Combinatorial indexing for single-cell nucleic acid sequencing

Abstract

The present disclosure provides methods for combinatorial indexing of nucleic acids of single cells or nuclei. In particular, the methods of the present disclosure result in the incorporation of at least two index sequences within the nucleic acids of single cells or nuclei. The present disclosure further provides methods for generating and sequencing libraries of such indexed nucleic acids.

Description

【Technical Field】 【0001】 Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 349,184, filed Jun. 6, 2022; U.S. Provisional Application No. 63 / 441,744, filed Jan. 27, 2023; and U.S. Provisional Application No. 63 / 499,689, filed May 2, 2023, the contents of each of which are hereby incorporated by reference in their entirety. 【0002】 The present disclosure relates to methods for combinatorial indexing of nucleic acids of single cells or nuclei. The present disclosure further provides methods for generating and sequencing libraries of such indexed nucleic acids. 【Background Art】 【0003】 Sequencing of nucleic acids of single cells has emerged as a powerful tool for analyzing the genetic heterogeneity of cell populations. For example, single - cell nucleic acid sequencing can be used to analyze cancer progression, map cell heterogeneity in diseased and healthy tissues, understand the development of immune or autoimmune disorders, and gain a better understanding of natural biological processes. 【0004】 Single - cell sequencing typically involves adding unique barcodes to the nucleic acids of single cells to enable parallel profiling of many single cells. There are several techniques for adding barcodes to single - cell nucleic acids, typically involving the use of multi - well plates or microfluidic devices. However, such techniques are limited by the capacity of the plates or microfluidic devices, avoiding the loading of two or more cells within the wells of a multi - well plate or droplets generated by a microfluidic device and preventing the labeling of nucleic acids of multiple cells with the same barcode. Thus, there is a need in the art for improved high - throughput methods for indexing single - cell nucleic acids. 【Summary of the Invention】 【0005】 The present disclosure provides methods for the combinatorial indexing of nucleic acids of single cells, as well as methods for generating and sequencing libraries of such indexed nucleic acids. 【0006】 In one aspect, the present disclosure provides a method for the combinatorial indexing of nucleic acids of single cells. In certain embodiments, the method comprises: (a) providing pooled cells or nuclei associated with or comprising a nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acids of the pooled cells or nuclei in emulsion droplets; (b) partitioning a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; and (c) incorporating the second index sequence into the nucleic acids within each compartment to generate doubly-indexed nucleic acids. In certain embodiments, the first index sequence is incorporated into the nucleic acids of the cells or nuclei in emulsion droplets by a method comprising: (i) contacting a plurality of cells or nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index sequence into the nucleic acids of the cells or nuclei to generate indexed nucleic acids; and (iv) combining the cells or nuclei associated with or comprising the indexed nucleic acids from the plurality of emulsion droplets to generate pooled cells or nuclei associated with or comprising a nucleic acid comprising the first index sequence. 【0007】 In certain embodiments, a method for combinatorial indexing of nucleic acids of single cells comprises: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles comprising a first index sequence; (b) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; and (c) incorporating the first index sequence into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; (d) combining cells or nuclei containing or associated with the indexed nucleic acids from the plurality of emulsion droplets to generate pooled cells or nuclei; (e) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; and (f) incorporating the second index sequence into the indexed nucleic acid within each compartment to generate a dual-indexed nucleic acid. 【0008】 In another aspect, the present disclosure provides a method for generating a sequencing library comprising nucleic acids from a plurality of single cells or nuclei. For example, without limitation, a method for generating a sequencing library can include the following: (a) providing a pooled cell or nucleus associated with or comprising a nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet; (b) partitioning a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acid within each compartment to generate a dual-indexed nucleic acid; and (d) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from a plurality of cells. In certain embodiments, the first index sequence is incorporated into the nucleic acid of the cells or nuclei in an emulsion droplet by a method comprising: (i) contacting a plurality of cells or nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cells or nuclei to generate an indexed nucleic acid; and (iv) combining the cells or nuclei associated with or comprising the indexed nucleic acid from the plurality of emulsion droplets to generate a pooled cell or nucleus associated with or comprising a nucleic acid comprising the first index sequence. 【0009】 In certain embodiments, a method for generating a sequencing library can include the following: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles comprising a first index sequence; (b) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; (d) combining the cells or nuclei containing or associated with the indexed nucleic acid from the plurality of emulsion droplets to generate pooled cells or nuclei; (e) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (f) incorporating the second index sequence into the indexed nucleic acid within each compartment to generate a dual-indexed nucleic acid; and (g) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from the plurality of cells. 【0010】 In another aspect, the present disclosure further provides a method for sequencing a library of nucleic acids from a plurality of single cells or nuclei. For example, but not limited to, a method for sequencing a library of nucleic acids from a plurality of single cells or nuclei includes: (a) providing a pooled cell or nucleus associated with or containing a nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet; (b) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acid within each compartment to generate a dual-indexed nucleic acid; (d) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from a plurality of cells; and (e) sequencing all or a subset of the dual-indexed nucleic acids. In certain embodiments, the first index sequence is incorporated into the nucleic acid of the cell or nucleus in an emulsion droplet by a method comprising: (i) contacting a plurality of cells or nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; and (iv) combining the cells or nuclei associated with or containing the indexed nucleic acid from the plurality of emulsion droplets to generate a pooled cell or nucleus associated with or containing a nucleic acid comprising the first index sequence. 【0011】 In certain embodiments, a method for sequencing a library comprising nucleic acids from a plurality of single cells or nuclei comprises: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles comprising a first index sequence; (b) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; (d) combining the cells or nuclei containing or associated with the indexed nucleic acid from the plurality of emulsion droplets to generate pooled indexed cells or nuclei; (e) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (f) incorporating the second index sequence into the indexed nucleic acid within each compartment to generate a dual-indexed nucleic acid; (g) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from the plurality of cells; and (h) sequencing all or a subset of the dual-indexed nucleic acids. 【0012】 In certain embodiments, the methods of the disclosure can further comprise incorporating a third index sequence into the dual-indexed nucleic acid to generate a triple-indexed nucleic acid. 【0013】 In certain embodiments, the particles are beads, such as gel microbeads. 【0014】 In certain embodiments, the emulsion droplets are generated within an emulsion droplet generation device or using emulsion droplet generation techniques. 【0015】 In certain embodiments, the emulsion droplets comprise at least two or more cells or nuclei. 【0016】 In certain embodiments, the first index array of each particle or subset of particles is unique with respect to the first index arrays of other particles. In certain embodiments, the second index array within one compartment or subset of compartments is unique with respect to the second index arrays of other compartments. 【0017】 In certain embodiments, the first index array is incorporated into a nucleic acid by performing an amplification process, a reverse transcription process, or a ligation process. In certain embodiments, the second index array is incorporated into an indexed nucleic acid by performing an amplification process or a ligation process. In certain embodiments, the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. 【0018】 In certain embodiments, a cell or nucleus is treated, for example, with a transposase in a transpososome complex before or during the step of contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles comprising a first index array. In certain embodiments, the transposase is Tn5 transposase or a variant thereof. In certain embodiments, a cell or nucleus is treated with a fixative prior to the step of contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles comprising a first index array. In certain embodiments, a plurality of cells are permeabilized or lysed prior to the step of contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles comprising a first index array. In certain embodiments, a cell or nucleus is treated with a multiplexing reagent before or during the step of contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles comprising a first index array. 【0019】 In certain embodiments, the plurality of compartments is a multi-well plate. In certain embodiments, the plurality of compartments is not an emulsion droplet. 【0020】 In certain embodiments, the indexed nucleic acids using the disclosed methods include RNA, such as mRNA. In certain embodiments, the indexed nucleic acids using the disclosed methods include DNA, such as genomic DNA. 【0021】 In certain embodiments, the plurality of cells includes at least about 100,000 cells. 【0022】 In certain embodiments, the plurality of cells includes pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or combinations thereof. In certain embodiments, the plurality of cells includes cells modified with a gene editing system, antibody-producing cells, cells in different developmental states, cells in different disease states, and / or cells treated with an agent, such as a therapeutic agent. 【0023】 In certain embodiments, the methods of the present disclosure further include performing a lineage tracing method. In certain embodiments, the methods of the present disclosure further include performing a genomic screening. For example, but not limited to, the genomic screening is a CRISPR-based screening, such as a gene editing CRISPR screening, a CRISPRi screening, or a CRISPRa screening. 【0024】 In certain embodiments, the methods of the present disclosure further comprise performing cell hashing techniques. In certain embodiments, a plurality of cells or nuclei isolated from a plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide comprising a barcode. For example, but not limited to, the methods of the present disclosure can further comprise contacting, prior to (a), a plurality of cells or nuclei isolated from a plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides comprising barcodes. In certain embodiments, the protein binding reagent binds to a protein located on the surface of at least one cell of the plurality of cells or nuclei isolated from a plurality of cells. In certain embodiments, the protein binding reagent is an antibody or a fragment thereof. In certain embodiments, the methods of the present disclosure further comprise determining the expression level of one or more proteins bound by the protein binding reagent. 【0025】 In certain embodiments, the present disclosure further provides a kit for performing the methods of the present disclosure. For example, but not limited to, the kits of the present disclosure can comprise a container comprising a plurality of particles comprising a first index sequence. In certain embodiments, the kit can further comprise a container comprising a plurality of second index sequences, such as a microtiter plate. In certain embodiments, the kits of the present disclosure comprise a container comprising one or more protein binding reagents coupled to an oligonucleotide comprising a barcode. In certain embodiments, the protein binding reagent is an antibody or a fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS 【0026】 【Figure 1】Schematic diagrams showing exemplary embodiments of an overloading and unpacking (“OAK”) method for combinatorial indexing of nucleic acids in single cells or nuclei. The OAK method involves overloading cells or nuclei into an emulsion droplet generation device together with gel beads to generate multi-cell gel beads-in-emulsion (“GEMS”) (left) for incorporating a first index sequence into the nucleic acids of the cells or nuclei. Subsequently, the multi-cell GEMS are unpacked to generate pooled cells or nuclei with indexed nucleic acids (center). The pooled indexed cells or nuclei are then distributed into different compartments to obtain a second index sequence (right), generating doubly indexed nucleic acids. 