Methods, kits and compositions for barcoding cells

Abstract

Provided are methods of barcoding cells of a sample of dispersed cells. Aspects of the methods include contacting the sample with an array of barcoded nucleic acids such that barcoded nucleic acids of the array enter cells of the sample to barcode cells of the sample. In some aspects, the method comprises employing a barcode of the barcoded cell to determine a location of the array contacted by the barcoded cell. Aspects of the methods further include pooling the barcoded cells or the nucleic acids from the cells, preparing a sequencing library from the barcoded cells and / or sequencing the sequencing library. Also provided are kits, compositions and devices that find use in practicing the disclosed methods.

Description

[0001] Atty. Docket No.: CLON-185WO

[0002] Methods, Kits and Compositions for Barcoding Cells

[0003] Cross-Reference to Related Applications

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 736,311 filed on December 19, 2024. The entire contents of the above-identified application are incorporated herein by reference.

[0005] Field of the Invention

[0006] This application generally relates to methods for barcoding cells, including cells present in heterogenous populations. Further, the present application provides kits and compositions for performing said methods.

[0007] Sequence Listing

[0008] A Sequence Listing is provided herewith as a Sequence Listing XML, “CLON- 185WO_SEQ_LISTING” created on December 17, 2025, and having a size of 11 ,515 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

[0009] Background

[0010] The development of next generation sequencing (NGS) technologies has allowed for the rapid extraction of valuable genomic and transcriptomic information from produced nucleic acid libraries. High throughput NGS technologies, such as the sequencing platforms provided by: Illumina® (e.g., the iSeq™, MiniSeq™, HiSeq™, MiSeq™, NextSeq™, NovaSeqTMand / or Genome Analyzer™ sequencing systems); Ion Torrent™ (e.g., the Ion PGM™ and / or Ion Proton™ sequencing systems); Pacific Biosciences (e.g., the PACBIO RS II Sequel sequencing system); Life Technologies™ (e.g., a SOLiD™ sequencing system); Roche (e.g., the 454 GS FLX+ and / or GS Junior sequencing systems); Element AVITI systems; Complete Genomics DNBSEQ Platforms (e.g., E25, G99, G400 and T7); and the like, allow for the sequencing of nucleic acid molecules more quickly and cheaply than previously used Sanger sequencing, and as such, these techniques have revolutionized biotechnology and biomedical research. In Atty. Docket No.: CLON-185WO addition, as these technologies have matured and become more user-friendly, their presence in clinical applications has continued to increase.

[0011] These powerful sequencing technologies place a particular emphasis on library preparation. Well-prepared and efficiently produced complementary DNA (cDNA) libraries made from RNA templates or DNA (e.g., genomic DNA) templates can be analyzed using NGS technologies for a diverse range of purposes.

[0012] In current NGS workflows, libraries prepared from samples obtained from bulk cell populations or single cells can be sequenced. Sequencing bulk cell populations does not allow for the analysis of genomic and / or transcriptomic changes at single cell resolution, which can mask underlying heterogeneity of different cell types in a bulk population. Where nucleic acid libraries are prepared from individual cells, NGS technologies allow for the analysis of genomic, transcriptomic, and / or epigenetic changes at single cell resolution. While single cell sequencing provides many benefits over bulk cell sequencing, in single cell sequencing one needs to be able to trace a given nucleic acid back to its original source.

[0013] While sample barcoding technologies have been developed to address this requirement, there remain upper limits with respect to the number of different cells that can be realistically processed in a given experiment. In some instances, the number of cells that are desirably processed in a single experiment, e.g., where all of the cells are ultimately pooled in a single sequencing ready library composition, exceeds the number that can readily be processed using current protocols.

[0014] Summary

[0015] In such instances, the inventors have realized that what is needed is an approach that allows for high-throughput analysis of cells without requiring specialized equipment beyond the instrumentation known in the current state of the art. In addition, the inventors also recognize that there is need for single-cell analysis methods that can be combined with other methods, e.g., Cleavage Under Targets and Tagmentation (CUT&Tag), ATAC-Seq, etc. Also of interest is the development of high throughput methods that provide for evaluation of cell-cell interactions. Atty. Docket No.: CLON-185WO

[0016] Embodiments of the present technology satisfy the above, and other, needs in the art by providing a spatial approach to uniquely identify nucleic acids produced from individual cells of dispersed cellular samples. In addition, the methods provided herein allow for the practical analysis of many cells in parallel, and accommodate a wide range of parameters (e.g., live cells, fixed cells, combination with other analysis methods) to provide convenient, high-throughput single cell analysis.

[0017] Provided are methods of barcoding cells of a sample of dispersed cells. Aspects of the methods include contacting the sample with an array of barcoded nucleic acids such that barcoded nucleic acids of the array enter cells of the sample to barcode cells of the sample. In some aspects, the array of barcoded cells includes distinct populations of barcoded cells stably associated with different locations of a solid support. The method may further include employing a barcode of the barcoded cell to determine a location of the array contacted by the barcoded cell. Aspects of the methods further include pooling the barcoded cells or the nucleic acids from the cells, preparing a sequencing library from the barcoded cells and / or sequencing the sequencing library. Also provided are kits, compositions and devices for use in performing embodiments of the methods as described herein.

[0018] Methods of barcoding cells of a sample of dispersed cells are provided. Aspects of the methods include: contacting the sample with an array of barcoded nucleic acids such that barcoded nucleic acids of the array enter cells of the sample to barcode cells of the sample, wherein the array of barcoded nucleic acids comprises a plurality of barcoded nucleic acids, wherein each barcoded nucleic acid of the plurality of barcoded nucleic acids comprises a barcode domain. In some instances, the sample of dispersed cells comprises dispersed live cells, where the method may, in some instances further include culturing the dispersed live cells contacted with the array of barcoded nucleic acids. In some instances, the sample of dispersed cells comprises fixed cells, which may be permeabilized. In some instances, the barcoded nucleic acids further comprise a cleavage domain, which domain may be cleaved by an enzymatic stimulus, a chemical stimulus or an electromagnetic stimulus, which domain may function as a cleavable linker, wherein the cleavable linker connects the barcoded nucleic acid to a solid support, e.g., where the cleavable linker comprises a photocleavable linker. In Atty. Docket No.: CLON-185WO some instances, the barcoded nucleic acids further comprise a capture domain, which capture domain may specifically bind to a ribonucleic acid, e.g., where the capture domain comprises a polyT sequence, a gene-specific sequence or a random sequence. In some instances, the capture domain binds to an exogeneous nucleic acid added to cells of the sample, e.g., where the exogeneous nucleic acid is conjugated to a specific binding member. In some instances, the capture domain binds to a transposon, a bridge oligonucleotide or a cDNA. In embodiments, the barcoded nucleic acids further comprise an adapter domain, e.g., where the adapter domain is a sequencing adapter domain, e.g., where the sequencing adapter domain comprises any one of SEQ ID NOs: 01 -12. In some instances, the barcoded nucleic acids further comprise a second barcode domain, and in some instances the barcoded nucleic acids further comprise a third barcode domain, and in some instances the barcoded nucleic acids further comprise a fourth barcode domain, and in some instances the barcoded nucleic acids further comprise a fifth barcode domain, and in some instances the barcoded nucleic acids further comprise a sixth barcode domain. In embodiments, the array of barcoded nucleic acids comprises distinct populations of barcoded nucleic acids, e.g., where the distinct populations of barcoded nucleic acids differ by one or more of: the barcode domain, a cleavage domain, a capture domain and an adapter domain, such as where the distinct populations of barcoded nucleic acids are functionally distinct populations of barcoded nucleic acids that differ by one or more of: a cleavage domain and a capture domain. In some instances, the array of barcoded nucleic acids comprises: a first functionally distinct population of barcoded nucleic acids comprising a first capture domain and a first cleavage domain; and a second functionally distinct population of barcoded nucleic acids comprising a second capture domain and a second cleavage domain, e.g., where the first capture domain is different from the second capture domain, where in some instances the first cleavage domain is different from the second cleavage domain. In some instances, the distinct populations of barcoded nucleic acids stably associated with different locations of a solid support. In some instances, each location of the different locations of the array comprises a population of barcoded nucleic acids with a unique barcode, such as where a location of the array comprises two or more functionally distinct populations of barcoded nucleic acids. In some Atty. Docket No.: CLON-185WO instances, the solid support comprises a planar solid support. In some instances, the different locations abut each other. In some instances, the different locations are separated from each other by an intervening region, e.g., where the intervening region is free of nucleic acids, such as where the intervening region is configured to impede cell adherence, e.g., where the intervening region comprises an anti-adherence coating. In some instances, the solid support comprises a multi-well array. In some instances, the solid support comprises a bead array. In embodiments, the sample has a cell concentration sufficient to provide upon contact with the array a confluence ranging from 25 to 100%, such as where the sample has a cell concentration sufficient to provide a confluence ranging from 25 to 75%, such as where the sample has a cell concentration sufficient to provide a confluence ranging from 75 to 100%. In embodiments, the barcoded nucleic acids enter the cells by transfection, e.g., where the transfection is mediated by a transfection agent, e.g., where the transfection agent is a nanoparticle agent. In some instances, the transfection is mediated by a physical stimulus, e.g., where the physical stimulus comprises electroporation. In some instances, the barcoded nucleic acids are attached to a cell uptake moiety that allows for the barcoded nucleic acids to enter the cells, e.g., where the cell uptake moiety is a cell penetrating peptide (CPP). In some instances, the CPP comprises transportan, TP10, penetratin, Tat or a series of arginine residues, and in some instances, the CPP comprises nine arginine residues. In some instances, the barcoded nucleic acid functions as a primer. In some instances, the barcoded nucleic acid functions as a template switch oligonucleotide. In some instances, the barcoded nucleic acid functions as a template nucleic acid. In some instances, the method further comprises employing the barcode domain of a barcoded cell to determine a location of the array contacted by the barcoded cell. In some instances, the method further includes pooling the barcoded cells or the nucleic acids from the barcoded cells. In some instances, the method further includes preparing a sequencing library from the barcoded cells. In some instances, the method further includes sequencing the sequencing library. In some instances, the sample of dispersed cells is not derived from a tissue or tissue section. In some instances, the sample of dispersed cells comprises one or more of: immune cells, neuronal cells, cardiac cells, endothelial cells, fibroblasts, liver cells and tumor cells. Atty. Docket No.: CLON-185WO

[0019] Also provided are kits, where such kits include kit comprising an array of barcoded nucleic acids comprising distinct populations of barcoded nucleic acids stably associated with different locations of a solid support, wherein the barcoded nucleic acid array is configured to be contacted with a sample of dispersed cells, and wherein each barcoded nucleic acid of the barcoded nucleic acid array comprises a barcode domain. In some instances, the kits further include one or more reverse transcription reagents, e.g., where the one or more reverse transcription reagents include a reverse transcriptase. In some instances, the solid support comprises a planar solid support. In some instances, the different locations abut each other. In some instances, the different locations are separated from each other by an intervening region, e.g., where the intervening region is free of nucleic acids. In some instances, the intervening region is configured to impede cell adherence, e.g., where the intervening region comprises an anti-adherence coating. In some instances, the solid support comprises a multi-well array. In some instances, the solid support comprises a bead array. In some instances, the barcoded nucleic acids further comprise a cleavage domain, which domain may be cleaved by an enzymatic stimulus, a chemical stimulus or an electromagnetic stimulus, which domain may function as a cleavable linker, wherein the cleavable linker connects the barcoded nucleic acid to a solid support, e.g., where the cleavable linker comprises a photocleavable linker. In some instances, the barcoded nucleic acids further comprise a capture domain, which capture domain may specifically bind to a ribonucleic acid, e.g., where the capture domain comprises a polyT sequence, a gene-specific sequence or a random sequence. In some instances, the capture domain binds to an exogeneous nucleic acid added to cells of the sample, e.g., where the exogeneous nucleic acid is conjugated to a specific binding member. In some instances, the capture domain binds to a transposon, a bridge oligonucleotide or a cDNA. In embodiments, the barcoded nucleic acids further comprise an adapter domain, e.g., where the adapter domain is a sequencing adapter domain, e.g., where the sequencing adapter domain comprises any one of SEQ ID NOs: 01 -12. In some instances, the barcoded nucleic acids further comprise a second barcode domain, and in some instances the barcoded nucleic acids further comprise a third barcode domain, and in some instances the barcoded nucleic acids further comprise a fourth barcode domain, and in some instances the barcoded Atty. Docket No.: CLON-185WO nucleic acids further comprise a fifth barcode domain, and in some instances the barcoded nucleic acids further comprise a sixth barcode domain. In embodiments, the array of barcoded nucleic acids comprises distinct populations of barcoded nucleic acids, e.g., where the distinct populations of barcoded nucleic acids differ by one or more of: the barcode domain, a cleavage domain, a capture domain and an adapter domain, such as where the distinct populations of barcoded nucleic acids are functionally distinct populations of barcoded nucleic acids that differ by one or more of: a cleavage domain and a capture domain. In some instances, the array of barcoded nucleic acids comprises: a first functionally distinct population of barcoded nucleic acids comprising a first capture domain and a first cleavage domain; and a second functionally distinct population of barcoded nucleic acids comprising a second capture domain and a second cleavage domain, e.g., where the first capture domain is different from the second capture domain, where in some instances the first cleavage domain is different from the second cleavage domain. In some instances, the distinct populations of barcoded nucleic acids stably associated with different locations of a solid support. In some instances, each location of the different locations of the array comprises a population of barcoded nucleic acids with a unique barcode, such as where a location of the array comprises two or more functionally distinct populations of barcoded nucleic acids. In some instances, the solid support comprises a planar solid support. In some instances, the different locations abut each other. In some instances, the different locations are separated from each other by an intervening region, e.g., where the intervening region is free of nucleic acids, such as where the intervening region is configured to impede cell adherence, e.g., where the intervening region comprises an anti-adherence coating. In some instances, the solid support comprises a multi-well array. In some instances, the solid support comprises a bead array. In embodiments, the sample has a cell concentration sufficient to provide upon contact with the array a confluence ranging from 25 to 100%, such as where the sample has a cell concentration sufficient to provide a confluence ranging from 25 to 75%, such as where the sample has a cell concentration sufficient to provide a confluence ranging from 75 to 100%. In embodiments, the barcoded nucleic acids enter the cells by transfection, e.g., where the transfection is mediated by a transfection agent, e.g., where the transfection Atty. Docket No.: CLON-185WO agent is a nanoparticle agent. In some instances, the transfection is mediated by a physical stimulus, e.g., where the physical stimulus comprises electroporation. In some instances, the barcoded nucleic acids are attached to a cell uptake moiety that allows for the barcoded nucleic acids to enter the cells, e.g., where the cell uptake moiety is a cell penetrating peptide (CPP). In some instances, the CPP comprises transportan, TP10, penetratin, Tat or a series of arginine residues, and in some instances, the CPP comprises nine arginine residues. In some instances, the barcoded nucleic acid functions as a primer. In some instances, the barcoded nucleic acid functions as a template switch oligonucleotide. In some instances, the barcoded nucleic acid functions as a template nucleic acid.

[0020] Also provided are compositions that include an array of barcoded nucleic acids comprising distinct populations of barcoded nucleic acids stably associated with different locations of a solid support contacted with a sample of dispersed cells, and wherein each barcoded nucleic acid of the barcoded nucleic acid array comprises a barcode domain. In some instances, the sample of dispersed cells comprises dispersed live cells. In some instances, the sample of dispersed cells comprises fixed cells. In some instances, the fixed cells are permeabilized. In some instances, the barcoded nucleic acids further comprise a cleavage domain, which domain may be cleaved by an enzymatic stimulus, a chemical stimulus or an electromagnetic stimulus, which domain may function as a cleavable linker, wherein the cleavable linker connects the barcoded nucleic acid to a solid support, e.g., where the cleavable linker comprises a photocleavable linker. In some instances, the barcoded nucleic acids further comprise a capture domain, which capture domain may specifically bind to a ribonucleic acid, e.g., where the capture domain comprises a polyT sequence, a gene-specific sequence or a random sequence. In some instances, the capture domain binds to an exogeneous nucleic acid added to cells of the sample, e.g., where the exogeneous nucleic acid is conjugated to a specific binding member. In some instances, the capture domain binds to a transposon, a bridge oligonucleotide or a cDNA. In embodiments, the barcoded nucleic acids further comprise an adapter domain, e.g., where the adapter domain is a sequencing adapter domain, e.g., where the sequencing adapter domain comprises any one of SEQ ID NOs: 01 -12. In some instances, the barcoded nucleic acids further Atty. Docket No.: CLON-185WO comprise a second barcode domain, and in some instances the barcoded nucleic acids further comprise a third barcode domain, and in some instances the barcoded nucleic acids further comprise a fourth barcode domain, and in some instances the barcoded nucleic acids further comprise a fifth barcode domain, and in some instances the barcoded nucleic acids further comprise a sixth barcode domain. In embodiments, the array of barcoded nucleic acids comprises distinct populations of barcoded nucleic acids, e.g., where the distinct populations of barcoded nucleic acids differ by one or more of: the barcode domain, a cleavage domain, a capture domain and an adapter domain, such as where the distinct populations of barcoded nucleic acids are functionally distinct populations of barcoded nucleic acids that differ by one or more of: a cleavage domain and a capture domain. In some instances, the array of barcoded nucleic acids comprises: a first functionally distinct population of barcoded nucleic acids comprising a first capture domain and a first cleavage domain; and a second functionally distinct population of barcoded nucleic acids comprising a second capture domain and a second cleavage domain, e.g., where the first capture domain is different from the second capture domain, where in some instances the first cleavage domain is different from the second cleavage domain. In some instances, the distinct populations of barcoded nucleic acids stably associated with different locations of a solid support. In some instances, each location of the different locations of the array comprises a population of barcoded nucleic acids with a unique barcode, such as where a location of the array comprises two or more functionally distinct populations of barcoded nucleic acids. In some instances, the solid support comprises a planar solid support. In some instances, the different locations abut each other. In some instances, the different locations are separated from each other by an intervening region, e.g., where the intervening region is free of nucleic acids, such as where the intervening region is configured to impede cell adherence, e.g., where the intervening region comprises an anti-adherence coating. In some instances, the solid support comprises a multi-well array. In some instances, the solid support comprises a bead array. In embodiments, the sample has a cell concentration sufficient to provide upon contact with the array a confluence ranging from 25 to 100%, such as where the sample has a cell concentration sufficient to provide a confluence ranging from 25 to 75%, such as where Atty. Docket No.: CLON-185WO the sample has a cell concentration sufficient to provide a confluence ranging from 75 to 100%. In embodiments, the barcoded nucleic acids enter the cells by transfection, e.g., where the transfection is mediated by a transfection agent, e.g., where the transfection agent is a nanoparticle agent. In some instances, the transfection is mediated by a physical stimulus, e.g., where the physical stimulus comprises electroporation. In some instances, the barcoded nucleic acids are attached to a cell uptake moiety that allows for the barcoded nucleic acids to enter the cells, e.g., where the cell uptake moiety is a cell penetrating peptide (CPP). In some instances, the CPP comprises transportan, TP10, penetratin, Tat or a series of arginine residues, and in some instances, the CPP comprises nine arginine residues. In some instances, the sample of dispersed cells is not derived from a tissue or tissue section. In some instances, the sample of dispersed cells comprises one or more of: immune cells, neuronal cells, cardiac cells, endothelial cells, fibroblasts, liver cells and tumor cells.

[0021] Brief Description of the Figures

[0022] FIG. 1A - FIG. 1 D provide schematics of barcoding cells in accordance with certain embodiments. FIG. 1A shows a surface bound barcoded nucleic acid that is part of an array finding use in embodiments disclosed herein. FIGS. 1 B-1 C illustrate a workflow in which dispersed cells are contacted with an array of barcoded nucleic acids present on a surface of a planar support, according to certain embodiments. FIG. 1 D provides a schematic illustrating reverse transcription of barcoded nucleic acids in accordance with an embodiment.

[0023] FIG. 2 provides a schematic illustrating an embodiment for analysis of protein (e.g., intracellular and / or cell surface protein) expression.

[0024] FIG. 3 provides a schematic illustrating an embodiment for performing tagmentation mediated workflows.

[0025] FIG. 4A - FIG. 4B provide schematics illustrating an embodiment where barcoded template switch oligonucleotides are introduced into dispersed cells.

[0026] FIG. 5A - FIG. 5B provide schematics illustrating an embodiment where barcoded template nucleic acids are introduced into dispersed cells. Atty. Docket No.: CLON-185WO

[0027] FIG. 6A - FIG. 6B provide schematics illustrating an embodiment where the array of barcoded nucleic acids includes two functionally distinct populations of barcoded nucleic acids in the same location of the array.

[0028] FIG. 7A - FIG. 7B show the detection of spatial barcodes incorporated into single cells of dispersed cells according to methods of the present technology. FIG. 7A shows spatial barcodes incorporated into representative single cells without the addition of cell penetrating peptides (CPPs). FIG. 7B shows spatial barcodes incorporated into representative single cells with the addition of CPPs.

[0029] Definitions

[0030] As used herein, the term “hybridization conditions” means conditions in which a primer, or other polynucleotide, specifically hybridizes to a region of a target nucleic acid with which the primer or other polynucleotide shares some complementarity. Whether a primer specifically hybridizes to a target nucleic acid is determined by such factors as the degree of complementarity between the polymer and a region or domain of the target nucleic acid and the temperature at which the hybridization occurs, which may be informed by the melting temperature (TM) of the primer. The melting temperature refers to the temperature at which half of the primer-target nucleic acid duplexes remain hybridized and half of the duplexes dissociate into single strands. The Tm of a duplex may be experimentally determined or predicted using the following formula Tm = 81.5 + 16.6(log 10[Na+]) + 0.41 (fraction G+C) - (60 / N), where N is the chain length and [Na+] is less than 1 M. See Sambrook and Russell (2001 ; Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor N.Y., Ch. 10). Other more advanced models that depend on various parameters may also be used to predict Tm of primer / target duplexes depending on various hybridization conditions. Approaches for achieving specific nucleic acid hybridization may be found in, e.g., Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I, chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier (1993).

[0031] The terms “complementary” and “complementarity” as used herein refer to a nucleotide sequence that base-pairs by non-covalent bonds to all or a region of a target Atty. Docket No.: CLON-185WO nucleic acid (e.g., a region of the nucleic acid product). In the canonical Watson-Crick base pairing, adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (II). As such, A is complementary to T and G is complementary to C. In RNA, A is complementary to II and vice versa. Typically, “complementary” refers to a nucleotide sequence that is at least partially complementary. The term “complementary” may also encompass duplexes that are fully complementary such that every nucleotide in one strand is complementary to every nucleotide in the other strand in corresponding positions. In certain cases, a nucleotide sequence may be partially complementary to a target, in which not all nucleotides are complementary to every nucleotide in the target nucleic acid in all the corresponding positions. For example, a primer may be perfectly (i.e., 100%) complementary to a region or domain of the target nucleic acid, or the primer and the target nucleic acid may share some degree of complementarity which is less than perfect (e.g., 70%, 75%, 85%, 90%, 95%, 99%).

