or coupling an adapter array to a protein, a peptide, and / or an antigen can be used, for example, for protein quantification, peptide quantification, and / or antigen quantification. In various embodiments, the adapter array can be attached (e.g., chemically attached) to an antibody. For example, the adapter array can be attached to an antibody using chemistries known to those skilled in the art for mediating DNA-protein binding. Antibodies against different proteins can be labeled with a nucleic acid sequence or nucleic acid strand containing a unique identifier sequence in addition to the adapter array. Subsequently, the antibody or a set of antibodies may be used in an immunostaining experiment to label the proteins or a set of proteins in fixed and / or permeabilized cells or tissues (see Figure 6). Subsequently, the cells may be subjected to the labeling or barcoding procedures disclosed herein.

[0042] In some embodiments, a nucleic acid sequence (e.g., a DNA molecule) attached or bound to an antibody can be released from the antibody and / or the adapter array. Sequencing reactions can reveal the unique identifier sequence associated with a given protein as well as the label or barcode associated with one or more unique cells. In certain embodiments, such methods can reveal or identify the number and / or type of proteins present in one or more cells.

[0043] In various embodiments, DNA aptamers and / or RNA aptamers can be used instead of or in addition to the nucleic acid-modified (or DNA-modified) antibodies as described above (see Figure 7). An adapter sequence (and a target protein-specific antibody) can be incorporated (e.g., directly incorporated) into the sequence of a given aptamer.

[0044] Another aspect of the present disclosure relates to a method of barcoding nucleic acids within cells. In some embodiments, the method is a method of barcoding nucleic acids within cells, comprising: (a) generating cDNA in a plurality of cells by reverse transcribing RNA using a reverse transcription primer comprising a 5' overhang sequence; (b) dividing the plurality of cells into at least two aliquots; (c) providing a plurality of nucleic acid tags to each of the at least two aliquots, wherein each barcode sequence of the plurality of nucleic acid tags introduced into a given aliquot is the same and different barcode sequences are introduced into each aliquot; (d) binding at least one of the cDNA of each of the at least two aliquots to the nucleic acid tag; (e) combining the at least two aliquots; and (f) repeating steps (b), (c), (d), and (e) at least once using the combined aliquots.

[0045] In certain embodiments, each nucleic acid tag may comprise a first strand comprising a 3' hybridization sequence extending from the 3' end of the barcode sequence and a 5' hybridization sequence extending from the 5' end of the barcode sequence. Each nucleic acid tag may also comprise a second strand comprising an overhang sequence, the overhang sequence comprising: (i) a first portion complementary to at least one of the 5' hybridization sequence and the 5' overhang sequence, and (ii) a second portion complementary to the 3' hybridization sequence.

[0046] Figure 8 shows cell splitting, tagging, and pooling according to an embodiment of the present disclosure. The reverse transcribed cells may be split into reaction vessels or wells. Four wells are shown in Figure 8. However, as discussed above, any suitable number of reaction vessels or wells can be used. One cell is highlighted to show its path through this process. As shown, the highlighted cell first enters well "a", where a first tag that hybridizes to the overhangs of all cDNA transcripts is added (shown within the square frame). This tag holds a unique barcode region "a" that identifies the well the cell was in. After hybridization, all cells are washed to remove excess tags, regrouped, and then redistributed into the same number of wells. The highlighted cell then enters well "c", where a second tag is added to identify the well the cell was in. After the second round, the cells are 4 2 = 16 possible paths through the tubes. This process can be repeated to add more tags to the cDNA transcripts and increase the number of possible paths the cells can take. Figures 9A and 9B show two exemplary workflows according to embodiments of the present disclosure.

[0047] Another aspect of the present disclosure relates to a kit for labeling nucleic acids in at least a first cell. In some embodiments, the kit may include at least one reverse transcription primer that includes a 5' overhang sequence. The kit may also include a plurality of first nucleic acid tags. Each first nucleic acid tag may include a first strand. The first strand may include a 3' hybridization sequence extending from the 3' end of the first labeling sequence and a 5' hybridization sequence extending from the 5' end of the first labeling sequence. Each first nucleic acid tag may further include a second strand. The second strand may include an overhang sequence, and the overhang sequence may include (i) a first portion complementary to at least one of the 5' hybridization sequence of the reverse transcription primer and the 5' overhang sequence, and (ii) a second portion complementary to the 3' hybridization sequence.

[0048] The kit may further include a plurality of second nucleic acid tags. Each second nucleic acid tag may include a first strand. The first strand may include a 3' hybridization sequence extending from the 3' end of the second labeled sequence and a 5' hybridization sequence extending from the 5' end of the second labeled sequence. Each second nucleic acid tag may further include a second strand. The second strand may include a protruding sequence, and the protruding sequence may include (i) a first portion complementary to at least one of the 5' hybridization sequence and the 5' protruding sequence of the reverse transcription primer, and (ii) a second portion complementary to the 3' hybridization sequence. In some embodiments, the first labeled sequence may be different from the second labeled sequence.

[0049] Also, in some embodiments, the kit may include one or more additional plurality of nucleic acid tags. Each nucleic acid tag of the one or more additional plurality of nucleic acid tags may include a first strand. The first strand may include a 3' hybridization sequence extending from the 3' end of the labeled sequence and a 5' hybridization sequence extending from the 5' end of the labeled sequence. Also, each nucleic acid tag of the one or more additional plurality of nucleic acid tags may include a second strand. The second strand may include a protruding sequence, and the protruding sequence may include (i) a first portion complementary to at least one of the 5' hybridization sequence and the 5' protruding sequence of the reverse transcription primer, and (ii) a second portion complementary to the 3' hybridization sequence. In some embodiments, the labeled sequence may be different for each given additional plurality of nucleic acid tags.

[0050] In various embodiments, the kit may further include at least one of a reverse transcriptase, a fixative, a permeabilizing agent, a ligation agent, and / or a lysing agent.

[0051] Another aspect of the present disclosure relates to a kit for labeling at least a first intracellular molecule. For example, the kit disclosed above may be adapted to label one or more of RNA, cDNA, DNA, protein, peptide, or antigen in at least a first cell.

[0052] Another aspect of the present disclosure relates to a method for uniquely labeling RNA molecules in a plurality of cells.The method comprises: (a) a step of fixing and permeabilizing a first plurality of cells before step (b), wherein the first plurality of cells are fixed and permeabilized at a temperature of less than about 8°C; (b) a step of reverse transcribing RNA molecules in the first plurality of cells to form complementary DNA (cDNA) molecules in the first plurality of cells, wherein the step of reverse transcribing the RNA molecules comprises a step of coupling a primer to the RNA molecules, and the primer comprises at least one of a poly(T) sequence or a random sequence; (c) a step of dividing the first plurality of cells containing the cDNA molecules into at least two primary aliquots, wherein the at least two primary aliquots comprise a first primary aliquot and a second primary aliquot; (d) a step of providing primary nucleic acid tags to the at least two primary aliquots, wherein the primary nucleic acid tag provided to the first primary aliquot is different from the primary nucleic acid tag provided to the second primary aliquot; (e) a step of coupling the cDNA molecules in each of the at least two primary aliquots to the provided primary nucleic acid tags; (f) a step of combining the at least two primary aliquots; (g) a step of dividing the combined primary aliquots into at least two secondary aliquots, wherein the at least two secondary aliquots comprise a first secondary aliquot and a second secondary aliquot; (h) a step of providing secondary nucleic acid tags to the at least two secondary aliquots, wherein the secondary nucleic acid tag provided to the first secondary aliquot is different from the secondary nucleic acid tag provided to the second secondary aliquot; (i) a step of coupling the cDNA molecules in each of the at least two secondary aliquots to the provided secondary nucleic acid tags; (j) a step of repeating steps (f), (g), (h), and (i) using subsequent aliquots, wherein the final nucleic acid tag comprises a capture agent; (k) a step of combining the final aliquots; (l) a step of lysing the first plurality of cells to release the cDNA molecules from the first plurality of cells to form a lysate; and / or (m) optionally including a step of adding a protease inhibitor and / or a binder to the lysate to cause the cDNA molecules to bind to the binder.

[0053] The method includes the step of further dividing the combined final aliquot into at least two final aliquots, where the at least two final aliquots include a first final aliquot and a second final aliquot. In some embodiments, the first plurality of cells may be fixed and permeabilized at less than about 8°C, less than about 7°C, less than about 6°C, less than about 5°C, about 4°C, less than about 4°C, less than about 3°C, less than about 2°C, less than about 1°C, or another suitable temperature. In certain embodiments, the method may include the step of dispensing the cells. For example, after the last or final round of barcoding (by ligation), the cells may be pooled and then lysed, and then the cells may be dispensed into different lysate aliquots. Each lysate aliquot may contain a predetermined number of cells.

[0054] For example, referring to step (m), the protease inhibitor may include phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), combinations thereof, and / or another suitable protease inhibitor. For example, referring to steps (j), (k), (l), and / or (m), the capture agent may include biotin or another suitable capture agent. Further, the binder may include avidin (e.g., streptavidin) or another suitable binder.

[0055] In certain embodiments, a method of uniquely labeling RNA molecules in a plurality of cells may (e.g., after step (m)) further comprise: (n) performing a template switch of a cDNA molecule bound to a binder using a template switch oligonucleotide; (o) amplifying the cDNA molecule to form an amplified cDNA molecule solution; and / or (p) introducing a solid phase reversible immobilization (SPRI) bead solution into the amplified cDNA molecule solution to remove polynucleotides less than about 200 base pairs, less than about 175 base pairs, or less than about 150 base pairs (see DeAngelis, MM et al., Nucleic Acids Research (1995) 23(22):4742). In other words, the cDNA molecule may be bound to streptavidin beads in the lysate. Template switching of the cDNA molecules attached to the beads may be performed (e.g., to add an adapter to the 3' end of the cDNA molecules). Subsequently, PCR amplification of the cDNA molecules may be performed, followed by addition of SPRI beads to remove polynucleotides less than about 200 base pairs. The ratio of the SPRI bead solution to the amplified cDNA molecule solution may be about 0.9:1 to about 0.7:1, about 0.875:1 to about 0.775:1, about 0.85:1 to about 0.75:1, about 0.825:1 to about 0.725:1, about 0.8:1, or another suitable ratio. Additionally, the SPRI bead solution may contain about 1M to 4M NaCl, about 2M to 3M NaCl, about 2.25M to 2.75M NaCl, about 2.5M NaCl, or another suitable amount of NaCl. The SPRI bead solution may also contain about 15 w / v% to 25 w / v% polyethylene glycol (PEG), and the molecular weight of the PEG may be about 7,000 g / mol to 9,000 g / mol (PEG8000). In various embodiments, the SPRI bead solution may contain about 17 w / v% to 23 w / v% PEG8000, about 18 w / v% to 22 w / v% PEG8000, about 19 w / v% to 21 w / v% PEG8000, about 20 w / v% PEG8000, or another suitable w / v% of PEG8000.

[0056] Methods for uniquely labeling RNA molecules in multiple cells may further include the step of adding a common adapter sequence to the 3' end of the released cDNA molecules. The common adapter sequence may be an adapter sequence that is the same or substantially the same for each of the cDNA molecules (i.e., within a given experiment). The addition of the common adapter may be performed or carried out in a solution containing up to about 10 w / v% PEG, and the molecular weight of the PEG is about 7,000 g / mol to 9,000 g / mol. In certain embodiments, the common adapter sequence can be added to the 3' end of the released cDNA molecules by performing template switching (see Picelli, S et al., Nature Methods 10, 1096 - 1098 (2013)).

[0057] Step (j) can be repeated a sufficient number of times to generate a unique set of nucleic acid tags for the nucleic acids of a single cell. For example, the number of times can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100.

[0058] In various embodiments, the primer of step (b) may further include a first specific barcode. Stated otherwise, the first barcode added to the cDNA molecules in a particular container, mixture, reaction, vessel, sample, well, or chamber may be pre - determined (e.g., specific to a given container, mixture, reaction, vessel, sample, well, or chamber). For example, 48 sets of different well - specific RT primers may be used (e.g., in a 48 - well plate). Thus, if 48 samples (e.g., cells, tissues, etc.) are present, each sample can obtain a unique well - specific barcode. However, if there are only 4 samples, each sample may have 12 different sets of well - specific RT primers. The user can determine which 12 correspond to each sample can be known, and thus the user can retrieve the sample identity. Other numbers of first specific barcodes (or well-specific RT primers) are also within the scope of the present disclosure. Such configurations can enable or provide multiplexing of the method as further described in Example 16.

[0059] The method comprises the steps of: (q) reverse transcribing RNA molecules in a second plurality of cells to form cDNA molecules in the second plurality of cells, wherein the step of reverse transcribing the RNA molecules comprises coupling a specific primer to the RNA molecules, the primer comprising at least one of a second specific barcode and a poly(T) sequence or a random sequence, and the first specific barcode is different from the second specific barcode such that cDNA molecules derived from a first plurality of cells can be distinguished from cDNA molecules derived from the second plurality of cells; (r) dividing a second plurality of cells containing the cDNA molecules into at least two primary aliquots, the at least two primary aliquots comprising a first primary aliquot and a second primary aliquot; (s) providing primary nucleic acid tags to the at least two primary aliquots, wherein the primary nucleic acid tag provided to the first primary aliquot is different from the primary nucleic acid tag provided to the second primary aliquot; (t) coupling the cDNA molecules within each of the at least two primary aliquots to the provided primary nucleic acid tags; (u) combining the at least two primary aliquots; (v) dividing the combined primary aliquots into at least two secondary aliquots, the at least two secondary aliquots comprising a first secondary aliquot and a second secondary aliquot; (w) providing secondary nucleic acid tags to the at least two secondary aliquots, wherein the secondary nucleic acid tag provided to the first secondary aliquot is different from the secondary nucleic acid tag provided to the second secondary aliquot; (x) coupling the cDNA molecules within each of the at least two secondary aliquots to the provided secondary nucleic acid tags; and / or (y) repeating steps (u), (v), (w), and (x) using subsequent aliquots, wherein the final nucleic acid tag may further comprise a capture agent. The above steps (e.g., steps (k), (l), and (m)) used in the first plurality of cells may be adapted to also be used in the second plurality of cells.