【Figure 2】 Provide an image showing multi-cell GEMs generated by loading a sample containing 280,000 Jurkat cells into an emulsion droplet generation device. 【Figure 3】 Provide an image showing intact Jurkat cells recovered after unpacking of multi-cell GEMs. 【Figure 4】 Provide a schematic diagram of an exemplary method including single cell CRISPR screening and the OAK method of the present disclosure. 【Figure 5】 Provide a schematic diagram of an exemplary method including lineage tracing techniques and the OAK method of the present disclosure. 【Figure 6】 Provide a schematic diagram showing the loading of different types of gel beads (e.g., 3’ gel beads, 5’ gel beads or multi-ome gel beads) together with cells into an emulsion droplet generation device for generating multi-cell GEMS for implementing the exemplary OAK-multi-ome method or the exemplary OAK-scRNAseq method of the present disclosure. 【Figure 7A】 Provide an image showing multi-cell GEMs generated by loading a sample containing 150,000 cells into an emulsion droplet generation device. 【Figure 7B】Provide an image showing multi - cell GEMs generated by loading a sample containing 450,000 cells into an emulsion droplet generation device. 【Figure 8】 Provide performance metrics of an exemplary OAK - multi - omics method of the present disclosure. 【Figure 9】 Provide a schematic diagram of an exemplary method of implementing cell hashing technology in combination with the OAK method of the present disclosure. Created with BioRender.com. 【Figure 10】 Provide a graph showing the abundance of cells assigned nine different hash tags in sequencing data obtained using the OAK method, compared to a control method for the same cell sample. 【Figure 11】 Provide a ridge plot showing the hash tag expression levels of depleted cells for each hash tag identity. 【Figure 12】 Provide the quality of transcriptome sequencing data from the experiment shown in Figure 9. 【Figure 13】 Figures 13A - 13B provide a UMAP of scRNA - Seq showing hash tag assignment (Figure 13A), indicating that the hash tags are evenly distributed among different cell types (Figure 13B). Figure 13C provides a graph showing the cell type frequencies obtained using the OAK method compared to a control method for the same cell sample. 【Figure 14】 Provide a schematic diagram of an exemplary method of implementing cell hashing technology in combination with the OAK method of the present disclosure for two cell lines, namely Jurkat and K562 cell lines. Created with BioRender.com. 【Figure 15】 Provide the quality of transcriptome sequencing data obtained from the experiment shown in Figure 14. 【Figure 16】 Provide a UMAP showing the clustering of scRNA - Seq data from Jurkat and K562 cell lines. 【Figure 17】 Provide a plot showing signals derived from antibody hash tags of Jurkat and K562 cell lines. 【Figure 18】 Shows the frequency of CDR3 clone types recovered from a homogeneous population of Jurkat cells. 【Mode for Carrying Out the Invention】 【0027】 The present disclosure relates to an improved method for combinatorial indexing of nucleic acids from single cells. In certain embodiments, the methods of the present disclosure include incorporation of at least two index sequences into the nucleic acids of single cells or nuclei. By using at least two index sequences, the combinatorial indexing method of the present disclosure enables identification of nucleic acids from single cells or nuclei by the presence of unique combinations of the two index sequences. In certain embodiments, the methods of the present disclosure can be used to generate libraries of indexed nucleic acids, such as dual-indexed nucleic acids. In certain embodiments, the present disclosure further provides methods for sequencing libraries generated from indexed nucleic acids, such as dual-indexed nucleic acids. 【0028】 The present disclosure is based in part on the discovery that the throughput of an indexing method is increased by more than 20-fold by overloading emulsion droplets to contain at least two cells or nuclei for incorporation of a first index array into the nucleic acid of the cell or nucleus. For example, by overloading emulsion droplets with two or more cells or nuclei for incorporation of a first index array, the methods of the present disclosure enable combinatorial indexing of nucleic acids from over 200,000 single cells. Further, by using emulsion droplets as a first step in adding an index array to the nucleic acid of a single cell, the disclosed combinatorial indexing process enables cells to remain intact for addition of a second index array to the indexed nucleic acid. Further, use of emulsion droplets as a first step in the indexing process ensures compatibility with different emulsion-based kits, reagents, and methods and enables subsets of cells to be sequenced independently of one another. 【0029】 In certain embodiments, the nucleic acids to be indexed using the methods of the disclosure can be DNA or RNA. In certain embodiments, the methods for the disclosure can be used to index the RNA of a single cell. For example, but not limited to, by using the methods for the disclosure to index the mRNA of a single cell, all or part of the transcriptome of a single cell can be analyzed. In certain embodiments, the methods of the disclosure can be used to index the genomic DNA of a single cell or nucleus. In certain embodiments, by using the methods of the disclosure to index the genomic DNA of a single cell, gene regulation in a single cell can be analyzed. In certain embodiments, by using the methods of the disclosure to index open chromatin sites in the genomic DNA of a single cell, chromatin accessibility in a single cell can be analyzed. In certain embodiments, the methods of the disclosure can be used to analyze samples that have been pooled and indexed using sample-specific oligonucleotides prior to pooling, or genetically distinct samples. In certain embodiments, the methods of the disclosure can be used to quantify the presence of a protein after protein detection using an oligonucleotide-labeled antibody. 【0030】 For clarity, and not by way of limitation, the detailed description of the subject matter disclosed in the present invention is divided into the following sections: I. Definitions; II. Cells; III. Methods of Use; IV. Kits; and V. Exemplary Embodiments. 【0031】 I. Definitions Unless otherwise defined, all technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs. The following references provide one of ordinary skill in the art with general definitions of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used in this specification, the following terms have the meanings set forth below unless otherwise specified. 【0032】 As used herein, in the claims, and / or in the specification, when the term "comprising" is used, the use of the phrase "a" or "an" can mean "one," which is also consistent with the meaning of "one or more," "at least one," and "one or two or more." 【0033】 The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within three standard deviations, or more than three standard deviations, depending on the technology field. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within one order of magnitude, preferably within five-fold, and more preferably within two-fold of a value. 【0034】 The term "amplification process" generally refers to any process in which a portion of a nucleic acid is copied or replicated into at least one additional nucleic acid molecule. 【0035】 The term "antibody" as used herein is used in the broadest sense and includes, without limitation, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity, encompassing various antibody structures. 【0036】 "Antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabody, linear antibody, single-chain antibody molecule (e.g., scFv), and multispecific antibodies formed from antibody fragments. 【0037】 As used herein, the term "biological sample" refers to a sample of biological material obtained from a subject and includes body fluids such as blood, plasma, serum, urine, sputum, cerebrospinal fluid, pleural fluid, nipple aspirate fluid, lymph fluid, respiratory, intestinal, and urogenital fluids, tears, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, fluid within an organ system, ascites, tumor cyst fluid, amniotic fluid, bronchoalveolar fluid, bile, and combinations thereof. 【0038】 The term "combinatorial indexing" refers to the process of incorporating two or more index sequences into a nucleic acid as an identification sequence. 【0039】 As used herein, the term "compartment" refers to a region or volume that separates or isolates one or more components from other components. Non-limiting examples of compartments include vials, tubes, wells, droplets, boluses, vessels, or regions or volumes separated by physical forces such as fluid flow, magnetism, electric current, etc. 【0040】 As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude additional acts or structures. Also, in the present disclosure, other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein are contemplated, whether or not explicitly recited. 【0041】 The term "coupled" can refer to connecting or joining two or more components by interaction, coupling, linking, force, or binding to hold the two or more components together. In certain embodiments, the term "coupled" includes, for example, a direct or indirect coupling where a first component is directly coupled to a second component or one or more intervening molecules are disposed between the first and second components. Exemplary couplings include covalent bonds, ionic bonds, van der Waals interactions, hydrogen bonds, and other bonds identifiable by one of ordinary skill in the art. 【0042】 As used herein, the term "depletion" refers to the separation of different data obtained during a single experiment. In certain embodiments, the term "depletion" refers to the separation of scRNA-SEQ data from hashtag sequencing and / or expression data. 【0043】 As used herein, the terms "detect" or "detection" refer to the determination of the presence and / or existence of a target, such as a protein target or a nucleic acid target, in a limited portion of a space, including but not limited to a sample. As used herein, the terms "detect" or "detection" can include, but are not limited to, the ability to interact with, particularly bind to, another compound, the ability to activate another compound, and additional properties identifiable by one of ordinary skill in the art upon reading the present disclosure, including the determination of the chemical and / or biological properties of the target. Detection can be quantitative or qualitative. Detection is "quantitative" (also referred to as quantification) when it refers to, relates to, or includes the measurement of the quantity or amount of a target or signal, including any analysis designed to determine the quantity or proportion of a target or signal, but not limited thereto. Detection is "qualitative" when it refers to, relates to, or includes the identification of the quality or type of a target or signal with respect to the relative abundance of another target or signal that has not been quantified. 【0044】 As used herein, the term "doubly-indexed nucleic acid" refers to a nucleic acid having two index sequences. 【0045】 As used herein, the term "emulsion droplet" refers to a droplet formed by at least two immiscible phases, such as a continuous phase and a dispersed phase. In certain embodiments, emulsion droplets can be formed within an emulsion droplet generation device using the addition of oil as the continuous phase into the emulsion droplet generation device. In certain embodiments, emulsion droplets can be formed by techniques that produce emulsion droplets, such as, but not limited to, vortexing. 【0046】 As used herein, the term "hybridization" refers to the process by which two single-stranded polynucleotides non-covalently bind to form a stable double-stranded polynucleotide. 【0047】 As used herein, the term "index array" refers to a nucleotide sequence used to identify a single cell or nucleus to which the index array relates. 【0048】 As used herein, the term "individual" or "subject" refers to a vertebrate or invertebrate, such as a human or non-human animal, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, livestock, sport animals, rodents, pets, etc. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, apes and monkeys. In certain embodiments, the individual or subject is human. 【0049】 As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur within the artificial environment. In vitro environments are exemplified by, but not limited to, test tubes and cell cultures. 