[0032] The percent identity of two nucleotide sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment). The nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity= # of identical positions / total # of positionsx O). When a position in one sequence is occupied by the same nucleotide as the corresponding position in the other sequence, then the molecules are identical at that position. A non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873- 5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et al., Nucleic Acids Res. 25:389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. In one aspect, parameters for sequence comparison can be set at score=100, wordlength=12, or can be varied (e.g., wordlength=5 or wordlength=20).

[0033] As used herein, an “oligonucleotide” is a single-stranded multimer of nucleotides from 2 to 500 nucleotides, e.g., 2 to 200 nucleotides. Oligonucleotides may be synthetic Atty. Docket No.: CLON-185WO or may be made enzymatically, and, in some embodiments, are 10 to 50 nucleotides in length. Oligonucleotides may contain ribonucleotide monomers (i.e., may be oligoribonucleotides or “RNA oligonucleotides”) or deoxyribonucleotide monomers (i.e., may be oligodeoxyribonucleotides or “DNA oligonucleotides”). In some cases, oligonucleotides may contain a mixture of ribonucleotides and deoxyribonucleotides. In some cases, Oligonucleotides may contain modified, i.e., non-natural nucleotides or modifications, including for example, LNA, FANA, 2'-O-Me RNA, 2'-fluoro RNA, or the like, linkage modifications (e.g., phosphorothioates, 3’-3’ and 5’-5’ reversed linkages), 5’ and / or 3’ end modifications (e.g., 5’ and / or 3’ amino, biotin, DIG, phosphate, thiol, dyes, quenchers, etc.), one or more fluorescently labeled nucleotides, or any other feature that provides a desired functionality to the oligonucleotides. Oligonucleotides may be 6 to10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200, up to 500 or more nucleotides in length, for example.

[0034] A “domain” when used in reference to nucleic acids refers to a stretch or length of a nucleic acid made up of a plurality of nucleotides, where the stretch or length provides a defined function to the nucleic acid. Examples of domains include capture domains, barcode domains, primer binding domains, hybridization domains, unique molecular identifier (UMI) domains, adapter domains (e.g., Next Generation Sequencing (NGS) adapter domains), NGS indexing domains, etc. In some instances, the terms “domain” and “region” may be used interchangeably. While the length of a given domain may vary, in some instances the length ranges from 2 to 100 nt, such as 5 to 50 nt, e.g., 5 to 30 nt. Amplification primer binding domains (e.g., PGR binding domains) are domains that are configured to bind via hybridization to an amplification primer (e.g., PGR primer). Adapter domains may be used as an amplification primer binding domain.

[0035] As used herein, the expression “derived from” describes a composition that results from a process whereby a first component (e.g., a first nucleic acid molecule), or information from that first component, is used to isolate, derive or construct a different second component (e.g., a second nucleic acid molecule that is different in structure, sequence or character from the first nucleic acid molecule from which it was derived). For example, a cDNA molecule is derived from a corresponding DNA template (e.g., genomic DNA) or RNA template (e.g., mRNA). Similarly, a cDNA library is derived from Atty. Docket No.: CLON-185WO

[0036] RNA or DNA that is collected from a cell or population of cells. Also for example, a cDNA library can be derived from mRNA that is collected from a cell or population of cells.

[0037] As used herein, the expression “barcode” describes most broadly a short nucleotide sequence, for example from 6 to 12 nucleotide sequence, which when appended to a larger polynucleotide, serves to tag that larger polynucleotide, thereby providing a means for counting or distinguishing individual nucleic acids in a larger pool of nucleic acids. As used herein, and as recognized by one of skill in the art, a broad array of barcodes and barcoding strategies are widely utilized and described in the prior art, all of which find use in the presently described technology. As used herein, the terms “barcode” or “index” may be used interchangeably with the terms tags, identifier tags, cell barcode, cell barcode sequences, sample barcodes and sample barcode sequences, well barcodes, source barcode sequences, identifiers, molecular identifiers and other similar and equivalent expressions and technologies. In some aspects of the technology, one or more barcodes may be appended to a larger polynucleotide. In embodiments where two or more barcodes are appended to a larger polynucleotide, said barcodes may be distinct from the other barcodes present on the larger polynucleotide. It is further noted that one or more barcodes may be appended to a larger polynucleotide at the 5’ and / or the 3’ end of the larger polynucleotide. In some embodiments, the one or more barcodes are appended to the 5’ end of the larger polynucleotide. In some embodiments, the one or more barcodes are appended to the 3’ end of the larger polynucleotide. In embodiments wherein two or more barcodes are utilized, the two or more barcodes may be appended to both the 5’ end and the 3’ end of the larger polynucleotide.

[0038] As used herein, the expression “product nucleic acid” or “nucleic acid product” refers to the nucleic acid that is produced as a result of a chemical process (e.g., a polymerization reaction, a hydrolysis reaction, a ligation reaction, a tagmentation reaction, etc.) Polymerization reactions include any chemical processes that employ a polymerase, including reverse transcription reactions, primer extension reactions, and template switching reactions. Nucleic acid products include cDNA, amplified cDNA and amplified nucleic acids produced from polymerase chain reactions (PGR). In some Atty. Docket No.: CLON-185WO embodiments, the product nucleic acid may be generated via a reverse transcription reaction using a template nucleic acid. The template nucleic acid may be RNA, DNA, cDNA, or amplified cDNA. For example, and not by way of limitation, a product nucleic acid may be generated via a reverse transcription reaction using an RNA template nucleic acid. The product nucleic acid may be further amplified using methods known in the art, such as PCR.

[0039] As used herein, the term “specific binding member” refers to one member of a pair of molecules which have binding specificity for one another. One member of the pair of molecules may have a region or domain, or an area on its surface, or a cavity, which specifically binds to a region or domain, or an area on the surface of, or a cavity in, the other member of the pair of molecules. Thus, the members of the pair have the property of binding specifically to each other to produce a binding complex. In some embodiments, the affinity between specific binding members in a binding complex is characterized by a Kd (dissociation constant) of 10-6M or less, such as 10’7M or less, including 10'8M or less, e.g., 10'9M or less, 1 O'10M or less, 10’11M or less, 10'12M or less, 10-13M or less, 10'14M or less, including 10'15M or less. In some embodiments, the specific binding members specifically bind with high avidity. By high avidity is meant that the binding member specifically binds with an apparent affinity characterized by an apparent Kd of 10 x 10'9M or less, such as 1 x 10'9M or less, 3 x 10’10M or less, 1 x 10’10M or less, 3 x 10'11M or less, 1 x 10-11M or less, 3 x 10’12M or less or 1 x 10’12M or less.

[0040] In certain cases, the specific binding member is a biomolecule. The specific binding member can be proteinaceous. As used herein, the term “proteinaceous” refers to a moiety that is composed of amino acid residues. A proteinaceous moiety can be a polypeptide. In certain cases, the proteinaceous specific binding member is an antibody. In certain embodiments, the proteinaceous specific binding member is an antibody fragment. As used herein, the terms “antibody” and “antibody molecule” are used interchangeably and refer to a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include the kappa (k), lambda (I), and heavy chain genetic loci, which together include the myriad variable Atty. Docket No.: CLON-185WO region genes, and the constant region genes mu (u), delta (d), gamma (g), sigma (e), and alpha (a) which encode the IgM, IgD, IgG, IgE, and IgA isotypes respectively. An immunoglobulin light or heavy chain variable region consists of a “framework” region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. The extent of the framework region and CDRs have been precisely defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, (1991 )). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. The term antibody is meant to include full length antibodies and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes as further defined below.

[0041] Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv (single chain Fv), a nanobodies, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments or other antigen-binding subsequences of antibodies, either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. For more details on antibody fragments see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Zapata et al., Protein Eng. 8(10): 1057-1062 (1995); Bates et al., Antibodies. 8(2): 28 (2019); Jin et al., Int J Mol Sci. 24(6): 5994 (2023); Ahmad et al., Clin Dev Immunol. 980250 (2012). Antibodies may be monoclonal or polyclonal and may have other specific activities on cells (e.g., antagonists, agonists, neutralizing, inhibitory, or stimulatory antibodies). The antibodies can be of any isotype, including IgG, IgA, IgM IgD, and IgE, and several of these may be further divided into subclasses, e.g., lgG1 , lgG2, lgG3, lgG4, IgA, and lgA2. It is understood that the antibodies may have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions. Additionally or alternatively, the Atty. Docket No.: CLON-185WO antibodies may have additional amino acid substitutions in the Fc region or regions outside of the ODR sequences responsible for antigen binding.

[0042] In certain embodiments, the specific binding member is a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, a triabody, or a nanobody. In certain embodiments, the specific binding member is an antibody. In some cases, the specific binding member is a murine antibody or binding fragment thereof. In certain instances, the specific binding member is a recombinant antibody or binding fragment thereof.

[0043] In certain embodiments, the specific binding member is composed of nucleic acids (e.g., DNA, RNA or modified and / or synthetic versions thereof). In some embodiments, the specific binding member is an aptamer.

[0044] Detailed Description

[0045] Provided are methods of barcoding cells of a sample of dispersed cells. Aspects of the methods include contacting the sample with an array of barcoded nucleic acids such that barcoded nucleic acids of the array enter cells of the sample to barcode cells of the sample. The barcoded nucleic acids of the array can be used to barcode the cells of the sample by incorporating a barcoded nucleic acid into a larger polynucleotide present in the cell. A barcoded nucleic acid may be incorporated into a larger polynucleotide via a reverse transcription reaction, a tagmentation reaction, a ligation reaction, or a polymerization reaction. The larger polynucleotide may be any polynucleotide present in the cell. In some embodiments, the larger polynucleotide may be RNA, such as an mRNA. In some embodiments, the larger polynucleotide may be DNA, such as genomic DNA. Alternatively, the barcoded nucleic acid may be retained in the cell without being incorporated into a larger polynucleotide. In such cases, the barcoded nucleic acid may serve as a template nucleic acid for subsequent polymerization reactions. Because the barcoded nucleic acids are initially present on an array, said barcoded nucleic acids may be assigned a spatial coordinate based on its position on said array. As such, in generating a barcoded cell by incorporating a barcoded nucleic acid into a cell, the spatial location of the cell can be determined based on the spatial location of the barcoded nucleic acid. Aspects of the methods described herein further include pooling the barcoded cells or the nucleic acids from the Atty. Docket No.: CLON-185WO cells, preparing a sequencing library from the barcoded cells and / or sequencing the sequencing library. Also provided are kits, compositions and devices for use in performing embodiments of the methods as described herein.

[0046] Before the present technology is described in greater detail, it is to be understood that this technology is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present technology will be limited only by the appended claims.

[0047] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the technology. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the technology, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the technology.

[0048] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present technology, representative illustrative methods and materials are now described.

[0050] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to Atty. Docket No.: CLON-185WO disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present technology is not entitled to antedate such publication by virtue of prior technology. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0051] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0052] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present technology. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0053] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §1 12, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §1 12 are to be accorded full statutory equivalents under 35 U.S.C. §1 12.

[0054] Methods of Barcodinq Cells

[0055] The methods described herein include methods of barcoding cells of a sample.

[0056] The methods comprise contacting the sample with an array of barcoded nucleic acids Atty. Docket No.: CLON-185WO such that the barcoded nucleic acids of the array enter the cells of the sample to barcode said cells, thereby generating barcoded cells. The barcode can serve to identify both the location of the array that was contacted by the cell (i.e., the spatial location of the cell) as well as to provide single cell resolution of nucleic acids derived from the sample, which may be achieved through use of the arrayed barcoded nucleic acid alone or in combination with additional barcoded nucleic acids provided at later steps in the workflow use to generate the final sequencing library.

[0057] In one aspect, the sample comprises dispersed cells. As used herein, the expression “dispersed cells” means cells that are dispersed, i.e., not connected together. Dispersed cells are discrete cells that are not connected by an extraceullar matrix (ECM). The dispersed cells may comprise groups of cells, such as clonal populations of cells that may touch other cells of the clone but be disperse - i.e. separated from other cells in the sample, e.g. from other clones or individual cells. In other words, dispersed cells are not present within a tissue or an organoid. As such, samples of dispersed cells are not tissue samples, tissue slices or tissue sections of any kind. Tissue samples, tissue slices and tissue sections include tissue samples, tissue slices and tissue sections that are frozen, fresh, fixed, unfixed, and / or permeabilized. Samples of dispersed cells are likewise not cryo-sectioned tissues or cryopreserved tissues. However, it is noted that dispersed cells may be individual cells that have been previously separated from tissues or cell cultures. In some embodiments, the dispersed cells are present in a cell suspension. In some embodiments, the dispersed cells are not derived from a tissue sample, tissue slice or tissue section.

[0058] The dispersed cells may be dispersed or suspended in a suitable liquid. Suitable liquids include cell culture media, buffers (e.g., phosphate buffered saline (PBS), Hank’s balanced salt solution (HBSS), Dulbecco's phosphate buffered saline (DPBS), etc.), etc. Cell culture media is well known in the art and may be tailored to accommodate specific cell types (see, e.g., Arora, M., “Cell Culture Media: A Review” Mater Methods. 2013, 3:175; ThermoFisher. “Recommended media types for common cells” https: / / www.thermofisher.com / us / en / home / references / gibco-cell-culture-basics / cell- culture-protocols / recommended-media-types-for-common-cells.html). A person of Atty. Docket No.: CLON-185WO ordinary skill in the art would readily be able to determine an appropriate liquid to disperse cells in.

[0059] Samples of dispersed cells may include any convenient concentration of cells. In some embodiments, samples of dispersed cells include a cell concentration ranging from about 1 ,000 cells / mL to about 1 x 109cells / mL including, e.g., 1 x 104cells / mL to 1 x 109cells / mL, 1 x 104cells / mL to 1 x 108cells / mL, 1 x 104cells / mL to 1 x 107cells / mL, 1 x 104cells / mL to 1 x 106cells / mL, 1 x 105cells / mL to 1 x 109cells / mL, 1 x 105cells / mL to 1 x 108cells / mL, 1 x 105cells / mL to 1 x 107cells / mL, 1 x 105cells / mL to 1 x 106cells / mL, 1 x 106cells / mL to 1 x 109cells / mL, 1 x 106cells / mL to 1 x 108cells / mL, 1 x 106cells / mL to 1 x 107cells / mL, 5 x 105cells / mL to 1 x 107cells / mL, 5 x 105cells / mL to 5 x 105cells / mL, 1 x 106cells / mL to 8 x 106cells / mL, and 1 x 106cells / mL to 5 x 106cells / mL. In some embodiments, samples of dispersed cells include a cell concentration of about 1 ,000 cells / mL, 1 x 104cells / mL, 1 x 105cells / mL, 1 x 106cells / mL, 2 x 106cells / mL, 3 x 106cells / mL, 4 x 106cells / mL, 5 x 106cells / mL, 6 x 106cells / mL, 7 x 106cells / mL, 8 x 106cells / mL, 9 x 106cells / mL, 1 x 107cells / mL, 1 x 108cells / mL or 1 x 109cells / mL

[0060] The dispersed cells may include any type of cell including, but not limited to, immune cells, neuronal cells, cardiac cells, endothelial cells, fibroblasts, liver cells, and tumor cells. Immune cells include, but are not limited to, lymphocytes, e.g., a T cell (e.g., a cytotoxic T cell (e.g., a CD8+ T cell), a helper T cell (e.g., a CD4+ T cell), a regulatory T cell (“Treg”), etc.) a natural killer (NK) cell, a B cell, and the like. The dispersed cells may be induced pluripotent stem cells (iPSCs). Subject immune cells may also include peripheral blood mononuclear cells, a macrophage, a dendritic cell, and a monocyte. The dispersed cells may be immortalized cells or primary cells. The dispersed cells may include cells derived from any of the 3 germ layers (e.g., endoderm, mesoderm, ectoderm). The dispersed cells may include any type of embryonic cells including, but not limited to, trophectoderm cells, inner cell mass cells, and cells from a blastocyst. Furthermore, cells from any population can be the source of a dispersed cells used in the subject methods, such as a population of prokaryotic or eukaryotic single celled organisms including bacteria or yeast. In some instances, the cells may be insect or plant cells. In some instances, the sample of dispersed cells utilized in the Atty. Docket No.: CLON-185WO subject methods may be a sample of mammalian dispersed cells, such as a rodent (e.g., mouse or rat) dispersed cells, a non-human primate dispersed cells, human dispersed cells, or the like. In some instances, the sample of mammalian dispersed cells may be mammalian blood cells, including but not limited to e.g., rodent (e.g., mouse or rat) blood cells, non-human primate cells, human blood cells, or the like. Where the dispersed cells originate from a sample where the cells are not initially isolated, such as when the cell is part of a tissue, the dispersed cells may be obtained from an initial cell sample using any convenient cell isolation protocol. For example, in some instances, single cells may be obtained through limiting dilution of cellular sample. In some instances, a single cell suspension can be obtained using standard methods known in the art including, for example, enzymatically using trypsin or papain to digest proteins connecting cells in tissue samples or releasing adherent cells in culture, or mechanically separating cells in a sample. In some instances, single cells may be obtained by sorting a cellular sample using a cell sorter instrument. By “cell sorter” as used herein is meant any instrument that allows for the sorting of individual cells into an appropriate vessel for downstream processes, such as those processes of library preparation as described herein. Useful cell sorters include flow cytometers, such as those instruments utilized in fluorescence activated cell sorting (FACS).

[0061] Barcoded Nucleic Acids

[0062] As summarized above, aspects of the technology include contacting a sample of dispersed cells with an array of barcoded nucleic acids. Barcoded nucleic acids are nucleic acids that include one or more barcode domains. In certain aspects, the barcoded nucleic acids are from 4 to 200 nucleotides in length. For example, the barcoded nucleic acids may be from 4 to 100 nucleotides in length, such as from 6 to 75, from 8 to 50, or from 10 to 40 nucleotides in length. The barcoded nucleic acids may include DNA, RNA or a combination thereof. In some embodiments, the barcoded nucleic acid consists of DNA. The barcoded nucleic acids may include one or more nucleotides (or analogs thereof) that are modified or otherwise non-naturally occurring. For example, the barcoded nucleic acid may include one or more nucleotide analogs (e.g., LNA, FANA, 2’-O-Me RNA, 2’-fluoro RNA, or the like), linkage modifications (e.g., Atty. Docket No.: CLON-185WO phosphorothioates, 3’-3’ and 5’-5’ reversed linkages), 5’ and / or 3’ end modifications (e.g., 5’ and / or 3’ amino, biotin, DIG, phosphate, thiol, dyes, quenchers, etc.), one or more fluorescently labeled nucleotides, or any other feature that provides a desired functionality to the barcoded nucleic acid. For more details regarding nucleotide analogs, see, e.g., Squires, Antiviral Therapy. (Suppl. 3): 1 -14 (2001 ); Hagedorn et al., Drug Discovery Today. 23(1 ): 101 -1 14 (2018); Duffy et al., BMC Biol. 18(1 12) (2020); Ochoa et al., Molecules. 25(20): 4659 (2020); and Kuwahara et al., Molecules. 15(8): 5423-5444 (2010).

[0063] Barcoded nucleic acids may include one or more barcode domains (e.g., one barcode domain, two barcode domains, three barcode domains, four barcode domains or five barcode domains) including, e.g., two or more barcode domains, three or more barcode domains, four or more barcode domains or five or more barcode domains.

[0064] In some embodiments, the barcoded nucleic acids include one barcode domain. In some embodiments, the barcoded nucleic acids include two barcode domains. In some embodiments, the barcoded nucleic acids include three barcode domains. In some embodiments, the barcoded nucleic acids include four barcode domains. In some embodiments, the barcoded nucleic acids include five barcode domains. In some embodiments, the barcoded nucleic acids include six barcode domains. In some embodiments, the barcoded nucleic acids include more than six barcode domains.

[0065] A barcode domain present on a barcoded nucleic acid comprises a short nucleotide sequence of about 4-16 nucleotides in length. In some embodiments, the barcode domain is 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or 16 nucleotides in length. In some embodiments, the barcode domain is 4 nucleotides in length. In some embodiments, the barcode domain is 5 nucleotides in length. In some embodiments, the barcode domain is 6 nucleotides in length. In some embodiments, the barcode domain is 7 nucleotides in length. In some embodiments, the barcode domain is 8 nucleotides in length. In some embodiments, the barcode domain is 9 nucleotides in length. In some embodiments, the barcode domain is 10 nucleotides in length. In some embodiments, the barcode domain is 1 1 nucleotides in length. In some embodiments, the barcode domain is 12 nucleotides in length. In some embodiments, the barcode domain is 13 nucleotides in length. In some embodiments, the barcode domain is 14 nucleotides in Atty. Docket No.: CLON-185WO length. In some embodiments, the barcode domain is 15 nucleotides in length. In some embodiments, the barcode domain is 16 nucleotides in length.

[0066] A barcoded nucleic acid may have two or more barcode domains that differ in nucleotide length. That is, the nucleotide length of the barcode domains present on a barcoded nucleic acid may be determined independently from each other. In some embodiments, a barcoded nucleic acid may comprise a first barcode domain that is between 4-16 nucleotides in length and a second barcode domain that is between 4-16 nucleotides in length. The barcoded nucleic acid may further comprise a third barcode domain that is between 4-16 nucleotides in length. The barcoded nucleic acid may further comprise a fourth barcode domain that is between 4-16 nucleotides in length. The barcoded nucleic acid may further comprise a fifth barcode domain that is between 4-16 nucleotides in length. The barcoded nucleic acid may further comprise a sixth barcode domain that is between 4-16 nucleotides in length.

[0067] The barcode domain may be present in a continuous stretch of nucleotides or it may be split into two or more regions or segments. In embodiments wherein the barcoded nucleic acid comprises two or more barcode domains, the barcode domains may differ by 1 or more nucleotides.

[0068] When appended to a larger polynucleotide, the barcode serves to tag that larger polynucleotide, thereby providing a means for counting or distinguishing individual nucleic acids in a larger pool of nucleic acids. For example, the barcode may be employed to determine the location on an array from which the nucleic acid comprising the barcode originated. Alternatively, the barcode may be employed to determine nucleic acids derived from a single cell present in the sample of dispersed cells. This may be achieved through the addition of a single barcode to the larger polynucleotide or through the addition of a combination of barcodes added at multiple steps in the workflow.