[0060] In various embodiments, each nucleic acid tag may comprise a first strand, the first strand comprising (i) a barcode sequence comprising a 3' end and a 5' end, and (ii) a 3' hybridization sequence and a 5' hybridization sequence adjacent to the 5' end and 3' end of the barcode sequence, respectively. Additionally, each nucleic acid tag may comprise a second strand, the second strand comprising (i) a first portion complementary to at least one of the 5' hybridization sequence and the adapter sequence, and (ii) a second portion complementary to the 3' hybridization sequence.

[0061] A method of uniquely labeling RNA molecules in multiple cells may further comprise the step of ligating at least two (or more thereof) nucleic acid tags bound to cDNA molecules. The ligation may be performed in a first plurality of cells.

[0062] The method may further comprise the step of removing unbound nucleic acid tags. In some embodiments, the method may comprise the step of ligating at least two nucleic acid tags bound to the released cDNA molecules. Most of the cDNA molecules bound to nucleic acid tags derived from a single cell may comprise the same set of bound nucleic acid tags. In various embodiments, the plurality of cells (e.g., a first and a second plurality of cells) may be selected from at least one of mammalian cells, yeast cells, and bacterial cells.

[0063] Another aspect of the disclosure is directed to a method of labeling nucleic acids in a first cell. A particular In an embodiment, the method comprises: (a) generating cDNA molecules in a plurality of cells including a first cell by reverse transcribing RNA using at least one of: (i) a first reverse transcription primer comprising a 5' overhang sequence and configured to reverse transcribe RNA having a poly(A) tail; and / or (ii) a second reverse transcription primer comprising a 5' overhang sequence and at least one of a random hexamer, a random heptamer, a random octamer, a random nonamer, and a random decamer; (b) dividing the plurality of cells into (n) aliquots; (c) providing a plurality of nucleic acid tags to each of the n aliquots; (d) binding at least one cDNA molecule of each of the n aliquots to the nucleic acid tags; (e) combining the n aliquots; (f) repeating steps (b), (c), (d), and (e) using the combined aliquots; (g) combining the final aliquots; (h) lysing the plurality of cells including the first cell to release cDNA molecules from the plurality of cells including the first cell to form a lysate; and / or (i) adding a protease inhibitor and / or a binder to the lysate such that the cDNA molecules bind to the binder.

[0064] For example, referring to step (c), each nucleic acid tag may include a first strand comprising: (i) a 3' hybridization sequence extending from the 3' end of a labeling sequence; and (ii) a 5' hybridization sequence extending from the 5' end of the labeling sequence. Each nucleic acid tag may also include a second strand comprising an overhang sequence, the overhang sequence including: (i) a first portion complementary to at least one of the 5' hybridization sequence and the 5' overhang sequence; and (ii) a second portion complementary to the 3' hybridization sequence. In some embodiments, the labeling sequences of the plurality of nucleic acid tags provided to a given aliquot may be the same, and different labeling sequences may be provided to each of the n aliquots.

[0065] In certain embodiments, step (f) can be repeated a sufficient number of times to generate a series of labeled sequences specific to the cDNA molecules of the first cell. For example, the number of times can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100.

[0066] The cDNA molecules may be formed or generated in an aliquot (e.g., reaction mixture). The concentration of the first reverse transcription primer in the aliquot may be from about 0.5 μM to about 10 μM, from about 1 μM to about 7 μM, from about 1.5 μM to about 4 μM, from about 2 μM to about 3 μM, about 2.5 μM, or another suitable concentration. The concentration of the second reverse transcription primer in the aliquot may be from about 0.5 μM to about 10 μM, from about 1 μM to about 7 μM, from about 1.5 μM to about 4 μM, from about 2 μM to about 3 μM, about 2.5 μM, or another suitable concentration.

[0067] In some embodiments, the method may include a step of fixing a plurality of cells prior to step (a). The plurality of cells may be fixed at less than about 8°C, less than about 7°C, less than about 6°C, less than about 5°C, about 4°C, less than about 4°C, less than about 3°C, less than about 2°C, less than about 1°C, or another suitable temperature. In certain embodiments, the method may include a step of permeabilizing a plurality of cells prior to step (a). The plurality of cells may be permeabilized at less than about 8°C, less than about 7°C, less than about 6°C, less than about 5°C, about 4°C, less than about 4°C, less than about 3°C, less than about 2°C, less than about 1°C, or another suitable temperature.

[0068] Also, the method of labeling nucleic acids in the first cell may include a step of ligating at least two nucleic acid tags bound to the cDNA molecule. In various embodiments, the ligation may be performed in a plurality of cells. The method removes unbound nucleic acid tags It may include a step of going. Further, at least one of the first and second reverse transcription primers may reverse transcribe a predetermined RNA or may be configured to reverse transcribe a predetermined RNA.

[0069] In various embodiments, the final nucleic acid tag may include a capture agent. Further, the method may include, after step (f), a step of lysing a plurality of cells to release cDNA molecules from within the plurality of cells to form a lysate. Also, the method may include a step of adding a protease inhibitor and / or a binder to the lysate to isolate the cDNA molecules. As discussed above, the protease inhibitor may include PMSF, AEBSF, combinations thereof, and / or another suitable protease inhibitor. The capture agent may include biotin or another suitable capture agent, and the binder may include avidin (e.g., streptavidin) or another suitable binder.

[0070] Also, the method for labeling nucleic acids in the first cell may include: (j) performing a template switch of the cDNA molecules bound to the binder; (k) amplifying the cDNA molecules to form an amplified cDNA molecule solution; and (l) introducing an SPRI beads solution into the amplified cDNA molecule solution to remove polynucleotides less than about 200 base pairs, less than about 175 base pairs, or less than about 150 base pairs. The ratio of the SPRI beads solution to the amplified cDNA molecule solution may be about 0.9:1 to about 0.7:1, about 0.875:1 to about 0.775:1, about 0.85:1 to about 0.75:1, about 0.825:1 to about 0.725:1, about 0.8:1, or another suitable ratio.

[0071] Furthermore, the SPRI beads solution may contain about 1M to 4M NaCl, about 2M to 3M NaCl, about 2.25M to 2.75M NaCl, about 2.5M NaCl, or another suitable amount of NaCl. Also, the SPRI beads solution may contain about 15 w / v% to 25 w / v% PEG, and the molecular weight of the PEG is about 7,000 g / mol to 9,000 g / mol. In various embodiments, the SPRI beads solution may contain about 17 w / v% to 23 w / v% PEG8000, about 18 w / v% to 22 w / v% PEG8000, about 19 w / v% to 21 w / v% PEG8000, about 20 w / v% PEG8000, or another suitable w / v% of PEG8000.

[0072] A method for uniquely labeling RNA molecules in multiple cells may further include the step of adding a common adapter sequence to the 3' end of the released cDNA molecules. As discussed above, the addition of the common adapter may be performed or carried out in a solution containing up to about 10 w / v% PEG, and the molecular weight of the PEG is about 7,000 g / mol to 9,000 g / mol. In certain embodiments, the common adapter sequence can be added to the 3' end of the released cDNA molecules by performing template switching.

[0073] In certain embodiments, any of the methods described above may be adapted to label nucleic acid molecules in the nucleus or multiple nuclei. For example, the method may include the step of uniquely labeling RNA molecules in multiple nuclei, or the step of labeling a nucleic acid in a first nucleus.

[0074] Another aspect of the present disclosure is directed to a kit for labeling a nucleic acid in a first cell. The kit may include a first reverse transcription primer containing a 5' overhang sequence and configured to reverse transcribe RNA having a poly(A) tail. Also, the kit may include a second reverse transcription primer containing a 5' overhang sequence and at least one of a random hexamer, a random heptamer, a random octamer, a random nonamer, and / or a random decamer. Good.

[0075] In some embodiments, the kit may include a plurality of first nucleic acid tags. As discussed above, each first nucleic acid tag may include a first strand comprising (i) a 3' hybridization sequence extending from the 3' end of the first labeling sequence, and (ii) a 5' hybridization sequence extending from the 5' end of the first labeling sequence. Additionally, each first nucleic acid tag may include a second strand comprising a protruding sequence. The protruding sequence may include (i) a first portion complementary to at least one of the 5' hybridization sequences and 5' protruding sequences of the first and second reverse transcription primers, and (ii) a second portion complementary to the 3' hybridization sequence.

[0076] Also, in certain embodiments, the kit may include a plurality of second nucleic acid tags. Each second nucleic acid tag may include a first strand comprising (i) a 3' hybridization sequence extending from the 3' end of the second labeling sequence, and (ii) a 5' hybridization sequence extending from the 5' end of the second labeling sequence. Additionally, each second nucleic acid tag may include a second strand comprising a protruding sequence. The protruding sequence may include (i) a first portion complementary to at least one of the 5' hybridization sequences and 5' protruding sequences of the first and second reverse transcription primers, and (ii) a second portion complementary to the 3' hybridization sequence. Further, the first labeling sequence may be different from the second labeling sequence.

[0077] Also, in various embodiments, the kit may include a plurality of final nucleic acid tags. Each final nucleic acid tag may include a first strand that includes (i) a 3' hybridization sequence extending from the 3' end of the final labeling sequence, and (ii) a 5' hybridization sequence extending from the 5' end of the final labeling sequence. Each final nucleic acid tag may also include a second strand that includes a protruding sequence. The protruding sequence may include (i) a first portion complementary to at least one of the 5' hybridization sequences and 5' protruding sequences of the first and second reverse transcription primers, and (ii) a second portion complementary to the 3' hybridization sequence. Each final nucleic acid tag may also include a capture agent. Further, the final labeling sequence may be different from the first and second labeling sequences (and / or any other labeling sequences). Also, in some embodiments, the kit may include at least one of a reverse transcriptase, a fixative, a permeabilizing agent, a ligation agent, a lysing agent, a protease inhibitor, and / or other suitable components.

[0078] As will be understood by those of skill in the art, each embodiment disclosed herein may include, consist essentially of, or consist of its specifically recited elements, steps, components, or ingredients. As used herein, the transitional term "comprise" or "comprises" means, without limitation, including, and permitting the inclusion of, elements, steps, components, or ingredients not expressly recited, even in large numbers, and intervening. The transitional phrase "consisting of" excludes any element, step, component, or ingredient not specified. The transitional phrase "consisting essentially of" limits the scope of an embodiment to the specified elements, steps, components, or ingredients, and those that do not materially affect the embodiment.

[0079] Unless otherwise indicated, all numerical values representing amounts of ingredients, properties such as molecular weights, and reaction conditions, etc. used in this specification and the claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by this disclosure. At a minimum and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be construed in light of the reported number of significant digits and by applying ordinary rounding techniques. When further clarity is desired, the term "about", when used in conjunction with a recited numerical value or range, has the meaning reasonably recognized by one of ordinary skill in the art, meaning slightly more or slightly less than the recited value or range, within ±20%, ±19%, ±18%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the recited value.

[0080] Notwithstanding the foregoing, the numerical ranges and parameter settings showing broad ranges of this disclosure are approximations, and the numerical values set forth in the specific examples are reported as accurately as possible. However, every numerical value inherently contains some degree of error necessarily resulting from the standard deviation found in its respective testing measurements.

[0081] The terms "a", "an", "the", and similar references used in the context of describing the present disclosure (in particular, in the context of the following claims) should be construed to include both the singular and the plural unless otherwise specified herein or clearly contradicted by the context. The description of a range of values herein is intended merely to serve as a convenient method of referring individually to each separate value within the range. Unless otherwise specified herein, each separate value is incorporated herein as if it were individually recited herein. All of the methods described herein can be performed in any suitable order unless otherwise specified herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is for illustrative purposes only and in no way limits the scope of the present disclosure as otherwise claimed. The language herein should not be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0082] The grouping of alternative elements or embodiments of the disclosure disclosed herein should not be construed as a limitation. Each member of each group is individually and, in combination with any other member of that group or other elements found herein, referenced and claimed. It is understood that one or more members of a group may be included in or deleted from the group for reasons of convenience and / or patentability. In the event of any such inclusion or deletion, the specification is deemed to include the modified group and thus meets the requirements of all Markush group recitations used in the appended claims.

[0083] The definitions and explanations used in this disclosure are intended and purposed to govern any future configuration in the following examples or whenever the meaning would not make sense or would essentially cease to make sense when applied, and unless explicitly and clearly modified, by the construction of the terms, in cases where the meaning would not make sense or would essentially cease to make sense, the definition should be by a dictionary known to those skilled in the art, such as Webster’s Dictionary, 3rd Edition, or Oxford Dictionary of Biochemistry and Molecular Biology (edited by Anthony Smith, Oxford University Press, Oxford 2004).

Examples

[0084] The following examples are illustrative of the methods and compositions of the present disclosure. In light of the present disclosure, one skilled in the art will recognize that variations of such examples and other examples of the methods and compositions of the present disclosure will be possible without undue experimentation.

[0085] Example 1 - Fixation and Reverse Transcription NIH / 3T3 (mouse) and Hela-S3 (human) cells can be grown to confluence in two separate 10 cm cell culture plates. The cells can be rinsed twice with 10 ml of 1× phosphate buffered saline (PBS), 1 ml of 0.05% trypsin can be added to each plate, and the plates can be incubated at 37 °C for 5 minutes. The cells can be detached by tilting each plate at a 45° angle while pipetting the trypsin over the entire plate, and this can be continued until all or substantially all of the cells are detached. Each cell line can be transferred to its own 15 ml conical centrifuge tube (FALCON (trademark)). 2 ml of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) can be added to each tube. The cell count of each tube can be calculated (e.g., with a hemocytometer or flow cytometer). For example, 200 μl of sample can be transferred from each tube to an individual 1.7 ml microcentrifuge tube (EPPENDORF (registered trademark)), and 100 μl of the sample can be run through an ACCURI (trademark) flow cytometer to calculate the cell concentration.