【0050】 As used herein, the term "in vivo" refers to a natural environment (e.g., an animal or cell) and processes or reactions that occur within the natural environment such as embryogenesis, cell differentiation, neurulation. 【0051】 As used herein, the term "isolated nucleus" refers to a nucleus separated from the components of its natural environment. 【0052】 "Isolated nucleic acid" refers to a nucleic acid molecule separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules that are normally contained within a cell that contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. 【0053】 As used herein, the term "ligation" refers to the formation of a covalent bond or linkage between two or more molecules, such as between the ends of two or more nucleic acid molecules. 【0054】 As used herein, the term "ligation process" generally refers to a process for covalently joining two or more molecules together by an enzyme. For example, two or more nucleic acid molecules can be covalently joined by a ligation process using a ligase. 【0055】 As used herein, "marker" refers to an agent that enables direct or indirect detection. Markers include, but are not limited to, fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels. Non-limiting examples of markers include green fluorescent protein ("GFP"), mCherry, dtTomato, or other fluorescent proteins known in the art (e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909(2005), which is incorporated herein by reference), 32P, 14C, 125I, 3H and 131I, fluorescent substances (such as rare earth chelates or lucifer yellow and its derivatives), rhodamine and its derivatives, dansyl, umbelliferone, luciferase (such as firefly luciferase and bacterial luciferase) (U.S. Patent No. 4,737,456), fluorescein, 2,3-dihydrophthalazine dione, and enzymes that produce a detectable signal, such as horseradish peroxidase (HRP), alkaline phosphorus sour enzyme, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase (G6PD)), and heterocyclic oxidases (such as uricase and xanthine oxidase). 【0056】 As used herein, the term "multi-indexed" refers to a nucleic acid having at least two index sequences. 【0057】 The term "nucleic acid" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine base or a pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by the sequence of bases, whereby the said bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. The term nucleic acid includes deoxyribonucleic acid (DNA), such as complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), such as messenger RNA (mRNA), synthetic forms of DNA or RNA, and hybrid polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition to this, the term nucleic acid includes both sense and antisense strands, as well as both single-stranded and double-stranded forms. Furthermore, the nucleic acids described herein can contain naturally occurring nucleotides or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides that contain derivatized sugars or phosphate backbone linkages or chemically modified residues include modified nucleotide bases. 【0058】 As used herein, the term "compartmentalize" generally refers to the separation of one or more components from other components within a region or volume. 【0059】 The term "plurality" refers to a number greater than one. In certain embodiments, the term "a plurality of cells" refers to a number of cells greater than one. For example, without limitation, a plurality of cells can include at least two cells. In certain embodiments, a plurality of cells can include about 10,000 or more cells, about 20,000 or more cells, about 30,000 or more cells, about 40,000 or more cells, about 50,000 or more cells, about 100,000 or more cells, about 150,000 or more cells, about 200,000 or more cells, about 300,000 or more cells, about 400,000 or more cells, or 500,000 or more cells. In certain embodiments, a plurality of cells can include at least about 100,000 cells. In certain embodiments, the term "a plurality of particles" refers to a number of particles greater than one. 【0060】 The term "reverse transcription process" refers to a process of generating a complementary strand of DNA using an enzyme called reverse transcriptase. 【0061】 As used herein, the term "specifically binds" refers to the preferential binding to a target molecule, such as a protein or nucleic acid, as compared to other molecules in a sample, such as a protein or nucleic acid. 【0062】 As used herein, the term "subset" refers to a small portion of a larger amount of material. 【0063】 As used herein, the term "triply-indexed" refers to a nucleic acid having three index sequences. 【0064】 II. Cells The present disclosure provides methods for indexing nucleic acids of a single cell or a nucleus obtained from a single cell, such as methods for doubly-indexing nucleic acids. In certain embodiments, the cells for use in the methods of the present disclosure can include any type of cell. In certain embodiments, the nuclei for use in the methods of the present disclosure can be obtained, such as isolated, from any type of cell. 【0065】 In certain embodiments, the cells are obtained from a subject. In certain embodiments, the subject can be a human, non-human primate such as an ape or monkey, livestock, mouse, rat, hamster, guinea pig, rabbit, dog, cat, sheep, pig, goat, cow or horse. In certain embodiments, the subject is a human. 【0066】 In certain embodiments, the cells can be obtained from a biological fluid. Non-limiting examples of biological fluids include whole blood, plasma, serum, sweat, urine, sputum, cerebrospinal fluid, pleural fluid, mucus, nipple aspirate, lymph fluid, fluids of the respiratory, intestinal and urogenital tracts, interstitial fluid, tears, saliva, breast milk, fluid from the lymphatic system, semen, vaginal secretions, cerebrospinal fluid, fluid within organ systems, ascites, tumor cyst fluid, amniotic fluid, bronchoalveolar fluid, bile fluid and combinations thereof. 【0067】 In certain embodiments, the cells can be obtained from a tissue, such as a tissue sample. Non-limiting examples of tissues include the eye (e.g., retina), muscle, skin, tendon, vein, artery, blood, heart, spleen, lymph node, bone, bone marrow, lung, bronchus, trachea, intestine, small intestine, large intestine, colon, rectum, salivary gland, tongue, gallbladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonad, testis, ovary, uterus, fallopian tube, thymus, pituitary gland, thyroid gland, adrenal gland or parathyroid tissue. 【0068】 In certain embodiments, the cells for use in the present disclosure can be fetal cells obtained from, for example, fetal tissue and / or amniotic fluid. In certain embodiments, the methods of the present disclosure can be used to analyze the health of a fetus and / or identify abnormalities in individual fetal cells. 【0069】 In certain embodiments, the cells can be obtained from an in vitro cell culture. For example, but not limited to, the cells can include a cell line. 【0070】 In certain embodiments, the cells can include primary cells, blood cells, somatic cells, cancer cells, cells derived from an organoid or xenograft, or stem cells such as pluripotent stem cells (iPSCs) or embryonic stem cells. In certain embodiments, the cells for use in the present disclosure can be derived from stem cells, such as stem cells that have undergone natural differentiation or artificially induced reprogramming or transdifferentiation. 【0071】 In certain embodiments, the cells can be obtained from a stored sample, such as a fixed sample, from a frozen sample, such as a frozen tissue sample, or from a fresh sample, such as a fresh tissue sample. 【0072】 In certain embodiments, the cells for use in the present disclosure can be treated with a drug. For example, without limitation, the drug can be a therapeutic agent. In certain embodiments, the cells for use in the present disclosure can be contacted with a drug, such as a therapeutic agent, prior to subjecting them to the combinatorial indexing methods described herein. In certain embodiments, the methods of the present disclosure can be used for drug screening, for example, to determine genomic and / or transcriptional changes associated with a test therapeutic agent, such as a newly identified therapeutic agent. In certain embodiments, the methods of the present disclosure can be used in determining genomic changes and / or transcriptional changes associated with resistance to a therapeutic agent. Non-limiting examples of such therapeutic agents include polypeptide therapeutic agents, such as antibody-based therapeutic agents, oligonucleotides, cell-based therapeutic agents, gene editing systems, and small molecule therapeutic agents. In certain embodiments, the therapeutic agent can be a cell cycle regulator, a kinase regulator (e.g., a kinase inhibitor or activator), a receptor regulator (e.g., a receptor inhibitor or activator), a chemotherapeutic agent, and / or an antibody (e.g., an agonist antibody or an antagonist antibody). In certain embodiments, the methods of the present disclosure include performing the combinatorial indexing methods described herein after performing single cell therapeutic agent screening. 【0073】 In certain embodiments, the cells are genetically modified to express and / or secrete a drug, such as a therapeutic agent. Non-limiting examples of therapeutic agents are described herein. For example, but not limited to, the cells used in the methods of the present disclosure express a polypeptide therapeutic agent, such as an antibody or antibody fragment. 【0074】 In certain embodiments, the cells can be immune cells. Non-limiting examples of immune cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer (NK) cells, and lymphocytes, such as B cells and T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and helper T cells). In certain embodiments, the cells can be T cells. In certain embodiments, the cells can be modified immune cells, such as modified T cells, e.g., chimeric antigen receptor (CAR) T cells and CAR NK cells, that are genetically engineered to express a CAR. 【0075】 In certain embodiments, the cells can be obtained from a malignant tumor of a tissue or tumor. Non-limiting examples of such malignant tumors include carcinomas, adenocarcinomas, sarcomas, and fibroadenomas. In certain embodiments, the cells are obtained from cancers such as bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, head and neck cancer, kidney cancer, leukemia, lung cancer, lymphoma, melanoma, pancreatic cancer, parathyroid cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer. In certain embodiments, the methods of the present disclosure can be used to identify mutations and / or genetic changes present in a single cancer cell. 【0076】 In certain embodiments, cells can be subjected to gene modification or regulation. For example, without limitation, cells for use in the present disclosure can be subjected to gene modification or regulation by an enzyme or a fusion protein containing such an enzyme, such as by contacting the cells with the enzyme or fusion protein. In certain embodiments, the enzyme or fusion protein containing such an enzyme can cleave a target gene, nick the target gene, edit the target gene, suppress the expression of the target gene, and / or activate the expression of the target gene in the cell. In certain embodiments, the enzyme or fusion protein containing such an enzyme is directed to a region of the target gene by a guide nucleic acid, for example, to modify or regulate the target gene. In certain embodiments, gene modification or regulation is achieved by nicking, cleaving, editing, suppressing, and / or activating the target gene by an enzyme or a fusion protein containing such an enzyme. Non-limiting examples of enzymes that can be used, for example, for use in a gene editing system to modify or regulate a gene include homing endonucleases or meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR enzymes, and fusion proteins containing such enzymes. In certain embodiments, cells for use in the present disclosure can be subjected to genomic screening in which a target gene is modified or regulated using, for example, an enzyme or a fusion protein containing such an enzyme disclosed herein. In certain embodiments, the methods of the present disclosure can be used in combination with single-cell genomic screening, for example, in combination with single-cell CRISPR-based screening as shown in FIG. 4. Non-limiting examples of CRISPR-based screening include gene editing CRISPR screening, CRISPR interference (CRISPRi) screening, and CRISPR activation (CRISPRa) screening. For example, without limitation, single-cell genomic screening can be performed followed by the combinatorial indexing method of the present disclosure.In certain embodiments, nucleic acids (e.g., guide nucleic acid molecules (guide RNAs (gRNAs)) and other nucleic acids) associated with the performance of single-cell genomic screening (e.g., single-cell CRISPR-based screening) in genetically modified cells are processed by the combinatorial indexing methods described herein. In certain embodiments, indexed guide nucleic acid molecules (e.g., gRNAs) can further undergo a secondary enrichment process prior to sequencing. In certain embodiments, the identity of the guide nucleic acid molecule (e.g., gRNA) can be determined by either direct sequencing of the guide nucleic acid molecule (e.g., gRNA) or sequencing of a surrogate barcode sequence associated with the guide nucleic acid molecule (e.g., gRNA). In certain embodiments, the combinatorial indexing methods described herein and methods including single-cell genomic screening, such as single-cell CRISPR-based screening, can further include modifying and / or manipulating the cells before and / or during the single-cell genomic screening. In certain embodiments, the combinatorial indexing methods described herein and methods including single-cell genomic screening can further include treating the cells with one or more therapeutic agents (e.g., drugs of interest), for example, prior to performing the single-cell genomic screening. For example, without limitation, as shown in FIG. 4, the combinatorial indexing methods described herein and methods including single-cell CRISPR-based screening can further include treating the cells with one or more drugs of interest, for example, prior to performing the single-cell CRISPR-based screening. In certain embodiments, the combinatorial indexing methods described herein and methods including single-cell genomic screening, such as single-cell CRISPR-based screening, can further include subjecting the cells to development and / or differentiation.In certain embodiments, information obtained from the combinatorial indexing methods and methods including single cell genomic screening described herein, such as the sequences of guide nucleic acid molecules (e.g., gRNAs) and associated cellular outcomes (e.g., gene editing, repression or activation and / or cell phenotypes), can be used to determine gene function (e.g., the genes targeted in the screening), drug responsiveness and / or mechanisms of drug resistance. In certain embodiments, the methods of the disclosure include performing a combinatorial indexing method described herein after performing a single cell genomic screening. 