[0069] In some embodiments, the barcoded nucleic acid includes a barcode domain and a capture domain. By capture domain it is meant a domain that specifically binds to a particular sequence of a nucleic acid (e.g., a particular sequence of a template nucleic acid, a particular sequence of an exogenous nucleic acid, a particular sequence of a transposon, a non-template nucleotide sequence of a nucleic acid, etc.). In some Atty. Docket No.: CLON-185WO embodiments, the barcoded nucleic acids include a capture domain that is 3’ of the barcode domain. In other words, the barcode domain is 5’ of the capture domain. The sequence of the capture domain may be independently defined or arbitrary. In certain aspects, the capture domain has a defined sequence, e.g., poly dT or a gene specific sequence. In other aspects, the capture domain has an arbitrary sequence (e.g., a random sequence, such as a random hexamer sequence). In yet other instances, the capture domain may be quasi-random, e.g., as described in U.S. Patent No. 8,206,913, the disclosure of which is herein incorporated by reference. While the length of the capture domain may vary, in some instances the length of this domain ranges from 5 to 50 nucleotides. In some embodiments, the length of the capture domain is between 5- 50 nucleotides in length, 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 10-15, 10-20, 10-25, 10-30, 10-35, 10-40, 10-45, 10-50, 15-20, 15-25, 15-30, 15-35, 15-40, 15-45, 15- 50, 20-25, 20-30, 20-35, 20-40, 20-45, 20-50, 25-30, 25-35, 25-40, 25-45, 25-50, 30-35, 30-40, 30-45, 30-50, 35-40, 35-45, 35-50, 40-45, 40-50, or 45-50 nucleotides in length.

[0070] In some embodiments, the capture domain may include one or more nucleotides (or analogs thereof) that are modified or otherwise non-naturally occurring. For example, the barcoded nucleic acid may include one or more nucleotide analogs (e.g., LNA, FANA, 2’-O-Me RNA, 2’-fluoro RNA, or the like), linkage modifications (e.g., phosphorothioates, 3’-3’ and 5’-5’ reversed linkages), 5’ and / or 3’ end modifications (e.g., 5’ and / or 3’ amino, biotin, DIG, phosphate, thiol, dyes, quenchers, etc.), one or more fluorescently labeled nucleotides, or any other feature that provides a desired functionality to the barcoded nucleic acid. For more details regarding nucleotide analogs, see, e.g., Squires, Antiviral Therapy. (Suppl. 3): 1 -14 (2001 ); Hagedorn et al., Drug Discovery Today. 23(1 ): 101 -1 14 (2018); Duffy et al., BMC Biol. 18(1 12) (2020); Ochoa et al., Molecules. 25(20): 4659 (2020); and Kuwahara et al., Molecules. 15(8): 5423-5444 (2010).

[0071] In some embodiments, the capture domain is a poly-dT sequence. In some embodiments, the poly-dT sequence is 5-50 nucleotides in length. In some embodiments, the poly-dT sequence is 5-10, 10-15, 15-20, 20-25, 20-30, 30-35, 35-40, 40-45, or 45-50 nucleotides in length. In some embodiments, each thymine residue of the poly-dT capture domain is connected via a phosphodiester bond. In some Atty. Docket No.: CLON-185WO embodiments, each thymine residue of the poly-dT capture domain is connected via a phosphorothioate bond. In some embodiments, the poly-dT capture domain comprises a mixture of phosphodiester and phosphorothioate bonds. For example, but not by way of limitation, a phosphorothioate bond may be used to connect every third thymine residue, every fourth thymine residue, every fifth thymine residue, or every sixth thymine residue. Additionally or alternatively, phosphorothioate bonds may be introduced between the last 3-5 thymine residues of the poly-dT capture domain. Additionally or alternatively, phosphorothioate bonds may be used at the 5’ end of the poly-dT capture domain. Additionally or alternatively, phosphorothioate bonds may be used at the 3’ end of the poly-dT capture domain. In some embodiments, phosphorothioate bonds may be used at both the 5’ and the 3’ end of the poly-dT capture domain.

[0072] In some embodiments, the capture domain may comprise a mixture of thymine and uracil residues. The thymine and uracil residues may be present in any ratio or order across the length of the capture domain. For example, and not by way of limitation, a capture domain may comprise 10% thymine and 90% uracil residues, 20% thymine and 80% uracil residues, 30% thymine and 70% uracil residues, 40% thymine and 60% uracil residues, 50% thymine and 50% uracil residues, 60% thymine and 40% uracil residues, 70% thymine and 30% uracil residues, 80% thymine and 20% uracil residues, 90% thymine and 10% uracil residues, or any range in between.

[0073] In some embodiments, the capture domain comprises only uracil residues and no thymine residues (i.e., 100% uracil residues and 0% thymine residues).

[0074] In some embodiments, the capture domain comprises one or more locked nucleic acids of thymine. In some embodiments, the barcoded nucleic acid may function as a primer for a subsequent polymerization reaction. In such embodiments wherein the barcoded nucleic acid is a primer, the capture domain may bind to a template nucleic acid (e.g., ribonucleic acid, deoxyribonucleic acid) present in a cell of the sample and serve as a primer for reverse transcription by a reverse transcriptase. Template nucleic acids include any nucleic acids that serve as a template for cDNA synthesis, including both RNA and DNA templates. In some embodiments, the capture domain specifically binds to a ribonucleic acid. In some embodiments, the capture domain specifically binds to a deoxyribonucleic acid. In some embodiments, the capture domain includes a polyT Atty. Docket No.: CLON-185WO sequence, a polyU sequence, a polyT / U sequence, a gene-specific sequence or a random sequence. Template nucleic acids in the dispersed cells may vary. According to certain embodiments, the template nucleic acids are template ribonucleic acids (template RNA). Template RNAs may be any type of RNA (or sub-type thereof) including, but not limited to, a messenger RNA (mRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a transacting small interfering RNA (ta-siRNA), a natural small interfering RNA (nat-siRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), a long non-coding RNA (IncRNA), a non-coding RNA (ncRNA), a transfer-messenger RNA (tmRNA), a precursor messenger RNA (pre-mRNA), a small Cajal body-specific RNA (scaRNA), a piwi-interacting RNA (piRNA), an endoribonuclease-prepared siRNA (esiRNA), a small temporal RNA (stRNA), a signal recognition RNA, a telomere RNA, a ribozyme, or any combination of RNA types thereof or subtypes thereof. According to certain embodiments, the template nucleic acids are template deoxyribonucleic acids (template DNA). Template DNAs may be any type of DNA (or sub-type thereof) including, but not limited to, genomic DNA (e.g., prokaryotic genomic DNA (e.g., bacterial genomic DNA, archaea genomic DNA, etc.), eukaryotic genomic DNA (e.g., plant genomic DNA, fungi genomic DNA, animal genomic DNA (e.g., mammalian genomic DNA (e.g., human genomic DNA, rodent genomic DNA (e.g., mouse, rat, etc.), etc.), insect genomic DNA (e.g., drosophila), amphibian genomic DNA (e.g., Xenopus), etc.)), viral genomic DNA, mitochondrial DNA, or any combination of DNA types thereof or subtypes thereof.

[0075] In embodiments where the barcoded nucleic acid functions as a primer, the capture domain may bind to an exogenous nucleic acid. By “exogenous nucleic acid” it is meant a nucleic acid that originates from outside of the cell. In some embodiments, the exogenous nucleic acid is added to cells of the sample. In some embodiments, the exogenous nucleic acid enters cells of the sample. Exogenous nucleic acids include DNA, RNA, and modified versions or combinations thereof. In some embodiments, the exogenous nucleic acid is conjugated to a specific binding member.

[0076] In embodiments where the barcoded nucleic acid functions as a primer, the capture domain may bind to a transposon. The transposon may be generated from a tagmentation reaction. Transposome complexes, employed in tagmentation reaction, Atty. Docket No.: CLON-185WO may include a transposase enzyme and a transposon nucleic acid (i.e., a transposon). In some embodiments, the transposase enzyme is Tn5 transposase. In a tagmentation reaction, DNA is simultaneously fragmented and modified by the transposome complex to generate fragmented DNA comprising transposons. DNA may include any of the types of template DNA discussed above. Tagmentation processes, transposition-based sequence manipulation, and components that may be employed in a tagmentation or transposition-based reactions include, but are not limited to, those described in, e.g., U.S. Pat. Nos. 10, 017, 759; 9, 790, 476; 9, 683, 230; 9, 388, 465; 9, 238, 671 ; 9, 193, 999; 8,383,345; 6,294,385; 6,159,736; 5,869,296 and 5,677,170; the disclosures of which are incorporated herein by reference in their entirety.

[0077] In some embodiments, the barcoded nucleic acid functions as a template switch oligonucleotide (TSO). In such embodiments where the barcoded nucleic acid functions as a template switch oligonucleotide (TSO), the capture domain binds to a non- templated stretch of nucleotides added by a polymerase (e.g., a reverse transcriptase) at the 3’ end of the cDNA strand. The template switch oligonucleotide may include one or more nucleotides (or analogs thereof) that are modified or otherwise non-naturally occurring. For example, the template switch oligonucleotide may include one or more nucleotide analogs (e.g., LNA, FANA, 2’-O-Me RNA, 2’-fluoro RNA, or the like), linkage modifications (e.g., phosphorothioates, 3’-3’ and 5’-5’ reversed linkages), 5’ and / or 3’ end modifications (e.g., 5’ and / or 3’ amino, biotin, DIG, phosphate, thiol, dyes, quenchers, etc.), one or more fluorescently labeled nucleotides, or any other feature that provides a desired functionality to the template switch oligonucleotide. The capture domain of the TSO may be referred to as a 3’ capture domain, where the 3' capture domain may vary in length, and in some instances ranges from 2 to 10 nucleotides in length, such as 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-

[0078] 6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8- 10, or 9-10 nucleotides in length. The capture domain of the TSO may be 2, 3, 4, 5, 6,

[0079] 7, 8, 9, or 10 nucleotides in length. The sequence of the 3' capture domain may be any convenient sequence, e.g., an arbitrary sequence, a heterpolymeric sequence (e.g., a hetero-trinucleotide) or homopolymeric sequence (e.g., a homo-trinucleotide, such as G-G-G), or the like. Examples of 3' capture domains and template switch Atty. Docket No.: CLON-185WO oligonucleotides are further described in U.S. Patent No. 5,962,272, the disclosure of which is herein incorporated by reference.

[0080] In some embodiments, the barcoded nucleic acid functions as a template nucleic acid. In other words, the barcoded nucleic acid serves as a template for cDNA synthesis. In such embodiments, the capture domain of the barcoded nucleic acid binds to a primer and the primer is extended to generate a cDNA strand that is complementary to the barcoded nucleic acid. In embodiments where the barcoded nucleic acid functions a template nucleic acid, the capture sequence may include any sequence that can bind to a primer including, e.g., a polyA sequence or a commercial capture sequence. By commercial capture sequence, it is meant a capture sequence that binds to a barcoded oligonucleotide used by a commercial single cell sequencing kit (e.g., a 10X Genomics® single cell sequencing kit). As would be understood by one of skill in the art, the capture sequence (which functions as a primer binding site) can be any sequence to which a primer can be designed to bind.

[0081] In some embodiments, the barcoded nucleic acids may comprise a capture domain that binds to cDNA synthesized in situ in the cells, such as a polyA capture domain. For example, cDNA may be synthesized from RNA present in the cells using in situ reverse transcription. The reverse transcription primer may be a polyT primer or a randomer primer. In some embodiments, the primer for reverse transcription further comprises a second primer domain which is subsequently incorporated into the cDNA. In said embodiments, the capture domain of the barcoded nucleic acids may specifically bind to the second primer domain.

[0082] In some embodiments, the barcoded nucleic acids may comprise a capture domain that has complementarity to a bridge oligonucleotide. The bridge oligonucleotide may be delivered to the cells as a reagent or attached to the array of barcoded nucleic acids. The bridge oligonucleotide may then be used to ligate the barcoded nucleic acid to RNA or cDNA present in the cells. Barcoded nucleic acids may include a capture domain having an arbitrary sequence that is suitable for and / or compatible with any desired downstream methods.

[0083] In some embodiments, the barcoded nucleic acids may comprise a cleavage domain. As used herein, the term “cleavage domain” refers to a domain that can be Atty. Docket No.: CLON-185WO cleaved, resulting in release of the barcoded nucleic acid from the substrate of the array. The cleavage domain may be designed to be cleaved using a variety of stimuli, including enzymatic stimuli, chemical stimuli, and / or electromagnetic stimuli. Cleavable linkages may be natural backbone linkages (e.g., phosphate backbone linkages of the DNA and / or RNA present in the barcoded nucleic acid), non-natural backbone linkages or linkages formed by the incorporation of a non-natural nucleotide (e.g., uracil in a DNA strand or nucleotide analogs). Examples of non-natural backbone linkages include, but are not limited to, backbone linkages between DNA and RNA nucleotides, phosphorothioate linkages, boranophosphate linkages, and phosphonate linkages.

[0084] When a cleavage domain is included in the barcoded nucleic acid, it is preferred that the cleavage domain is located 5’ of the barcode domain. In embodiments wherein the barcoded nucleic acid comprises a capture domain, it is preferred that the cleavage domain is located 5’ of the capture domain (and the barcode domain). In one aspect, described herein is a barcoded nucleic acid that comprises, from 5’ to 3’, a cleavage domain and one or more barcode domains. In one aspect, described herein is a barcoded nucleic acid that comprises, from 5’ to 3’, a cleavage domain, one or more barcode domains, and a capture domain as described herein. In some embodiments, the barcoded nucleic acid comprises, from 5’ to 3’, a cleavage domain and one or more barcode domains, wherein the cleavage domain may be cleaved by an enzymatic stimulus, an electromagnetic stimulus, or a chemical stimulus. In some embodiments, the cleavage domain may be an enzymatic stimulus. In some embodiments, the cleavage domain may be a chemical stimulus. In some embodiments, the cleavage domain may be an electromagnetic stimulus. In some embodiments, the barcoded nucleic acid comprising a cleavage domain may comprise 2, 3, 4, 5, 6, or more barcode domains.

[0085] In some embodiments, the cleavage domain may be cleaved by an enzymatic stimulus. In some embodiments, the enzymatic stimulus may be a nuclease. In some embodiments, the nuclease may be a ribonuclease, which cleaves ribonucleotide linkages present in the barcoded nucleic acid. In some embodiments, the nuclease may be an endonuclease. In such embodiments, the barcoded nucleic acid may be designed to have a restriction site that is recognized by the nuclease, such as the endonuclease. Atty. Docket No.: CLON-185WO

[0086] In some embodiments, the nuclease may be thermostable - that is the activity of the nuclease is stable across a range of temperatures. In some embodiments, the nuclease may be thermolabile - that is the nuclease only exhibits activity within a certain temperature range as would be readily determined by one of ordinary skill in the art, but can be inactivated above a certain temperature. Examples of enzymatic stimuli are described in the art, including PCT Publication No. WO 2019 / 040788, incorporated herein by reference.

[0087] In some embodiments, the cleavage domain may be cleaved by a chemical stimulus. In some embodiments, the chemical stimulus may specifically cleave a modified nucleotide present in the barcoded nucleic acid. For example, but not by way of limitation, nucleotide analogs 5-hydroxy-dCTP, 5-hydroxy-dUTP, 7-Deaza-7-nitro- dATP, 7-deaza-7-nitro-dGTP may be specifically cleaved by pyrrolidine. In some embodiments, the chemical stimulus is pyrrolidine. Examples of chemical stimuli are described in the art, including PCT Publication No. WO 2019 / 040788, incorporated herein by reference.

[0088] In some embodiments, the cleavage domain may be cleaved by an electromagnetic stimulus. Examples of electromagnetic stimuli include exposure to visible light, ultraviolet light, and infrared. For example, the cleavage domain may be a photocleavable linker. In some embodiments, the photocleavable linker may be cleaved when exposed to ultraviolet light, i.e., at wavelengths between 100-380nm. In some embodiments, the photocleavable linker may be cleaved when exposed to ultraviolet light at 100-380nm. In some embodiments, the photocleavable linker may be cleaved when exposed to ultraviolet light at about 280 nm. Examples of electromagnetic stimuli are described in the art, including PCT Publication No. WO 2019 / 040788, incorporated herein by reference.

[0089] In one aspect, the cleavage domain may function as a cleavable linker, connecting the barcoded nucleic acid to a solid support. In some cases, the cleavable linker reversibly connects the barcoded nucleic acid to the solid support. In some cases, the cleavable linker non-reversibly connects the barcoded nucleic acid to the solid support. Embodiments using cleavable linkers are described in more detail below. Atty. Docket No.: CLON-185WO

[0090] In some embodiments, the barcoded nucleic acids further include a unique molecular identifier. The terms “unique molecular identifiers” and “UMIs” refer to randomers of varying length, e.g., ranging in length in some instances from 6 to 12 nucleotides, that can be used for counting of individual molecules of a given molecular species. In some instances, counting is facilitated by attaching UMIs from a diverse pool of UMIs to individual molecules of a target of interest such that each individual molecule receives a unique UMI. In such instances, by counting individual transcript molecules, PCR bias generated during NGS library prep can be corrected for, and a more quantitative understanding of the sample population can be achieved.

[0091] In some embodiments, the barcoded nucleic acid includes an adapter domain. The adapter domain may serve various purposes in downstream applications. In some instances, the adapter domain may serve as a primer binding site for further amplification or, e.g., PCR, nested amplification or suppression amplification. In some instances, the adapter domain may be used for introducing additional domains, such as may be employed in NGS applications (such as described below in greater detail).

[0092] In some embodiments, the adapter domain includes a sequencing platform adapter domain. By “sequencing platform adapter domain” is meant a domain that includes at least a portion of a nucleic acid domain (e.g., a sequencing platform adapter nucleic acid sequence) utilized by a sequencing platform of interest, such as a sequencing platform provided by Illumina® (e.g., the iSeq™, MiniSeq™, HiSeq™, MiSeq™, NextSeq™, NovaSeqTMand / or Genome Analyzer™ sequencing systems); Ion Torrent™ (e.g., the Ion PGM™ and / or Ion Proton™ sequencing systems); Pacific Biosciences (e.g., the PACBIO RS II sequencing system); Thermo Fisher Scientific (e.g., a SOLiD sequencing system); Element AVITI systems; Complete Genomics DNBSEQ Platforms (e.g., E25, G99, G400 and T7); or any other sequencing platform of interest. In certain aspects, the sequencing platform adapter domain includes a nucleic acid domain selected from: a domain (e.g., a “capture site” or “capture sequence”) that specifically binds to a surface-attached sequencing platform oligonucleotide (e.g., the P5 or P7 oligonucleotides attached to the surface of a flow cell in an Illumina® sequencing system); a sequencing primer binding domain (e.g., a domain to which the Read 1 or Read 2 primers of the Illumina® platform may bind); or a combination of such Atty. Docket No.: CLON-185WO domains. The sequencing platform adapter domains may include nucleic acids of any length and sequence suitable for the sequencing platform of interest. According to certain embodiments, the sequencing platform adapter domain includes a nucleic acid domain that is from 2 to 8 nucleotides in length, such as from 9 to 15, from 16-22, from 23-29, or from 30-36 nucleotides in length. Examples of such sequencing platform adapter domains, that may be present include, but are not limited to, those described in U.S. Patent Nos. 9,719,136; 10,415,087; 10,781 ,443; 10,941 ,397; 10,954,510; and 1 1 ,124,828; the disclosures of which are herein incorporated by reference.

[0093] The sequencing adapter domain may have a length and sequence that enables a polynucleotide (e.g., an oligonucleotide) employed by the sequencing platform of interest to specifically bind to the adapter domain, e.g., for solid phase amplification and / or sequencing by synthesis of the cDNA insert flanked by the nucleic acid domains. Example adapter domains include the P5 (5’-AATGATACGGCGACCACOGA-3’)(SEQ ID NO:01 ), P7 (5’-CAAGCAGAAGACGGCATACGAGAT-3’)(SEQ ID NO:02), Read 1 primer (5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’)(SEQ ID NO:03) and Read 2 primer (5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATOT-3’)(SEQ ID NO:04) domains employed on the IlluminaO-based sequencing platforms. Other example nucleic acid domains include the A adapter (5’- CCATCTCATCCCTGCGTGTCTCCGACTCAG-3’)(SEQ ID NO:05) and P1 adapter (5’- CCTCTCTATGGGCAGTCGGTGAT-3’)(SEQ ID NO:06) domains employed on the Ion TorrentTM-based sequencing platforms. Other example nucleic acid domains include the SMRTBell adapter 1 (5’- ATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3’) (SEQ ID NO: 07) used in PacBio® sequencing systems. Other example nucleic acid domains include the sequencing adapters used in sequencing systems provided by Oxford Nanopore Technologies. These include the ligation adapter having a top strand sequence (5’- TTTTTTTTCCTGTACTTCGTTCAGTTACGTATTGCT-3’) (SEQ ID NO: 08) and a bottom strand sequence (5’-GCAATACGTAACTGAACGAAGTACAGG-3’) (SEQ ID NO: 09); the native adapter having a top strand sequence (5’- TTTTTTTTCCTGTACTTCGTTCAGTTACGTATTGCT-3’) (SEQ ID NO: 10) and a Atty. Docket No.: CLON-185WO bottom strand sequence (5’-AOGTAAOTGAACGAAGTACAGG-3’) (SEQ ID NO: 1 1 ); and the Rapid Adapter T sequence (5’- TTTTTTTTCCTGTACTTCGTTCAGTTACGTATTGCT-3’) (SEQ ID NO: 12).

[0094] The nucleotide sequences of adapter domains useful for sequencing on a sequencing platform of interest may vary and / or change over time. Adapter sequences are typically provided by the manufacturer of the sequencing platform (e.g., in technical documents provided with the sequencing system and / or available on the manufacturer’s website). Based on such information, the sequence of the sequencing platform adapter domain may be designed to include all or a portion of one or more nucleic acid domains in a configuration that enables sequencing the nucleic acid insert (corresponding to the template nucleic acid) on the platform of interest. Sequencing platform adapter constructs that may be included in a non-templated sequence as well as other nucleic acid reagents described herein, are further described in U.S. Patent Application Serial No. 14 / 478,978 published as US 2015-011 1789 A1 and issued as U.S. Patent No. 10,941 ,397, the disclosure of which is herein incorporated by reference.