[0086] Equal numbers of cells from each tube can be combined, using as many cells as possible, into a new single 15 ml conical centrifuge tube (FALCON (trademark)). Centrifugation can be performed at 500 × g for 5 minutes in a 15 ml conical centrifuge tube (FALCON (trademark)). It may be useful to use a bucket centrifuge so that the cells pellet at the bottom of the tube rather than on the side of the tube. The liquid can be aspirated without disrupting the cell pellet, and the cells can be resuspended in 500 μl of 4% formaldehyde. The cells can then be left at room temperature (i.e., 20 - 25 °C) for 10 minutes. 1.5 ml of 0.5% TRITON (trademark) X-100 can be added to the tube and gently mixed with a pipette. The tube can then be centrifuged at 500 × g for 5 minutes. Again, the liquid can be aspirated without disrupting the pellet, and the pellet can be washed twice with 1 ml of PBS without resuspending the pellet. If the pellet breaks during washing, the second wash can be skipped and the process can proceed to the next step. The pellet can then be resuspended in 1 ml of 0.1 N HCl and incubated at room temperature for 5 minutes.

[0087] 2 ml of Tris-HCl (pH 8.0) can be added to a new 15 ml conical centrifuge tube (FALCON (trademark)). The fixed cells in the above HCl can be transferred to the tube with Tris-HCl to neutralize the HCl. The number of cells in the tube can then be calculated as discussed above (e.g., with a hemocytometer or flow cytometer). The fixed cells in Tris-HCl can be centrifuged at 500 × g for 5 minutes, and the liquid can be aspirated without disrupting the pellet. Without disrupting the pellet, the pellet can be washed twice with 1 ml of RNase-free molecular grade water. The cells can then be resuspended at a concentration of 2.5 million cells / ml (the concentration calculated before the last centrifugation step can be used to do this).

[0088] A reverse transcription mix can be prepared (55 μl of M-MuLV reverse transcriptase buffer (ENZYMATICS®), 55 μl of M-MuLV reverse transcriptase (ENZYMATICS®), 5.5 μl of dNTP (25 mM per base), 3.44 μl of RNase inhibitor (ENZYMATICS®, 40 units / μl), 210.4 μl of nuclease-free water, and 2.75 μl of RT primer (BC_0055, 100 μM)). In the wells of a 24-well cell culture plate, 300 μl of the reverse transcription mix can be combined with 200 μl of fixed cells (about 500,000 cells) and pipetted to mix gently. Then, the mixture can be incubated at room temperature for 10 minutes to anneal the reverse transcription primer, and then the mixture can be incubated in a humidified incubator at 37 °C overnight (i.e., about 16 hours).

[0089] A primer (BC_0055) that can be used for reverse transcription is shown in FIG. 10. This is a tethered primer designed to bind to the start of the poly(A) tail of messenger RNA. Primers can be synthesized with a 3' end that is any of 4 bases (N) and a second position from the 3' that is any base other than T (V). Also, the primer can contain 15 consecutive dTs. In some embodiments, the primer may contain more than 15 dTs. In some other embodiments, the primer may contain less than 15 dTs. In embodiments where the primer contains less than 15 dTs, the melting temperature of the primer may decrease. Domain s0 does not have to hybridize to messenger RNA, but instead can provide an accessible binding domain for a linker oligo. Also, the primer contains a 5' phosphate that allows the primer to be ligated to another oligo by T4 DNA ligase.

[0090] Example 2 - Preparation of Barcodes Barcodes were arrayed in order in a 96-well plate at a concentration of 100 μM. Each barcode was annealed to its corresponding linker oligo (see FIGS. 10-12).

[0091] FIG. 11 shows the annealed first-round barcode oligos. 96 first-round barcode oligos having unique sequences in domain i8a were used. In the first round, the unique sequence of domain i8a is the region of the sequence used as the barcode. By varying 8 nucleotides, there are 65,536 possible unique sequences. In some embodiments, more than 8 nucleotides may be present in domain i8a. In some other embodiments, less than 8 nucleotides may be present in domain i8a. The first-round barcodes were pre-annealed to the linker strand (BC_0056) via the complementary sequence of domain s1. The linker strand can include a complementary sequence to a part (domain s0) of the reverse transcription primer that can bring the 3'-end of the first-round barcode close to and hybridize to the vicinity of the 5'-end of the reverse transcription primer. Subsequently, the phosphate of the reverse transcription primer can be ligated to the 3'-end of the first-round barcode by T4 DNA ligase. Domain s2 can provide an accessible binding domain for linker oligos to be used in another round of barcoding. Also, the first-round barcode oligo can include a 5'-phosphate that can be ligated to the 3'-end of another oligo by T4 DNA ligase.

[0092] Figure 12 shows the annealed second-round barcode oligos. 96 second-round barcode oligos having unique sequences for domain i8b were used. In the second round, the unique sequence of domain i8b is the region of the sequence used as the barcode. By varying 8 nucleotides, there are 65,536 possible unique sequences. In some embodiments, more than 8 nucleotides may be present in domain i8b. In some embodiments, less than 8 nucleotides may be present in domain i8b. The second-round barcode can be pre-annealed to the linker strand (BC_0058) via the complementary sequence of domain s3. The linker strand can contain a complementary sequence to a part (domain s2) of the first-round barcode oligo that can bring the 3’ end of the first-round barcode close to and hybridize to the vicinity of the 5’ end of the second-round barcode oligo. Thereafter, the phosphate of the first-round barcode oligo can be ligated to the 3’ end of the second-round barcode by T4 DNA ligase. Domain s4 is a linker for use in another round of barcoding An accessible binding domain for the oligo can be provided. Also, the second-round barcode oligo can contain a 5’ phosphate that can be ligated to the 3’ end of another oligo by T4 DNA ligase.

[0093] FIG. 13 shows the annealed third-round barcode oligos. 96 third-round barcode oligos having unique sequences for domain i8c were used. In the third round, the unique sequence of domain i8c is the region of the sequence used as the barcode. By varying 8 nucleotides, there are 65,536 possible unique sequences. In some embodiments, more than 8 nucleotides may be present in domain i8c. In some other embodiments, less than 8 nucleotides may be present in domain i8c. The third-round barcode can be pre-annealed to the linker strand (BC_0060) via the complementary sequence of domain s5. The linker strand can contain a complementary sequence to a part (domain s4) of the second-round barcode oligo that can bring the 3' end of the second-round barcode close to and hybridize to the vicinity of the 5' end of the third-round barcode oligo. Subsequently, the phosphate of the second-round barcode oligo can be ligated to the 3' end of the third-round barcode by T4 DNA ligase. Third-round barcode oligos can be synthesized having unique molecular identifiers (UMIs; see Islam et al., Nature Methods, 2014) consisting of 10 random nucleotides (domain UMI: NNNNNNNNNN). Due to PCR amplification bias, multiple sequencing reads may result from a cDNA. By using UMIs, each cDNA can be counted only once. Also, the third-round barcode can contain a domain corresponding to a part of the ILLUMINA® TruSeq adapter. Third-round barcodes having biotin molecules at the 5' end can be synthesized so that the barcoded cDNA can be sufficiently isolated with streptavidin-coated magnetic beads.

[0094] Starting from a 100 μM stock of each barcode oligo (i.e., in one 96-well plate per round), 11 μl of the barcode oligo was transferred to a 96-well PCR plate. 9 μl of BC_0056 (100 μM stock) was added to each well of the plate with round 1 barcode. 9 μl of BC_0058 (100 μM stock) was added to each well of the plate with round 2 barcode. 9 μl of BC_0060 (100 μM stock) was added to each well of the plate with round 3 barcode. Then, each plate was placed in a thermocycler with a program of heating to 90 °C, reducing the heat at 0.1 °C / second, and stopping when the temperature reached 25 °C, annealing the barcode to the corresponding linker oligo. 2.2 μl was transferred from each well with round 1 barcode to a new 96-well plate (referred to as plate L1). 3.8 μl was transferred from each well with round 2 barcode to a new 96-well plate (referred to as plate L2). 6.1 μl was transferred from each well with round 3 barcode to a new 96-well plate (referred to as plate L3).

[0095] Example 3 - Preparation of Ligation-Stop Oligos After ligation in each round, ligation can be stopped by adding an excess amount of oligo complementary to the linker strand (see Figure 14). To stop each barcode ligation, an oligo strand completely complementary to the linker oligo can be added. Such oligos can bind to the linker strand attached to the unligated barcode and can replace the unligated barcode by a strand displacement reaction. Thereafter, the unligated barcode can be completely single-stranded. Since T4 DNA ligase cannot ligate single-stranded DNA to other single-stranded DNA, the ligation reaction will stop progressing. To ensure that all linker oligos bind to the complementary oligos, a molar excess of complementary oligos (relative to the linker oligo Add (te). To stop the first round of ligation, add BC_0064 (complementary to BC_0056). To stop the second round of ligation, add BC_0065 (complementary to BC_0058). To stop the third round of ligation, add BC_0066 (complementary to BC_0060).

[0096] Dilutions of each stop ligation strand (BC_0064, BC_0065, BC_0066) can be prepared as follows: 264 μl of stop ligation strand (BC_0064, BC_0065, BC_0066), 300 μl of 10× T4 DNA ligase buffer, and 636 μl of nuclease-free water.

[0097] Example 4 - Ligation of Barcodes to cDNA 5 μl of 10% TRITON™ X-100 can be added to the reverse transcription reaction in the 24-well plate described above (to a final concentration of 0.1%). The reverse transcription (RT) reaction with cells can be transferred to a 15 ml conical centrifuge tube (FALCON™). The RT reaction can be centrifuged at 500×g for 10 minutes and resuspended in 2 ml of nuclease-free water. The cells can be combined with the ligase mix (in a disposable pipette reservoir (10 ml), 600 μl of 10× T4 ligase buffer, 2040 μl of nuclease-free water, all of the resuspended cells (2000 μl), 100 μl of T4 DNA ligase (NEW ENGLAND BIOLABS®, 400,000 units / ml), and 60 μl of 10% TRITON™ X-100). The cells and ligase mix can be mixed by gently tilting the reservoir back and forth several times. Using a multi-channel pipette, 40 μl of the cells in the ligase mix can be added to each well of the annealed round 1 barcodes (plate L1). Each well can be mixed by gently pipetting up and down 2 - 3 times. The cells in the ligase mix can be incubated at 37°C for 60 minutes.

[0098] 10 μl of diluted BC_0064 can be added to each well to stop the ligation. Then, the sample can be incubated at 37 °C for 30 minutes. All of the cells can be collected into a new disposable pipette reservoir (10 ml). Using a 1 ml pipette, the cells can be passed through a 40 μM strainer and into a new disposable pipette reservoir (10 ml). 100 μl of T4 DNA ligase (NEW ENGLAND BIOLABS®, 400,000 units / ml) can be added to the cells in the reservoir. The cells and ligase mix can be mixed by gently tilting the reservoir back and forth several times, and using a multichannel pipette, 40 μl of the cells in the ligase mix can be added to each well of the annealed round 2 barcode (plate L2). Each well can be mixed by gently pipetting up and down 2 - 3 times, and then the sample can be incubated at 37 °C for 60 minutes.

[0099] 10 μl of diluted BC_0065 can be added to each well to stop the ligation. The sample can be incubated at 37 °C for 30 minutes, and then the cells can be collected into a new disposable pipette reservoir (10 ml). Using a 1 ml pipette, the cells can be passed through a 40 μM strainer and into a new disposable pipette reservoir (10 ml). 100 μl of T4 DNA ligase (NEW ENGLAND BIOLABS®, 400,000 units / ml) can be added to the cells in the reservoir. The cells and ligase mix can be mixed by gently tilting the reservoir back and forth several times. Using a multichannel pipette, 40 μl of the cells in the ligase mix can be added to each well of the annealed round 3 barcode (plate L3). Then, each well can be mixed by gently pipetting up and down 2 - 3 times, and the sample can be incubated at 37 °C for 60 minutes.

[0100] 10 μl of diluted BC_0066 can be added to each well to stop ligation. The sample can be incubated at 37°C for 30 minutes. All cells can be collected into a new disposable pipette reservoir (10 ml). The cells can be transferred to a 15 ml conical centrifuge tube (FALCON™), and the tube can be filled with wash buffer (nuclease-free water, 0.05% Tween 20, and 25% formamide) to 15 ml. The sample can be incubated at room temperature for 15 minutes. Then the cells can be pelleted at 500 × g for 10 minutes, and the liquid can be removed without disrupting the pellet. The cells in each tube can be resuspended in 100 μl of PBS, and the cells can be counted (e.g., with a hemocytometer or flow cytometer). In one example, 57,000 cells were retained. The number of cells to sequence can be selected. In one example, the cells were distributed into aliquots of 25 cells, 250 cells, 2,500 cells, and 25,000 cells. 300 μl of lysis buffer (10 mM NaF, 1 mM Na 3 VO 4 , 0.5% DOC buffer, and 0.5% TRITON™ X-100) can be added to each of the cell aliquots, and each of the cell aliquots can be passed through an 8-gauge syringe needle 8 times.

[0101] Example 5 - Binding of Barcoded cDNA to Streptavidin-Coated Beads First, DYNABEADS® MYONE™ Streptavidin C1 beads can be resuspended. 20 μl of resuspended DYNABEADS® MYONE™ Streptavidin C1 beads can be added to a 1.7 ml microcentrifuge tube (EPPENDORF®) (for each cell aliquot). The beads can be washed three times with 1× phosphate buffered saline Tween20 (PBST) and resuspended in 20 μl of PBST. 900 μl of PBST can be added to the cell aliquot, and 20 μl of washed C1 beads can be added to the aliquot of lysed cells. The sample can be placed on a gentle roller at room temperature for 15 minutes and then washed three times with 800 μl of PBST using a magnetic tube rack (EPPENDORF®). Thereafter, the beads can be resuspended in 100 μl of PBS.