【0077】 In certain embodiments, the cells for use in the disclosure can be genetically modified with a gene editing system. Non-limiting examples of gene editing systems include homing endonucleases or meganucleases, ZFNs, TALENs, and CRISPR gene editing systems. For example, but not limited to, the methods of the disclosure can be used to identify and / or quantify nucleic acid modifications generated by a gene editing system. 【0078】 In certain embodiments, the cells can include one or more lineage barcodes. For example, without limitation, the combinatorial indexing methods of the present disclosure that use cells having lineage barcodes can be implemented in combination with single-cell lineage tracing techniques. In certain embodiments, single-cell lineage tracing techniques can be used to obtain information regarding lineage identity and to associate cells over multiple time points during development, differentiation, and / or manipulation (e.g., drug treatment). In certain embodiments, the combinatorial indexing methods described herein and methods that include single-cell lineage tracing techniques are used to determine cell type, the cell's genome, or specific gene mutations associated with cells that confer resistance to a drug of interest, as shown in FIG. 5. Non-limiting examples of single-cell lineage tracing techniques are TraCe-seq as disclosed in International Publication No. WO 2021 / 188973 and Chang et al., Nature Biotechnology 40:86-93 (2022), the contents of each of which are hereby incorporated by reference in their entirety. In certain embodiments, the lineage barcode can be included in an exogenous nucleic acid construct that is introduced into the cell, as described, for example, in Section III. Alternatively, in certain embodiments, the lineage barcode can be an endogenous gene marker. In certain embodiments, the lineage barcodes and other nucleic acids present in the cell that are associated with the implementation of the single-cell lineage tracing technique are processed by the combinatorial indexing methods described herein. In certain embodiments, the lineage barcode can further undergo a secondary enrichment process prior to sequencing. In certain embodiments, the methods of the present disclosure include performing a lineage tracing technique and performing the combinatorial indexing methods described herein at different time points during the treatment of a cell population with a drug of interest. In certain embodiments, the methods of the present disclosure include performing a lineage tracing technique and performing the combinatorial indexing methods described herein at different time points during the development and / or differentiation of a cell population.In certain embodiments, information obtained from the combinatorial indexing methods and methods involving single-cell lineage tracing techniques described herein can be used to determine, for example, how different lineages undergo diverse processes of development and / or differentiation using information on lineage identity and molecular phenotypes at the cellular level. In certain embodiments, information obtained from the combinatorial indexing methods and methods involving single-cell lineage tracing techniques described herein can be used to determine how a particular lineage shares similar programs in development and / or differentiation. In certain embodiments, information obtained from the combinatorial indexing methods and methods involving single-cell lineage tracing techniques described herein can be used to determine, as shown for example in FIG. 5, how existing gene programs in each cell and lineage affect their responses to external stimuli, including but not limited to drug treatment. 【0079】 In certain embodiments, a cell can comprise one or more hashtags. For example, without limitation, the combinatorial indexing methods of the present disclosure can use cells bound by a protein-binding reagent coupled to an oligonucleotide comprising a barcode. In certain embodiments, a protein-binding reagent coupled to an oligonucleotide comprising a barcode can be bound to a cell for use in the combinatorial indexing methods of the present disclosure as described in Section III. In certain embodiments, the methods of the present disclosure include performing cell hashing techniques and performing the combinatorial indexing methods described herein. A non-limiting example of a cell hashing technique is CITE-seq as disclosed in Stoeckius et al., Nature Methods 14:865-868 (2017), the entire contents of which are incorporated herein by reference. In certain embodiments, protein-binding reagents and barcodes coupled to other nucleic acids associated with performing cell hashing techniques are processed by the combinatorial indexing methods described herein. 【0080】 In certain embodiments, the cells can be bacterial cells. For example, without limitation, the methods of the present disclosure can be used to analyze the microbiota of a subject, such as the gut microbiota of a subject. 【0081】 In certain embodiments, the cells can include cells having different developmental stages. In certain embodiments, the cells can include cells in different disease states. For example, without limitation, the methods of the present disclosure can be used to analyze the genetic regulation of different developmental stages. In certain embodiments, the methods of the present disclosure can be used to analyze the molecular dynamics of different cell types during different developmental stages. In certain embodiments, the methods of the present disclosure can be used for the identification of rare and / or transient developmental stages and / or disease states. 【0082】 In certain embodiments, the cells can be derived from any model organism. For example, without limitation, the model organisms can be Escherichia coli, yeast, Arabidopsis thaliana, Xenopus laevis, zebrafish, Drosophila melanogaster, ascidians, Caenorhabditis elegans, mice, and monkeys. 【0083】 In certain embodiments, the cells can be cells infected with an infectious agent. Non-limiting examples of infectious agents include viruses, bacteria, fungi, and protozoa. 【0084】 In certain embodiments, the cells of interest can be concentrated to produce a concentrated cell sample, which can be subjected to the methods of the present disclosure. Any technique known in the art can be used to concentrate the cells of interest. 【0085】 In certain embodiments, nuclei can be isolated from any cell type. For example, without limitation, nuclei can be isolated from any one of the cells disclosed herein. Methods for isolating nuclei from cells are known to those of skill in the art and can be used to isolate nuclei for use in the present disclosure. 【0086】 III. Usage Method The present disclosure relates to an improved method for combinatorial indexing of nucleic acids from single cells or nuclei. In certain embodiments, the methods of the present disclosure include incorporating at least two index sequences into the nucleic acids of single cells or nuclei. Figure 1 provides a flowchart of an exemplary method of the present disclosure. 【0087】 In certain embodiments, the methods of the present disclosure can include contacting a cell or a nucleus isolated from a cell with a particle comprising a first index sequence. Figure 6 is a schematic diagram showing exemplary particles, such as gel beads, that can be used in the present disclosure. In certain embodiments, contacting the cell or nucleus with the particle can include contacting the cell or nucleus with the particle to produce a mixture of the cell or nucleus and the particle. For example, without limitation, the cell or nucleus is present in an aqueous phase and the particle is present in a second aqueous phase, and the two aqueous phases are combined to produce a single aqueous phase of the cell or nucleus and the particle. 【0088】 In certain embodiments, a plurality of cells or nuclei isolated from a plurality of cells are contacted with the particles. In certain embodiments, the plurality of cells can comprise at least about 5,000 cells. For example, without limitation, the plurality of cells can comprise about 10,000 or more cells, about 20,000 or more cells, about 30,000 or more cells, about 40,000 or more cells, about 50,000 or more cells, about 100,000 or more cells, about 150,000 or more cells, about 200,000 or more cells, about 300,000 or more cells, about 400,000 or more cells, or 500,000 or more cells. In certain embodiments, the plurality of cells comprise at least about 100,000 cells. In certain embodiments, the plurality of cells comprise at least about 200,000 cells. In certain embodiments, the plurality of cells comprise from about 5,000 to about 200,000 cells, such as from about 10,000 to about 200,000 cells, from about 50,000 to about 200,000 cells, from about 100,000 to about 200,000 cells, from about 150,000 to about 200,000 cells, from about 5,000 to about 150,000 cells, from about 5,000 to about 100,000 cells, from about 5,000 to about 50,000 cells, from about 5,000 to about 10,000 cells, from about 10,000 to about 150,000 cells, or from about 50,000 to about 100,000 cells. 【0089】 In certain embodiments, a plurality of particles can be contacted with cells or nuclei, such as a plurality of cells or nuclei isolated from a plurality of cells. In certain embodiments, the plurality of particles, such as beads, includes from about 5,000 to about 1,000,000 particles.For example, but not limited to, the plurality of particles include from about 10,000 to about 1,000,000 particles, from about 20,000 to about 1,000,000 particles, from about 30,000 to about 1,000,000 particles, from about 40,000 to about 1,000,000 particles, from about 50,000 to about 1,000,000 particles, from about 60,000 to about 1,000,000 particles, from about 70,000 to about 1,000,000 particles, from about 80,000 to about 1,000,000 particles, from about 90,000 to about 1,000,000 particles, from about 100,000 to about 1,000,000 particles, from about 200,000 to about 1,000,000 particles, from about 300,000 to about 1,000,000 particles, from about 400,000 to about 1,000,000 particles, from about 500,000 to about 1,000,000 particles, from about 600,000 to about 1,000,000 particles, from about 700,000 to about 1,000,000 particles, from about 800,000 to about 1,000,000 particles, from about 900,000 to about 1,000,000 particles, from about 10,000 to about 500,000 particles, from about 20,000 to about 500,000 particles, from about 30,000 to about 500,000 particles, from about 40,000 to about 500,000 particles, from about 50,000 to about 500,000 particles, from about 60,000 to about 500,000 particles, from about 70,000 to about 500,000 particles, from about 80,000 to about 500,000 particles, from about 90,000 to about 500,000 particles, from about 100,000 to about 500,000 particles, from about 200,000 to about 500,000 particles, from about 300,000 to about 500,000 particles, from about 400,000 to about 500,000 particles, from about 10,000 to about 400,000 particles, from about 10,000 to about 300,000 particles, from about 10,000 to about 200,000 particles, from about 10,000 to about 100,000 particles, from about 50,000 to about 200,000 particles, from about 60,000 to about 190,000 particles, from about 70,000 to about 180,000 particles, from about 80,000 to about 170,000 particles, from about 90,000 to about 150,000 particles, or from about 90,000 to about 110,000 particles. In certain embodiments, the plurality of particles include from about 50,000 to about 450,000 particles.In certain embodiments, the plurality of particles comprises from about 400,000 to about 1,000,000 particles. In certain embodiments, the plurality of particles comprises from about 200,000 to about 800,000 particles. In certain embodiments, the plurality of particles comprises from about 300,000 to about 800,000 particles. In certain embodiments, the plurality of particles comprises from about 400,000 to about 800,000 particles. In certain embodiments, the plurality of particles comprises about 50,000 particles. In certain embodiments, the plurality of particles comprises about 100,000 particles. In certain embodiments, the plurality of particles comprises about 400,000 particles. 