[0095] Arrays of Barcoded Nucleic Acids

[0096] Aspects of embodiments of the technology include arrays of barcoded nucleic acids. In some embodiments, the array of barcoded nucleic acids includes distinct barcoded nucleic acids stably associated with different locations of a solid support. The barcoded nucleic acids may be directly or indirectly attached to the solid support. For example, the solid support may be functionalized such that the barcoded nucleic acids are directly conjugated to the surface of the solid support. Alternatively, the barcoded nucleic acids may be conjugated to a particle, such as a bead, which is adhered to the surface of the solid support.

[0097] In some embodiments, the array of barcoded nucleic acids may further include other nucleic acids (e.g., primer oligonucleotides, template switch oligonucleotides). In some embodiments, the array of barcoded nucleic acids may further include peptides or proteins stably associated with the solid support. In some embodiments, the peptides are cell-penetrating peptides. In some embodiments, the proteins are antibodies or antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof Atty. Docket No.: CLON-185WO may specifically bind to a target expressed on the surface of a cell in the dispersed cells. Alternatively, the antibodies or antigen-binding fragments thereof may specifically bind to an intracellular target. Antibody or antigen-binding fragments may access intracellular targets via permeabilization of the cells and / or due to the size of the antibody or antigen-binding fragment (e.g., a nanobody). In some embodiments, the antibodies or antigen-binding fragments thereof are conjugated to a cell-penetrating peptide.

[0098] In some embodiments, each location of the different locations of the array includes barcoded nucleic acids with a unique barcode. In other words, each location comprises barcoded nucleic acids, wherein at least one of the barcode domains is the same for all barcoded nucleic acids within said location, but distinct from at least one of the barcode domains for barcoded nucleic acids outside of said location. Because the specific barcode domain is associated with the location of the array, the barcode can be used to assign a spatial location to a cell (or nucleic acids derived therefrom) or group of cells at that location that has been tagged with said barcode. In some embodiments, two or more different locations, up to and including all of the locations, of the array may abut each other (e.g., the different locations may share a boundary with each other). In embodiments wherein the different locations abut each other, the locations may be separated by a distance of 50 pm or less. In some embodiments, the different locations are separated from each other by an intervening region. In some embodiments wherein the locations are separated from each other by an intervening region, the intervening regions are free of nucleic acids (e.g., the intervening regions are not stably associated with barcoded nucleic acids). In some embodiments, the intervening regions are configured to impede cell adherence, such as with an anti-adherence coating. Examples of anti-adherence coatings are known in the art, and include, but are not limited to, polyethylene glycol (PEG) coatings, silane coatings, and wax coatings. Additional antiadherence coatings are described in Chen, L, et al. “Functional polymer surfaces for controlling cell behaviors” Materials Today 2018 21 :38-59. In some embodiments, the different locations are configured to promote cell adherence, such as with an adherence-promoting coating (see, e.g., adherence-promoting coatings described below). In some embodiments, the intervening regions comprise a compound(s) that Atty. Docket No.: CLON-185WO results in cell lysis and / or cell death, such as a toxin. Examples of compounds that result in cell lysis and / or cell death include, but are not limited to, vanadium dioxide (VO2, silver nanoparticles, graphitic carbon-coated nanoparticles, hydrophobic cationic coatings and organic surface agents (see, e.g., Haidar et al., Appl Bio Sci. 103(47): 17667-17671 (2006); Li et al., Global Challenges. 3(1800058): 1 -14 (2019);

[0099] Mohammadinejad et al., Autophagy. 15(1 ): 4-33 (2018); Zhang et al., Toxicol In Vitro. 29(4): 762-768 (2015); and Kim et al., J Biomed Mater Res A. 103(9): 2875-2887 (2015).

[0100] In some embodiments, the array of barcoded nucleic acids includes distinct populations of barcoded nucleic acids. As used herein, “distinct populations of barcoded nucleic acids” refers to two or more populations of barcoded nucleic acids that differ by any one of the domains present (e.g., barcode domain, capture domain, adapter domain, cleavage domain, etc.).

[0101] In some embodiments, the array of barcoded nucleic acids includes functionally distinct populations of barcoded nucleic acids. As used herein, “functionally distinct populations of barcoded nucleic acids” refers to two or more populations of barcoded nucleic acids that differ by a domain other than the barcode domain (and, if present, a UMI). That is, the barcoded nucleic acids may differ by either a cleavage domain, a capture domain, or both. It may be advantageous to have functionally distinct populations of barcoded nucleic acids for several reasons. If the functionally distinct populations of barcoded nucleic acids differ by capture domain, the functionally distinct populations allow for the detection of different targets. If the functionally distinct populations of barcoded nucleic acids differ by cleavage domain, the functionally distinct populations may allow for temporal detection of targets. For example, a first functionally distinct population of barcoded nucleic acids may have a cleavage domain that is cleaved by a first stimulus (e.g., an enzymatic stimulus) and a second functionally distinct population of barcoded nucleic acids may have a cleavage domain that is cleaved by a second stimulus (e.g., an electromagnetic stimulus). In such an example, the barcoded nucleic acids may be released from the array and enter into the dispersed cells in a temporally regulated manner by first using the first stimulus (e.g., the enzymatic stimulus) and later using the second stimulus (e.g., the electromagnetic Atty. Docket No.: CLON-185WO stimulus). In some embodiments, an array may comprise two, three, four, five, six, or more functionally distinct populations of barcoded nucleic acids. The functionally distinct populations of barcoded nucleic acids may have different cleavage domains, different capture domains, or both. Functionally distinct populations of barcoded nucleic acids may have a barcode domain that is the same or different. For example, in cases where two functionally distinct populations of barcoded nucleic acids are present in the same location on an array, the two functionally distinct populations of barcoded nucleic acids may have the same barcode domain (e.g., a barcode domain correlated to a location on an array), but different cleavage domains. In another example, in cases where two functionally distinct populations of barcoded nucleic acids are present in two different locations on an array, the two functionally distinct populations of barcoded nucleic acids may have the different barcode domains and different cleavage domains. In some embodiments, functionally distinct populations of barcoded nucleic acids may have two or more barcode domains, wherein the two or more barcode domains are the same or different. For example, in some cases, two functionally distinct populations of barcoded nucleic acids may have the same first barcode domain (e.g., a first barcode domain correlated to location of the array), different second barcode domains (e.g., a second barcode domain correlated with the cleavage domain present in the barcoded nucleic acid) and different cleavage domains. Functionally distinct populations of barcoded nucleic acids may be interspersed across the array or in defined locations. In some embodiments, the functionally distinct populations of barcoded nucleic acids are present in the same location of the array.

[0102] Locations that include barcoded nucleic acids (which may be referred to as features) may be of any convenient size or area. In some embodiments, each location is approximately the same size as a single cell (e.g., a mammalian cell). In some embodiments, each location is smaller than the size of a cell. In some embodiments, each location is about the same area as the 5%-90% the area of the cell (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% the area of the cell) including, e.g., 10%-50% the area of the cell, 15%-40% the area of the cell 20%-40% the area of the cell and 25%-50% the area of a cell. In some embodiments, each location is significantly larger than the size of a single cell. In some embodiments, each Atty. Docket No.: CLON-185WO location is about the same area as the size of 10 or more cells, 50 or more cells, 100 or more cells, 500 or more cells, 1000 or more cells, 5000 or more cells, 10000 or more cells, 50000 or more cells or 100,000 or more cells. When the number of cells at any barcode location is greater than 1 , then the barcode provided by the array can be used to identify individual cells at the location in combination with the additional barcodes added at later steps. In some embodiments, the shape of each location is approximately circular. In some embodiments, the shape of each location is rectangular. In some embodiments, each location includes an area with a diameter of about 1 pm to about 10 mm (e.g., a diameter of 1 to 10pm, 10 pm to 50 pm, 10 pm to 40 pm, 10 pm to 30 pm, 10 pm to 1000 pm, 10 pm to 750 pm, 10 pm to 500 pm, 30 pm to 500 pm, 50 pm to 500 pm, 50 pm to 250 pm, 50 pm to 200 pm, 75 pm to 200 pm, 100 pm to 200 pm, 50 pm to 150 pm, 75 pm to 225 pm, 1 pm to 1000 pm, 500 pm to 10 mm, 500 pm to 5 mm, 500 pm to 2 mm, 500 pm to 1000 pm or 1 mm to 10 mm). In some embodiments, each location includes an area with a diameter of 2 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 60 pm, 70 pm, 75 pm, 80 pm, 90 pm, 100 pm, 1 10 pm, 120 pm, 125 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 175 pm, 180 pm, 190 pm, 200 pm, 210 pm, 220 pm, 225 pm, 230 pm, 240 pm or 250 pm. In some embodiments, each location has an area of about 500 pm2to 25 mm2(e.g., an area of 500 pm2to 10,000 pm2, 500 pm2to 5,000 pm2, 500 pm2to 3,000 pm2, or 1 ,000 pm2to 2,000 pm2). In some embodiments, each location has an area of about 500 pm2, 1 ,000 pm2, 1 ,500 pm2, 2,000 pm2, 2,500 pm2, 3,000 pm2, 3,500 pm2, 4,000 pm2, 4,500 pm2, 5,000 pm2, 10,000 pm2, 20,000 pm2, 25,000 pm2or 50,000 pm2.

[0103] In embodiments where different locations are separated from each other by an intervening region, locations may be from about 50 pm to 1000 pm apart from each other (e.g., 100 pm to 1000 pm, 100 to 700 pm, 200 pm to 1000 pm, 200 pm to 700 pm, 100 pm to 500 pm, or 200 pm to 500 pm apart from each other). In some embodiments, locations are 50 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 850 pm, 900 pm, 950 pm or 1000 pm apart from each other.

[0104] In some cases, the solid support includes a planar solid support. The solid support may be made of any suitable material including, but not limited to, glass, silicon, Atty. Docket No.: CLON-185WO metal, membranes (e.g., nitrocellulose, methylcellulose, cellulose, polyvinylidene fluoride) and plastic (e.g., polytetrafluoroethylene, polyvinylidene difluoride, polystyrene, polycarbonate, polypropylene). Solid supports include, but are not limited to, a multi-well array, a bead array, a coverslip, a cell culture chamber or a petri dish. In some embodiments, the solid support includes a multi-well array (e.g., a 6 well array, a 12 well array, a 24 well array, a 48 well array, a 96 well array, a 384 well array or a 1536 well array).

[0105] In some embodiments, the solid support includes a bead array. The solid support may be associated with a population of beads to form a bead array. In some embodiments, each bead of the population of beads has a diameter of about 1 pm to about 100 pm (including, e.g., 1 pm to 50 pm, 5 pm to 50 pm, 10 pm to 40 pm and 10 pm to 30 pm). In some embodiments, each bead of the population of beads has a diameter of about 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 1 1 pm, 12 pm, 13 pm, 14 pm, 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, or 100 pm. Beads may be porous or non-porous. Beads may be made of any suitable material including, but not limited to, polystyrene, polymethacrylate, silica, and polyacrylamide. In some embodiments, the population of beads forms a layer of beads on top of the solid support. In some embodiments, the beads are packed at an inter-bead spacing of 20 pm or less, including, e.g., 100 pm or less. Bead arrays suitable to be used in the methods of the present technology include those described in U.S. Patent Application No. 17 / 051 ,793 published as United States Published Application Publication No. US20210123040A1 , the disclosure of which is incorporated by reference. In embodiments where the solid support includes a bead array, each bead may include a different location (i.e., each bead includes a unique barcoded nucleic acid).

[0106] Solid supports may be treated and / or coated to prepare the substrates for attachment to other components using any variety of methods. Substrate treatments include, but are not limited to, acid treatment, base treatment, oxidizing treatment, plasma treatment, detergent treatment, heat treatment or combinations thereof. In some embodiments, the solid supports are configured to promote cell adherence. In some embodiments, the solid supports are treated with a coating that promotes cell Atty. Docket No.: CLON-185WO adherence (i.e., an adherence-promoting coating). Adherence-promoting coatings include, but are not limited to, polylysine coating, glutaraldehyde coating, extracellular matrix coating (e.g., collagen coating, gelatin coating, fibronectin coating, Matrigel® coating, laminin coating, vitronectin coating), and combinations thereof. In some cases, the solid support may be coated with an adherence-promoting coating in a pattern such that the different locations are configured to promote cell adherence, while intervening regions are not coated with an adherence-promoting coating. In some embodiments, the solid supports are configured to impede cell adherence. In some embodiments, the solid supports are treated with an anti-adherence coating. For example, intervening regions of the solid support are coated with an anti-adherence coating. In some embodiments, the intervening regions described above (e.g., the intervening regions positioned between the different locations on the solid support) are configured to impede cell adherence. In some embodiments, the intervening region includes an antiadherence coating. Anti-adherence coatings include, but are not limited to, polyethylene glycol (PEG) coatings, silane coatings, wax coatings, etc. Anti-adherence coatings are well-known in the art and can be found in, e.g., Chen, L, et al. “Functional polymer surfaces for controlling cell behaviors” Materials Today 2018 21 :38-59. In some embodiments, the intervening regions are configured to impede cell adherence by not coating the areas with a coating that promotes cell adherence.

[0107] As described above, in some embodiments, the barcoded nucleic acids are stably associated with different locations of a solid support. In some embodiments, a particular end (e.g., the 5’ end or the 3’ end) of the barcoded nucleic acid may be stably associated with the solid support. Barcoded nucleic acids may be stably associated with the different locations of a solid support due to physical adsorption, covalent bonding and non-covalent bonding. Physical adsorption attachment methods include, but are not limited to, hydrogen bonding, van der Waals forces, electrostatic forces and hydrophobic interactions. Covalent bonding attachment methods include, but are not limited to, click chemistry, phosphate chemistry, NHS-ester chemistry, thiol-chemistry and silane-chemistry. Non-covalent bonding includes, but is not limited to, streptavidinbiotin interactions, antibody-antigen interactions and the like. In some embodiments, the barcoded nucleic acids are mixed with a carrier material (e.g., a carrier protein or a Atty. Docket No.: CLON-185WO carrier lipid) and subsequently dried in particular locations on the solid support, such that the barcoded nucleic acids are stably associated with the solid support (see, e.g., U.S. Patent No. 6,951 ,757, the disclosure of which is incorporated by reference).

[0108] As discussed above, in some embodiments, the barcoded nucleic acid includes a cleavage domain. In some embodiments, the cleavage domain functions as a cleavable linker, wherein the barcoded nucleic acids are bound to the solid support by the cleavable linker. In other words, the cleavable linker connects (e.g., reversibly or non- reversibly connects) the barcoded nucleic acid to the solid support. In some embodiments, a particular end of the barcoded nucleic acid (e.g., the 5’ end or the 3’ end) is bound to the solid support by the cleavable linker. The term “cleavable linker” refers to a linker containing a moiety (e.g., bond) that can be selectively cleaved by a specific stimulus. Cleavable linkers may include an electrophilically cleavable moiety, an enzymatically cleavable moiety, a nucleophilically cleavable moiety, an electromagnetically cleavable moiety (e.g., a photocleavable moiety), a metal cleavable moiety, an electrolytically-cleavable moiety, and a moiety that is cleavable under reductive and oxidative conditions. In some embodiments, the cleavable linker includes a photocleavable linker. Photocleavable linkers are linkers comprising a moiety that may be cleaved by light (e.g., light of a particular wavelength). Suitable photocleavable groups and linkers for use include, but are not limited to, ortho-nitrobenzyl-based linkers, phenacyl linkers, alkoxybenzoin linkers, chromium arene complex linkers, NpSSMpact linkers and pivaloylglycol linkers, as described in Guillier et al. (Chem. Rev. 2000 1000:2091 -2157).

[0109] Contacting the Cellular Sample with an Array of Barcoded Nucleic Acids

[0110] Aspects of the methods provided herein include contacting the cellular sample of dispersed cells with an array of barcoded nucleic acids, e.g., as described above. Contacting the sample of dispersed cells with the array of barcoded acids may include plating or placing the dispersed cells on top of the array, centrifuging the dispersed cells and the array together such that cells contact the array, or combinations thereof.

[0111] In some embodiments, the sample of dispersed cells has a cell concentration sufficient to provide upon contact with the array, a desired confluence. Cell confluency Atty. Docket No.: CLON-185WO refers to the proportion (e.g., percentage) of the array that is covered by adherent cells. In some embodiments, the sample has a cell concentration sufficient to provide upon contact with the array a confluence ranging from 10 to 100% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) including, e.g., 10 to 90%, 20 to 100%, 25 to 100%, 25 to 95%, 25 to 90%, 25 to 85%, 25 to 80%, 25 to 75%, 25 to 60%, 25 to 50%, 40 to 75%, 40 to 60%, 50 to 100%, 50 to 90%, 50 to 80%, 50 to 70%, 60 to 100%, 60 to 90%, 60 to 80%, 70 to 100%, 70 to 90% and 80 to 100%. In some embodiments, the sample has a cell concentration sufficient to provide a confluence ranging from 25% to 100%. In some embodiments, the sample has a cell concentration sufficient to provide a confluence ranging from 25% to 75%. In some embodiments, the sample has a cell concentration sufficient to provide a confluence ranging from 75% to 100%.

[0112] In some embodiments, the dispersed cells include dispersed live cells. In some embodiments, the method further includes culturing the dispersed live cells contacted with the array of barcoded nucleic acids. The cells may be cultured using appropriate cell culture conditions. Cell culture conditions for particular cell types are well known in the art. The dispersed live cells may be cultured for any length of time. In some embodiments, the dispersed live cells are cultured for 10 minutes to one week after the dispersed live cells are contacted with the array of barcoded nucleic acids including, e.g., 10 minutes to 24 hours, 10 minutes to 12 hours, 1 hour to 24 hours, 1 hour to 12 hours, 2 hours to 24 hours, 2 hours to 12 hours, 2 hours to 8 hours, 2 hours to 6 hours, 1 day to 2 days and 1 to 3 days.

[0113] In one aspect, dispersed cells (including dispersed live cells) may be contacted to an array of barcoded nucleic acids, wherein the array comprises functionally distinct populations of barcoded nucleic acids. In some embodiments, the array comprises a first functionally distinct population of barcoded nucleic acids and a second functionally distinct population of barcoded nucleic acids, wherein the first functionally distinct population of barcoded nucleic acids comprises a first capture domain and a first cleavage domain and the second functionally distinct population of barcoded nucleic acids comprises a second capture domain and a second cleavage domain. In some embodiments, the first capture domain and the second capture domain are different. In Atty. Docket No.: CLON-185WO some embodiments, the first cleavage domain and the second cleavage domain are different. In some embodiments, the array may comprise a third functionally distinct population of barcoded nucleic acids, a fourth functionally distinct population of barcoded nucleic acids, or more.

[0114] In some embodiments, the barcoded nucleic acids of the array enter the cells (e.g., live cells) of the sample by transfection. In some embodiments, the transfection is mediated by a transfection agent. Transfection reagents include, but are not limited to, lipid-based transfection agents (e.g., Lipofectamine™), polymeric transfection agents, cationic transfection agents, peptide-based transfection agents, calcium phosphate and dendrimers. In some embodiments, the transfection agent is a nanoparticle agent. Nanoparticle agents include, but are not limited to, lipid nanoparticles, polymeric nanoparticles, cationic nanoparticles and inorganic nanoparticles (e.g., Au nanoparticles). Transfection methods and transfection reagents are well known in the art, see, e.g., Chong, Z. X., et al “Transection types, methods and strategies: a technical review” PeerJ 202~\ 9:e1 1165; Mendes, B. B., et al “Nanodelivery of nucleic acids” Nature Reviews Methods Primers 2022 24, the disclosures of which is incorporated by reference. In some embodiments, the transfection is mediated by a physical stimulus. In some embodiments, the physical stimulus is electroporation (i.e., the application of an electrical pulse). In some embodiments, the physical stimulus is laser optoporation (i.e., the application of a laser pulse). In some embodiments, the physical stimulus is sonoporation (i.e., the application of ultrasonic waves). In some embodiments, the physical stimulus is a microstructure or a nanostructure (e.g., contacting the cell with one or more microneedles, nanoneedles, micropillars, nanopillars, etc.). In some embodiments, the physical stimulus is a combination of the physical stimuli listed above (e.g., nanostructure electroporation). In some embodiments, the barcoded nucleic acids of the array enter the cells passively (i.e., without the use of a transfection agent or any additional stimuli). In some embodiments, the array of barcoded nucleic acids is first contacted by a transfection reagent and then contacted by the sample of dispersed cells. In some embodiments, the transfection reagent is deposited on and dried in the locations (i.e., features) of the array. In some embodiments, the excess transfection reagent is removed prior to contacting the sample with the array. For further details Atty. Docket No.: CLON-185WO regarding such transfection methods, see, e.g., U.S. Patent No. 6,951 ,757, the disclosure of which is herein incorporated by reference.

[0115] In some embodiments, the barcoded nucleic acids are attached to a cell uptake moiety that allows for the barcoded nucleic acids to enter the cells. A cell uptake moiety is any moiety that facilitates uptake and / or intake of the moiety and the nucleic acid it is attached to into a cell. In some embodiments, the cell uptake moiety facilitates uptake and / or intake into the cell via cellular mechanisms involved in membrane translocation (e.g., endocytosis-mediated translocation). Cell uptake moieties include, but are not limited to peptides, nanoparticles, and small molecules. In some embodiments, the cell uptake moiety is a cell-penetrating peptide (CPP). In some embodiments, cellpenetrating peptides include one or more positively charged amino acids. Cellpenetrating peptides include those discussed in U.S. Patent No. 9,540,634, U.S. Patent No. 8,632,972, and U.S. Patent No. 9,260,703, the disclosures of which are herein incorporated by reference. In some embodiments, the CPP includes transportan, TP10, penetratin, Tat or a series of arginine residues. In some embodiments, the CPP comprises a series of arginine residues. In some embodiments, the CPP comprises 9 arginine residues (9R).

[0116] In some embodiments, the cell uptake moiety is a nanoparticle (e.g., a functionalized nanoparticle). Barcoded nucleic acids may be attached to cell uptake moieties using any convenient method. Barcoded nucleic acids may be attached to the cell uptake moieties due to physical adsorption, covalent bonding and non-covalent bonding. Physical adsorption attachment methods include, but are not limited to, hydrogen bonding, van der Waals forces, electrostatic forces and hydrophobic interactions. Covalent bonding attachment methods include, but are not limited to, click chemistry, phosphate chemistry, NHS-ester chemistry, thiol-chemistry and silane- chemistry. Non-covalent bonding includes, but is not limited to, streptavidin-biotin interactions, antibody-antigen interactions and the like.