[0102] Example 6 - RNase Treatment of Beads The microcentrifuge tube (EPPENDORF®) containing the sample can be placed in a magnetic tube rack (EPPENDORF®) for 2 minutes and then the liquid can be aspirated. The beads can be resuspended in an RNase reaction mixture (3 μl RNase mix (ROCHE®), 1 μl RNaseH (NEW ENGLAND BIOLABS®), 5 μl RNaseH 10× buffer (NEW ENGLAND BIOLABS®), and 41 μl nuclease-free water). The sample can be incubated at 37°C for 1 hour, removed from 37°C, and placed in a magnetic tube rack (EPPENDORF®) for 2 minutes. Without resuspending the beads, while the tube is placed in the magnetic tube rack, the sample can be washed with 750 μl of nuclease-free water + 0.01% Tween20 (H 2 O-T). Then the liquid can be aspirated. Without resuspending the beads, while the tube is placed in the magnetic tube rack, the sample can be washed with 750 μl of H 2It can be washed with O-T. Next, the liquid can be aspirated while the tube remains placed in the magnetic tube black. Thereafter, the tube can be removed from the magnetic tube black, and the sample can be resuspended in 40 μl of nuclease-free water.

[0103] Example 7 - 3’ Adapter Ligation Referring to FIG. 15, to promote PCR amplification, a single-stranded DNA adapter oligo (BC_0047) can be ligated to the 3'-end of the cDNA. To prevent concatemers of the adapter oligo, dideoxycytidine (ddC) can be included at the 3'-end of the adapter oligo. BC_0047 having a phosphate at the 5'-end and ddC at the 3'-end was generated. Some enzymes are capable of ligating single-stranded oligos to the 3'-end of single-stranded DNA. Herein, T4 RNA ligase 1 (NEW ENGLAND BIOLABS (registered trademark)) was used. Thermostable 5'AppDNA / RNA ligase (NEW ENGLAND BIOLABS (registered trademark)) can also be used with a preadenylated adapter oligo.

[0104] Specifically, 20 μl of RNase-treated beads can be added to a single PCR tube. 80 μl of ligase mix (5 μl of T4 RNA ligase 1 (NEW ENGLAND BIOLABS (registered trademark)), 10 μl of 10× T4 RNA ligase buffer, 5 μl of 50 μM BC_0047 oligo, 50 μl of 50% PEG8000, and 10 μl of 10 mM ATP) can be added to the 20 μl of beads in the PCR tube. The 50 μl of ligase mixed with the beads can be transferred to a new PCR tube to prevent too many beads from precipitating at the bottom of a single tube, and the sample can be incubated at 25° C. for 16 hours.

[0105] Example 8 - Generation of Illumina®-Compatible Sequencing Products The ligation reactions from both PCR tubes can be combined and placed into a single 1.7 ml microcentrifuge tube (EPPENDORF®). 750 μl of H 2 O-T can be added to each sample. Each tube can be placed in a magnetic tube rack (EPPENDORF®) for 2 minutes, the liquid can be aspirated, and the sample can be resuspended in 40 μl of water. The sample can be transferred to a PCR tube. 60 μl of PCR mix can be added to each tube (50 μl of 2×PHUSION® DNA polymerase master mix (THERMO FISHER™ Scientific), 5 μl of BC_0051 (10 μM), and 5 μl of BC_0062 (10 μM)). 10 cycles of PCR can be performed (3 minutes at 98 °C, 10 repetitions of (10 seconds at 98 °C, 15 seconds at 65 °C, and 60 seconds at 72 °C), and 5 minutes at 72 °C). Figure 16 shows the PCR products. After ligating the 3’ adapter oligo (BC_0047) to the barcoded cDNA, the cDNA can be amplified using PCR. As shown in Figure 16, primers BC_0051 and BC_0062 were used.

[0106] The PCR samples can be procured from the previous step, and the magnetic beads can be moved to the bottom of each tube using a magnet. Without transferring any of the magnetic beads, 90 μl of the PCR reaction can be transferred to a new 1.7 ml. 10 μl of nuclease-free water can be added to each of the 1.7 ml tubes to bring the total volume to 100 μl. 60 μl of AMPURE® beads can be added to the 100 μl of PCR reaction (0.6×SPRI) and allowed to bind for 5 minutes. The tube can be placed on the magnet for 2 minutes, and without resuspending the beads, the sample can be washed with 200 μl of 70% ethanol (wait 30 seconds). Without resuspending the beads, the sample can be washed again with 200 μl of 70% ethanol (wait 30 seconds), and then the sample can be air-dried for 5 - 10 minutes until the ethanol has evaporated.

[0107] Each sample can be resuspended in 40 μl of nuclease-free water. The tube can be placed on a magnetic rack for 2 minutes. While still leaving the microcentrifuge tube (EPPENDORF (registered trademark)) on the magnetic rack, 38 μl of the solution can be transferred to a new 1.7 ml tube without moving the beads. 62 μl of nuclease-free water can be added to the sample to make the total volume 100 μl. Then, 60 μl of AMPURE (trademark) beads can be added to 100 μl of the PCR reaction (0.6×SPRI) and allowed to bind for 5 minutes. The tube can be placed on the magnet for 2 minutes, and then the sample can be washed with 200 μl of 70% ethanol without resuspending the beads (wait for 30 seconds). Without resuspending the beads, the sample can be washed again with 200 μl of 70% ethanol (wait for 30 seconds), and then the sample can be air-dried for 5 - 10 minutes until the ethanol evaporates.

[0108] ​The sample can be resuspended in 40 μl of nuclease-free water, and each tube can be placed on a magnetic rack for 2 minutes. While the tube remains placed on the magnetic rack, 38 μl of the solution can be transferred to a new 1.7 ml tube without transferring any of the beads. 20 μl of the 38 μl eluate can be added to an optical PCR tube. Further, the PCR mix can be added to this tube (25 μl of PHUSION® DNA Polymerase Master Mix (THERMO FISHER® Scientific), 2.5 μl of BC_0027 (10 μM), 2.5 μl of BC_0063 (10 μM), and 2.5 μl of 20× EVAGREEN® (BIOTIUM®)). After PCR as shown in Figure 16, the full-length ILLUMINA® adapter sequence can be introduced by another round of PCR. As shown in Figure 17, BC_0027 contains a flow cell binding sequence and a binding site for the TRUSEQ® Read 1 primer. BC_0063 contains a flow cell binding sequence, and TruSeq multiplex Read 2, and an index binding sequence. There is also a region for the sample index, which is GATCTG in this example.

[0109] The above sample can be subjected to a qPCR machine under the following cycle conditions: 1) 98°C for 3 minutes, 2) 98°C for 10 seconds, 3) 65°C for 15 seconds, 4) 72°C for 60 seconds, and 5) repeat steps 2 - 4 (for example, 10 - 40 times depending on when the exponential increase in fluorescence stops). The tube can be transferred to a thermocycler set at 72°C for 5 minutes. The qPCR reaction product can be run on a 1.5% agarose gel for 40 minutes, and a band of 450 - 550 bp can be excised and gel-extracted (QIAQUICK® Gel Extraction Kit). The product can be sequenced using paired-end sequencing on an ILLUMINA® MISEQ™. The sequencing primers may be standard TRUSEQ™ multiplex primers. Read 1 can sequence the cDNA sequence, and Read 2 can cover the unique molecular identifier as well as three barcode sequences (8 nucleotides each). The sample barcode can be sequenced using Index Read 1, and thus multiple samples can be sequenced together.

[0110] Example 9 - Data Analysis Sequencing reads were grouped by cell barcodes (each of three barcodes of 8 nucleotides, with a total of 96×96×96 = 884,736 combinations). Each barcode combination should correspond to cDNA derived from a single cell. Only reads with valid barcodes were retained. Sequencing reads with each barcode combination were aligned against both the human genome and the mouse genome. Reads that aligned to both genomes were discarded. Multiple reads with the same unique molecular identifier were counted as a single read. Reads with unique molecular identifiers having two or fewer mismatches were considered to be generated by sequencing errors and counted as a single read. For each unique barcode combination, the number of reads aligned to the human genome (x-axis) and the number of reads aligned to the mouse genome (y-axis) were plotted (see Figure 18). Since each cell should ideally contain only one type of RNA, either mouse or human, the ideal plot would have all points along either the x-axis or the y-axis. The fact that most of the points in the plot of Figure 18 are near the axes indicates that the method is feasible. The fact that most of the points in the plot are near the axes indicates that the method is feasible.

[0111] Each point in the plot corresponds to cDNA with the same combination of barcodes and should represent cDNA derived from a single cell. For each point, the number of reads uniquely mapped to the mouse genome is plotted on the y-axis, and the number of reads uniquely mapped to the human genome is plotted on the x-axis. If the cDNA with a particular combination of barcodes is derived from a single cell, all cDNA with that particular combination of barcodes should map either completely to the human genome or completely to the mouse genome. As described above, the fact that most barcode combinations map near either the x-axis (human cells) or the y-axis (mouse cells) indicates that the method can actually yield single-cell RNA sequencing data.

[0112] Example 10 - Method for Uniquely Labeling Molecules of Multiple Cells For the protocol disclosed below, the predicted experimental time is two (2) days. As shown below, RNase inhibitor may be added to the buffer. Thus, if any buffer contains the term "+RI", this indicates that ENZYMATICS® RNase inhibitor should be added at a final concentration of 0.1 U / μL. The centrifugation step may be performed using a swinging bucket rotor. In some embodiments, the use of a fixed angle centrifuge may be associated with more cell loss. Depending on the tissue type, it may be necessary to vary the centrifugation speed to optimize cell retention (e.g., smaller cells = higher speed).

[0113] For DNA barcode plate generation, the following may be required: i) three IDT® 96-well plates, reverse transcription barcode primers, ligation round 1, and ligation round 2 stock DNA oligo plates (100 μM); ii) two linker oligos, BC_0215 and BC_0060 (note: these are assumed to have a stock concentration of 1 mM, so if a different stock concentration is used (e.g., 100 μM stock), correct the volume); and iii) six 96-well PCR plates (e.g., three (3) stock plates for at least 10 experiments to be continued, and three (3) plates for the first experiment). Note that this allows for the generation of 100 μL of DNA barcode per well. Generally, each experiment requires only 4 μL / well of reverse transcription primer solution, with which 25 experiments can be continued. Generally, each experiment requires only 10 μL / well of barcode / linker solution, and a total of 10 experiments can be continued with such plates.

[0114] Round 1 Reverse Transcription Barcode Primer (final concentration in each of the 48 wells: 12.5 μM random hexamer and 12.5 μM 15dT primer): 1) Using a multi-channel pipette, add 12.5 μL of the IDT® reverse transcription barcode primer in columns A - D to columns A - D of the BC stock 96-well PCR plate; 2) Using a multi-channel pipette, add 12.5 μL of the IDT® reverse transcription barcode primer in columns E - H to columns A - D of the BC stock 96-well PCR plate (here, mix poly dT with the random hexamer primer); 3) Add 75 μl of water to columns A - D of the BC stock 96-well PCR plate.

[0115] Round 2 Ligation Round (final concentration: 12 μM barcode, 11 μM linker - BC_0215): 1) Using a multi-channel pipette, add 12 μL of the IDT® round 2 barcode to the R1 stock 96-well PCR plate; 2) Add 138.6 μl of BC_0215 (1 mM) to the 10.9494 mL of water in the recess (BC_0215_dil); and 3) Using a multi-channel pipette, add 88 μL of BC_0215_dil to each well of the R2 stock 96-well PCR plate. to the water (BC_0215_dil); and 3) Using a multi-channel pipette, add 88 μL of BC_0215_dil to each well of the R2 stock 96-well PCR plate.

[0116] Ligation Round 3 (final concentration: 14 μM barcode, 13 μM linker - BC_0060): 1) Using a multi-channel pipette, add 14 μL of the round 3 barcode to the R3 stock 96-well PCR plate; 2) Add 163.8 μl of BC_0060 (1 mM) to the 10.6722 mL of water in the recess (BC_0060_dil); and 3) Using a multi-channel pipette, add 86 μL of BC_0060 to each well of the R3 stock 96-well PCR plate.

[0117] Each ligation plate (without R2 and R3, reverse transcription barcode) anneals barcode and linker oligos with the following thermocycling protocol: 1) heat at 95°C for 2 minutes, and 2) cool to 20°C at a rate of 0.1°C / second; and 3) 4°C.

[0118] Aliquot 10 μL of each barcode / linker stock into three new 96-well PCR plates. These are the plates to be used for DNA barcoding in the dispense-pool ligation step of this protocol.

[0119] I. Nuclear extraction (optional): 1) Prepare the following: a) maintain the dounce-type homogenizer at 4°C until use, b) 15 ml of 1×PBS + 37.5 SUPERASE-IN™ + 19 μl of ENZYMATICS® RNase inhibitor (maintain on ice), and c) pre-cool the centrifuge to 4°C.

[0120] 2) Prepare NIM1 buffer (Table 1):

[0121]

Table 1

[0122] 3) Prepare homogenization buffer (Table 2):

[0123]

Table 2

[0124] 4) Dance-type homogenizer: a) Add the tissue / cell sample to the dance-type homogenizer; in the case of cells, resuspend in 700 μl of homogenization buffer; b) Add approximately 700 μl of homogenization buffer; c) Perform 5 reciprocations with a loose pestle; d) Perform 10 - 15 reciprocations with a tight pestle; e) Add homogenization buffer to make it 1 ml; and f) Examine the cell lysate with 5 μl of trypan blue and 5 μl of cells using a hemocytometer to confirm whether nuclei have been released.

[0125] 5) Filter the homogenate through a 40-μm strainer and place it in a 5-ml EPPENDORF (trademark) tube (or a 15-ml FALCON (trademark) tube). By tilting the filter at 45° while filtering above the tube, it can be ensured that the lysate passes through as intended. Note: This filtration process is different from the following filtration process.

[0126] 6) Centrifuge at 600×g for 4 minutes (4 °C) and remove the supernatant (it may be okay to leave about 20 μL to avoid pellet aspiration). 7) Resuspend in 1 ml of 1×PBS + RI. 8) Add 10 μl of BSA. 9) Centrifuge at 600×g for 4 minutes. 10) Resuspend in 200 μl of 1×PBS + RI.

[0127] 11) Take 50 μl of the resuspended cells from Step 4 and add 150 μl of 1×PBS + RI. Count the sample using a hemocytometer and / or a flow cytometer. The volume of the resuspended cells in Step 4 can be varied based on the user's discretion. 12) Pass the cells through a 40-μm strainer into a new 15-ml Falcon (trademark) tube and place on ice (see the following note regarding Step 4 of the fixation and permeabilization process). 13) Resuspend the desired number of nuclei (typically 2M) in 1 ml of 1×PBS + RI and proceed to Step 5 of the following fixation and permeabilization protocol.