【0090】 In certain embodiments, the particles combined with the cells or nuclei can be glass, plastic, gel or metal particles. In certain embodiments, the particles can be beads, such as microbeads. In certain embodiments, the particles are gel beads, such as gel microbeads. In certain embodiments, the particles can be of any shape, for example, the particles can have a spherical, non-spherical, elliptical, oblong, amorphous, circular or cylindrical shape. In certain embodiments, the particles can have a diameter of about 0.5 to about 500 μm. In certain embodiments, the particles have a diameter of less than about 0.5 μm, less than about 1.0 μm, less than about 1.5 μm, less than about 2.0 μm, less than about 5.0 μm, less than about 10.0 μm, less than about 20.0 μm, less than about 30.0 μm, less than about 40.0 μm, less than about 50.0 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, less than about 100 μm, less than about 250 μm or less than about 500 μm. 【0091】 In certain embodiments, the first index array is coupled to particles, such as beads. In certain embodiments, the first index array is coupled to particles, such as beads, using a linker. In certain embodiments, the first index array is releasably attached to particles, such as beads. In certain embodiments, each particle may have a plurality of index arrays attached thereto, such as about 10 index arrays, about 100 index arrays, about 1,000 index arrays, about 10,000 index arrays, or about 100,000 index arrays, or more. In certain embodiments, each particle may have about 10 to about 100,000 index arrays attached thereto, such as, for example, about 50 to about 100,000, about 100 to about 100,000, about 1,000 to about 100,000, about 10,000 to about 100,000, about 50,000 to about 100,000, about 10 to about 50,000, about 10 to about 10,000, about 10 to about 1,000, about 10 to about 100, about 10 to about 50, about 100 to about 50,000, about 1,000 to about 50,000, about 100 to about 10,000, or about 1,000 to about 100,000 index arrays. In certain embodiments, all of the index arrays attached to a particular particle, such as a bead, can have the same nucleotide sequence. In certain embodiments, all of the index arrays attached to a particular particle, such as a bead, can have the same nucleotide sequence and can represent a number of diverse index arrays across a plurality of particles, such as beads, used.For example, and not by way of limitation, the plurality of beads can include a diverse library of index arrays that includes at least about 100 different index arrays, at least about 1,000 different index arrays, at least about 10,000 different index arrays, at least about 100,000 index arrays, at least about 1,000,000 different index arrays, at least about 1,500,000 different index arrays, at least about 2,000,000 different index arrays, at least about 2,500,000 different index arrays, at least about 3,000,000 different index arrays, at least about 3,500,000 different index arrays, at least about 4,000,000 different index arrays, at least about 4,500,000 different index arrays, or at least about 5,000,000 different index arrays. In certain embodiments, the plurality of beads can include from about 100 to about 5,000,000 different index arrays, such as, for example, from about 100 to about 1,000,000, from about 100 to about 500,000, from about 100 to about 50,000, from about 100 to about 10,000, from about 100 to about 1,000, from about 100 to about 500, from about 500 to about 5,000,000, from about 1,000 to about 5,000,000, from about 10,000 to about 5,000,000, from about 50,000 to about 5,000,000, from about 500,000 to about 5,000,000, from about 1,000,000 to about 5,000,000, from about 10,000 to about 1,000,000, from about 100,000 to about 1,000,000 or from about 500,000 to about 1,000,000 different index arrays. 【0092】 In certain embodiments, the first index array of each particle, e.g., bead, is unique with respect to other particles among the plurality of particles. For example, but not limited to, the first index array attached to the particles present in the emulsion droplets is unique as compared to the array of the first index arrays attached to the particles present in different emulsion droplets. Alternatively, about 50% or less of the particle population among the plurality of particles, e.g., about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less, are attached to index arrays having the same nucleic acid sequence as different particles. In certain embodiments, about 10% or less of the particle population among the plurality of particles are attached to index arrays having the same nucleic acid sequence as different particles. In certain embodiments, about 5% or less of the particle population among the plurality of particles are attached to index arrays having the same nucleic acid sequence as different particles. In certain embodiments, about 1% to about 50% (e.g., about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 10%, about 1% to about 5%, about 5% to about 50%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 5% to about 40%, about 5% to about 20%, or about 5% to about 10%) of the particle population among the plurality of particles are attached to index arrays having the same nucleic acid sequence as different particles. 【0093】 In certain embodiments, the index arrays for use in the present disclosure, such as the first index array and / or the second index array, can be up to 200 nucleotides in length. In certain embodiments, the index array can be from about 5 to about 200 nucleotides in length, such as from about 5 to about 150 nucleotides in length, from about 5 to about 100 nucleotides in length, from about 5 to about 50 nucleotides in length, from about 10 to about 150 nucleotides in length, from about 20 to about 100 nucleotides in length, or from about 10 to about 100 nucleotides in length. In certain embodiments, the index array can be from about 5 to about 50 nucleotides in length, such as from about 5 to about 45 nucleotides in length, from about 5 to about 40 nucleotides in length, from about 5 to about 35 nucleotides in length, from about 5 to about 30 nucleotides in length, from about 5 to about 25 nucleotides in length, from about 5 to about 20 nucleotides in length, from about 5 to about 15 nucleotides in length, from about 5 to about 10 nucleotides in length, from about 10 to about 50 nucleotides in length, from about 15 to about 50 nucleotides in length, from about 20 to about 50 nucleotides in length, from about 25 to about 50 nucleotides in length, from about 30 to about 50 nucleotides in length, from about 35 to about 50 nucleotides in length, from about 40 to about 50 nucleotides in length, from about 10 to about 40 nucleotides in length, or from about 10 to about 30 nucleotides in length. 【0094】 In certain embodiments, the first index array is provided in the first polynucleotide. For example, without limitation, the first polypeptide comprising the first index array is coupled to the particles described herein, such as beads. In certain embodiments, the first polynucleotide can further comprise a universal array. For example, without limitation, the universal array can be a sequence common to two or more first polynucleotides. In certain embodiments, the universal array can be a sequence common to all of the first polynucleotides. In certain embodiments, the universal array can be used, for example, as a primer binding site for binding primers used in an amplification process or as a primer. In certain embodiments, the first polynucleotide can further comprise a sequence that is at least partially complementary to the sequence of a cellular or nuclear nucleic acid. In certain embodiments, the universal array can comprise a sequence that binds to an RNA poly A tail. For example, without limitation, the universal array can comprise a poly T sequence that binds to the poly A tail of mRNA. In certain embodiments, the first polynucleotide comprises a universal array and a first index array. In certain embodiments, the first polynucleotide comprises a first index array and a sequence complementary to the sequence of a cellular or nuclear nucleic acid. In certain embodiments, the first polynucleotide comprises a universal array, a first index array, and a sequence complementary to the sequence of a cellular or nuclear nucleic acid. In certain embodiments, the first polynucleotide comprises a universal array, a first index array for identifying a compartment (e.g., an emulsion droplet), and an additional sequence, such as, without limitation, a unique molecular identifier. 【0095】 In certain embodiments, the universal array can be designed to capture chromatin fragments and any other DNA fragments resulting from transposome processing. Non-limiting examples of such capture mechanisms can include the binding and ligation of complementary sequences. In certain embodiments, the universal array can be designed to capture nucleic acids introduced into cells or nuclei by incubation, electroporation, infection, editing, or other methods. Non-limiting examples of such capture mechanisms include the binding and ligation of complementary sequences. In certain embodiments, the nucleic acids introduced can include, but are not limited to, guide RNAs (gRNAs), lineage barcodes, oligonucleotides conjugated to antibodies, cell hashing oligonucleotides, and other oligonucleotides used for sample multiplexing. 【0096】 In certain embodiments, the first polynucleotide can exist in multiple forms, and the use of multiple forms of the universal array can enable the simultaneous capture of multiple molecular types. For example, without limitation, the molecular types can be RNAs, DNAs, and synthetic oligonucleotides associated with antibodies, peptides, proteins, molecular complexes, lipids, or cholesterol. In certain embodiments, the first polynucleotide includes additional universal sequences that assist in the integration of the second index sequence. 【0097】 In certain embodiments, the cells and / or nuclei can be fixed prior to use in the methods of the present disclosure. For example, but not limited to, the cells or nuclei are fixed prior to contacting the cells or nuclei with particles coupled to a first index array. Any technique known in the art for fixing cells and / or nuclei can be used in the present disclosure. For example, but not limited to, the cells and / or nuclei can be contacted with a fixative. In certain embodiments, the fixation process can include chemical cross-linking, for example, by using glutaraldehyde and / or formaldehyde. In certain embodiments, the cells or nuclei can be fixed with an alcohol, such as methanol and / or ethanol. 【0098】 In certain embodiments, the cells can be permeabilized prior to use in the methods of the present disclosure. For example, but not limited to, the cells are permeabilized prior to contacting the cells or nuclei with particles coupled to a first index array. Any technique known in the art for permeabilizing cells can be used in the present disclosure. For example, but not limited to, the cells can be permeabilized with an alcohol, such as methanol, and / or a detergent, such as Tween-20. In certain embodiments, the cells can be fixed and permeabilized. 【0099】 In certain embodiments, the cells, such as cells of a tissue, can be dissociated prior to use in the methods of the present disclosure. For example, but not limited to, the cells can be dissociated to produce a single cell suspension for use in the present disclosure. In certain embodiments, the cells (or nuclei obtained from such cells) can be dissociated prior to contacting the cells with particles coupled to a first index array. Methods for dissociating cells, such as cells of a tissue, are known in the art. 【0100】 In certain embodiments, the cells or nuclei can be cross-linked before use in the methods of the present disclosure. For example, without limitation, the cells or nuclei can be cross-linked before contacting the cells or nuclei with particles coupled to a first index sequence. Alternatively or additionally, the cells or nuclei can be cross-linked in emulsion droplets. In certain embodiments, the cell membranes for use in the present disclosure can be cross-linked. Cross-linking of cells, such as cross-linking of cell membranes or nuclei, can be achieved by exposing the cells to any cross-linking agent. In certain embodiments, cross-linking can be performed using a fixative as disclosed herein. In certain embodiments, cross-linking can be performed by exposing the cells to dithiobis(succinimidyl propionate) (DSP). 