[0117] In some embodiments, the dispersed cells include fixed cells. Cells may be fixed prior to or after contacting the array of barcoded nucleic acids. In some embodiments, the cells are fixed after contacting the array of barcoded nucleic acids. In some embodiments, cells are fixed after culturing the dispersed live cells for a particular Atty. Docket No.: CLON-185WO length of time. Cells may be fixed using any convenient fixation method including, but not limited to, formaldehyde (e.g., 4% paraformaldehyde), dimethyl suberimidate, acetone, methanol, freezing or any combination thereof.

[0118] In some embodiments, the fixed cells are permeabilized. Any convenient protocol for permeabilizing the dispersed cells may be employed. The term "permeabilize" as used herein means to render permeable the membrane, e.g., cell membrane to reagents employed in the method, e.g., barcoded nucleic acids, etc. The term "permeable" as used herein refers to the ability of enzymes, oligonucleotides, etc., or other material to pass through a lipid bilayer membrane such as a cell membrane or a nuclear envelope, which is the membrane that encloses the nucleus. The term "permeable" can be a relative term to indicate permeability to specific reagents (e.g., of a particular size) with respect to other reagents. In some embodiments, the permeabilized cells remain structurally intact. That is, the cell does not rupture or lyse following permeabilization. Methods of determining permeabilization and monitoring for lysed cells are known in the art and include microscopy, fluorescence microscopy, or the use of other dye indicators such as live / dead stains. In embodiments described herein, permeabilization can be performed by contacting the cells with a chemical agent capable of porating a cell and / or an organelle membrane. In some instances, the chemical agent is a detergent and permeabilization can be performed by contacting the cells with a buffer comprising one or more detergents. The term "detergent" as used herein refers to an amphiphilic (partly hydrophilic / polar and partly hydrophobic / non- polar) surfactant or a mixture of amphiphilic surfactants. Detergents can be broadly categorized according to the charge of their polar portion. Anionic detergents, classified as having a negative charge, include but are not limited to alkylbenzenesulfonates and bile acids, such as deoxycholic acid. Cationic detergents, classified as having a positive charge, include but are not limited to quaternary ammonium and pyridinium-based detergents. Nonionic detergents, classified as having no charge, include but are not limited to polyoxyethylene glycol-based detergents such as polysorbates (e.g., polysorbate-20, polysorbate-80) and f-octylphenoxypolyethoxyethanol (sold as Triton™), and glycoside-based detergents such as nonanoyl-N-Hydroxyethylglucamide and nonanoyl-N-methylglucamide. Zwitterionic detergents include, but are not limited to, Atty. Docket No.: CLON-185WO

[0119] 3-((3-cholamidopropyl) dimethylammonio)-1 -propanesulfonate (CHAPS) and amidosulfobetaine-type detergents.

[0120] A detergent may be used to permeabilize the cell membrane of dispersed cells present in a sample. A detergent may further be used to permeabilize the nucleus of dispersed cells present in a sample. The detergent may be the same or different. In some embodiments, the detergent is an ionic detergent, a nonionic detergent, or a zwitterionic detergent. In some embodiments, the ionic detergent is a cationic detergent or a anionic detergent. In some embodiments, the detergent is sodium dodecyl sulfate (SDS) or sodium lauryl sulfate (SLS). In some embodiments, the detergent is a saponin. In some embodiments, the saponin is digitonin. In some embodiments, the detergent is polysorbate-20 or polysorbate-80. In some embodiments, the detergent is deoxycholate. In some embodiments, the detergent is sarkosyl. Selection of the appropriate detergent is dependent on the cell type and would be readily determined by one of ordinary skill in the art.

[0121] Suitable concentration of detergents for permeabilizing cells and organelles such as nuclei include various concentrations depending on the detergent (see e.g., sodium dodecyl sulfate at a final concentration up to 1 %). Additional information on common detergents including their critical micelle concentration values (CMCs) and other properties can be found in "Detergents: Handbook & Selection Guide to Detergents & Detergent Removal" available from G-Biosciences (2018); Neugebauer, Detergents: An overview, in Methods in Enzymology, M. P. Deutscher, Editor (1990) Academic Press, p. 239-253; and Schramm et al., Surfactants and their applications. Annual Reports Section "C" (Physical Chemistry), 2003. 99(0): p. 3-48; where such information readily allows the skilled user to fine-turn their detergent's concentrations to make sure it doesn't exceed the CMC and cause full lysis of the cell / organelle. As will be understood by a person skilled in the art, permeabilization allows for enzymes and other reagents to passively cross the cell membrane into the cytoplasm and / or nucleoplasm and subsequently perform enzymatic reactions in-cell or in-organelle. As the permeabilized cells are not lysed, intracellular components, such as nucleic acids (e.g., mRNAs and genomic DNA), remain within the cell or nucleus and do not diffuse out. Atty. Docket No.: CLON-185WO

[0122] To carry out subsequent reactions in situ, dispersed cells, including fixed dispersed cells, live dispersed cells, and / or permeabilized dispersed cells, may be contacted with any additional reagents needed for said subsequent reactions such that the additional reagents enter the cells. Such reagents include, but are not limited to, reagents needed for reverse transcription reactions, reagents needed for primer extension reactions, reagents needed for template switching reactions, reagents needed for tagmentation reactions, and combinations thereof. Such reagents include, but are not limited to enzymes (e.g., reverse transcriptase, polymerase, transposase), primers, dNTPs, buffers, and the like. Such reagents may come in contact with the dispersed cells at any convenient time including prior to contacting the sample with the array of barcoded nucleic acids, after contacting the sample with the array of barcoded nucleic acids but before pooling the barcoded cells, or after pooling the barcoded cells.

[0123] An embodiment of the method where the barcoded nucleic acid functions as a primer is shown in FIGS. 1A-1 D. In this embodiment, the method includes producing barcoded nucleic acid products via a reverse transcription reaction. FIG. 1 A shows barcoded nucleic acid 100A that includes capture domain 102A (e.g., a polyT sequence), barcode domain 104A, adapter domain 106A and cleavage domain 108A. Barcoded nucleic acid 100A is stably associated with solid support 110A. For example, as depicted in FIG. 1A, cleavage domain 108A functions as a cleavable linker (e.g., a photocleavable linker) that connects barcoded nucleic acid 100A to solid support 110A.

[0124] FIG. 1 B depicts an array of barcoded nucleic acids 120B attached to solid support 110B. While the solid support is depicted as a planar solid support in FIG. 1 B, any solid support such as those discussed above may be used (e.g., a multi-well array, a bead array, etc.). Next, dispersed cells 130B come into contact with the solid surface that includes the array of barcoded nucleic acids. Then, as shown in FIG. 1C, the barcoded nucleic acids are released from the solid surface (e.g., the cleavage domain is cleaved to release the barcoded nucleic acids). Barcoded nucleic acids that are released from the solid surface are able to enter the cell that is in close proximity to the region of the barcoded nucleic acids. For example, cell 130C contains barcoded nucleic acids that include a barcode defining a location on the surface. A first location 122C includes a population of barcoded nucleic acids with the same barcode. A second Atty. Docket No.: CLON-185WO location 124C includes a population of barcoded nucleic acids with the same barcode, where the barcode is distinct from the barcode in location 122C. A third location 126C includes a population of barcoded nucleic acids with the same barcode, where the barcode is distinct from the barcodes in locations 122C and 124C. As such, as shown in FIG. 1C, each cell will contain a particular barcode since each cell contacts a different location of the surface of the barcoded nucleic acid array. Next, as shown in FIG. 1 D, barcoded nucleic acids 100D inside of cell 130D hybridize to RNA 132D (e.g., mRNA) within the cell. The barcoded nucleic acids each include adapter domain 106D, barcode domain 104D and capture domain 102D (e.g., a polyT sequence). The cells are contacted with reagents needed for reverse transcription (e.g., reverse transcriptase, dNTPs, buffers, etc.). Capture domain 102D binds to RNA 132D and is extended into a cDNA strand via an in situ reverse transcription reaction to form barcoded nucleic acid product 134D. Each barcoded nucleic acid product 134D includes a barcode domain that identifies a particular location (e.g., an individual cell). Once barcoded, the cDNAs from multiple cells are pooled and further processed to generate a NGS library for sequencing. By sequencing the captured RNA sequence as well as the barcode sequence, it is possible to i) associate the RNAs to a particular location of the array, and ii) associate the RNAs to an individual cell that contacted that particular location.

[0125] Another embodiment of the methods is shown in FIG. 2. This method incorporates barcoded binding members and finds use in multi-omics workflows. This embodiment follows the same steps shown in FIGS. 1A-1C where the barcoded nucleic acids enter the cell. Next, as shown in FIG. 2, barcoded nucleic acids 200 inside of cell 230 hybridize to RNA 232 within the cell. Barcoded nucleic acid 200 includes adapter domain 206, barcode domain 204 and capture domain 202. The cells are contacted with reagents needed for reverse transcription (e.g., reverse transcriptase, dNTPs, buffers, etc.). Capture domain 202 binds to RNA 232 and is extended into a cDNA strand via an in situ reverse transcription reaction to form barcoded nucleic acid product 234. Each barcoded nucleic acid product 234 includes a barcode domain that identifies a particular location (e.g., an individual cell). The cell is also contacted with barcoded specific binding member 250 (e.g., a barcoded antibody) that includes target barcode domain 252. The cell may be contacted with barcoded binding member 250 at any convenient Atty. Docket No.: CLON-185WO time (e.g., before contacting the sample with the array of barcoded nucleic acids or after contacting the sample with the array of barcoded nucleic acids). Barcoded binding member 250 binds to target protein 254 based on the specificity of the specific binding member. Target barcode domain 252 defines the identity of the target protein that is bound. Target barcode domain 252 is also capable of hybridizing to capture domain 202 of barcoded nucleic acid 200, and thus enables copying of the target barcode domain into cDNA 256 with the barcode (e.g., a barcoded target nucleic acid product). In other words, the capture domain of the barcoded nucleic acid binds to an exogenous nucleic acid (i.e., the nucleic acid comprising the target barcode domain). Once barcoded, the cDNAs from multiple cells are pooled and further processed to generate an NGS library for sequencing. By sequencing the captured RNA sequences and the target barcodes (i.e., the barcodes that identify the bound target protein) as well as the barcode from the barcoded nucleic acids, it is possible to i) associate the RNAs and target protein to a particular location of the array, and ii) associate the RNAs and target protein to an individual cell that contacted that particular location. This method is similarly applicable to other multi-omics analyses such that different combinations of one or more target proteins, RNA and DNA can be barcoded and analyzed simultaneously.

[0126] Another embodiment of the methods is shown in FIG. 3. This method incorporates tagmentation reactions for barcoding DNA (e.g., genomic DNA). This embodiment follows the same steps shown in FIGS. 1 A-1C where the barcoded nucleic acids enter the cell. As shown in FIG. 3, barcoded nucleic acid 300 includes adapter domain 306, barcode domain 304 and capture domain 302. The cells are contacted with reagents needed for tagmentation (e.g., transposomes comprising transposase (e.g., Tn5) and transposons). The cells are also contacted with reagents needed for primer extension (e.g., polymerase, dNTPs, buffers, etc.). Next, a tagmentation reaction is performed in cell 330 in situ. Tagmentation reactions are carried out by transposases 360 on DNA 362 from the cell nucleus to generate tagged nucleic acid fragments 364. In other words, the tagmentation reaction results in the transposons being tagged onto the ends of the nucleic acid fragments. Capture domain 302 binds to the transposon of tagged nucleic acid fragments 364, and thus serves as a primer which can be extended into a cDNA strand that includes the barcode. Once barcoded, the cDNAs from multiple Atty. Docket No.: CLON-185WO cells are pooled and further processed to generate an NGS library for sequencing. By sequencing the DNA fragments as well as the cell barcode sequence, it is possible to associate the DNA fragments to a particular location (e.g., an individual cell). Such embodiments are compatible with transposase mediated applications, e.g., CUT & Tag and ATAC-seq methods, see, e.g., U.S. Patent No. 1 1 ,885,814 and U.S. Patent No. 10,059,989, the disclosures of which are herein incorporated by reference. For example, in embodiments employing CUT & Tag, specific binding members (e.g., antibodies, including chromatin specific antibodies) bind to chromatin-associated proteins of interest. A fusion protein that includes a transposase (e.g., protein A-Tn5 fusion protein) is able to associate with the specific binding members. The transposase (e.g., Tn5 transposase) can be activated (e.g., by the addition of an activation agent (e.g., Mg2+) to cleave (e.g., tagment) DNA local to the sites where it is bound. In other embodiments, such as those employing ATAC-Seq, a transposase (e.g., Tn5 transposase) cleaves (e.g., tagments) DNA at random within regions of open chromatin. Tagmented DNA produced from methods employing tagmentation (e.g., CUT & Tag, ATAC-Seq, etc.) can be used in the same manner as depicted in FIG. 3 to produce barcoded nucleic acid products.

[0127] In some embodiments, as depicted in FIG. 3, the sample is contacted with an array such that the barcoded nucleic acids of the array enter the cells of the sample, and then the tagmentation reaction occurs. However, in some embodiments, the tagmentation reaction may be carried out before the sample is contacted with the array. For example, the cells of the sample may first be contacted with reagents necessary for a tagmentation reaction (e.g., transposome comprising transposase and transposons) to generate tagged nucleic acid fragments, then the cells may be contacted with the array of barcoded nucleic acids such that the barcoded nucleic acids of the array enter the cells of the sample, and then the barcoded nucleic acids may bind to the transposon of tagged nucleic acid fragments, where the barcoded nucleic acids may then serve as a primer which can be extended into a cDNA.

[0128] Another embodiment of the methods is shown in FIGS. 4A-4B. In this method, the barcoded nucleic acid functions as a template switch oligonucleotide (TSO). As shown in FIG. 4A, the array may include barcoded nucleic acids 400A and primers Atty. Docket No.: CLON-185WO

[0129] 470A. Barcoded nucleic acid 400A includes capture domain 402A (e.g., rGrGrG), barcode domain 404A, adapter domain 406A, and cleavage domain 408A. Barcoded nucleic acid 400A functions as a template switch oligonucleotide (TSO). Barcoded nucleic acid 400A is stably associated with solid support 41 OA. For example, as depicted in FIG. 4A, cleavage domain 408A functions as a cleavable linker (e.g., a photocleavable linker) that connects barcoded nucleic acid 400A to solid support 41 OA. Also attached to solid support 41 OA is primer oligonucleotide 470A. Primer oligonucleotide 470A includes binding domain 472A, an adapter domain 476A, and cleavage domain 478A. Primer oligonucleotide 470A is stably associated with solid support 410A. For example, as depicted in FIG. 4A, cleavage domain 478A functions as a cleavable linker (e.g., a photocleavable linker) that connects barcoded nucleic acid 470A to solid support 41 OA. Next, dispersed cells come into contact with an array of barcoded nucleic acids and primer oligonucleotides (as depicted in FIG. 1 B, except that the array includes both barcoded nucleic acids and separate primer oligonucleotides). The barcoded nucleic acids and primer oligonucleotides are then released from the solid surface (e.g., the cleavage domain is cleaved). Barcoded nucleic acids and primer oligonucleotides that are released from the solid surface enter the cell based on proximity and diffusion. As such, each cell contains barcoded nucleic acids that include a barcode defining a location on the surface and primer oligonucleotides. The cells are also contacted with reagents needed for a template switching reaction (e.g., polymerase (e.g., reverse transcriptase), dNTPs, buffers, etc.). Next, as shown in FIG. 4B, a template switching reaction occurs in situ within cell 430B. Primer oligonucleotide 470B includes binding domain 472B and adapter domain 476B. Primer oligonucleotide 470B binds to RNA 432B within the cell via binding domain 472B, and is extended into a cDNA strand via an in situ template switching reaction within the cell. The polymerase (e.g., reverse transcriptase with template switching ability) adds non-template nucleotides to the 3’ end of the cDNA. The TSO 400B includes capture domain 402B, barcode domain 404B and adapter domain 406B. Capture domain 402B of the TSO hybridizes with the non-template nucleotides on the 3’ end of the cDNA, allowing the polymerase to then template switch to TSO 400B. The polymerase continues to extend the cDNA to form barcoded nucleic acid product 434B. Each barcoded nucleic acid Atty. Docket No.: CLON-185WO product 434B includes a barcode that identifies a particular location (e.g., an individual cell). Once barcoded, the cDNAs from multiple cells are pooled and further processed to generate an NGS library for sequencing. By sequencing the captured RNA sequence as well as the barcode sequence, it is possible to i) associate the RNAs to a particular location of the array, and ii) associate the RNAs to an individual cell that contacted that particular location.

[0130] In some embodiments, as depicted in FIG. 4A, the array includes both barcoded nucleic acids and primers that are stably associated with the solid support. In such instances, each location of the array may include primer nucleic acids that are identical in sequence, even though the barcoded nucleic acids of different locations differ from each other. However, in some embodiments, the array may solely include barcoded nucleic acids. In such embodiments, the sample is contacted with primers prior to the template switching reaction (e.g., prior to contacting the sample with the array of barcoded nucleic acids or after contacting the sample with the array of barcoded nucleic acids). The sample may be contacted with primers and other reagents for the template switching reaction simultaneously.

[0131] Polymerases appropriate for template switching reactions include reverse transcriptases. In some instances, the polymerase is a reverse transcriptase capable of template-switching including, e.g., a retroviral reverse transcriptase, a retrotransposon reverse transcriptase, a retroplasmid reverse transcriptase, a retron reverse transcriptase, a bacterial reverse transcriptase, a group II intron-derived reverse transcriptase, and mutants, variants derivatives, or functional fragments thereof, e.g., RNase H minus or RNase H reduced enzymes. For example, the reverse transcriptase may be a Moloney Murine Leukemia Virus reverse transcriptase (MMLV-RT) or a Bombyx mori reverse transcriptase (e.g., Bombyx mori R2 non-LTR element reverse transcriptase). Polymerases capable of template switching that find use in practicing the subject methods are commercially available and include SMARTScribe™ reverse transcriptase and PrimeScript™ reverse transcriptase available from Takara Bio USA (San Jose, CA). In addition to a template switching capability, the polymerase may include other useful functionalities. For example, the polymerase may have terminal transferase activity, where the polymerase is capable of catalyzing the addition of Atty. Docket No.: CLON-185WO deoxyribonucleotides to the 3’ hydroxyl terminus of an RNA or DNA molecule. In certain aspects, when the polymerase reaches the 5’ end of the template, the polymerase is capable of incorporating one or more additional nucleotides at the 3’ end of the nascent strand not encoded by the template. For example, when the polymerase has terminal transferase activity, the polymerase may be capable of incorporating 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional nucleotides at the 3’ end of the nascent strand. All of the nucleotides may be the same (e.g., creating a homonucleotide stretch at the 3’ end of the nascent strand) or one or more of the nucleotides may be different from the other(s) (e.g., creating a heteronucleotide stretch at the 3’ end of the nascent strand). In certain aspects, the terminal transferase activity of the polymerase results in the addition of a homonucleotide stretch of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the same nucleotides (e.g., all dCTP, all dGTP, all dATP, or all dTTP). In some embodiments, the polymerase is an MMLV reverse transcriptase (MMLV RT). MMLV RT incorporates additional nucleotides (predominantly three dCTPs) at the 3’ end of the nascent strand.

[0132] Another embodiment of the methods is shown in FIGS. 5A-5B. In this method, the barcoded nucleic acid is a template nucleic acid. In other words, the barcoded nucleic acids may be used as a template in a primer extension reaction. In some embodiments, the primer extension reaction can occur in combination with other barcoding methods (e.g., a 10x Genomics® barcoding system), allowing for increased throughput screening of samples. This embodiment follows similar steps shown in FIGS. 1A-1C where the barcoded nucleic acids enter the cell. However, multiple cells (e.g., 100 or 1000 cells) are plated within each location. As such, there are multiple cells associated with each location of multiple locations. Each location may be a well of a multi-well array (e.g., a 96 well plate, a 384 well plate, etc.). As shown in FIG. 5A, barcoded nucleic acids 500A that enter cell 530A each include adapter domain 506A, barcode domain 504A and capture domain 502A. Also within cell 530A is RNA 532A (e.g., mRNA) that includes a polyA tail 533A. In some embodiments, the polyA tail is added to the RNA in situ prior to the delivery of the barcoded nucleic acids. For example, a polyA tail may be added to RNA present in a cell via an in situ ligation reaction or in situ terminal transferase or polymerization reaction. Next, multiple cells from multiple locations are pooled. As shown in FIG. 5B, each cell is then encapsulated Atty. Docket No.: CLON-185WO in a droplet 580B together with a bead 582B. Bead 582B includes first oligonucleotide 590B and second oligonucleotide 592B. First oligonucleotide 590B includes a capture domain, a UMI domain and a cell barcode. The capture domain of first oligonucleotide 590B binds to barcoded nucleic acid 500B and is extended into cDNA strand 594B. Second oligonucleotide 592B includes a capture domain (e.g., a polyT sequence), a UMI domain, and a cell barcode. The capture domain of second oligonucleotide 592B binds to RNA 532B and is extended into cDNA strand 596B. This allows for both the RNA of the cell and the barcoded nucleic acid to become encoded with the cell barcode from the bead in the droplet. Next, the cDNAs from multiple cells are pooled and further processed to generate an NGS library for sequencing. The cell barcode labeling the RNA and the barcoded nucleic acid enable the association of the cell to a location on the original array, defined by the barcode in the barcoded nucleic acid. Accordingly, a single cell can be associated with a distinct location (e.g., a well of a multi-well array).

[0133] Another embodiment of the methods is shown in FIGS. 6A-6B. In this embodiment, the method includes producing barcoded nucleic acid products via a reverse transcription reaction, wherein the array of barcoded nucleic acids includes two functionally distinct populations of barcoded nucleic acids. FIG. 6A shows first barcoded nucleic acid 600A and second barcoded nucleic acid 601 A. First barcoded nucleic acid 600A includes first capture domain 602A, barcode domain 604A, adapter domain 606A and cleavage domain 608A. First barcoded nucleic acid 600A is stably associated with solid support 61 OA. For example, as depicted in FIG. 6A, cleavage domain 608A functions as a cleavable linker that connects barcoded nucleic acid 600A to solid support 610A. Second barcoded nucleic acid 601 A includes second capture domain 603A, barcode domain 605A, an adapter domain 607A and cleavage domain 609A. Barcoded nucleic acid 601 A is stably associated with solid support 610A. For example, as depicted in FIG. 6A, cleavage domain 608A functions as a cleavable linker that connects barcoded nucleic acid 601A to solid support 610A. First capture domain 602A and second capture domain 603A each bind to different target regions. In some embodiments, cleavage domain 608A and cleavage domain 608B are cleaved via distinct stimuli. Accordingly, barcoded nucleic acid 600A and barcoded nucleic acid Atty. Docket No.: CLON-185WO

[0134] 601 A can be temporally regulated and released at different times throughout an experiment.