[0128] II. Fixation and permeabilization treatment: 1) Prepare the following buffers (calculated for two experiments): a) 1.33% formalin (360 μL of 37% aqueous formaldehyde solution (SIGMA (registered trademark)) + 9.66 ml of PBS) solution, store at 4 °C; b) 6 mL of 1×PBS + RI (15 μL of SUPERASE-IN (trademark) and 7.5 μL of ENZYMATICS (registered trademark) RNase inhibitor); c) 2 mL of 0.5×PBS + RI (5 μL of SUPERASE-IN (trademark) and 2.5 μL of ENZYMATICS (registered trademark) RNase inhibitor); d) 500 μL of 5% TRITON (trademark) X-100 + RI (2 μL of SUPERASE-IN (trademark)); e) 500 μL of 100 mM Tris pH 8.0 + 2 μL of SUPERASE-IN (trademark); and f) Set the centrifuge to 4 °C.

[0129] 2) Pellet the cells by centrifuging at 500×g for 3 minutes at 4°C (for some cells, faster centrifugation may be required). 3) Resuspend the cells in 1 mL of cold PBS+RI. Keep the cells on ice between these steps. 4) Pass the cells through a 40-μm strainer into a new 15-mL Falcon (trademark) tube and place on ice. Note: The cell resuspension is likely to not pass through the strainer passively, which may cause cell loss. Instead, fill a 1-ml pipette with the resuspension, press the tip directly against the strainer, and actively extrude the liquid. This action should take about 1 (one) second. 5) Add 3 mL of cold 1.33% formaldehyde (final concentration is 1% formaldehyde). Fix the cells on ice for 10 minutes. 6) Add 160 μL of 5% TRITON (trademark) X-100+RI to the fixed cells and mix by gently pipetting up and down 5 times using a 1-mL pipette. Permeabilize the cells on ice for 3 minutes. 7) Centrifuge the cells at 500×g for 3 minutes at 4°C. 8) Carefully aspirate the cells and resuspend in 500 μL of cold PBS+RI. 9) Add 500 μL of cold 100 mM Tris-HCl, pH 8.0. 10) Add 20 μL of 5% TRITON (trademark) X-100. 11) Centrifuge the cells at 500×g for 3 minutes at 4°C. 12) Aspirate the cells and resuspend in 300 μL of cold 0.5×PBS+RI.

[0130] 13) Pass the cells through a 40 μM strainer and place them into a new 1.7 mL tube (see the above annotation regarding Step 4 of the fixation and permeabilization process). 14) Count the cells using a hemocytometer or flow cytometer and dilute the cell suspension to 1,000,000 cells / mL. Maintain the cell suspension on ice during the cell counting. Note: This step will determine the number of cells that will proceed to the distribution-pool round. It will be possible to sequence only a subset of the cells that proceed to the distribution-pool round (which can be done during the sub-library generation in the lysis step). The total number of barcode combinations to be used should be calculated, and the maximum number of cells that can be sequenced with minimal barcode collisions should be determined. Without being bound by any one particular theory, the number of cells to be processed should not exceed 5% of the total barcode combinations. Generally, a dilution of 500k - 1M cells / mL can be used here (equivalent to 4 - 8k cells entering each well in the reverse transcription barcoding round).

[0131] III. Reverse Transcription: 1) Aliquot 4 μL of the RT barcode stock plate into the upper four (IV) rows (48 wells) of a new 96-well plate. Cover this plate with an adhesive plate seal until ready for use.

[0132] 2) Generate the following reverse transcription (RT) mix on ice (Table 3):

[0133]

Table 3

[0134] 3) Add 8 μL of RT mix to each of the upper 48 wells. Each well should contain a volume of 12 μL here. 4) Add 8 μL of cells in 0.5×PBS + RI to each of the upper 48 wells. Each well should contain a volume of 20 μL here. 5) Add the plate to a thermocycler with the following protocol: a) 50 °C for 10 minutes; b) i) 8 °C for 12 seconds, ii) 15 °C for 45 seconds, iii) 20 °C for 45 seconds, and iv) 30 °C for 30 seconds, v) 42 °C for 2 minutes, vi) 50 °C for 3 minutes for 3 (three) cycles; c) 50 °C for 5 minutes; and d) then always at 4 °C.

[0135] 6) Place the RT plate on ice. 7) Prepare 2 mL of 1×NEB buffer 3.1 with 20 μL of ENZYMATICS® RNase inhibitor. 8) Transfer each RT reaction to a 15 mL FALCON® tube (also on ice in this case). 9) Add 9.6 μL of 10% TRITON® X-100 to obtain a final concentration of 0.1%. 10) Centrifuge the pooled RT reactions at 500×g for 3 minutes. 11) Aspirate the supernatant and resuspend it in 2 mL of 1×NEB buffer 3.1 + 20 μL of ENZYMATICS® RNase inhibitor.

[0136] IV. Ligation barcoding: 1) Prepare the following ligation master mix on ice (Table 4):

[0137]

Table 4

[0138] 2) Add 2 mL of cells in NEB Buffer 3.1 to the ligation mix. This mix should have a volume of 4.04 mL here. 3) Add the mix to the recessed part. 4) Using a multi-channel pipette, add 40 μL of the ligation mix (with cells) to each well of the Round 1 DNA barcode plate. 5) Cover the Round 1 DNA barcode plate with an adhesive plate seal and incubate at 37 °C for 30 minutes with gentle rotation (50 rpm). 6) Prepare the Round 1 blocking solution and add it to a new recessed part (Table 5).

[0139]

Table 5

[0140] 7) Remove the Round 1 DNA barcoded plate from the incubator and remove the cover. 8) Using a multi-channel pipette, add 10 μL of the Round 1 blocking solution to each of the 96 wells of the Round 1 DNA barcoded plate. 9) Cover the Round 1 DNA barcode plate with an adhesive plate seal and incubate at 37 °C for 30 minutes with gentle rotation (50 rpm). 10) Remove the Round 1 DNA barcoded plate from the incubator, remove the cover, and pool all the cells into a new recessed part. 11) Pass all the cells in this recessed part through a 40 μm strainer into another recessed part (see the above note regarding Step 4 of the fixation and permeabilization process). 12) Add 100 μL of T4 DNA ligase to this recessed part and mix by pipetting approximately 20 times. 13) Using a multi-channel pipette, add 50 μL of the cell / ligation solution to each well of the Round 2 DNA barcode plate. 14) Cover the Round 2 DNA barcode plate with an adhesive plate seal and incubate at 37 °C for 30 minutes with gentle rotation (50 rpm).

[0141] 15) Prepare the Round 2 blocking solution and add it to a new recessed part (Table 6).

[0142]

Table 6

[0143] 16) Remove the round 2 DNA barcoding plate from the incubator and remove the cover. 17) Using a multi-channel pipette, add 20 μL of round 2 blocking and stop solution to each of the 96 wells of the round 2 DNA barcoding plate. 18) Pool all the cells into a new well (do not incubate in the final blocking step). 19) Pass all the cells in this well through a 40 μm strainer into a 15 mL FALCON (trademark) tube (see the above note regarding step 4 of the fixation and permeabilization process). 19) Count the cells using a flow cytometer. Before aliquoting the sample for counting, ensure that the cells are well mixed.

[0144] V. Lysis: 1) Prepare 2× lysis buffer (Table 7).

[0145]

Table 7

[0146] 2) If a white precipitate appears, warm it at 37 °C until the precipitate returns to the solution (about 10 - 15 minutes).

[0147] 3) Prepare the following wash buffer (Table 8).

[0148]

Table 8

[0149] 4) Add 70 μL of 10% TRITON™ X-100 to the cells (final concentration of approximately 0.1%). 5) Centrifuge at 1000 × g for 5 minutes in a 15 ml tube. Note: The pellet in the following steps may be very small and may not be visible. 6) Aspirate the supernatant, leaving approximately 30 μl to avoid disturbing the pellet. a) If possible, use a 20 μL pipette to remove as much of the supernatant as possible. 7) Resuspend in 4 mL of wash buffer. 8) Centrifuge at 1000 × g for 5 minutes. 9) Aspirate the supernatant and resuspend in 50 μl of 1× PBS + RI.

[0150] 10) Dilute 5 μl with 195 μL of 1× PBS and count by flow cytometry. Alternatively, take 5 μl and place it in 5 μl of 1× PBS and count using a hemocytometer (it may be difficult to distinguish debris from cells). 11) Determine how many sublibraries to generate (number of sublibraries = number of tubes required) and how many cells each of those sublibraries will have. 12) Aliquot the desired number of cells from each sublibrary into new 1.7 mL tubes. Add 1× PBS to each tube to bring the final volume to 50 μL.

[0151] 13) Add 50 μL of 2× lysis buffer to each tube. 14) Add 10 μL of proteinase K (20 mg / mL) to each lysate. 15) Incubate at 55 °C for 2 hours with shaking at 200 rpm. 16) Stop point (optional): Freeze the lysates at -80 °C.

[0152] VI. Prepare the buffers. First, prepare the following stock solutions (Tables 9 - 11).

[0153]

Table 9

[0154]

Table 10

[0155]

Table 11

[0156] Thereafter, prepare the following smaller aliquots (RNase inhibitor is added; Tables 12 - 14):

[0157]

Table 12

[0158]

Table 13

[0159]

Table 14

[0160] VII. Purification of cDNA Note: The stirring step is carried out using a foam 1.7 mL tube holder and a vortexer at a low setting (2 / 10).

[0161] Wash MYONE (trademark) C1 DYNABEADS (registered trademark): 1) For each lysate to be processed, add 44 μL of MYONE (trademark) C1 DYNABEADS (registered trademark) to a 1.5 mL tube (e.g., 1 lysate = 44 μL, 2 lysates = 88 μL, 3 lysates = 132 μL, etc.). 2) Add 800 μL of 1×B&WT buffer. 3) Place the sample on the magnetic rack and wait until the liquid becomes clear (1 - 2 minutes). 4) Remove the supernatant and resuspend the beads in 800 μL of 1×B&W-T buffer. 5) Repeat steps 3 - 4 two more times for a total of three washes. 6) Place the sample on the magnetic rack and wait until the liquid becomes clear. 7) Resuspend the beads in 100 μL (per sample) of 2×B&W buffer + RI.

[0162] Binding of the sample to streptavidin: 1) Add 5 μL of 100 μM PMSF to each sample and leave at room temperature for 10 minutes. 2) Add 100 μl of resuspended C1 beads to each tube. 3) Stir at room temperature for 60 minutes to bind the cDNA to the C1 beads. 4) Place the sample on a magnetic rack and wait until the liquid becomes clear (1 - 2 minutes). 5) Remove the supernatant and resuspend the beads in 250 μL of 1×B&W. 6) Stir the beads at room temperature for 5 minutes. 7) Repeat steps 5 and 6. 8) Remove the supernatant and resuspend the beads in 250 μL of 10 mM Tris+T. 9) Stir the beads at room temperature for 5 minutes. 10) Leave the beads in the final wash solution on ice.

[0163] Template switch. Prepare the following mix according to the number of samples (Table 15).

[0164] [Table 15]

[0165] 1) Place the sample on a magnetic rack and wait until the liquid becomes clear. 2) While still on the magnetic rack, remove the supernatant and wash with 250 μL of water (do not resuspend the beads this time). 3) Resuspend the sample in 200 μl of the template switch mix. 4) Incubate at room temperature for 30 minutes with stirring or rotation. 5) Incubate at 42°C for 90 minutes with stirring or rotation (shake the incubator at 100 rpm).

[0166] 6) Potential stop point. If stopping, perform the following (otherwise skip to the next section): a) Place the sample on a magnetic rack and wait until the liquid becomes clear, b) Resuspend in 250 μL of Tris-T.

[0167] VIII. cDNA amplification. Prepare the following PCR mix according to the number of samples (Table 16).

[0168]

Table 16

[0169] 1) Place the sample on a magnetic rack and wait until the liquid becomes clear. 2) While the sample is on the magnetic rack, wash it with 250 μL of nuclease-free water (without resuspending). 3) Resuspend the sample in 220 μL of PCR mix and aliquot it equally into four different PCR tubes.

[0170] 4) Run the following thermocycling program: a) 95 °C for 3 minutes; b) 98 °C for 20 seconds; c) 65 °C for 45 seconds; d) 72 °C for 3 minutes; e) Repeat (b - d) 4 times (total of 5 cycles); and f) Hold at 4 °C.

[0171] 5) Combine all four reactions in a single 1.7 mL tube. Before combining the reactions, ensure that any beads that may be attached to the bottom or side of the PCR tubes are thoroughly resuspended. 6) Place the sample on a magnetic rack and wait until the liquid becomes clear. 7) Transfer 200 μL of the supernatant into four optical-grade qPCR tubes (50 μL in each tube). 8) Add 2.5 μL of 20× EVAGREEN® to each qPCR tube. 9) Run the following qPCR program (to prevent overamplification, ensure that the sample is removed as soon as the signal begins to exceed the logarithmic phase): a) 95 °C for 3 minutes; b) 98 °C for 20 seconds; c) 67 °C for 20 seconds; d) 72 °C for 3 minutes; e) Repeat (b - d) until the signal transitions from exponential amplification to a plateau; f) 72 °C for 5 minutes; and g) Hold at 4 °C.

[0172] 10) Optional: Run an agarose gel or bioanalyze the resulting qPCR. There will likely be a combination of cDNA and dimers.

[0173] SPRI Size Selection (0.8x). 1) Combine the qPCR reactants in a single tube. 2) Remove 180 μL of the pooled qPCR reactants and place them in a new 1.7 mL tube. 3) Add 144 μL of KAPA™ Pure Beads to the tube and briefly vortex to mix. Wait 5 minutes to allow binding to the DNA. 4) Place the tube on a magnetic rack and wait until the liquid becomes clear. 5) Remove the supernatant. 6) While still attached to the magnetic rack, wash the beads with 750 μL of 85% ethanol. Do not resuspend the beads. 7) Repeat Step 6.