【0101】 In certain embodiments, the nuclei for use in the present disclosure can be nucleosome-free. In certain embodiments, the nuclei can be treated to deplete nucleosomal nuclei. In certain embodiments, the nuclei can be treated to deplete nucleosomal nuclei before use in any one of the disclosed methods. For example, without limitation, the nuclei can be treated to deplete nucleosomal nuclei before contacting the nuclei with particles coupled to a first index sequence. Non-limiting examples of methods for depleting nucleosomal nuclei include contacting isolated nuclei with a chaotropic agent or surfactant that can disrupt the interaction between nucleic acids and proteins. In certain embodiments, nucleosome-free or depleted nuclei can be used in the methods of the present disclosure to index genomic DNA. 【0102】 In certain embodiments, cells or nuclei for use in the present disclosure can be contacted with a transposase. Non-limiting examples of transposases include Tn5, Mu, and Tn7, or variants thereof. For example, but not by way of limitation, the transposase can be a hyperactive mutant of the transposase, such as a hyperactive Tn5 transposase. Non-limiting examples of hyperactive Tn5 variants are disclosed in Picelli et al., Genome Res. 24(12):2033-2040 (2014), the contents of which are hereby incorporated by reference in their entirety. In certain embodiments, nuclei can be treated with a transposase or a transpososome (e.g., a transposase complexed with DNA containing a transposase recognition sequence) prior to use in the present disclosure. For example, but not by way of limitation, nuclei can be treated with a transposase or a transpososome prior to contacting the nuclei with particles coupled to a first index sequence. Alternatively or additionally, the transposase or transpososome can be compartmentalized into emulsion droplets together with the particles and two or more nuclei. In certain embodiments, the transpososome is complexed with a DNA sequence containing a transposon adjacent to a transposase recognition sequence, and the transposon is inserted into genomic DNA. In certain embodiments, the transposon can include a primer binding sequence and / or an adapter. In certain embodiments, the treatment of nuclei with a transposase or a transpososome can be used in the methods of the present disclosure for an assay for transposase-accessible chromatin (ATAC) sequencing. 【0103】 In certain embodiments, the method can further include compartmentalizing individual particles and one or more cells or nuclei into compartments. In certain embodiments, each compartment contains one particle and two or more cells or nuclei. In certain embodiments, the compartment is an emulsion. For example, but not by way of limitation, the compartment is an emulsion droplet. 【0104】 In certain embodiments, the compartment is not a well of a multi-well plate, such as a well of a microtiter plate. 【0105】 In certain embodiments, one or more of the individual beads and cells or nuclei can be compartmentalized into emulsion droplets. In certain embodiments, compartmentalizing the individual particles and one or more cells or nuclei into emulsion droplets can include contacting a plurality of particles from a first aqueous phase and cells or nuclei from a second aqueous phase with a different phase, such as a non-aqueous phase immiscible with the first and / or second aqueous phase, to form emulsion droplets. Alternatively, compartmentalizing the individual particles and one or more cells or nuclei into emulsion droplets can include contacting a first aqueous phase containing a plurality of particles and cells or nuclei with a different phase, such as a non-aqueous phase immiscible with the first aqueous phase, to form emulsion droplets. 【0106】 In certain embodiments, the emulsion droplets can be generated by providing cells and / or nuclei in a first fluid, such as a first aqueous phase, combining the first fluid with a second fluid, such as a second aqueous phase or a non-aqueous phase, and shearing the fluids to produce a plurality of emulsion droplets containing one or more of the individual particles and cells or nuclei. In certain embodiments, the shearing of the fluids can be performed using any known method, technique, or device for mixing solutions. For example, without limitation, shearing the fluids can include vortexing, shaking, flicking, stirring, and / or pipetting. In certain embodiments, shearing the fluids includes vortexing the fluids to produce emulsion droplets. 【0107】 In certain embodiments, the emulsion droplets can be generated using any emulsion droplet generation device. In certain embodiments, the emulsion droplet generation device can be a microfluidic device. For example, without limitation, the droplets can be generated on the Chromium™ platform commercially available from 10xGenomics. In certain embodiments, a first aqueous phase containing an index array carried by beads flows through a channel segment, and a second aqueous phase containing cells or nuclei flows through a second channel segment towards the channel junction. As shown in FIG. 1, a partitioning fluid (e.g., oil) is introduced from one or more side channels into the channel junction, and the combined flow enters an outlet channel. Within the channel junction, the two aqueous flows are combined with the partitioning oil and partitioned into emulsion droplets (referred to herein as "GEMs") that contain co-partitioned nuclei or cells and beads (FIG. 1). In certain embodiments, by controlling the flow characteristics of each fluid combined at the channel junction and the shape of the channel junction, the desired occupancy levels of beads, cells (or nuclei), or both within the generated droplets can be achieved. Additional disclosure regarding microfluidic devices for use in the present disclosure is provided in International Publication No. WO 2017 / 096158 and U.S. Patent No. 11,193,122, the contents of each of which are incorporated herein by reference in their entireties. 【0108】 In certain embodiments, multiple emulsion droplets can be generated by partitioning individual particles and one or more cells or nuclei. In certain embodiments, the methods of the present disclosure can include generating at least about 1,000 droplets, at least about 2,000 droplets, at least about 3,000 droplets, at least about 4,000 droplets, at least about 5,000 droplets, at least about 6,000 droplets, at least about 7,000 droplets, at least about 8,000 droplets, at least about 9,000 droplets, at least about 10,000 droplets, at least about 20,000 droplets, at least about 30,000 droplets, at least about 40,000 droplets, at least about 50,000 droplets, at least about 60,000 droplets, at least about 70,000 droplets, at least about 80,000 droplets, at least about 90,000 droplets, at least about 100,000 droplets, at least about 200,000 droplets, at least about 300,000 droplets, at least about 400,000 droplets, at least about 500,000 droplets, at least about 600,000 droplets, at least about 700,000 droplets, at least about 800,000 droplets, at least about 900,000 droplets, or at least about 1,000,000 droplets. In certain embodiments, from about 5,000 to about 200,000 droplets are generated. 【0109】 In certain embodiments, each compartment, e.g., droplet, contains two or more cells or nuclei along with a single bead. For example, without limitation, the number of cells or nuclei in each droplet can be from about 2 to about 20. In certain embodiments, the number of cells or nuclei in each droplet is from about 3 to about 20, from about 4 to about 20, from about 5 to about 20, from about 6 to about 20, from about 7 to about 20, from about 8 to about 20, from about 9 to about 20, from about 10 to about 20, from about 11 to about 20, from about 12 to about 20, from about 13 to about 20, from about 14 to about 20, from about 15 to about 20, from about 16 to about 20, from about 17 to about 20, from about 18 to about 20, from about 19 to about 20, from about 2 to about 19, from about 2 to about 18, from about 2 to about 17, from about 2 to about 16, from about 2 to about 15, from about 2 to about 14, from about 2 to about 13, from about 2 to about 12, from about 2 to about 11, from about 2 to about 10, from about 2 to about 9, from about 2 to about 8, from about 2 to about 7, from about 2 to about 6, from about 2 to about 5, from about 2 to about 4, from about 2 to about 3, or from about 3 to about 10 cells or nuclei. In certain embodiments, each droplet contains at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 cells or nuclei. In certain embodiments, the number of cells or nuclei in each droplet can be from about 2 to about 10. In certain embodiments, from about 5,000 to about 200,000 droplets are generated, and each droplet can contain from about 2 to about 10 cells or nuclei. 【0110】 By incorporating two or more cells or nuclei into each emulsion droplet along with a single bead, indexing of the nucleic acids of the cells or nuclei present in the emulsion droplets with the same index array becomes possible. As described herein, incorporating two or more cells or nuclei into each emulsion droplet increases the throughput of the indexing technology and increases the volume of the emulsion droplet generation device, e.g., microfluidic device, used to generate the droplets by at least 20-fold. 【0111】 In certain embodiments, other reagents can be co-compartmentalized into the emulsion droplets. In certain embodiments, such reagents can be used, for example, for an amplification process or a ligation process to incorporate a first index array into a nucleic acid. For example, without limitation, the reagents can include polymerase, reverse transcriptase, nucleoside triphosphates or NTP analogs, primers, cofactors, ligation reaction reagents, endonucleases, lysis reagents, dyes, markers or labels. In certain embodiments, additional reagents can include proteases for removing proteins bound to nucleic acids of cells or nuclei, and / or transposases or transposomes for fragmenting or inserting known sequences into nucleic acids of cells or nuclei. 【0112】 In certain embodiments, the method can further include incorporating a first index array disposed on particles, such as beads, into the nucleic acid of a cell or nucleus to generate an indexed nucleic acid. In certain embodiments, incorporating the first index array into the nucleic acid is performed within a droplet, for example, using reagents co-compartmentalized in the droplet. Any suitable nucleic acid amplification method known in the art can be used to incorporate the first index array into the nucleic acid of a cell or nucleus. Non-limiting examples of such amplification methods include polymerase chain reaction (PCR), reverse transcriptase PCR, real-time PCR, rolling circle amplification (RCA), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), transcription-mediated amplification (TMA), single primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase polymerase amplification (RPA), nicking enzyme amplification reaction (NEAR), nicking endonuclease-assisted nanoparticle activation (NENNA), and ligase chain reaction (LCR). In certain embodiments, the amplification process is an isothermal amplification process, such as TMA, NEAR, and / or RPA. Fakruddin et al., J. Pharm. Bioallied. Sci. 5(4):245-252(2013) and Yan et al., Mol. BioSyst. 10:970-1003(2014) disclose additional amplification methods for use in the present disclosure, the content of each of which is hereby incorporated by reference in its entirety. In certain embodiments, the amplification process uses, for example, a universal sequence present in a first polynucleotide to bind primers used in the amplification process. Alternatively, a ligation process can be used to incorporate the first index array into the nucleic acid of a cell or nucleus. For example, without limitation, the index array can be ligated to the nucleic acid of a single cell or nucleus by a ligase. 【0113】 In certain embodiments, reverse transcription is used to incorporate a first index array into the nucleic acid of a cell or nucleus. For example, but not limited to, reverse transcription is used to incorporate a first index array into cellular RNA, such as cellular mRNA. In certain embodiments, within a compartment, such as an emulsion droplet, a first polynucleotide providing the first index array binds to cellular RNA, such as mRNA. Reverse transcriptase binds to the RNA complexed with the first polynucleotide and initiates the synthesis of a complementary DNA (cDNA) strand. Any reverse transcriptase known in the art can be used in the methods of the present disclosure. In certain embodiments, RNase H can be present within the compartment, such as within an emulsion droplet, to degrade the RNA of the cDNA:RNA complex. In certain embodiments, the reverse transcriptase can lack RNase H activity. In certain embodiments, the reverse transcriptase can have terminal transferase activity and template switching activity to incorporate a selected universal sequence at the end of the cDNA strand. In certain embodiments, a compartment, such as an emulsion droplet, can further comprise a DNA-dependent polymerase that uses the single-stranded cDNA as a template to synthesize a complementary cDNA strand to form double-stranded cDNA. 【0114】 In certain embodiments, PCR is used to incorporate a first index array into the nucleic acid of a cell or nucleus. For example, but not limited to, when the nucleic acid to be indexed is DNA, such as genomic DNA, PCR is used. 