[0135] FIG. 6B depicts array of barcoded nucleic acids 620B that includes a plurality of barcoded acids including a first population of first barcoded nucleic acids 600B and second population of second barcoded nucleic acids 601 B. Next, dispersed cells 630B come into contact with the solid surface that includes the array of barcoded nucleic acids. Then, as shown in FIG. 6B, the barcoded nucleic acids are released from the solid surface (e.g., the cleavage domain is cleaved). Barcoded nucleic acids that are released from the solid surface are able to enter the cell that is in close proximity to the region of the barcoded nucleic acids. As such, each cell 630B contains barcoded nucleic acids from a first functionally distinct population of nucleic acids 600B and barcoded nucleic acids form a second functionally distinct population of nucleic acids 601 B. In this embodiment, the capture domains of barcoded nucleic acids 600B and 601 B bind to different target regions, and thus nucleic acids corresponding to these different target regions are both able to barcoded.

[0136] FIGS. 1-6 illustrate representative embodiments of the methods described herein. In some embodiments, the barcoded nucleic acid comprises a capture domain that is a polyT capture domain. In some embodiments, the barcoded nucleic acid comprises a capture domain having a degenerate sequence. In some embodiments, the barcoded nucleic acid comprises one barcode domain. In some embodiments, the barcoded nucleic acid comprises two, three, four, five, six, or more barcode domains.

[0137] Reactions described above (e.g., primer extension reactions, template switching reactions, reverse transcription reactions, tagmentation reactions, etc.) are carried out under conditions sufficient to produce the desired barcoded nucleic acids. By “conditions sufficient to produce the desired barcoded nucleic acid products " is meant reaction conditions that permit the relevant nucleic acids and / or other reaction components in the reaction to interact with one another in the desired manner. For example, in some instances, the conditions may be sufficient for nucleic acids of the reaction mixture to hybridize. In some instances, the conditions may be sufficient for an enzyme of the reaction mixture to catalyze a chemical process such as e.g., polymerization, hydrolysis, ligation, tagmentation, etc. Achieving suitable reaction Atty. Docket No.: CLON-185WO conditions may include selecting reaction mixture components, concentrations thereof, and a reaction temperature to create an environment in which the relevant processes proceed, including e.g., the relevant nucleic acids hybridize with one another in a sequence specific manner, the relevant polymerase polymerizes resulting in elongation of a nucleic acid, etc. In addition to specific nucleic acids (e.g., template nucleic acids, oligonucleotides, primers, etc.) of a reaction the reaction mixture may include buffer components that establish an appropriate pH, salt concentration (e.g., KOI concentration), etc. Conditions sufficient to produce a double stranded nucleic acid complex may include those conditions appropriate for hybridization, also referred to as “hybridization conditions”.

[0138] Achieving suitable reaction conditions may include selecting reaction mixture components, concentrations thereof, and a reaction temperature to create an environment in which one or more polymerases are active and / or the relevant nucleic acids in the reaction interact (e.g., hybridize) with one another in the desired manner. In suitable reaction conditions, in addition to reaction components, the reaction mixture may include buffer components that establish an appropriate pH, salt concentration (e.g., KCI concentration), metal cofactor concentration (e.g., Mg2+or Mn2+concentration), and the like, for the extension reaction(s), for example second strand synthesis reactions, and / or template switching to occur. Other components may be included, such as one or more nuclease inhibitors (e.g., an RNase inhibitor and / or a DNase inhibitor), one or more additives for facilitating amplification / replication of GC rich sequences (e.g., GC-Melt™ reagent (Takara Bio USA, Inc. (San Jose, GA)), betaine, DMSO, ethylene glycol, 1 ,2-propanediol, or combinations thereof), one or more molecular crowding agents (e.g., polyethylene glycol, or the like), one or more enzymestabilizing components (e.g., DTT present at a final concentration ranging from 1 to 10 mM (e.g., 5 mM)), and / or any other reaction mixture components useful for facilitating polymerase-mediated extension reactions and / or template-switching.

[0139] One or more reaction mixtures may have a pH suitable for amplification (e.g., PCR amplification), ligation, second strand synthesis, or tagmentation. In certain embodiments, the pH of the reaction mixture ranges from 5 to 9, such as from 7 to 9, including from 8 to 9, e.g., 8 to 8.5. In some instances, the reaction mixture includes a Atty. Docket No.: CLON-185WO pH adjusting agent. pH adjusting agents of interest include, but are not limited to, sodium hydroxide, hydrochloric acid, phosphoric acid buffer solution, citric acid buffer solution, and the like. For example, the pH of the reaction mixture can be adjusted to the desired range by adding an appropriate amount of the pH adjusting agent.

[0140] The temperature range suitable for primer extension reactions may vary according to factors such as the particular polymerase employed, the melting temperatures (Tm) of any primers employed, etc. In some instances, a reverse transcriptase (e.g., an MMLV reverse transcriptase) may be employed and the reaction mixture conditions sufficient for reverse transcriptase-mediated extension of a hybridized primer include bringing the reaction mixture to a temperature ranging from 4° C to 72° C, such as from 16° C to 70° C, e.g., 37° C to 50° C, such as 40° C to 45° C, including 42° C.

[0141] Amplification mediated reactions may employ a suitable polymerase for producing barcoded nucleic acid products. Any convenient amplification polymerase may be employed including but not limited to DNA polymerases including thermostable polymerases. Useful amplification polymerases include e.g., Taq DNA polymerases, Pfu DNA polymerases, Terra™ DNA polymerase, those described in U.S. Patent No. 6,127,155 (the disclosure of which is incorporated herein by reference in its entirety), derivatives thereof and the like. In some instances, the amplification polymerase may be a hot start polymerase including but not limited to e.g., a hot start Taq DNA polymerase, a hot start Pfu DNA polymerase, and the like. An amplification polymerase may be combined into a reaction mixture such that the final concentration of the amplification polymerase is sufficient to produce a desired amount of product nucleic acid. In certain aspects, the amplification polymerase (e.g., a thermostable DNA polymerase, a hot start DNA polymerase, etc.) is present in the reaction mixture at a final concentration of from 0.1 to 200 units / pL (U / pL), such as from 0.5 to 100 U / pL, such as from 1 to 50 U / pL, including from 5 to 25 U / pL, e.g., 20 U / pL. Nucleic acid reactions, e.g., amplification reactions, of the subject methods may include combining dNTPs into a reaction mixture. In certain aspects, each of the four naturally-occurring dNTPs (dATP, dGTP, dCTP and dTTP) are added to the reaction mixture. For example, dATP, dGTP, dCTP and dTTP may be added to the reaction mixture such that the final concentration of each dNTP is Atty. Docket No.: CLON-185WO from 0.01 to 100 mM, such as from 0.1 to 10 mM, including 0.5 to 5 mM (e.g., 1 mM). In some instances, one or more types of nucleotide added to the reaction mixture may be a non-naturally occurring nucleotide, e.g., a modified nucleotide having a binding or other moiety (e.g., a fluorescent moiety, a biotin moiety) attached thereto, a nucleotide analog, or any other type of non-naturally occurring nucleotide that finds use in the subject methods or a downstream application of interest.

[0142] Reaction mixtures may be subjected to various temperatures to drive various aspects of the reaction including but not limited to e.g., denaturing / melting of nucleic acids, hybridization / annealing of nucleic acids, polymerase-mediated elongation / extension, etc. Temperatures at which the various processes are performed may be referred to according to the process occurring including e.g., melting temperature, annealing temperature, elongation temperature, etc. The optimal temperatures for such processes will vary, e.g., depending on the polymerase used, depending on characteristics of the nucleic acids, etc. Optimal temperatures for particular polymerases, including reverse transcriptases and amplification polymerases, may be readily obtained from reference texts. Optimal temperatures related to nucleic acids, e.g., annealing and melting temperatures may be readily calculated based on known characteristics of the subject nucleic acid including e.g., overall length, hybridization length, percent G / C content, secondary structure prediction, etc.

[0143] In some embodiments, the method further includes using protocols such as amplification protocols, ligation protocols, tagmentation protocols, second strand synthesis reactions, further barcoding protocols (e.g., cell barcoding protocols) etc. The reaction mixture components in such protocols are combined under conditions sufficient to produce the desired barcoded nucleic acid products of the reaction. For example, in some instances, the reaction components of an amplification reaction are combined under conditions sufficient to produce barcoded nucleic acid products via one or more rounds of amplification, e.g., one or more PCR rounds. In some instances, the reaction components of a ligation reaction are combined under conditions sufficient to produce ligated barcoded cell nucleic acid products.

[0144] With any protocol employed to produce barcoded nucleic acid products, reagents employed in such protocols may be employed to introduce additional features into the Atty. Docket No.: CLON-185WO barcoded nucleic acid products, where such additional features may be features that find use in downstream processing of the barcoded cells. For example, where generation of barcoded nucleic acids is part of a sequencing library generation protocol, additional features (domains) may be incorporated into the barcoded nucleic acid products. Suitable domains include those described above, such as adapter domains, sequencing platform adapter construct domains, barcode domains, and primer binding domains.

[0145] Further Processing

[0146] In some embodiments, the method further includes pooling the barcoded cells. Following production of cDNAs comprising barcodes that identify the location of the cell, the barcoded cells are combined or pooled to produce a first pool of cells comprising barcoded cDNAs. The barcoded cells may be combined or pooled using any convenient protocol. The number of barcoded cells in the resultant first pool of barcoded cells may vary, and in some instances ranges from 2 to 10,000,000 cells, such as 10,000 to 1 ,000,000 cells or 10,000 to 100,000 cells. In some embodiments, a barcode from a barcoded nucleic acid is associated with a single cell (i.e., the barcoded cells each have a unique barcode). In some embodiments, a barcode from a barcoded nucleic acid is associated with a group of cells (i.e. a group of cells share of barcode, but the barcode differs from that of other groups of cells).

[0147] In some embodiments, the pooled cells are split into subsequent pools. Splitting the pooled cells into two or more pools of cells allows for combinatorial barcoding of the cells in the sample. “Combinatorial barcoding" in the context of single-cell analysis refers to a technique wherein cells are labeled with unique combinations of short DNA sequences (e.g., barcode domains) by splitting the initial pool of cells into two or more pools of cells and labeling the cells in the pools of cells with a barcode domain. This process can be repeated (splitting, labeling, pooling, splitting, labeling, pooling). This approach allows for the identification and analysis of thousands to millions of individual cells in a single experiment, without the need for isolating each cell separately. The process effectively creates a unique barcode signature for each cell by combining the different barcodes added during the multiple rounds of pooling. Atty. Docket No.: CLON-185WO

[0148] The splitting of cells can be performed using any suitable partition. In some embodiments, the partition is a well in a multi-well plate (such as a 6, 12, 24, 48, 96, or 384 well plate or a nanowell chip, such as an ICELL8® chip, available from Takara Bio USA, Inc., containing 5184 nanowells of about 150 - 350 nanoliters in volume). In some embodiments, the partition is a droplet (such as an oil-in-water emulsion droplet). In some embodiments, the partition is an array described herein. In some embodiments, the partition is a tube. In some instances, the cells are subjected to a combinatorial barcoding protocol as described in United States Published Patent Application Publication No. US 2024-0352448, the disclosure of which is herein incorporated by reference.

[0149] In some embodiments, the method further includes lysing the barcoded cells. Lysis can be achieved by any suitable method, including heat, freeze-thaw cycles, detergents or chemical lysis, or any combination thereof. In some instances, a mild lysis procedure can advantageously be used to prevent the release of nuclear chromatin, thereby avoiding genomic contamination of a cDNA library, and to minimize degradation of mRNA. Examples of suitable lysis methods include heating the cells at between 65- 802C for between 1 -10 minutes. The cells may be re-suspended in any suitable buffer. The buffer may comprise a detergent. For example, heating the cells at 72QC for 2 minutes in the presence of polysorbate-20 is sufficient to lyse the cells while resulting in no detectable genomic contamination from nuclear chromatin. Lysis can also be achieved with a protease such as Proteinase K or by the use of chaotropic salts such as guanidine isothiocyanate (U.S. Publication No. 2007 / 0281313).

[0150] In some embodiments, the method includes isolating and pooling the nucleic acids from the barcoded cells. Nucleic acids may be isolated using methods known in the art, including lysis of said cells to release the nucleic acids into solution. Cells may be lysed prior to pooling the cells or after pooling the cells. In some embodiments, the method includes first lysing the cells and then pooling the nucleic acids from the cells. In some embodiments, the method includes first pooling the cells and then lysing the cells to release the nucleic acids into solution.

[0151] In some embodiments, the method further includes preparing a sequencing library from the barcoded cells. Barcoded nucleic acid products may be prepared for Atty. Docket No.: CLON-185WO sequencing applications, such as next generation sequencing applications. The barcoded nucleic acids are prepared to be sequencing ready in that all the necessary domains for next-generation sequencing are incorporated into the nucleic acids. Necessary domains include the sequencing platform adapter domains as described above. Sequencing platform adapter domains may be added to the barcoded nucleic acids at any appropriate step throughout the methods described herein. For example, sequencing platform adapter domains may be added to nucleic acids in cells via the barcoded nucleic acids of the array. In such embodiments, the barcoded nucleic acids of the array would comprise a sequencing platform adapter domain. Additionally or alternatively, sequencing platform adapter domains may be added during amplification wherein the primers used for amplification of nucleic acids (derived from barcoded cells) comprise one or more sequencing platform adapter domains. Additionally or alternatively, sequencing platform adapter domains may be ligated to nucleic acids (derived from barcoded cells). Additionally or alternatively, sequencing platform adapter domains may be added to nucleic acids (derived from barcoded cells) via a transposon reaction, wherein the transposon comprises one or more sequencing platform adapter domains. Additionally or alternatively, sequencing platform adapter domains may be added to nucleic acids (derived from barcoded cells) using a template switch oligonucleotide, wherein the template switch oligonucleotide comprises one or more sequencing platform adapter domains.

[0152] For example, sequencing platform adapter constructs that may be necessary for use in a given sequencing application may be incorporated into the barcoded nucleic acid products during preparation of the barcoded nucleic acid products, e.g., by inclusion of such on the components employed in the preparation of barcoded nucleic acid products, e.g., template switch oligonucleotides, amplification primers, transposon nucleic acids, etc.

[0153] In some embodiments, the barcoded nucleic acid products may be further processed to generate a sequencing ready library, where any convenient approach may be employed in such instances. In such embodiments, one or more of such constructs may be incorporated into the barcoded nucleic acid products after the preparation thereof. Where desired, such adapter constructs may be added to a nucleic acid of Atty. Docket No.: CLON-185WO interest, e.g., a barcoded nucleic acid product, by a variety of means. For example, adapter sequences may be added through the action of a polymerase with terminal transferase activity. Adapter sequences may be incorporated into a nucleic acid during an amplification reaction. In some instances, adapter sequences may be directly attached to a nucleic acid, e.g., to a barcoded nucleic acid product. Methods of directly attaching an adapter sequence to a nucleic acid will vary and may include but are not limited to e.g., ligation, chemical synthesis / linking, enzymatic nucleotide addition (e.g., by a polymerase with terminal transferase activity), tagmentation, and the like.

[0154] In some instances, the methods may include attaching sequencing platform adapter constructs, and / or adapters comprising any sequence for any use, to ends of a nucleic acid. For example, in some instances, oligonucleotides and / or primers utilized in the subject methods may not include sequencing platform adapter constructs and thus desired sequencing platform adapter constructs may be attached following the production of a barcoded nucleic acid product of interest. Adapter constructs attached to the ends of a barcoded nucleic acid of interest or a derivative thereof may include any sequence elements useful in a downstream sequencing application, including any of the elements described above with respect to the optional sequencing platform adapter constructs of the oligonucleotides and / or primers of the herein described methods. For example, the adapter constructs attached to the ends of nucleic acid of interest or a derivative thereof may include a nucleic acid domain or complement thereof selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, and combinations thereof.

[0155] Attachment of the sequencing platform adapter constructs may be achieved using any suitable approach. In certain aspects the adapter constructs are attached to the ends of the product nucleic acid or a derivative thereof using an approach that is the same or similar to “seamless” cloning strategies. Seamless strategies eliminate one or more rounds of restriction enzyme analysis and digestion, DNA end-repair, dephosphorylation, ligation, enzyme inactivation and clean-up, and the corresponding loss of nucleic acid material. Seamless attachment strategies of interest include: the In- Atty. Docket No.: CLON-185WO

[0156] Fusion® cloning systems available from Takara Bio USA, Inc. (San Jose, CA), SLIC (sequence and ligase independent cloning) as described in Li & Elledge (2007) Nature Methods 4:251 -256; Gibson assembly as described in Gibson et al. (2009) Nature Methods 6:343-345; OPEC (circular polymerase extension cloning) as described in Quan & Tian (2009) PLoS ONE 4(7): e6441 ; SLiCE (seamless ligation cloning extract) as described in Zhang et al. (2012) Nucleic Acids Research 40(8): e55, and the GeneArt® seamless cloning technology by Life Technologies (Carlsbad, CA).

[0157] Any suitable approach may be employed for providing additional nucleic acid sequencing domains to a nucleic acid of interest or derivative thereof having less than all of the useful or necessary sequencing domains for a sequencing platform of interest. For example, a nucleic acid of interest or derivative thereof could be amplified using PCR primers having adapter sequences at their 5’ ends (e.g., 5’ of the region of the primers complementary to the nucleic acid of interest or derivative thereof), such that the amplicons include the adapter sequences in the original nucleic acid as well as the adapter sequences in the primers, in any desired configuration. Other approaches, including those based on seamless cloning strategies, restriction digestion / ligation, tagmentation, or the like may be employed. Methods for adding nucleic acid domains to a Next Generation Sequencing library are known in the art, for example but not limited to, those described in Pat. No. US11 ,124,828, the entirety of which is hereby incorporated by reference.

[0158] Following prescribed library preparation and / or amplification steps, e.g., as described above, prepared libraries may be considered ready for sequencing. In some embodiments, the method further includes sequencing the sequencing library using a next-generation sequencing platform. Suitable NGS sequencing platforms include, but are not limited to, a sequencing platform provided by Illumina® (e.g., the iSeq™, MiniSeq™, HiSeq™, MiSeq™, NextSeq™, NovaSeqTMand / or Genome Analyzer™ sequencing systems); Ion Torrent™ (e.g., the Ion PGM™ and / or Ion Proton™ sequencing systems); Pacific Biosciences (e.g., the PACBIO RS II Sequel sequencing system); Oxford Nanopore Technologies (ONT); Life Technologies™ (e.g., a SOLiD sequencing system); Roche (e.g., the 454 GS FLX+ and / or GS Junior sequencing systems); Element AVITI systems; Complete Genomics DNBSEQ Platforms (e.g., E25, Atty. Docket No.: CLON-185WO

[0159] G99, G400 and T7).The NGS protocol will vary depending on the particular NGS sequencing system employed. Detailed protocols for sequencing an NGS library, including any further amplification (e.g., solid-phase amplification), sequencing of the amplicons, and analyzing the sequencing data are available from the manufacturer of the NGS sequencing system employed.

[0160] The present methods are compatible with other sequencing methods including, but not limited to, Drop-seq, DroNc-seq, Nx1 -seq, Seq-Well, sci-RNA-seq, scifi- RNAseq, sci-RNA- seq3, SCRB-seq, CEL-Seq, CEL-Seq2, SPLiT-seq, MARS-seq, Smart-seq / C1 , C1 -CAGE, RamDa-seq, Microwell-seq, Smart-seq2, Smart-seq3, bead- seq, LIBRA-seq, G&T-seq, Quartz-Seq, Perturb-seq, MULTI-seq, ChlP-seq, ATAC- seq, SNES, scBS-seq, scRRBS, Smart-RRBS, Drop-ChIP, scDam&T-seq, ScNallmi-seq, DR-seq, CITE-seq, TotalSeq, CytoSeq, STRT-seq, STRT / C1 , SUPeR-seq, REAP-seq, MATQ-seq, scM&T-seq, scMT-seq, scCAT-seq, Paired- seq, SNARE-seq, T-ATAC-seq, Slide-seq, RAGE-Seq, FB5P-seq, TEA-seq, SUGAR-seq, CRISP-seq, single-cell RNA- seq, single-cell sequencing using Fluent, multiplexed scRNA-seq via e.g. cell hashing or CellPlex, and / or CROP- seq. For more details regarding compatible sequencing methods, see, e.g., U.S. Patent No. 10,059,989, U.S. Patent No. 10,689,643, U.S. Patent No. 9,938,524, U.S. Patent No. 11 ,597,964, U.S. Patent No. 1 1 ,767,557, U.S. Patent No. 10,400,235, U.S. Patent No. 11 ,885,814 and U.S. Patent No. 1 1 ,543,417, US Patent No. 10,266,894, Sasagawa et al. Genome Biol. 2013 Apr 17;14(4):R31 , Hayashi et al. Nat Commun 9, 619 (2018), Macaulay et al. Nat Methods 12, 519-522 (2015), the disclosures of which are herein incorporated by reference.

[0161] For example, the present methods may be used with ATAC-seq as follows. ATAC-seq or “Assay for Transposase-Accessible Chromatin using Sequencing” uses transposases to detect accessible chromatin. Briefly, chromatin of a cell nucleus is contacted with a transpose (e.g., Tn5). The transposase tagments (i.e. , cleaves and tags) DNA within regions of open chromatin (i.e., accessible chromatin). As a part of the tagmentation reaction, the transposase adds nucleic acid sequences (i.e., transposons) to the ends of the fragmented DNA. Prior to or after tagmentation, the cell is further contacted with barcoded nucleic acids such that the barcoded nucleic acids enter the cell. As depicted in FIG. 3, a capture domain of the barcoded nucleic acids described Atty. Docket No.: CLON-185WO herein may bind to the transposon of the tagmented nucleic acid fragments. As such, the barcoded nucleic acid can function as a primer which can be extended into a cDNA strand that includes a barcode. Once barcoded, the cDNAs from multiple cells are pooled and further processed to generate an NGS library for sequencing. By sequencing the DNA fragments as well as the cell barcode sequence, it is possible to associate the DNA fragments from regions of open chromatin to a particular location (e.g., an individual cell). For more details on ATAC-seq, see, e.g., U.S. Patent No. 10,059,989, the disclosure of which is herein incorporated by reference.