[0174] 8) Remove the ethanol and air dry the beads (for about 5 minutes). Do not over-dry the beads to cause cracking. 9) Resuspend the beads in each tube in 20 μL of water. Once the beads are completely resuspended in water, incubate the tubes at 37 °C for 10 minutes. 10) Place the tubes on the magnetic rack and wait until the liquid becomes clear. 11) Transfer 18.5 μL of the eluate to a new optical grade PCR tube. 12) Run a Bioanalyzer trace on 10 μL of the eluate.

[0175] 13) If no dimers are present after size selection, proceed directly to the "Tagmentation and ILLUMINA® Amplicon Generation" section below. If dimers are still present, proceed to Step 14 and perform a second amplification and size selection step. This may be necessary for cells with low RNA content, but should not be necessary for cells with high RNA content (e.g., HeLa-S3, NIH / 3T3, etc.).

[0176] Second qPCR (Optional). 14) Prepare the following PCR mix according to the number of samples (Table 17).

[0177]

Table 17

[0178] 15) Add 31.5 μL of qPCR master mix to each optical PCR tube containing the previous PCR sample. Gently flick the tubes by hand to mix and briefly centrifuge in a tabletop centrifuge to remove air bubbles. 16) Run the following qPCR program (to ensure the sample is removed once the signal begins to pass the logarithmic phase to prevent overamplification): a) 95 °C for 3 minutes; b) 98 °C for 20 seconds; c) 67 °C for 20 seconds; d) 72 °C for 3 minutes; e) Repeat (b - d) until the signal transitions from exponential amplification to a plateau; f) 72 °C for 5 minutes; and g) Hold at 4 °C.

[0179] 17) Run an agarose gel or bioanalyze the resulting qPCR. The amplified cDNA should be clearly visible between 500 bp and 2500 bp while dimers may still be present (see the left side of Figure 19 for the expected size distribution).

[0180] Second SPRI size selection (0.8×). 18) Combine the qPCR reactions in a single tube. 19) Remove 40 μL of the qPCR reaction and place it in a new 1.7 mL tube. 20) Add 32 μL of KAPA™ Pure Beads to each tube and briefly vortex to mix. Wait 5 minutes for the DNA to bind.

[0181] 21) Place the tubes on a magnetic rack and wait until the liquid becomes clear. 22) Remove the supernatant. 23) While the tubes are still on the magnetic rack, wash with 750 μL of 85% ethanol. 24) Repeat step 5. 25) Remove the ethanol and air dry the beads (approx. 5 minutes). Do not over - dry the beads to cause cracking. 26) Resuspend the beads in each tube in 20 μL of water and wait 5 minutes. 27) Place the tubes on the magnetic rack and wait until the liquid becomes clear. 28) Transfer 18.5 μL of the eluate to a 1.7 mL tube. 29) Run an agarose gel or bioanalyze the resulting qPCR. At this point, there should be few, if any, dimers. If dimers remain, perform another round of qPCR followed by another 0.8× SPRI size selection.

[0182] IX. Tagmentation and ILLUMINA® Amplicon Generation. 1) Quantify the amplified cDNA with Quibit and dilute it to 0.12 ng / μL. 2) Preheat the thermocycler to 55 °C. 3) For each sample, combine 600 pg of purified cDNA with H 2 O to make a total volume of 5 μl. 4) Add 10 μl of Nextera TD buffer and 5 μl of Amplicon Tagment enzyme to each tube (the total volume of the reaction is 20 μl here). Pipette approximately 5 times to mix and centrifuge. 5) Incubate at 55 °C for 5 minutes. 6) Add 5 μl of neutralization buffer. Pipette approximately 5 times to mix and centrifuge (bubbles are not abnormal). 7) Incubate at room temperature for 5 minutes.

[0183] 8) Add each PCR to the tube in the following order: i) 15 μl of Nextera PCR mix; ii).8 μl of H 2 O; iii) 1 μl of 10 μM primer with N7 index, one of BC_0076 - BC_0083; and iv) 1 μl of 10 μM Nextera (BC_0118) N501 oligo. 9) Run the following thermocycle program: i) 95 °C, 30 seconds; ii) a) 95 °C, 10 seconds; b) 55 °C, 30 seconds; and c) 72 °C, 30 seconds for 12 cycles; and iii) then 72 °C for 5 minutes and then always 4 °C.

[0184] 10) Transfer 40 μl of the 50 μL reaction to a 1.7 mL tube. 11) Add 28 μL of KAPA® Pure Beads to perform 0.7× cleanup. Elute 20 μl. 12) Bioanalyze the resulting sample and quantify it with Quibit before sequencing (see the right side of Figure 19 for the expected size distribution).

[0185] X. ILLUMINA® Array Determination. 1) Use paired-end array determination performed with the 150 bp kit. 2) Set Read 1 to 66 nt (transcript array). 3) Set Read 2 to 94 nt (cell-specific barcode and UMI). 4) Prepare a sub-library index including a 6 nt Read 1 index.

[0186] Example 11 - Maintenance of Cells on Ice for Cell Fixation and Permeabilization on Ice Unique Molecular Index (UMI) is a random molecular barcode added to each transcript / cDNA prior to amplification. The unique molecular index enables the exclusion of PCR duplicates by computer. This is possible because such duplicates have the same UMI sequence. Thus, even if multiple PCR duplicates are sequenced, the original transcripts will each be counted only once.

[0187] The measure of UMI per cell indicates how many unique RNA molecules per cell can be detected. This is directly related to the degree of efficiency in barcoding and processing the molecules to enable detection by next-generation sequencing. This measure is generally the ultimate criterion in the art, and most researchers in the single-cell RNA sequencing space determine how well an experiment is going based on the number of UMI per cell detected with a given amount of raw sequencing reads (e.g., for 50,000 sequence reads per cell, 10,000 UMI per cell).

[0188] For each of Examples 11 - 16, all conditions were sequenced to the same saturation level within the same table (i.e., Tables 18, 19, 20, 21, and 22), enabling an accurate comparison across conditions. Each condition had the same number of raw sequencing reads compared to another condition. This indicated that the detected differences in UMI detected per cell were due to changes in conditions rather than the depth of sequencing.

[0189] In other protocols (see, e.g., Rosenberg, AB et al., BioRxiv (2017): p. 105163), formaldehyde fixation is carried out at room temperature. In this specification, experiments were conducted showing that there is a significant improvement in maintaining cells on ice (e.g., at 4 °C) during fixation and permeabilization (see Table 18).

[0190]

Table 18

[0191] Example 12 - Addition of Protease Inhibitor (PMSF) and Direct Binding to Streptavidin Beads In other protocols (see, e.g., Rosenberg, AB et al., BioRxiv (2017): p. 105163), first, nucleic acids are isolated from the lysis solution using SPRI bead cleanup, and then the desired nucleic acids (including 5'-biotin) are bound to streptavidin beads. In this specification, by adding PMSF to the lysate and then directly adding streptavidin beads (thereby skipping the initial SPRI isolation of nucleic acids and proceeding to the next step), an improvement in the number of detected unique molecules per cell was found (see Table 19).

[0192]

Table 19

[0193] Example 13 - 0.6× vs 0.8× SPRI Cleanup In other protocols (see, e.g., Rosenberg, AB et al., BioRxiv (2017): p. 105163), after cDNA amplification, 0.6× SPRI cleanup was performed. After cDNA amplification, SPRI cleanup can be used to remove short-chain products (i.e., products less than about 200 base pairs). In this specification, by adding SPRI beads at a ratio of 0.8:1 to the PCR product, it was found that the detected unique RNA molecules per cell were more than in the case of the previous 0.6:1 ratio of SPRI to PCR product (see Table 20).

[0194]

Table 20

[0195] Example 14 - Concentrations of Random Hexamer and Poly dT RT Primers In other protocols (see, e.g., Rosenberg, A. B. et al., BioRxiv (2017): p. 105163), reverse transcription is carried out using only poly dT primers for reverse transcription. Herein, it has been found that by combining barcoded random hexamer primers and varying the concentration, a significant increase in UMI / cell was brought about. Referring to Table 21, for protocol, version 2.1, condition number 4 (729 UMI / cell) is used. Continuing to refer to Table 21, herein, condition number 10 was used (1263 UMI / cell). Thus, by combining specific concentrations of random hexamers and poly dT reverse transcription primers together, the efficiency of in situ reverse transcription is increased.

[0196]

Table 21

[0197] Example 15 - Use of First Round Barcodes as Sample Identifiers Barcoded reverse transcription primers were used to generate first-round barcodes on cDNA molecules. Subsequently, such cDNA molecules were further barcoded by adding the barcode to the 5’ end by ligation as described above.

[0198] The proof of concept is described in Rosenberg, AB et al., (2018) Science, 360(6385); 176 - 182, which is hereby incorporated by reference in its entirety. As described herein, the experiment included four (4) unique samples. Which cells belonged to which sample was essentially tracked by observing the first barcode (incorporated by RT). This sample identification may require the barcode sequence corresponding to each well and knowledge of which sample was placed in which well. Using this information, a reference table from the first round barcode sequence to the sample ID can be created.

[0199] Figures 20 and 21 show the experimental setup for multiplexing samples. In this particular case, four samples were multiplexed across 48 wells of a 96 - well plate. By extending this same theory, it can be understood that up to 96 samples can be multiplexed in a single experiment.

[0200] Referring to Figure 22, an analysis of the transcriptome using the T - distributed stochastic neighbor embedding method (t - SNE) in an experiment using four different biological samples is shown. Each point represents the transcriptome from one individual cell. The sample identity of each transcriptome can be determined using the identity of the barcode incorporated during RT (using a barcoded RT primer).

[0201] Example 16 - PEG Incorporation for Template Switching of DNA / RNA Bound to Magnetic Beads The biotinylated oligo (cDNA / RNA duplex) attached to the reverse transcribed RNA is bound to magnetic beads coated with streptavidin. Subsequently, a template switch is performed on this cDNA / RNA duplex so that a single common adapter sequence can be incorporated at the 3'-end of the cDNA. Incorporating PEG (molecular weight 7000 - 9000) up to 10 w / v% into the template switch reaction containing the cDNA / RNA duplex bound to the streptavidin-coated magnetic beads can improve the efficiency of this step as measured by transcript detection per cell (Table 22).

[0202]

Table 22

[0203] Example 18 - Exemplary Method for Uniquely Labeling RNA Molecules Panel A of Figure 23 shows the labeling of the transcriptome by partition-pool barcoding. In each partition-pool round, fixed cells or nuclei can be randomly distributed into wells, and transcripts can be labeled with well-specific barcodes. Barcoded RT primers can be used in the first round. Second and third round barcodes can be added to the cDNA by ligation. The fourth barcode can be added to the cDNA molecules by PCR during sequencing library preparation. The bottom scheme shows the final barcoded cDNA molecules.

[0204] Panel B of Figure 23 shows a species mixing experiment with libraries prepared from 1,758 total cells. Human UBC extends horizontally along the x-axis, mouse UBC extends vertically along the y-axis, and mixed-species UBC is distributed between human UBC and mouse UBC. The estimated barcode collision frequency is 0.2%, but the species purity is >99%.

[0205] Panel C of Figure 23 shows UMI counts from a mixed experiment performed on fresh frozen (-80°C stored for 2 weeks) cells and nuclei. Median human UMI counts for fresh cells: 15,365; frozen cells: 15,078; nuclei: 12,113; frozen nuclei: 13,636.

[0206] Panel D of Figure 23 shows that gene expression measured by the method provided here is highly correlated between frozen cells and cells processed immediately (Pearson-r: 0.987). Frozen fresh cells were processed in two different Split-seq experiments.

[0207] As shown in Figure 24, fixed and permeabilized cells can be randomly distributed into wells each containing a reverse transcription primer with a well-specific barcode. In situ reverse transcription adds the well-specific barcode and converts RNA to cDNA. The cells can then be pooled and randomly distributed again into a second set of wells each containing a unique well-specific barcode. Such barcodes can be hybridized and ligated to the 5' end of the barcoded reverse transcription primer to add a second round of barcoding. The cells can be pooled and brought together again, and subsequent distribution-ligation-pool rounds can be performed. After the final round of ligation, the cDNA molecules contain a cell-specific combination of barcode, unique molecular identifier, and universal PCR handle at the 5' end. During the PCR step of library preparation, a fourth barcoding round can be performed. The oligonucleotides listed in Table 23 were used in the above examples. "rG" is an RNA base and "+G" is a locked nucleic acid base.

[0208] The oligonucleotides listed in Table 23 were used in the above examples. "rG" is an RNA base and "+G" is a locked nucleic acid base.

[0209]

Table 23-1

[0210]

Table 23-2

[0211] Certain embodiments of the disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations to these described embodiments will be apparent to those skilled in the art upon reading the above description. Applicants expect those skilled in the art to use such variations as appropriate, and Applicants intend that the various embodiments of the disclosure be practiced otherwise than as specifically described herein. Accordingly, the disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, any combination of the above-described elements in all possible variations thereof is included in the disclosure unless otherwise specified or clearly inconsistent with the context.

[0212] Furthermore, throughout this specification, numerous references are made to patents and printed publications. Each of the references and printed publications cited above is hereby incorporated by reference in its entirety into this specification.

[0213] It should be understood that the embodiments of the disclosure are illustrative of the principles of the disclosure. Other modifications that can be made are within the scope of the disclosure. Accordingly, by way of example and not limitation, alternative configurations of the disclosure can be used in accordance with the teachings herein. Accordingly, the disclosure is not limited to exactly what is illustrated and described.

[0214] The details shown herein are by way of example and are for purposes of illustrative discussion of the preferred embodiments of the disclosure only, and are presented in order to provide what is considered to be the most useful and readily understood description of the principles and conceptual aspects of the various embodiments of the disclosure.

[0215] It will be apparent to those skilled in the art that numerous modifications can be made to the details of the embodiments described above without departing from the fundamental principles of the present disclosure. Accordingly, the scope of the present invention should be determined solely by the following claims.