【0115】 In certain embodiments, the first index array is released from particles, such as gel beads. In certain embodiments, the index array is released from the particles before incorporating the index array into the nucleic acid. Alternatively or additionally, the index array is released from the particles after incorporating the index array into the nucleic acid. In certain embodiments, the first index array is released from the particles by lysing the particles, such as gel beads. In certain embodiments, the particles can be lysed or degraded using a reducing agent, changing the temperature, such as heating a droplet, changing the pH, and / or exposing to light. In certain embodiments, the first index array is released from the particles by cleaving a linker that couples the index array to the particles. In certain embodiments, the first index array is released from the particles within a droplet. 【0116】 In certain embodiments, the method can further include combining indexed nucleic acids from a plurality of compartments, such as emulsion droplets, to generate pooled indexed nucleic acids. For example, without limitation, the method includes combining cells and / or nuclei that contain or are associated with indexed nucleic acids from a plurality of compartments, such as emulsion droplets, to generate pooled cells and / or nuclei. In certain embodiments, nucleic acids containing a first index sequence are removed from a compartment, such as an emulsion droplet. For example, without limitation, nucleic acids containing a first index sequence can be separated from the emulsion droplet by an unpacking process, in which case the indexed nucleic acids present in the emulsion droplet are released. Any technique known in the art for disrupting an emulsion can be used in the unpacking process. Non-limiting examples of techniques used to disrupt an emulsion include acidification, centrifugation, filtration, and addition of salts. In certain embodiments, cells containing the indexed nucleic acids remain intact during the incorporation of the first index sequence, and the unpacking process releases the intact cells from the emulsion droplets. 【0117】 In certain embodiments, subsets of the released indexed nucleic acids are pooled together. In certain embodiments, subsets of cells or nuclei containing the indexed nucleic acids released from a compartment, such as an emulsion droplet, are pooled together. For example, without limitation, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the compartment, such as the indexed nucleic acids (or cells containing or related to the indexed nucleic acids) released from an emulsion droplet are pooled together. In certain embodiments, at least about 1% of the compartment, such as the indexed nucleic acids (or cells containing or related to the indexed nucleic acids) released from an emulsion droplet are pooled together. In certain embodiments, at least about 2% of the compartment, such as the indexed nucleic acids (or cells containing or related to the indexed nucleic acids) released from an emulsion droplet are pooled together. In certain embodiments, at least about 10% of the compartment, such as the indexed nucleic acids (or cells containing or related to the indexed nucleic acids) released from an emulsion droplet are pooled together. In certain embodiments, at least about 50% of the compartment, such as the indexed nucleic acids (or cells containing or related to the indexed nucleic acids) released from an emulsion droplet are pooled together.In certain embodiments, about 1% to about 5% of the compartments, such as the indexed nucleic acids (or cells containing or related to the indexed nucleic acids) released from emulsion droplets, are pooled together. In certain embodiments, about 1% to about 10% of the compartments, such as the indexed nucleic acids (or cells containing or related to the indexed nucleic acids) released from emulsion droplets, are pooled together. In certain embodiments, all of the indexed nucleic acids released from compartments, such as emulsion droplets, are pooled together. In certain embodiments, all of the cells containing the indexed nucleic acids released from compartments, such as emulsion droplets, are pooled together. 【0118】 In certain embodiments, the method can further include distributing a subset of the pooled indexed nucleic acids into a plurality of compartments containing a second index sequence. Indexed cells or nuclei can be partitioned into a plurality of compartments to obtain a second index sequence. Non-limiting examples of compartments include tubes, plates, vials, and wells. In certain embodiments, the compartment can be a well of a multi-well plate, such as a 48-well, 96-well, 384-well, or 1536-well plate, e.g., a well of a microtiter plate. In certain embodiments, the compartment in which the second index sequence is obtained by the indexed nucleic acid is not an emulsion droplet. 【0119】 In certain embodiments, each compartment, e.g., well, contains two or more cells or nuclei comprising indexed nucleic acids. For example, without limitation, the number of cells or nuclei in each compartment can be from about 2 to about 20,000. In certain embodiments, the number of cells or nuclei in each compartment, e.g., well, is from about 10 to about 20,000, from about 100 to about 20,000, from about 1,000 to about 20,000, from about 2,000 to about 20,000, from about 3,000 to about 20,000, from about 4,000 to about 20,000, from about 5,000 to about 20,000, from about 6,000 to about 20,000, from about 7,000 to about 20,000, from about 8,000 to about 20,000, from about 9,000 to about 20,000, from about 10,000 to about 20,000, from about 11,000 to about 20,000, from about 12,000 to about 20,000, from about 13,000 to about 20,000, from about 14,000 to about 20,000, from about 15,000 to about 20,000, from about 16,000 to about 20,000, from about 17,000 to about 20,000, from about 18,000 to about 20,000, from about 19,000 to about 20,000, from about 2 to about 19,000, from about 2 to about 18,000, from about 2 to about 17,000, from about 2 to about 16,000, from about 2 to about 15,000, from about 2 to about 14,000, from about 2 to about 13,000, from about 2 to about 12,000, from about 2 to about 11,000, from about 2 to about 10,000, from about 2 to about 9,000, from about 2 to about 8,000, from about 2 to about 7,000, from about 2 to about 6,000, from about 2 to about 5,000, from about 2 to about 4,000, from about 2 to about 3,000, from about 2 to about 2,000, from about 2 to about 1,000, or from about 2 to about 500 cells or nuclei.In certain embodiments, the number of cells or nuclei in each compartment, such as in a well, can be from about 100 to about 15,000, from about 5,000 to about 15,000, from about 1,000 to about 15,000, from about 2,000 to about 15,000, from about 3,000 to about 15,000, from about 4,000 to about 15,000, from about 5,000 to about 15,000, from about 6,000 to about 15,000, from about 7,000 to about 15,000, from about 8,000 to about 15,000, from about 9,000 to about 15,000, from about 9,000 to about 12,000, from about 500 to about 10,000, from about 1,000 to about 10,000, from about 2,000 to about 10,000, from about 3,000 to about 10,000, from about 4,000 to about 10,000, from about 5,000 to about 10,000, from about 6,000 to about 10,000, from about 7,000 to about 10,000, from about 8,000 to about 10,000, or from about 9,000 to about 10,000. In certain embodiments, each compartment contains at least about 2, at least about 10, at least about 100, at least about 500, at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, at least about 8,000, at least about 9,000, at least about 10,000, at least about 11,000, at least about 12,000, at least about 13,000, at least about 14,000, at least about 15,000, at least about 16,000, at least about 17,000, at least about 18,000, at least about 19,000, or at least about 20,000 cells or nuclei. In certain embodiments, the pooled indexed nucleic acids, e.g., cells or nuclei containing the pooled, indexed nucleic acids, are well mixed such that cells or nuclei having nucleic acids indexed with the same first index sequence are not assigned to the same compartment. As disclosed herein, the use of two index sequences in the methods of the present disclosure enables the labeling of nucleic acids from single cells using unique combinations of the two index sequences. 【0120】 In certain embodiments, the method can further include incorporating a second index sequence into the indexed nucleic acids within each compartment to generate a dual-indexed nucleic acid. Any suitable nucleic acid sequence amplification method known in the art can be used to incorporate the second index sequence into the cell or nuclear indexed nucleic acids. Non-limiting examples of such amplification methods are provided herein. For example, but not limited to, the amplification process is an isothermal amplification process. In certain embodiments, the second index sequence can be incorporated into the indexed nucleic acids using a ligation process. In certain embodiments, the second index sequence can be incorporated into the indexed nucleic acids using a polymerase chain reaction. 【0121】 In certain embodiments, each compartment contains a second index sequence that is unique with respect to the index sequences in other compartments. Alternatively, about 50% or less of the compartments, such as about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less contain a second index sequence having the same nucleic acid sequence as the second index sequence of other compartments. In certain embodiments, 10% or less of the compartments contain a second index sequence having the same nucleic acid sequence as the second index sequence of other compartments. In certain embodiments, 5% or less of the compartments contain a second index sequence having the same nucleic acid sequence as the second index sequence of other compartments. 【0122】 In certain embodiments, the second index array is provided in the second polynucleotide. In certain embodiments, the second polynucleotide further comprises a sequence that is reverse complementary to the sequence of the indexed nucleic acid. In certain embodiments, the second polynucleotide further comprises a sequence that is reverse complementary to the sequence of the first polynucleotide. In certain embodiments, the reverse complementarity enables amplification by polymerase chain reaction or linear amplification or other amplification methods. In certain embodiments, the second polynucleotide comprises a molecular handle that mediates its ligation to the indexed nucleic acid. In certain embodiments, the second polynucleotide can comprise a primer, e.g., a sequence that can bind to a primer binding site or function as a primer. For example, without limitation, the second polynucleotide can comprise a sequence that can bind to a primer for use in the sequencing and / or amplification process of the indexed nucleic acid. 【0123】 In certain embodiments, each compartment contains a reagent for incorporating the second index array into the indexed nucleic acid. For example, without limitation, the reagent can include reagents for performing an amplification or ligation process within the compartment. Non-limiting examples of such reagents are disclosed herein and include polymerases, nucleoside triphosphates or NTP analogs, primers, cofactors, ligation reaction reagents, endonucleases, dyes, markers, and labels. In certain embodiments, each compartment can further comprise a lysis reagent. 【0124】 In certain embodiments, the method can further include lysing the cells. For example, but not limited to, sodium hydroxide, potassium hydroxide, sodium dodecyl sulfate, nonionic surfactants, saponin, proteinase, lysozyme, freeze-thaw processes, ultraviolet light, and / or heat can be used to lyse the cells. In certain embodiments, the cells are lysed after incorporating a second index sequence into the indexed nucleic acid to generate a dual-indexed nucleic acid. Alternatively, the cells are lysed within a compartment containing the second index sequence but before incorporating the second index sequence into the indexed nucleic acid. In certain embodiments, the cells are lysed after the second compartmentalization but before the addition of reagents for incorporating the second index sequence. 【0125】 In certain embodiments, the method can further include incorporating one or more additional index arrays into the dual-indexed nucleic acid. In certain embodiments, one or more additional index arrays, such as two or more, three or more, or four or more additional index arrays, can be incorporated into the dual-indexed nucleic acid. For example, without limitation, the methods of the present disclosure can further include incorporating a third index array into the dual-indexed nucleic acid to generate a triple-indexed nucleic acid. In certain embodiments, incorporating the third index array into the dual-indexed nucleic acid can be performed in another plurality of compartments containing the third index array, as described for the second index array. In certain embodiments, the methods of the present disclosure can further include incorporating a fourth index array into the triple-indexed nucleic acid to generate a quadruple-indexed nucleic acid. In certain embodiments, incorporation of one or more additional index arrays into the dual-indexed nucleic acid can include providing an additional index array, such as a third index array, that can be coupled to the first and / or second index arrays to generate a triple-indexed nucleic acid. In certain embodiments, an additional index array, such as a third index array, can be ligated to the first and / or second index arrays to generate a triple-indexed nucleic acid. In certain embodiments, the fourth index array can be coupled to the first, second, and / or third index arrays, for example, the fourth index array can be ligated to the third index array to generate a quadruple-indexed nucleic acid. 