[0162] For example, the present methods may be used with CUT&Tag as follows. CUT&Tag or “Cleavage Under Targets and Tagmentation” uses specific binding members (e.g., antibodies) to detect DNA at target protein sites of interest on chromatin. Briefly, chromatin of a cell nucleus is contacted with a specific binding member (e.g., antibody) that binds to a chromatin-associated protein of interest. A fusion protein that includes a transposase (e.g., protein A-Tn5 fusion protein) is able to associate with the specific binding members. The transposase (e.g., Tn5 transposase) can be activated (e.g., by the addition of an activation agent (e.g., Mg2+) to tagent (e.g., cleave and tag) DNA local to the sites where it is bound. As a part of the tagmentation reaction, the transposase adds nucleic acid sequences (i.e. , transposons) to the ends of the fragmented DNA. Prior to or after tagmentation, the cell is further contacted with barcoded nucleic acids such that the barcoded nucleic acids enter the cell. As depicted in FIG. 3, a capture domain of the barcoded nucleic acids described herein may bind to the transposon of the tagmented nucleic acid fragments. As such, the barcoded nucleic acid can function as a primer which can be extended into a cDNA strand that includes a barcode. Once barcoded, the cDNAs from multiple cells are pooled and further processed to generate an NGS library for sequencing. By sequencing the DNA fragments as well as the cell barcode sequence, it is possible to associate the DNA fragments from a chromatin-associated protein of interest to a particular location (e.g., an individual cell). For more details on CUT&Tag, see, e.g., U.S. Patent No. 1 1 ,885,814, the disclosure of which is herein incorporated by reference. Atty. Docket No.: CLON-185WO

[0163] Methods described herein of barcoding cells of a sample of dispersed cells may be used to produce barcoded cells for a variety of different purposes. In certain embodiments, the subject methods may be used to generate a sequencing library for sequencing on a next-generation sequencing platform.

[0164] The methods of the present application find use in high-throughput single cell assays. Barcoded arrays can be tailored to include any desired number of barcoded nucleic acids using any desired spatial distribution. The spatial distribution of barcoded nucleic acids may be referred to by their location (X,Y coordinates) on the array. In some embodiments, barcoded nucleic acids may be distributed on the array such that distinct populations of barcoded nucleic acids (e.g., functionally distinct populations of barcoded nucleic acids and / or populations of barcoded nucleic acids with distinct barcodes) are separated by between 1 pm to 100 pm. In some cases, each population of the distinct populations of barcoded nucleic acids includes nucleic acids with a unique barcode. In some cases, the distinct populations of barcoded nucleic acids are functionally distinct populations of barcoded nucleic acids that differ by one or more of: a cleavage domain and a capture domain. In some embodiments, barcoded nucleic acids are distributed on the array such that distinct barcoded nucleic acids are separated by between 1 -5 pm, 5-10 pm, 10-15 pm, 15-20 pm, 20-25 pm, 25-30 pm, SO- 35 pm, 35-40 pm, 40-45 pm, 45-50 pm, 50-55 pm, 55-60 pm, 60-65 pm, 65-70 pm, 70- 75 pm, 75-80 pm, 80-85 pm, 85-90 pm, 90-95 pm, or 95-100 pm. Distinct populations of barcoded nucleic acids may differ by any one of the domains present (e.g., barcode domain, capture domain, adapter domain, cleavage domain). Barcoded nucleic acids within each population of barcoded nucleic acids may further include unique molecular identifiers (UMIs). In other words, each barcoded nucleic acid of the population of barcoded nucleic acids has a different UMI. In some embodiments, the distinct barcoded nucleic acids may be distributed on the array such that the distinct barcoded nucleic acids are separated by about the average size of the cell of the sample being analyzed. As an example, for an average cell size of 20 pm, the barcoded nucleic acids may be separated on the array by about 20 pm. In this way, each cell is labeled with a unique barcode. In some embodiments, the barcoded nucleic acids may be distributed Atty. Docket No.: CLON-185WO such that the barcoded nucleic acids are separated by a distance that is larger than the cell size. For example, if barcoded nucleic acids are distributed across wells of a multiwell array, each well may comprise distinct barcoded nucleic acids. In some embodiments, each location of the different locations of the array includes two or more (e.g., three or more, four or more, etc.) functionally distinct populations of barcoded nucleic acids.

[0165] The methods described herein are compatible with other single-cell barcoding methods (e.g., commercial barcoded bead methods such as a 10x Genomics® barcoding system), allowing for association of a “spatial” barcode and a cell barcode. The methods of the present application are compatible with combinatorial barcoding strategies (e.g., Sci-Seq), where the method of the present application provides the first round of barcoding and an additional barcoding method provides a second round of barcoding. As such individual cells can be localized to a single location (e.g., a single well) among many locations, allowing for massively parallel screening of samples.

[0166] In one aspect, the method comprises first using the barcoded nucleic acids as a primer. After contacting the array of barcoded nucleic acids with a sample of dispersed cells, the barcoded nucleic acids are released such that the barcoded nucleic acids enter the cells. The barcoded nucleic acids may comprise any desired capture domain that is compatible with the method to be carried out. For example, the capture domain may be a capture domain that binds to RNA present in the cell, such as a polyT capture domain or a randomer capture domain. Alternatively, the barcoded nucleic acids may comprise a capture domain that binds to cDNA synthesized in situ in the cells, such as a polyA capture domain. For example, cDNA may be synthesized from RNA present in the cells using in situ reverse transcription. The reverse transcription primer may be a polyT primer or a randomer primer. In some embodiments, the primer for reverse transcription further comprises a second primer domain which is subsequently incorporated into the cDNA. In said embodiments, the capture domain of the barcoded nucleic acids may specifically bind to the second primer domain. Alternatively, the barcoded nucleic acids may comprise a capture domain that has complementarity to a bridge oligonucleotide. The bridge oligonucleotide may be delivered to the cells as a reagent, or attached to the array of barcoded nucleic acids. The bridge oligonucleotide Atty. Docket No.: CLON-185WO may then be used to ligate the barcoded nucleic acid to RNA or cDNA present in the cells. Alternatively, the barcoded nucleic acids may comprise a capture domain having an arbitrary sequence that is suitable for downstream methods.

[0167] The barcoded nucleic acids are utilized as a primer to label the RNA or cDNA in the cells of the sample. After, nuclei are isolated from the cells using methods known in the art. The nuclei can subsequently be distributed across a second form of barcoding. For example, the second form of barcoding may be a second array of barcoded nucleic acids. Alternatively, the second form of barcoding may be multiple wells, wherein each well comprises a unique barcode. The barcode from the second form of barcoding can then be incorporated into the barcoded RNA or cDNA using methods known in the art and described herein. The combination of barcodes (from the first array of barcoded nucleic acids and the second form of barcoding) allows for the assignment of a unique barcode signature for each cell.

[0168] The methods of the present application find use in high-throughput drug screening. For example, in the embodiments wherein wells of multi-well plates include an array of barcoded nucleic acids, each well of the plate can be treated with a different drug and the barcoding thus defines not only the individual cells but the drug treatment / sample.

[0169] The methods of the present application are compatible with various additional assays, allowing for high-throughput RNA-Seq, protein detection, CUT & Tag, ATAC- Seq or combinations thereof. This enables high-throughput analysis of RNA, DNA, protein and combinations thereof. In the case of RNA-Seq, 3’end capture or full-length RNA sequencing can be carried out. For example, polyA capture and / or template switching can be used to obtain full length cDNA. Random priming of RNA or gene specific targeting is also possible, by changing the sequence of the capture domain of the barcoded nucleic acid. Full-length molecules can be sequenced using long-read methods such as Oxford Nanopore Technologies (ONT) or Pacific Biosciences (PacBio).

[0170] The methods of the present application find use in sequencing immune receptors such as T cells or B cells. In embodiments wherein the barcoded nucleic acid is a TSO, barcoded nucleic acid products may include a barcode on the 5’ end of the nucleic acid Atty. Docket No.: CLON-185WO product. 5’ end barcode labeling may find use in immune receptor analysis (e.g., T cell receptor (TOR) analysis and B cell receptor (BCR) analysis).

[0171] The methods of the present application are compatible with a wide variety of cells, including, e.g., fixed cells and live cells. As described previously, cells may be labeled with specific binding members that include a target barcode. Specific binding members may be used that bind to any protein (intracellular, extracellular, membrane- associated, etc.) that may be desired. As such, specific proteins or epitopes or combinations of specific proteins or epitopes can be associated with a specific barcode, where the specific barcode may also be associated with other nucleic acids.

[0172] Further, the methods of the present application provide localization information of barcoded cells. As such, assessment of cell :cell interactions is possible since barcodes can be traced back to a location of origin. Thereby, multi-omic single-cell analysis can be carried out in relation to the spatial relationships of cells.

[0173] A prepared library may be utilized in various downstream analyses and, in some instances, the preparation of the library may be specifically reconfigured for a desired type of downstream analysis. For example, in some instances, a prepared library may be subjected to whole transcriptome analysis (WTA) that includes analysis of mRNA as well as non-mRNA RNA species such as non-coding RNA (e.g., Long noncoding RNAs (IncRNAs), non-poly adenylated RNAs, snRNA and snoRNA). Therefore, in some instances, library preparation may be specifically configured to allow for analysis of non- mRNA RNAs within the transcriptome, e.g., by utilizing primers that do not rely on hybridization to the poly(A) tail (e.g., random primers) or by the addition of a tailing reaction, e.g., by adding a poly(A) tail to RNA species that are not naturally polyadenylated prior to production of product double stranded cDNA.

[0174] In some instances, preparation of a library, e.g., a library for WTA, may include a step of reducing the amount of ribosomal RNAs within the sample and / or library. This may be done either with the original cell source template nucleic acids before any indexing step of the technology, e.g., using a RiboGone™ product (Takara Bio USA Inc., San Jose, CA) or post-generation of indexed fragments (e.g., ZapR technology, for example as described in U.S. Patent No. 10,150,985), for example, after any indexing step including at the end of all indexing steps prior to sequencing. Any convenient Atty. Docket No.: CLON-185WO method of reducing and / or removing unwanted ribosomal RNAs may be employed for selective removal, including using affinity purification, degradation of the contaminating nucleic acid (e.g., using a RiboGone™ product (Takara Bio USA Inc., San Jose, GA) and those methods described in U.S. Patent Nos. 9,428,794 and 10,150,985, the disclosures of which are incorporated herein by reference in their entirety), combinations thereof, and the like.

[0175] In certain embodiments, a prepared library may be utilized in a differential expression analysis, including determining the relative expression (i.e. , the up or down regulation) of one or more genes. Differential expression may be qualitatively or quantitatively determined, and such analyses may be transcriptome wide or may be targeted. As such, the number of expressed transcripts evaluated in a subject differential expression analysis will vary. A differential expression analysis as used herein is not limited in regard to the number of expressed transcripts that are analyzed in a subject genome. In some embodiments, a differential expression analysis may evaluate a limited number transcripts such as a panel of marker genes specifically targeted for analysis. Alternatively, the entire transcribed content of the cell may be assessed for differential expression.

[0176] Transcript categories to which a targeted expression analysis may be limited will vary and may include but not be limited to e.g., immune gene transcripts, such as cytokines, chemokines or cell surface markers of immune cell subsets, kinases, G- protein coupled receptors, druggable genes, and others. Useful categories and subcategories of immune genes generally include those groups of genes responsible for functioning of the immune system and the successful defense against pathogens, including but not limited to e.g., those genes associated with immune system process (such as the genes identified by gene ontology (GO) accession number G0:0002376 (available online at geneontology(dot)org) including but not limited to e.g., those genes associated with B cell mediated immunity, B cell selection, T cell mediated immunity, T cell selection, activation of immune response, antigen processing and presentation, antigen sampling in mucosal-associated lymphoid tissue, basophil mediated immunity, eosinophil mediated immunity, hemocyte differentiation, hemocyte proliferation, immune effector process, immune response, immune system development, immunological Atty. Docket No.: CLON-185WO memory process, leukocyte activation, leukocyte homeostasis, leukocyte mediated immunity, leukocyte migration, lymphocyte co-stimulation, lymphocyte mediated immunity, mast cell mediated immunity, myeloid cell homeostasis, myeloid leukocyte mediated immunity, natural killer cell mediated immunity, negative regulation of immune system process, neutrophil mediated immunity, positive regulation of immune system process, production of molecular mediator of immune response, regulation of immune system process, somatic diversification of immune receptors, tolerance induction, embryo development (e.g., blastocyst implantation and development), and the like. Specific genes of interest include, but are not limited to: cytokines, interleukins, interleukin receptors, CD4, CD8, CD3, PD-1 , etc.

[0177] Kits, Compositions and Devices

[0178] Aspects of the present disclosure also include compositions and kits as well as devices for use therewith or therein.

[0179] Most generally, the term "kit" is used to describe any assemblage of articles that facilitate the execution of a process, method, assay, analysis, manipulation of a sample, or the like. Kits can contain written instructions describing how to use the kit (e.g., instructions describing the methods of the present technology), chemical reagents or enzymes required for the method, primers, probes, buffer solutions, any type of containers (for example, containers for sample collection or sample manipulation) or reaction vessels, or any other components. A kit need not contain every component necessary to execute a method of the technology. The compositions and kits of the technology may include, e.g., one or more of any of the reaction components described above with respect to the subject methods.

[0180] In some embodiments, kits of the technology include a barcoded nucleic acid array comprising distinct populations of barcoded nucleic acids stably associated with different locations of a solid support, where the barcoded nucleic acid array is configured to be contacted with a sample of dispersed cells. In some embodiments, the kit further includes one or more reverse transcription reagents. In some embodiments, the solid support includes a planar solid support. In some embodiments, the distinct populations of barcoded nucleic acids are separated by about 1 -100 pm. In some Atty. Docket No.: CLON-185WO embodiments, the distinct barcoded nucleic acids are separated by 1 -5 pm, 5-10 pm, 10-15 pm, 15-20 pm, 20-25 pm, 25-30 pm, 30-35 pm, 35-40 pm, 40-45 pm, 45-50 pm, 50-55 pm, 55-60 pm, 60-65 pm, 65-70 pm, 70-75 pm, 75-80 pm, 80-85 pm, 85-90 pm, 90-95 pm, or 95-100 pm. In some cases, different locations (wherein each location includes a population of barcoded nucleic acids) may abut each other. In some cases, different locations (wherein each location includes a population of barcoded nucleic acids) may be separated by an intervening region. Said intervening region may be free of nucleic acids and / or configured to impede cell adherence. The solid support may be configured to impede cell adherence in the intervening regions of the array wherein there are no barcoded nucleic acids. This can include using an anti-adherence coating.

[0181] In some embodiments, the solid support includes a multi-well array or a bead array. In some embodiments, the different locations of the solid support further includes a transfection reagent (e.g. a transfection reagent as discussed above).

[0182] In embodiments, the barcoded nucleic acids include a barcode domain. In some embodiments, the barcoded nucleic acids further include a cleavage domain. The cleavage domain may be cleaved by an enzymatic stimulus, a chemical stimulus or an electromagnetic stimulus. In some cases, the cleavable domain functions as a cleavable linker, wherein the cleavable linker connects the barcoded nucleic acid to a solid support. In some cases, the cleavable linker comprises a photocleavable linker. In some embodiments, the barcoded nucleic acids further include a capture domain. In some embodiments, the capture domain specifically binds to a ribonucleic acid. In some embodiments, the capture domain includes a polyT sequence, a gene-specific sequence or a random sequence. In some embodiments, the capture domain binds to an exogeneous nucleic acid (e.g., an exogenous nucleic acid conjugated to a specific binding member) added to cells of the sample. In some embodiments, the capture domain binds to a transposon, a bridge oligonucleotide or a cDNA. In some embodiments, the barcoded nucleic acids further include an adapter domain. In some cases, the adapter domain is a sequencing adapter domain. In some cases, the sequencing adapter domain includes any one of SEQ ID NOs: 01 -12. In some embodiments, the barcoded nucleic acids further include a second barcode domain, a third barcode domain, a fourth barcode domain, a fifth barcode domain and / or a sixth Atty. Docket No.: CLON-185WO barcode domain. Barcoded nucleic acids may be attached to a cell uptake moiety (e.g., a CPP, including those discussed above).

[0183] In some embodiments, the distinct populations of barcoded nucleic acids differ by one or more of: the barcode domain, a cleavage domain, a capture domain and an adapter domain. In some embodiments, the distinct populations of barcoded nucleic acids are functionally distinct populations of barcoded nucleic acids that differ by one or more of: a cleavage domain and a capture domain. In some embodiments, the array of barcoded nucleic acids includes a first functionally distinct population of barcoded nucleic acids comprising a first capture domain and a first cleavage domain and a second functionally distinct population of barcoded nucleic acids comprising a second capture domain and a second cleavage domain. In such embodiments, the first capture domain may be different from or the same as the second capture domain. In such embodiments, the first cleavage domain may be different from or the same as the second cleavage domain. In some embodiments, each location of the different locations of the array comprises a population of barcoded nucleic acids with a unique barcode. In some embodiments, a location of the array comprises two or more functionally distinct populations of barcoded nucleic acids.

[0184] The kits may further include one or more additional reagents employed in embodiments of the technology, such as one or more polymerases (e.g., a template switching polymerase, a reverse transcriptase, an amplification polymerase, etc.), ligases, transposases, primers, buffers, dNTPs (including e.g., dATP, dCTP, dGTP, dTTP, dllTP, etc. or any one or any combination thereof), and the like. The subject kits may include, or the compositions and devices may be provided with, one or more test reagents, including control nucleic acids (e.g., control nucleic acid templates), and the like. In some instances, the reagents may be provided in lyophilized form, such as lyophilized enzymes, lyophilized reverse transcriptase, lyophilized DNA polymerase, etc. In some embodiments, the kit further includes one or more reverse transcription reagents. In some cases, the one or more reverse transcription reagents include a reverse transcriptase.

[0185] Compositions of the present technology include compositions comprising an array of barcoded nucleic acids including distinct populations of barcoded nucleic acids Atty. Docket No.: CLON-185WO stably associated with different locations of a solid support contacted with a sample of dispersed cells. Barcoded nucleic acids and arrays of barcoded nucleic acids include those described above. Samples of dispersed cells include those described above. In some instances, the sample of dispersed cells includes live cells. In some instances, the sample of dispersed cells includes fixed cells. In some cases, the fixed cells are permeabilized. In some embodiments, the dispersed cells have a confluence ranging from 25 to 100% (including, e.g., 25 to 75% and 75 to 100%). In some instances, the different locations of the solid support abut each other. In other instances, the different locations of the solid support are separated from each other by an intervening region (e.g., an intervening region free of nucleic acids). In some instances, the intervening region of the solid support is configured to impede cell adherence (e.g., by comprising an anti-adherence coating). In some embodiments, the solid support includes a multiwell array or a bead array.

[0186] In some instances, components of the subject compositions and / or kits may be presented as a “cocktail” where, as used herein, a cocktail refers to a collection or combination of two or more different but similar components in a single vessel. Components of the kits may be present in separate containers, or multiple components may be present in a single container, as desired. The subject compositions may be present in any suitable environment. According to one embodiment, the composition is present in a reaction tube (e.g., a 0.2 ml_ tube, a 0.6 ml_ tube, a 1 .5 ml_ tube, or the like) or a well or microfluidic chamber or droplet or other suitable container. In certain aspects, the composition is present in two or more (e.g., a plurality of) reaction tubes or wells (e.g., a plate, such as a 96-well plate, a multi-well plate, e.g., containing about 1000, 5000, or 10,000 or more wells). The tubes and / or plates may be made of any suitable material, e.g., polypropylene, or the like, PDMS, or aluminum. The containers may also be treated to reduce adsorption of nucleic acids to the walls of the container. In certain aspects, the tubes and / or plates in which the composition is present provide for efficient heat transfer to the composition (e.g., when placed in a heat block, water bath, thermocycler, and / or the like), so that the temperature of the composition may be altered within a short period of time, e.g., as necessary for a particular enzymatic reaction to occur. According to certain embodiments, the composition is present in a Atty. Docket No.: CLON-185WO thin-walled polypropylene tube, or a plate having thin-walled polypropylene wells or materials such as aluminum having high heat conductance.

[0187] In some instances, the components or reagents of the kit may be provided in a collection of individual vessels (e.g., separate tubes) or multiple vessels, e.g., a multiwell device, in which the components or reagents may be provided in liquid or dried form.

[0188] Any suitable reaction vessel(s) may be employed in the subject kits or devices and / or to contain a subject composition. Useful reaction vessels include but are not limited to e.g., tubes (e.g., single tubes, multi-tube strips, etc.), wells (e.g., of a multi-well plate (e.g., a 96-well plate, 384 well plate, or a plate with any number of wells such as 2000, 4000, 6000, or 10000 or more). Multi-well plates may be independent or may be part of a chip and / or device, e.g., as described in greater detail below. As such, in certain embodiments, the reaction vessel employed is a well or wells of a multi-well device. The present disclosure is not limited by the type of multi-well devices (e.g., plates or chips) employed. In general, such devices have a plurality of wells that contain, or are dimensioned to contain, liquid (e.g., liquid that is trapped in the wells such that gravity alone cannot make the liquid flow out of the wells). One exemplary chip is the 5184-well SMARTCHIP™ (Takara Bio USA, San Jose CA). Other exemplary chips are provided in U.S. Patents 8,252,581 ; 7,833,709; and 7,547,556, all of which are herein incorporated by reference in their entireties including, for example, for the teaching of chips, wells, thermocycling conditions, and associated reagents used therein). Other exemplary chips include the OPENARRAY™ plates used in the QUANTSTUDIO™ real-time PCR system (sold by Applied Biosystems). Another exemplary multi-well device is a 96-well or 384-well plate.

[0189] In addition to the above-mentioned components, a subject kit may further include instructions for using the components of the kit, e.g., to practice the subject methods as described above. The instructions are generally recorded on a suitable recording medium. The instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e. , associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic Atty. Docket No.: CLON-185WO storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, CD-ROM, diskette, Hard Disk Drive (HDD) etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0190] As can be appreciated from the disclosure provided above, embodiments of the present disclosure have a wide variety of applications. Accordingly, the examples presented herein are offered for illustration purposes and are not intended to be construed as a limitation on the embodiments of the present disclosure in any way. Those of ordinary skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. Thus, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0191] Examples

[0192] Example 1 : Barcoding a Sample of Live Dispersed Cells

[0193] K562 cells were cultured using standard cell culture techniques. The cells were washed 3 times using 1X saline-sodium citrate (SSC) buffer. Cells were resuspended in 1 X SSC at a concentration of about 4-5M / mL.