Claims

1. 1. A method for uniquely labeling RNA molecules in a plurality of cells, comprising: (a) fixing and permeabilizing a first plurality of cells prior to step (b), wherein the first plurality of cells is fixed and permeabilized at less than about 8° C.; (b) reverse transcribing the RNA molecules in the first plurality of cells to form complementary DNA (cDNA) molecules in the first plurality of cells, wherein reverse transcribing the RNA molecules comprises coupling a primer to the RNA molecules, the primer comprising at least one of a poly(T) sequence or a random sequence; (c) dividing the first plurality of cells comprising cDNA molecules into at least two primary aliquots, the at least two primary aliquots comprising a first primary aliquot and a second primary aliquot; (d) providing primary nucleic acid tags to the at least two primary aliquots, wherein the primary nucleic acid tag provided to the first primary aliquot is different from the primary nucleic acid tag provided to the second primary aliquot; (e) coupling said cDNA molecules in each of said at least two primary aliquots with said provided primary nucleic acid tags; (f) combining said at least two primary aliquots; (g) dividing the combined primary aliquot into at least two secondary aliquots, the at least two secondary aliquots comprising a first secondary aliquot and a second secondary aliquot; (h) providing secondary nucleic acid tags to the at least two secondary aliquots, wherein the secondary nucleic acid tag provided to the first secondary aliquot is different from the secondary nucleic acid tag provided to the second secondary aliquot; (i) coupling said cDNA molecules in each of said at least two secondary aliquots with said secondary nucleic acid tags provided; (j) repeating steps (f), (g), (h), and (i) with a subsequent aliquot, wherein the final nucleic acid tag comprises the capture agent; (k) combining the final aliquots; (l) lysing the first plurality of cells to release the cDNA molecules from within the first plurality of cells to form a lysate; and (m) adding a protease inhibitor and a binding agent to the lysate such that the cDNA molecules bind to the binding agent; The method includes:

2. 10. The method of claim 1, further comprising splitting the combined final aliquot into at least two final aliquots, wherein the at least two final aliquots include a first final aliquot and a second final aliquot.

3. 3. The method of claim 1 or claim 2, wherein the first plurality of cells is fixed and permeabilized at less than about 7°C, less than about 6°C, less than about 5°C, less than about 4°C, less than about 3°C, less than about 2°C, or less than about 1°C.

4. The method of any one of claims 1 to 3, wherein the protease inhibitor comprises phenylmethanesulfonyl fluoride (PMSF) or 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF).

5. The method of any one of claims 1 to 4, wherein the capture agent comprises biotin and the binding agent comprises avidin.

6. The method of claim 5 , wherein the binding agent comprises streptavidin.

7. (n) performing template switching of the cDNA molecule bound to the binding agent; (o) amplifying the cDNA molecules to form a solution of amplified cDNA molecules; and (p) introducing a solution of solid phase reversible immobilization (SPRI) beads into said solution of amplified cDNA molecules to remove polynucleotides less than 200 base pairs, wherein the ratio of SPRI bead solution to amplified cDNA molecule solution is about 0.9:1 to about 0.7:

1. The method of any one of claims 1 to 6, further comprising:

8. 8. The method of claim 7, wherein the ratio of SPRI bead solution to lysate is about 0.875:1 to about 0.775:1, about 0.85:1 to about 0.75:1, or about 0.825:1 to about 0.725:

1.

9. 9. The method of claim 7 or claim 8, wherein the SPRI bead solution comprises about 2M to 3M NaCl, and about 15% to 25% w / v polyethylene glycol (PEG), the molecular weight of the PEG being about 7,000 g / mol to 9,000 g / mol.

10. adding a common adaptor sequence to the 3' end of the released cDNA molecules, said adding step being carried out in a solution containing up to about 10% w / v polyethylene glycol, said polyethylene glycol having a molecular weight of about 7,000 g / mol to 9,000 g / mol. The method of any one of claims 1 to 9, further comprising:

11. The method of claim 10, wherein the common adaptor sequence is added to the 3' end of the released cDNA molecule by performing template switching.

12. 12. The method of any one of claims 1 to 11, wherein step (j) is repeated a sufficient number of times to generate a set of unique nucleic acid tags for the nucleic acids of a single cell.

13. 13. The method of claim 12, wherein the number of times is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100.

14. The primers of step (b) further comprise a first specific barcode, and the method further comprises: (q) reverse transcribing RNA molecules in a second plurality of cells to form cDNA molecules in the second plurality of cells, wherein reverse transcribing the RNA molecules comprises coupling a specific primer to the RNA molecules, the primer comprising a second specific barcode and at least one of a poly(T) sequence or a random sequence, the first specific barcode being different from the second specific barcode such that the cDNA molecules derived from the first plurality of cells can be distinguished relative to the cDNA molecules derived from the second plurality of cells; (r) dividing the second plurality of cells comprising cDNA molecules into at least two primary aliquots, the at least two primary aliquots comprising a first primary aliquot and a second primary aliquot; (s) providing primary nucleic acid tags to the at least two primary aliquots, the primary nucleic acid tags provided to the first primary aliquot being at least one primary nucleic acid tag provided to the second primary aliquot; different from the primary nucleic acid tag provided to the (t) coupling said cDNA molecules in each of said at least two primary aliquots with said provided primary nucleic acid tags; (u) combining said at least two primary aliquots; (v) dividing the combined primary aliquot into at least two secondary aliquots, the at least two secondary aliquots comprising a first secondary aliquot and a second secondary aliquot; (w) providing secondary nucleic acid tags to the at least two secondary aliquots, wherein the secondary nucleic acid tag provided to the first secondary aliquot is different from the secondary nucleic acid tag provided to the second secondary aliquot; (x) coupling the cDNA molecules in each of the at least two secondary aliquots to the provided secondary nucleic acid tags; and (y) repeating steps (u), (v), (w), and (x) with a subsequent aliquot, wherein the final nucleic acid tag comprises a capture agent. The method of any one of claims 1 to 13, further comprising:

15. Each of the nucleic acid tags comprises: a barcode sequence comprising a 3' end and a 5' end; and a 3' hybridization sequence and a 5' hybridization sequence adjacent to the 3' end and the 5' end, respectively, of the barcode sequence; a first strand comprising: a first portion complementary to at least one of the 5' hybridization sequence and the adapter sequence; and a second portion complementary to the 3' hybridization sequence A second strand comprising The method according to any one of claims 1 to 14, comprising:

16. Ligating at least two of said nucleic acid tags attached to said cDNA molecules. The method of any one of claims 1 to 15, further comprising:

17. 17. The method of claim 16, wherein the ligation is performed within the first plurality of cells.

18. Removing unbound nucleic acid tags The method of any one of claims 1 to 17, further comprising:

19. Ligating at least two of the nucleic acid tags attached to the released cDNA molecules. The method of any one of claims 1 to 15, further comprising:

20. 20. The method of any one of claims 1 to 19, wherein the majority of said nucleic acid tag-bound cDNA molecules derived from a single cell contain the same set of bound nucleic acid tags.

21. 21. The method of any one of claims 1 to 20, wherein the first plurality of cells is selected from at least one of mammalian cells, yeast cells, and bacterial cells.

22. 1. A method for labeling a nucleic acid in a first cell, comprising: (a) (i) a first reverse transcription primer comprising a 5′ overhanging sequence, a first reverse transcription primer, the first reverse transcription primer being configured to reverse transcribe RNA having a poly(A) tail; and (ii) a second reverse transcription primer comprising a 5′ overhanging sequence and at least one of a random hexamer, a random heptamer, a random octamer, a random nonamer, and a random decamer. generating complementary DNA (cDNA) molecules in a plurality of cells, including the first cell, by reverse transcribing RNA using at least one of: (b) dividing the plurality of cells into (n) aliquots; (c) providing a plurality of nucleic acid tags to each of the n aliquots, each nucleic acid tag comprising: a first strand comprising a 3' hybridization sequence extending from the 3' end of the labeled sequence and a 5' hybridization sequence extending from the 5' end of the labeled sequence; and a second strand comprising an overhanging sequence, the overhanging sequence comprising (i) a first portion complementary to at least one of the 5' hybridization sequence and the 5' overhanging sequence, and (ii) a second portion complementary to the 3' hybridization sequence; each labeling sequence of the plurality of nucleic acid tags provided in a given aliquot is the same, and each of the n aliquots is provided with a different labeling sequence; (d) binding at least one of the cDNA molecules of each of the n aliquots to a nucleic acid tag; (e) combining the n aliquots; and (f) repeating steps (b), (c), (d), and (e) with the combined aliquots; (g) combining the final aliquots; (h) lysing the plurality of cells, including the first cell, to release the cDNA molecules from within the plurality of cells, including the first cell, to form a lysate; and (i) adding a protease inhibitor and a binding agent to the lysate such that the cDNA molecules bind to the binding agent; The method includes:

23. 23. The method of claim 22, wherein step (f) is repeated a number of times sufficient to generate a series of labeled sequences that are unique to the cDNA molecule of the first cell.

24. 24. The method of claim 23, wherein the number of times is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100.

25. 25. The method of any one of claims 22 to 24, wherein the cDNA molecules are produced in aliquots, and the concentration of the first reverse transcription primer in the aliquot is from about 0.5 μM to about 10 μM, and the concentration of the second reverse transcription primer in the aliquot is from about 0.5 μM to about 10 μM.

26. Fixing the plurality of cells prior to step (a), wherein the plurality of cells is fixed at less than about 8° C. The method of any one of claims 22 to 25, further comprising:

27. 27. The method of claim 26, wherein the plurality of cells is fixed at less than about 7°C, less than about 6°C, less than about 5°C, less than about 4°C, less than about 3°C, less than about 2°C, or less than about 1°C.

28. Permeabilizing the plurality of cells prior to step (a), wherein the plurality of cells is permeabilized at less than about 8° C. The method of any one of claims 22 to 27, further comprising:

29. 29. The method of claim 28, wherein the plurality of cells is permeabilized at less than about 7°C, less than about 6°C, less than about 5°C, less than about 4°C, less than about 3°C, less than about 2°C, or less than about 1°C.

30. Ligating at least two of said nucleic acid tags attached to said cDNA molecules. The method of any one of claims 22 to 29, further comprising:

31. 31. The method of claim 30, wherein the ligation is performed within the plurality of cells.

32. Removing unbound nucleic acid tags The method of any one of claims 22 to 31, further comprising:

33. 33. The method of any one of claims 22 to 32, wherein at least one of the first and second reverse transcription primers is configured to reverse transcribe a predetermined RNA.

34. The final nucleic acid tag comprises a capture agent, and the method further comprises: After step (f), lysing the plurality of cells to release the cDNA molecules from within the plurality of cells to form a lysate; and adding protease inhibitors and binding agents to the lysate to isolate the cDNA molecules; The method of any one of claims 22 to 33, further comprising:

35. 35. The method of claim 34, wherein the protease inhibitor comprises phenylmethanesulfonyl fluoride (PMSF) or 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF).

36. 36. The method of claim 34 or claim 35, wherein the capture agent comprises biotin and the binding agent comprises avidin.

37. 37. The method of claim 36, wherein the binding agent comprises streptavidin.

38. (j) performing template switching of the cDNA molecule bound to the binding agent; (k) amplifying the cDNA molecules to form a solution of amplified cDNA molecules; and (l) introducing a solution of solid phase reversible immobilization (SPRI) beads into said solution of amplified cDNA molecules to remove polynucleotides less than 200 base pairs, wherein the ratio of SPRI bead solution to amplified cDNA molecule solution is about 0.9:1 to about 0.7:

1. The method of any one of claims 22 to 37, further comprising:

39. 39. The method of claim 38, wherein the ratio of SPRI bead solution to lysate is about 0.875:1 to about 0.775:1, about 0.85:1 to about 0.75:1, or about 0.825:1 to about 0.725:

1.

40. The SPRI bead solution contains about 2M to 3M NaCl, and about 15% to 25% w / v 40. The method of claim 38 or 39, comprising: / v% polyethylene glycol (PEG), the molecular weight of the PEG being about 7,000 g / mol to 9,000 g / mol.

41. adding a common adaptor sequence to the 3' end of the released cDNA molecules, said adding step being carried out in a solution containing up to about 10% w / v polyethylene glycol, said polyethylene glycol having a molecular weight of about 7,000 g / mol to 9,000 g / mol. The method of any one of claims 22 to 40, further comprising:

42. 42. The method of claim 41 , wherein the common adaptor sequence is added to the 3′ end of the released cDNA molecule by performing template switching.

43. Ligating at least two of the nucleic acid tags attached to the released cDNA molecules. The method of any one of claims 22 to 42, further comprising:

44. 44. The method of any one of claims 22 to 43, wherein the plurality of cells is selected from at least one of mammalian cells, yeast cells, and bacterial cells.

45. 1. A kit for labeling a nucleic acid in a first cell, comprising: a first reverse transcription primer comprising a 5' overhanging sequence, said first reverse transcription primer configured to reverse transcribe RNA having a poly(A) tail; a second reverse transcription primer comprising a 5' overhanging sequence and at least one of a random hexamer, a random heptamer, a random octamer, a random nonamer, and a random decamer; A plurality of first nucleic acid tags, each of the first nucleic acid tags comprising: a first strand comprising a 3' hybridization sequence extending from the 3' end of a first labeled sequence and a 5' hybridization sequence extending from the 5' end of said first labeled sequence; and a plurality of first nucleic acid tags comprising a second strand comprising an overhanging sequence, the overhanging sequence comprising (i) a first portion complementary to at least one of the 5' hybridization sequence and the 5' overhanging sequence of the first and second reverse transcription primers, and (ii) a second portion complementary to the 3' hybridization sequence; a plurality of second nucleic acid tags, each second nucleic acid tag comprising: a first strand comprising a 3' hybridization sequence extending from the 3' end of a second labeled sequence and a 5' hybridization sequence extending from the 5' end of said second labeled sequence; and a second strand comprising an overhanging sequence, the overhanging sequence comprising (i) a first portion complementary to at least one of the 5′ hybridization sequence and the 5′ overhanging sequence of the first and second reverse transcription primers, and (ii) a second portion complementary to the 3′ hybridization sequence, the first labeling sequence being different from the second labeling sequence; and A plurality of final nucleic acid tags, each final nucleic acid tag comprising: a first strand comprising a 3' hybridization sequence extending from the 3' end of the final labeled sequence, and a 5' hybridization sequence extending from the 5' end of said final labeled sequence; a second strand comprising an overhanging sequence, the overhanging sequence comprising (i) a first portion complementary to at least one of the 5' hybridization sequence and the 5' overhanging sequence of the first and second reverse transcription primers, and (ii) a second portion complementary to the 3' hybridization sequence of the first and second reverse transcription primers; a second strand comprising a complementary second portion; and a plurality of final nucleic acid tags comprising a capture agent, the final labeling sequence being different from the first and second labeling sequences; Kit including:

46. 46. ​​The kit of claim 45, further comprising at least one of a reverse transcriptase, a fixation agent, a permeabilization agent, a ligation agent, a lysis agent, and a protease inhibitor.