[0194] A cleavage buffer was prepared using 1 .5 pL of SSC buffer, 18.5 pL cells, 9 pL of 0.4% Trypan blue, and 1 pL water (total 30 pL volume). The cleavage buffer with cells Atty. Docket No.: CLON-185WO was pipetted onto an array of barcoded nucleic acids comprising a barcode domain and a capture domain comprising a degenerate sequence. About 74,000 cells were added to the array. The barcoded nucleic acids were attached to the substrate via a cleavage domain that is cleaved following an electromagnetic stimulus (i.e., a photocleavable linker). The array was incubated on ice for 5 minutes.

[0195] After incubation, the array was irradiated using an ultraviolet (UV) lamp at 260 nm for 60 seconds. The array was incubated on ice for another 15 minutes. After incubation, the cells were carefully extracted from the array by pipetting up and down and transferred to a clean tube with 1 mL 1 X SSC. Cells were pelleted via centrifugation and re-suspended in 500 pL of 1 X PBS.

[0196] Cells were distributed into nanowells of an ICELL8® chip using the ICELL8® ex Single Cell System (available from Takara Bio USA, San Jose CA). Libraries were prepared per manufacturer protocols. Briefly, cells were lysed in nanowells and the barcoded nucleic acids from the array were used as a primer for reverse transcription. The resultant cDNA was amplified using PCR, at which stage i5 / i7 barcodes were added to the amplicons. Each nanowell had a unique i5 and i7 combination. The amplified cDNA was pooled into one tube and purified using NucleoMag® NGS Clean- Up and Size Select (available from Takara Bio USA, San Jose CA). Amplified cDNA derived from ribosomal RNA was depleted from the sample using ZapR® ribosomal depletion (available from Takara Bio USA, San Jose CA). After the depletion step, the library was amplified to generate the sequencing library. The sequencing library was sequenced on a NextSeq® 500 (available from Illumina, San Diego CA). Sequencing reads were de-multiplexed by i5 / i7 index combinations to obtain single-cell information. Of the 689 cells that were sequenced, 89.6% of said cells contained a spatial barcode from the barcoded nucleic acid. This demonstrates that the barcoded nucleic acid can function as a primer for reverse transcription and incorporate spatial information into the nucleic acids of the cells applied to the array. Fig. 7A shows spatial barcode reads for six representative single cells, demonstrating that the spatial barcodes are affording single cell resolution. Approximately 382 genes (exon+intron) were detected at a sequencing depth of >70k reads per cell. Atty. Docket No.: CLON-185WO

[0197] Next, the addition of a cell penetrating peptide (OPP) was used to determine its effect on enhancing barcoded nucleic acid delivery. Cells were prepared and applied to the array of barcoded nucleic acids as described above, with the addition of 1 mM R9 CPP to the cleavage buffer (polyarginine 9R, available from GenScript). Sequencing libraries were prepared as described above. Of the 681 cells that were sequenced, 95.4% of said cells contained a spatial barcode from the barcoded nucleic acid. This demonstrates that the addition of CPP to the cleavage buffer enhanced delivery of the barcoded nucleic acids as compared to cleavage buffer without CPPs. Fig. 7B shows spatial barcode reads for six representative single cells demonstrating the spatial barcodes are affording single cell resolution (similar to the samples without CPPs). Approximately 482 genes (exon+intron) were detected at a sequencing depth of >60k reads per cell. Therefore, the addition of CPPs to the cleavage buffer not only increased the delivery of barcoded nucleic acids to the cells, but also resulted in significantly more genes being detected as shown in Table 2.

[0198] A summary of the spatial barcode incorporation into single cells with and without CPP treatment is provided in Table 1. The spatial reads ratio is determined as the ratio of reads for a given cell that contain a spatial barcode from a barcoded nucleic acid of the present technology.

[0199] Table 1 : Impact of CPP Treatment on Barcoded Nucleic Acid Delivery

[0200] A summary of the sequencing results is provided in Table 2.

[0201] Table 2: Sequencing Data with or without CPP Treatment Atty. Docket No.: CLON-185WO

[0202] Although the foregoing technology has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this technology that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0203] Accordingly, the preceding merely illustrates the principles of the technology. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the technology and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the technology and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the technology as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e. , any elements developed that perform the same function, regardless of structure. The scope of the present technology, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present technology is embodied by the appended claims.

Claims

Atty. Docket No.: CLON-185WOWHAT IS CLAIMED IS:1 . A method of barcoding cells of a sample of dispersed cells, the method comprising: contacting the sample with an array of barcoded nucleic acids such that barcoded nucleic acids of the array enter cells of the sample to barcode cells of the sample, wherein the array of barcoded nucleic acids comprises a plurality of barcoded nucleic acids, wherein each barcoded nucleic acid of the plurality of barcoded nucleic acids comprises a barcode domain.

2. The method according to Claim 1 , wherein the sample of dispersed cells comprises dispersed live cells.

3. The method according to Claim 2, wherein the method further comprises culturing the dispersed live cells contacted with the array of barcoded nucleic acids.

4. The method according to Claim 1 , wherein the sample of dispersed cells comprises fixed cells.

5. The method according to Claim 4, wherein the fixed cells are permeabilized.

6. The method according to any one of Claims 1 to 5, wherein the barcoded nucleic acids further comprise a cleavage domain.

7. The method according to Claim 6, wherein the cleavage domain is cleaved by an enzymatic stimulus, a chemical stimulus or an electromagnetic stimulus.

8. The method according to Claims 6 or 7, wherein the cleavable domain functions as a cleavable linker, wherein the cleavable linker connects the barcoded nucleic acid to a solid support.Atty. Docket No.: CLON-185WO9. The method according to Claim 8, wherein the cleavable linker comprises a photocleavable linker.

10. The method according to any one of Claims 1 to 9, wherein the barcoded nucleic acids further comprise a capture domain.11 . The method according to Claim 10, wherein the capture domain specifically binds to a ribonucleic acid.

12. The method according to Claim 11 , wherein the capture domain comprises a polyT sequence, a gene-specific sequence or a random sequence.

13. The method according to Claim 10, wherein the capture domain binds to an exogeneous nucleic acid added to cells of the sample.

14. The method according to Claim 13, wherein the exogeneous nucleic acid is conjugated to a specific binding member.

15. The method according to Claim 10, wherein the capture domain binds to a transposon, a bridge oligonucleotide or a cDNA.

16. The method according to any one of Claims 1 to 15, wherein the barcoded nucleic acids further comprise an adapter domain.

17. The method according to Claim 16, wherein the adapter domain is a sequencing adapter domain.

18. The method according to Claims 15 or 16, wherein the sequencing adapter domain comprises any one of SEQ ID NOs: 01 -12.Atty. Docket No.: CLON-185WO19. The method according to any of Claims 1 to 18, wherein the barcoded nucleic acids further comprise a second barcode domain.

20. The method according to Claim 19, wherein the barcoded nucleic acids further comprise a third barcode domain.21 . The method according to Claim 20, wherein the barcoded nucleic acids further comprise a fourth barcode domain.

22. The method according to Claim 21 , wherein the barcoded nucleic acids further comprise a fifth barcode domain.

23. The method according to Claim 22, wherein the barcoded nucleic acids further comprise a sixth barcode domain.

24. The method according to any one of Claims 1 to 23, wherein the array of barcoded nucleic acids comprises distinct populations of barcoded nucleic acids.

25. The method according to Claim 24, wherein the distinct populations of barcoded nucleic acids differ by one or more of: the barcode domain, a cleavage domain, a capture domain and an adapter domain.

26. The method according to Claims 24 or 25, wherein the distinct populations of barcoded nucleic acids are functionally distinct populations of barcoded nucleic acids that differ by one or more of: a cleavage domain and a capture domain.

27. The method according to Claim 26, wherein the array of barcoded nucleic acids comprises: a first functionally distinct population of barcoded nucleic acids comprising a first capture domain and a first cleavage domain; andAtty. Docket No.: CLON-185WO a second functionally distinct population of barcoded nucleic acids comprising a second capture domain and a second cleavage domain.

28. The method according to Claim 27, wherein the first capture domain is different from the second capture domain.

29. The method according to Claims 27 or 28, wherein the first cleavage domain is different from the second cleavage domain.

30. The method according to any one of Claims 24 to 29, wherein the distinct populations of barcoded nucleic acids stably associated with different locations of a solid support.31 . The method according to Claim 30, wherein each location of the different locations of the array comprises a population of barcoded nucleic acids with a unique barcode.

32. The method according to Claims 30 or 31 , wherein a location of the array comprises two or more functionally distinct populations of barcoded nucleic acids.

33. The method according to any one of Claims 30 to 32, wherein the solid support comprises a planar solid support.

34. The method according to any one of Claims 30 to 33, wherein the different locations abut each other.

35. The method according to any one of Claims 30 to 34, wherein the different locations are separated from each other by an intervening region.

36. The method according to Claim 35, wherein the intervening region is free of nucleic acids.Atty. Docket No.: CLON-185WO37. The method according to Claims 35 or 36, wherein the intervening region is configured to impede cell adherence.

38. The method according to Claim 37, wherein intervening region comprises an anti-adherence coating.

39. The method according to any one of Claims 30 to 38, wherein the solid support comprises a multi-well array.

40. The method according to any one of Claims 30 to 38, wherein the solid support comprises a bead array.41 . The method according to any one of Claims 1 to 40, wherein the sample has a cell concentration sufficient to provide upon contact with the array a confluence ranging from 25 to 100%.

42. The method according to Claim 41 , wherein the sample has a cell concentration sufficient to provide a confluence ranging from 25 to 75%.

43. The method according to Claim 41 , wherein the sample has a cell concentration sufficient to provide a confluence ranging from 75 to 100%.

44. The method according to any one of Claims 1 to 43, wherein the barcoded nucleic acids enter the cells by transfection.

45. The method according to Claim 44, wherein the transfection is mediated by a transfection agent.

46. The method according to Claim 45, wherein the transfection agent is a nanoparticle agent.Atty. Docket No.: CLON-185WO47. The method according to Claim 46, wherein the transfection is mediated by a physical stimulus.

48. The method according to Claim 47, wherein the physical stimulus comprises electroporation.

49. The method according to any one of Claims 1 to 48, wherein the barcoded nucleic acids are attached to a cell uptake moiety that allows for the barcoded nucleic acids to enter the cells.

50. The method according to Claim 49, wherein the cell uptake moiety is a cell penetrating peptide (CPF).51 . The method according to Claim 50, wherein the CPP comprises transportan, TP10, penetratin, Tat or a series of arginine residues.

52. The method according to Claim 51 , wherein the CPP comprises nine arginine residues.

53. The method according to any one of Claims 1 to 52, wherein the barcoded nucleic acid functions as a primer.

54. The method according to any one of Claims 1 to 52, wherein the barcoded nucleic acid functions as a template switch oligonucleotide.

55. The method according to any of Claims 1 to 52, wherein the barcoded nucleic acid functions as a template nucleic acid.Atty. Docket No.: CLON-185WO56. The method according to any one of Claims 1 to 55, wherein the method further comprises employing the barcode domain of a barcoded cell to determine a location of the array contacted by the barcoded cell.

57. The method according to any one of Claims 1 to 56, further comprising pooling the barcoded cells or the nucleic acids from the barcoded cells.

58. The method according to any one of Claims 1 to 57, further comprising preparing a sequencing library from the barcoded cells.

59. The method according to Claim 58, wherein the method further comprises sequencing the sequencing library.

60. The method according to any one of Claims 1 to 59, wherein the sample of dispersed cells is not derived from a tissue or tissue section.61 . The method according to any one of Claims 1 to 60, wherein the sample of dispersed cells comprises one or more of: immune cells, neuronal cells, cardiac cells, endothelial cells, fibroblasts, liver cells and tumor cells.

62. A kit comprising: an array of barcoded nucleic acids comprising distinct populations of barcoded nucleic acids stably associated with different locations of a solid support, wherein the barcoded nucleic acid array is configured to be contacted with a sample of dispersed cells, and wherein each barcoded nucleic acid of the barcoded nucleic acid array comprises a barcode domain.

63. The kit according to Claim 62, wherein the kit further comprises one or more reverse transcription reagents.Atty. Docket No.: CLON-185WO64. The kit according to Claim 63, wherein the one or more reverse transcription reagents include a reverse transcriptase.

65. The kit according to any one of Claims 62 to 64, wherein the solid support comprises a planar solid support.

66. The kit according to any one of Claims 62 to 65, wherein the different locations abut each other.

67. The kit according to any one of Claims 62 to 65, wherein the different locations are separated from each other by an intervening region.

68. The kit according to Claim 67, wherein the intervening region is free of nucleic acids.

69. The kit according to Claims 67 or 68, wherein the intervening region is configured to impede cell adherence.

70. The kit according to Claim 69, wherein intervening region comprises an antiadherence coating.71 . The kit according to any one of Claims 62 to 70, wherein the solid support comprises a multi-well array.

72. The kit according to any one of Claims 62 to 70, wherein the solid support comprises a bead array.

73. The kit according to any one of Claims 62 to 72, wherein the barcoded nucleic acids further comprise a cleavage domain.Atty. Docket No.: CLON-185WO74. The kit according to Claim 73, wherein the cleavage domain is cleaved by an enzymatic stimulus, a chemical stimulus or an electromagnetic stimulus.

75. The kit according to Claims 73 or 74, wherein the cleavable domain functions as a cleavable linker, wherein the cleavable linker connects the barcoded nucleic acid to a solid support.

76. The kit according to Claim 75, wherein the cleavable linker comprises a photocleavable linker.

77. The kit according to any one of Claims 62 to 76, wherein the barcoded nucleic acids further comprise a capture domain.

78. The kit according to Claim 77, wherein the capture domain specifically binds to a ribonucleic acid.

79. The kit according to Claim 78, wherein the capture domain comprises a polyT sequence, a gene-specific sequence or a random sequence.

80. The kit according to Claim 77, wherein the capture domain binds to an exogeneous nucleic acid added to cells of the sample.81 . The kit according to Claim 80, wherein the exogeneous nucleic acid is conjugated to a specific binding member.

82. The kit according to Claim 77, wherein the capture domain binds to a transposon, a bridge oligonucleotide or a cDNA.

83. The kit according to any one of Claims 62 to 82, wherein the barcoded nucleic acids further comprise an adapter domain.Atty. Docket No.: CLON-185WO84. The kit according to Claim 83, wherein the adapter domain is a sequencing adapter domain.

85. The kit according to Claims 83 or 84, wherein the sequencing adapter domain comprises any one of SEQ ID NOs: 01 -12.

86. The kit according to any of Claims 62 to 85, wherein the barcoded nucleic acids further comprise a second barcode domain.

87. The kit according to Claim 86, wherein the barcoded nucleic acids further comprise a third barcode domain.

88. The kit according to Claim 87, wherein the barcoded nucleic acids further comprise a fourth barcode domain.

89. The kit according to Claim 88, wherein the barcoded nucleic acids further comprise a fifth barcode domain.

90. The kit according to Claim 89, wherein the barcoded nucleic acids further comprise a sixth barcode domain.91 . The kit according to any one of Claims 62 to 90, wherein the distinct populations of barcoded nucleic acids differ by one or more of: the barcode domain, a cleavage domain, a capture domain and an adapter domain.

92. The kit according to Claim 91 , wherein the distinct populations of barcoded nucleic acids are functionally distinct populations of barcoded nucleic acids that differ by one or more of: a cleavage domain and a capture domain.

93. The kit according to Claim 92, wherein the array of barcoded nucleic acids comprises:Atty. Docket No.: CLON-185WO a first functionally distinct population of barcoded nucleic acids comprising a first capture domain and a first cleavage domain; and a second functionally distinct population of barcoded nucleic acids comprising a second capture domain and a second cleavage domain.

94. The kit according to Claim 93, wherein the first capture domain is different from the second capture domain.

95. The kit according to Claims 93 or 94, wherein the first cleavage domain is different from the second cleavage domain.

96. The kit according to any one of Claims 62 to 95, wherein each location of the different locations of the array comprises a population of barcoded nucleic acids with a unique barcode.

97. The kit according to any one of Claims 62 to 96, wherein a location of the array comprises two or more functionally distinct populations of barcoded nucleic acids.

98. The kit according to any one of Claims 62 to 97, wherein the different locations further comprise a transfection agent.

99. The kit according to any of Claims 62 to 98, wherein the barcoded nucleic acids are attached to a cell uptake moiety that allows for the barcoded nucleic acids to enter the cells.

100. The kit according to Claim 99, wherein the cell uptake moiety is a cell penetrating peptide (CPP).101 . The kit according to Claim 100, wherein the CPP comprises transportan, TP10, penetratin, Tat or a series of arginine residues.Atty. Docket No.: CLON-185WO102. The kit according to Claim 101 , wherein the CPP comprises nine arginine residues.

103. The kit according to any of Claims 62 to 102, wherein the barcoded nucleic acid functions as a primer.

104. The kit according to any of Claims 62 to 102, wherein the barcoded nucleic acid functions as a template switch oligonucleotide.

105. The kit according to any of Claims 62 to 102, wherein the barcoded nucleic acid functions as a template nucleic acid.

106. A composition comprising: an array of barcoded nucleic acids comprising distinct populations of barcoded nucleic acids stably associated with different locations of a solid support contacted with a sample of dispersed cells, and wherein each barcoded nucleic acid of the barcoded nucleic acid array comprises a barcode domain.

107. The composition according to Claim 106, wherein the sample of dispersed cells comprises dispersed live cells.

108. The composition according to Claim 106, wherein the sample of dispersed cells comprises fixed cells.

109. The composition according to Claim 108, wherein the fixed cells are permeabilized.

110. The composition according to any one of Claims 106 to 109, wherein the barcoded nucleic acids further comprise a cleavage domain.Atty. Docket No.: CLON-185WO111. The composition according to Claim 110, wherein the cleavage domain is cleaved by an enzymatic stimulus, a chemical stimulus or an electromagnetic stimulus.

112. The composition according to Claims 110 or 111 , wherein the cleavable domain functions as a cleavable linker, wherein the cleavable linker connects the barcoded nucleic acid to a solid support.

113. The composition according to Claim 112, wherein the cleavable linker comprises a photocleavable linker.

114. The composition according to any one of Claims 106 to 113, wherein the barcoded nucleic acids further comprise a capture domain.

115. The composition according to Claim 114, wherein the capture domain specifically binds to a ribonucleic acid.

116. The composition according to Claim 115, wherein the capture domain comprises a polyT sequence, a gene-specific sequence or a random sequence.

117. The composition according to Claim 114, wherein the capture domain binds to an exogeneous nucleic acid added to cells of the sample.

118. The composition according to Claim 117, wherein the exogeneous nucleic acid is conjugated to a specific binding member.

119. The composition according to Claim 114, wherein the capture domain binds to a transposon, a bridge oligonucleotide or a cDNA.

120. The composition according to any one of Claims 106 to 119, wherein the barcoded nucleic acids further comprise an adapter domain.Atty. Docket No.: CLON-185WO121 . The composition according to Claim 120, wherein the adapter domain is a sequencing adapter domain.

122. The composition according to Claims 120 or 121 , wherein the sequencing adapter domain comprises any one of SEQ ID NOs: 01 -12.

123. The composition according to any of Claims 106 to 119, wherein the barcoded nucleic acids further comprise a second barcode domain.

124. The composition according to Claim 123, wherein the barcoded nucleic acids further comprise a third barcode domain.

125. The composition according to Claim 124, wherein the barcoded nucleic acids further comprise a fourth barcode domain.

126. The composition according to Claim 125, wherein the barcoded nucleic acids further comprise a fifth barcode domain.

127. The composition according to Claim 126, wherein the barcoded nucleic acids further comprise a sixth barcode domain.

128. The composition according to any one of Claims 106 to 127, wherein the distinct populations of barcoded nucleic acids differ by one or more of: the barcode domain, a cleavage domain, a capture domain and an adapter domain.

129. The composition according to Claim 128, wherein the distinct populations of barcoded nucleic acids are functionally distinct populations of barcoded nucleic acids that differ by one or more of: a cleavage domain and a capture domain.Atty. Docket No.: CLON-185WO130. The composition according to Claim 129, wherein the array of barcoded nucleic acids comprises: a first functionally distinct population of barcoded nucleic acids comprising a first capture domain and a first cleavage domain; and a second functionally distinct population of barcoded nucleic acids comprising a second capture domain and a second cleavage domain.131 . The composition of Claim 130, wherein the first capture domain is different from the second capture domain.

132. The composition of Claims 130 or 131 , wherein the first cleavage domain is different from the second cleavage domain.

133. The composition according to any one of Claims 106 to 132, wherein each location of the different locations of the array comprises a population of barcoded nucleic acids with a unique barcode.

134. The composition according to any one of Claims 106 to 133, wherein a location of the array comprises two or more functionally distinct populations of barcoded nucleic acids.

135. The composition according to any one of Claims 106 to 134, wherein the solid support comprises a planar solid support.

136. The composition according to any one of Claims 106 to 135, wherein the different locations abut each other.

137. The composition according to any one of Claims 106 to 135, wherein the different locations are separated from each other by an intervening region.Atty. Docket No.: CLON-185WO138. The composition according to Claim 137, wherein the intervening region is free of nucleic acids.

139. The composition according to Claims 137 or 138, wherein the intervening region is configured to impede cell adherence.

140. The composition according to Claim 139, wherein intervening region comprises an anti-adherence coating.141 . The composition according to any one of Claims 106 to 140, wherein the solid support comprises a multi-well array.

142. The composition according to any one of Claims 106 to 140, wherein the solid support comprises a bead array.

143. The composition according to any one of Claims 106 to 142, wherein the sample has a cell concentration sufficient to provide upon contact with the array a confluence ranging from 25 to 100%.

144. The composition according to Claim 143, wherein the sample has a cell concentration sufficient to provide a confluence ranging from 25 to 75%.

145. The composition according to Claim 143, wherein the sample has a cell concentration sufficient to provide a confluence ranging from 75 to 100%.

146. The composition according to any one of Claims 106 to 145, wherein the different locations further comprise a transfection agent.

147. The composition according to any one of Claims 106 to 146, wherein the barcoded nucleic acids are attached to a cell uptake moiety that allows for the barcoded nucleic acids to enter the cells.Atty. Docket No.: CLON-185WO148. The composition according to Claim 147, wherein the cell uptake moiety is a cell penetrating peptide (CPP).

149. The composition according to Claim 148, wherein the CPP comprises transportan, TP10, penetratin, Tat or a series of arginine residues.

150. The composition according to Claim 149, wherein the CPP comprises nine arginine residues.151 . The composition according to any one of Claims 106 to 150, wherein the sample of dispersed cells is not derived from a tissue or tissue section.

152. The composition according to any one of Claims 106 to 151 , wherein the sample of dispersed cells comprises one or more of: immune cells, neuronal cells, cardiac cells, endothelial cells, fibroblasts, liver cells and tumor cells.

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