47. one or more additional plurality of nucleic acid tags, each nucleic acid tag comprising: a first strand comprising a 3' hybridization sequence extending from the 3' end of the labeled sequence and a 5' hybridization sequence extending from the 5' end of the labeled sequence; and a second strand comprising an overhanging sequence, the overhanging sequence comprising: (i) a first portion complementary to at least one of the 5' hybridization sequence and the 5' overhanging sequence of the first and second reverse transcription primers, and (ii) a second portion complementary to the 3' hybridization sequence; 47. The kit of claim 45 or claim 46, wherein the labeling sequence further comprises one or more additional plurality of nucleic acid tags, each given additional plurality of nucleic acid tags being different.

48. 1. A method for uniquely labeling RNA molecules in multiple nuclei, comprising: (a) fixing and permeabilizing a first plurality of nuclei prior to step (b), wherein said first plurality of nuclei is fixed and permeabilized at less than about 8° C.; (b) reverse transcribing the RNA molecules in the first plurality of nuclei to form complementary DNA (cDNA) molecules in the first plurality of nuclei, wherein reverse transcribing the RNA molecules comprises coupling a primer to the RNA molecules, the primer comprising at least one of a poly(T) sequence or a random sequence; (c) dividing the first plurality of nuclei comprising cDNA molecules into at least two primary aliquots, the at least two primary aliquots comprising a first primary aliquot and a second primary aliquot; (d) providing primary nucleic acid tags to the at least two primary aliquots, wherein the primary nucleic acid tag provided to the first primary aliquot is different from the primary nucleic acid tag provided to the second primary aliquot; (e) coupling said cDNA molecules in each of said at least two primary aliquots with said provided primary nucleic acid tags; (f) combining said at least two primary aliquots; (g) dividing the combined primary aliquot into at least two secondary aliquots, the at least two secondary aliquots comprising a first secondary aliquot and a second secondary aliquot; (h) providing secondary nucleic acid tags to the at least two secondary aliquots, wherein the secondary nucleic acid tag provided to the first secondary aliquot is different from the secondary nucleic acid tag provided to the second secondary aliquot; (i) coupling said cDNA molecules in each of said at least two secondary aliquots with said provided secondary nucleic acid tags; (j) repeating steps (f), (g), (h), and (i) with a subsequent aliquot, wherein the final nucleic acid tag comprises the capture agent; (k) combining the final aliquots; (l) lysing the first plurality of nuclei to release the cDNA molecules from within the first plurality of nuclei to form a lysate; and (m) adding a protease inhibitor and a binding agent to the lysate such that the cDNA molecules bind to the binding agent; The method includes:

49. 49. The method of claim 48, further comprising splitting the combined final aliquot into at least two final aliquots, wherein the at least two final aliquots include a first final aliquot and a second final aliquot.

50. 50. The method of claim 48 or claim 49, wherein the first plurality of nuclei is fixed and permeabilized at less than about 7°C, less than about 6°C, less than about 5°C, less than about 4°C, less than about 3°C, less than about 2°C, or less than about 1°C.

51. 51. The method of any one of claims 48 to 50, wherein the protease inhibitor comprises phenylmethanesulfonyl fluoride (PMSF) or 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF).

52. The method of any one of claims 48 to 51, wherein the capture agent comprises biotin and the binding agent comprises avidin.

53. 53. The method of claim 52, wherein the binding agent comprises streptavidin.

54. (n) performing template switching of the cDNA molecule bound to the binding agent; (o) amplifying the cDNA molecules to form a solution of amplified cDNA molecules; and (p) introducing a solution of solid phase reversible immobilization (SPRI) beads into said solution of amplified cDNA molecules to remove polynucleotides less than 200 base pairs, wherein the ratio of SPRI bead solution to amplified cDNA molecule solution is about 0.9:1 to about 0.7:

1. The method of any one of claims 48 to 53, further comprising:

55. 55. The method of claim 54, wherein the ratio of SPRI bead solution to lysate is about 0.875:1 to about 0.775:1, about 0.85:1 to about 0.75:1, or about 0.825:1 to about 0.725:

1.

56. 56. The method of claim 54 or claim 55, wherein the SPRI bead solution comprises about 2M to 3M NaCl, and about 15% to 25% w / v polyethylene glycol (PEG), the molecular weight of the PEG being about 7,000 g / mol to 9,000 g / mol.

57. 57. The method of any one of claims 48 to 56, further comprising the step of adding a common adaptor sequence to the 3' end of the released cDNA molecules, said adding step being carried out in a solution containing up to about 10% w / v polyethylene glycol, the molecular weight of said polyethylene glycol being between about 7,000 g / mol and 9,000 g / mol.

58. 58. The method of claim 57, wherein the common adaptor sequence is added to the 3' end of the released cDNA molecule by performing template switching.

59. 59. The method of any one of claims 48 to 58, wherein step (j) is repeated a sufficient number of times to generate a set of unique nucleic acid tags for the nucleic acids of a single cell.

60. The number of times is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 60. The method of claim 59, selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100.

61. The primers of step (b) further comprise a first specific barcode, and the method further comprises: (q) reverse transcribing RNA molecules in a second plurality of nuclei to form cDNA molecules in said second plurality of nuclei, wherein reverse transcribing said RNA molecules comprises coupling a specific primer to said RNA molecules, said primer comprising a second specific barcode and at least one of a poly(T) sequence or a random sequence, said first specific barcode being different from said second specific barcode such that said cDNA molecules derived from said first plurality of nuclei can be distinguished relative to said cDNA molecules derived from said second plurality of nuclei; (r) dividing the second plurality of nuclei comprising cDNA molecules into at least two primary aliquots, the at least two primary aliquots comprising a first primary aliquot and a second primary aliquot; (s) providing primary nucleic acid tags to the at least two primary aliquots, wherein the primary nucleic acid tag provided to the first primary aliquot is different from the primary nucleic acid tag provided to the second primary aliquot; (t) coupling said cDNA molecules in each of said at least two primary aliquots with said provided primary nucleic acid tags; (u) combining said at least two primary aliquots; (v) dividing the combined primary aliquot into at least two secondary aliquots, the at least two secondary aliquots comprising a first secondary aliquot and a second secondary aliquot; (w) providing secondary nucleic acid tags to the at least two secondary aliquots, wherein the secondary nucleic acid tag provided to the first secondary aliquot is different from the secondary nucleic acid tag provided to the second secondary aliquot; (x) coupling the cDNA molecules in each of the at least two secondary aliquots to the provided secondary nucleic acid tags; and (y) repeating steps (u), (v), (w), and (x) with a subsequent aliquot, wherein the final nucleic acid tag comprises a capture agent. The method of any one of claims 48 to 60, further comprising:

62. Each of the nucleic acid tags comprises: a barcode sequence comprising a 3' end and a 5' end; and a 3' hybridization sequence and a 5' hybridization sequence adjacent to the 3' end and the 5' end, respectively, of the barcode sequence; a first strand comprising: a first portion complementary to at least one of the 5' hybridization sequence and the adapter sequence; and a second portion complementary to the 3' hybridization sequence A second strand comprising The method of any one of claims 48 to 61, comprising:

63. Ligating at least two of said nucleic acid tags attached to said cDNA molecules. The method of any one of claims 48 to 62, further comprising:

64. 64. The method of claim 63, wherein said ligation is performed within said first plurality of nuclei. 。

65. Removing unbound nucleic acid tags The method of any one of claims 48 to 64, further comprising:

66. Ligating at least two of the nucleic acid tags attached to the released cDNA molecules. The method of any one of claims 48 to 62, further comprising:

67. 67. The method of any one of claims 48 to 66, wherein the majority of said nucleic acid tag-bound cDNA molecules derived from a single cell comprise the same set of bound nucleic acid tags.

68. 1. A method of labeling a nucleic acid in a first nucleus, comprising: (a) (i) a first reverse transcription primer comprising a 5′ overhanging sequence, wherein the first reverse transcription primer is configured to reverse transcribe RNA having a poly(A) tail; and (ii) a second reverse transcription primer comprising a 5′ overhanging sequence and at least one of a random hexamer, a random heptamer, a random octamer, a random nonamer, and a random decamer. generating complementary DNA (cDNA) molecules in a plurality of nuclei, including the first nucleus, by reverse transcribing RNA using at least one of: (b) dividing the plurality of nuclei into (n) aliquots; (c) providing a plurality of nucleic acid tags to each of the n aliquots, each nucleic acid tag comprising: a first strand comprising a 3' hybridization sequence extending from the 3' end of the labeled sequence and a 5' hybridization sequence extending from the 5' end of the labeled sequence; and a second strand comprising an overhanging sequence, the overhanging sequence comprising (i) a first portion complementary to at least one of the 5' hybridization sequence and the 5' overhanging sequence, and (ii) a second portion complementary to the 3' hybridization sequence; each labeling sequence of the plurality of nucleic acid tags provided in a given aliquot is the same, and each of the n aliquots is provided with a different labeling sequence; (d) binding at least one of the cDNA molecules of each of the n aliquots to a nucleic acid tag; (e) combining the n aliquots; and (f) repeating steps (b), (c), (d), and (e) with the combined aliquots; (g) combining the final aliquots; (h) lysing the plurality of nuclei, including the first nucleus, to release the cDNA molecules from within the plurality of nuclei, including the first nucleus, to form a lysate; and (i) adding a protease inhibitor and a binding agent to the lysate such that the cDNA molecules bind to the binding agent; The method includes:

69. 69. The method of claim 68, wherein step (f) is repeated a sufficient number of times to generate a series of labeled sequences that are unique to said cDNA molecule of said first nucleus.

70. The number of times is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 4 70, 75, 80, 85, 90, 95, and 100.

71. 71. The method of any one of claims 68-70, wherein the cDNA molecules are produced in aliquots, and the concentration of the first reverse transcription primer in the aliquot is from about 0.5 μM to about 10 μM, and the concentration of the second reverse transcription primer in the aliquot is from about 0.5 μM to about 10 μM.

72. Fixing the plurality of nuclei prior to step (a), wherein the plurality of nuclei is fixed at less than about 8° C. The method of any one of claims 68 to 71, further comprising:

73. 73. The method of claim 72, wherein said plurality of nuclei are fixed at less than about 7°C, less than about 6°C, less than about 5°C, less than about 4°C, less than about 3°C, less than about 2°C, or less than about 1°C.

74. permeabilizing the plurality of nuclei prior to step (a), wherein the plurality of nuclei is permeabilized at less than about 8° C. The method of any one of claims 68 to 73, further comprising:

75. 75. The method of claim 74, wherein the plurality of nuclei are permeabilized at less than about 7°C, less than about 6°C, less than about 5°C, less than about 4°C, less than about 3°C, less than about 2°C, or less than about 1°C.

76. Ligating at least two of said nucleic acid tags attached to said cDNA molecules. The method of any one of claims 68 to 75, further comprising:

77. 77. The method of claim 76, wherein said ligation is performed within said multiple nuclei.

78. Removing unbound nucleic acid tags The method of any one of claims 68 to 77, further comprising:

79. 79. The method of any one of claims 68 to 78, wherein at least one of the first and second reverse transcription primers is configured to reverse transcribe a predetermined RNA.

80. The final nucleic acid tag comprises a capture agent, and the method further comprises: After step (f), lysing the plurality of nuclei to release the cDNA molecules from within the plurality of nuclei to form a lysate; and adding protease inhibitors and binding agents to the lysate to isolate the cDNA molecules; 80. The method of any one of claims 68 to 79, further comprising:

81. 81. The method of claim 80, wherein the protease inhibitor comprises phenylmethanesulfonyl fluoride (PMSF) or 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF).

82. 82. The method of claim 80 or claim 81, wherein the capture agent comprises biotin and the binding agent comprises avidin.

83. 83. The method of claim 82, wherein the binding agent comprises streptavidin.

84. (j) performing template switching of the cDNA molecule bound to the binding agent; (k) amplifying the cDNA molecules to form a solution of amplified cDNA molecules; and (l) introducing a solution of solid phase reversible immobilization (SPRI) beads into said solution of amplified cDNA molecules to remove polynucleotides less than 200 base pairs, wherein the ratio of SPRI bead solution to amplified cDNA molecule solution is about 0.9:1 to about 0.7:

1. The method of any one of claims 68 to 83, further comprising:

85. 85. The method of claim 84, wherein the ratio of SPRI bead solution to lysate is about 0.875:1 to about 0.775:1, about 0.85:1 to about 0.75:1, or about 0.825:1 to about 0.725:

1.

86. 86. The method of claim 84 or claim 85, wherein the SPRI bead solution comprises about 2M to 3M NaCl, and about 15% to 25% w / v polyethylene glycol (PEG), the molecular weight of the PEG being about 7,000 g / mol to 9,000 g / mol.

87. 87. The method of any one of claims 68 to 86, further comprising the step of adding a common adaptor sequence to the 3' end of the released cDNA molecules, said adding step being carried out in a solution containing up to about 10% w / v polyethylene glycol, the molecular weight of said polyethylene glycol being between about 7,000 g / mol and 9,000 g / mol.

88. 88. The method of claim 87, wherein the common adaptor sequence is added to the 3' end of the released cDNA molecule by performing template switching.

89. Ligating at least two of the nucleic acid tags attached to the released cDNA molecules. The method of any one of claims 68 to 88, further comprising:

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