【0126】 In certain embodiments, the methods of the present disclosure can further include performing cell hashing techniques. For example, without limitation, the methods of the present disclosure can further include contacting cells and / or nuclei with a reagent that binds to a target protein (referred to herein as a "protein-binding reagent"). In certain embodiments, the protein-binding reagent is a reagent that specifically binds to a target protein, e.g., a reagent that specifically binds to a target protein of cells and / or nuclei in a sample. Non-limiting examples of protein-binding reagents include antibodies or antibody-binding fragments thereof, aptamers, peptides, and small molecules. In certain embodiments, the protein-binding reagent is an antibody or an antibody-binding fragment thereof. 【0127】 In certain embodiments, the protein bound by the protein-binding reagent can be any protein present intracellularly or on the cell surface. For example, without limitation, the target protein can be an intracellular protein, an extracellular protein, or a transmembrane protein. In certain embodiments, the target protein is a mutant form of a protein or the wild-type of a protein. In certain embodiments, the target protein is an exogenously expressed protein. In certain embodiments, the target protein is an endogenous protein. In certain embodiments, the protein is a lineage-specific protein. In certain embodiments, the protein is a cell-specific protein (e.g., capable of distinguishing between two or more cell types). 【0128】 In certain embodiments, the protein binding reagent can be coupled to an oligonucleotide. In certain embodiments, the oligonucleotide conjugated to the protein binding reagent can include a barcode (referred to herein as a "hash tag"). In certain embodiments, the protein binding reagent can be an antibody (or a fragment thereof) coupled to an oligonucleotide comprising a barcode. In certain embodiments, the barcode enables the identification of the protein binding reagent, such as an antibody, and the corresponding protein to which it binds. In certain embodiments, the protein binding reagent can be a TotalSeq™ antibody (BioLegend, San Diego, CA). In certain embodiments, the use of a protein binding reagent, such as an antibody, enables the detection of a protein in a single sample (e.g., in a single cell in a sample). In certain embodiments, the use of a protein binding reagent, such as an antibody, enables the quantification of a protein in a single sample (e.g., in a single cell in a sample). 【0129】 In certain embodiments, the methods of the disclosure can include contacting cells and / or nuclei with a plurality of protein binding reagents. For example, but not by way of limitation, each protein binding reagent of the plurality of protein binding reagents specifically binds to a single target protein. In certain embodiments, each protein binding reagent is coupled to a unique barcode. In certain embodiments, a plurality of protein binding reagents can be used to bind a plurality of different target proteins in a sample. In certain embodiments, the use of a plurality of protein binding reagents, such as antibodies, enables the detection of a plurality of proteins in a single sample (e.g., in a single cell in a sample). In certain embodiments, the use of a plurality of protein binding reagents, such as antibodies, enables the quantification of a plurality of proteins in a single sample (e.g., in a single cell in a sample). 【0130】 In certain embodiments, cells and / or nuclei can be contacted with a protein binding reagent before contacting the cells and / or nuclei with a plurality of particles (e.g., to implement the combinatorial indexing method of the present disclosure). In certain embodiments, cells and / or nuclei can be contacted with a protein binding reagent before fixing the cells and / or nuclei with a plurality of particles (e.g., methanol fixation). In certain embodiments, a subset of the cells and / or nuclei to be contacted with the plurality of particles can be contacted with a protein binding reagent (e.g., a protein binding reagent coupled to an oligonucleotide containing a barcode), and a different subset of the cells and / or nuclei to be contacted with the plurality of particles can be contacted with a different protein binding reagent (e.g., a different protein binding reagent coupled to an oligonucleotide containing a different barcode). Alternatively or additionally, the cells and / or nuclei to be contacted with the plurality of particles can be contacted with a plurality of protein binding reagents. In certain embodiments, at least 2 or more protein binding reagents, at least 3 or more protein binding reagents, at least 4 or more protein binding reagents, at least 5 or more protein binding reagents, at least 6 or more protein binding reagents, at least 7 or more protein binding reagents, at least 8 or more protein binding reagents, at least 9 or more protein binding reagents, at least 10 or more protein binding reagents, at least 50 or more protein binding reagents, at least 100 or more protein binding reagents, at least 150 or more protein binding reagents, at least 200 or more protein binding reagents, at least 250 or more protein binding reagents, or at least 300 or more protein binding reagents can be used in the present disclosure, each protein binding reagent specifically binds to a single target protein, and each protein binding reagent is coupled to a unique barcode. In certain embodiments, a sample (e.g., a plurality of cells and / or nuclei) can be contacted with a protein binding reagent, e.g., an antibody specific for a target protein coupled to a barcode, under time and conditions that support specific binding of the protein binding reagent to the target protein. 【0131】 In certain embodiments, the methods of the present disclosure can further include treating cells and / or nuclei with an agent, such as a therapeutic agent. For example, without limitation, the methods of the present disclosure can include contacting cells and / or nuclei with an agent (e.g., a therapeutic agent) prior to, for example, performing the combinatorial indexing methods described herein. In certain embodiments, the methods of the present disclosure include contacting cells and / or nuclei with an agent, such as a therapeutic agent, for at least about 15 minutes, at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, or at least about 48 hours prior to performing the combinatorial indexing methods described herein. In certain embodiments, the methods of the present disclosure include contacting cells and / or nuclei with an agent, such as a therapeutic agent, for about 15 minutes to about 48 hours (e.g., about 15 minutes to about 24 hours, about 15 minutes to about 18 hours, about 15 minutes to about 12 hours, about 15 minutes to about 10 hours, about 15 minutes to about 6 hours, about 15 minutes to about 2 hours, about 30 minutes to about 48 hours, about 1 hour to about 48 hours, about 2 hours to about 48 hours, about 6 hours to about 48 hours, about 10 hours to about 48 hours, about 12 hours to about 48 hours, about 18 hours to about 48 hours, about 24 hours to about 48 hours, about 1 hour to about 48 hours, about 2 hours to about 24 hours, or about 2 hours to about 12 hours). In certain embodiments, the therapeutic agent is from a library of therapeutic agents, and the methods of the present disclosure can be used to identify therapeutic agents that will have a therapeutic effect. Non-limiting examples of therapeutic agents are described herein and can include polypeptide therapeutics, such as antibody-based therapeutics, oligonucleotides, cell-based therapeutics, gene editing systems, and small molecule therapeutics. 【0132】 In certain embodiments, the methods of the present disclosure can further include subjecting cells and / or nuclei to genetic modification or regulation. In certain embodiments, the methods of the present disclosure can further include subjecting cells and / or nuclei to genomic screening, for example, prior to performing the combinatorial indexing methods described herein. For example, without limitation, the methods of the present disclosure can further include contacting cells and / or nuclei with a gene editing system, for example, prior to performing the combinatorial indexing methods. Non-limiting examples of gene editing systems include homing endonucleases or meganucleases, ZFNs, TALENs, and CRISPR gene editing systems. In certain embodiments, the methods of the present disclosure can further include subjecting cells and / or nuclei to CRISPR-based screening, for example, prior to performing the combinatorial indexing methods described herein. 【0133】 In certain embodiments, an exemplary method for combinatorially indexing the nucleic acids of single cells or nuclei can include the following: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles, such as microbeads, comprising a first index sequence; (b) partitioning individual particles and one or more of the cells or nuclei into an emulsion, such as an emulsion droplet; (c) incorporating the first index sequence into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; (d) combining cells or nuclei containing or associated with the indexed nucleic acids from a plurality of emulsions, such as emulsion droplets, to generate pooled cells or nuclei; (e) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; and (f) incorporating the second index sequence into the indexed nucleic acid within each compartment to generate a dual-indexed nucleic acid. In certain embodiments, the method can include: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of protein-binding reagents, such as a plurality of antibodies, coupled to oligonucleotides comprising barcodes; (b) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles, such as microbeads, comprising a first index sequence; (c) partitioning individual particles and one or more of the cells or nuclei into an emulsion, such as an emulsion droplet; and (d) incorporating the first index sequence into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; (e) combining cells or nuclei containing or associated with the indexed nucleic acids from a plurality of emulsions, such as emulsion droplets, to generate pooled cells or nuclei; (f) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; and (g) incorporating the second index sequence into the indexed nucleic acid within each compartment to generate a dual-indexed nucleic acid.In certain embodiments, the method can further include incorporating a third index sequence into the dual-indexed nucleic acid to generate a triple-indexed nucleic acid. In certain embodiments, the plurality of cells or nuclei are subjected to genomic screening, cell hashing techniques, lineage tracing methods, and / or treated with therapeutic agents before incorporating the first index sequence into the nucleic acids of the plurality of cells or nuclei. In certain embodiments, the plurality of cells or nuclei are subjected to genomic screening, cell hashing techniques, lineage tracing methods, and / or treated with therapeutic agents before contacting the cells or nuclei with the protein-binding reagent. 【0134】 In certain embodiments, an exemplary method for combinatorially indexing the nucleic acids of single cells or nuclei can include the following: (a) providing a pooled cell or nucleus associated with or containing a nucleic acid that includes a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet; (b) partitioning a subset of the pooled cells or nuclei into a plurality of compartments that each include a second index sequence; and (c) incorporating the second index sequence into the nucleic acids within each compartment to generate doubly-indexed nucleic acids. In certain embodiments, the first index sequence is incorporated into the nucleic acid of a cell or nucleus in an emulsion droplet by a method that includes: (i) contacting a plurality of cells or nuclei with a plurality of particles that each include the first index sequence; (ii) partitioning each particle and one or more of the cells or nuclei into an emulsion droplet; (iii) incorporating the first index sequence into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; and (iv) combining the cells or nuclei associated with or containing the indexed nucleic acid from the plurality of emulsion droplets to generate a pooled cell or nucleus associated with or containing a nucleic acid that includes the first index sequence. In certain embodiments, the pooled cell or nucleus is subjected to genomic screening, cell hashing techniques, lineage tracing methods, and / or treatment with a therapeutic agent prior to incorporating the first index sequence into the nucleic acid of the pooled cell or nucleus. 【0135】 In certain embodiments, the method can include: (a) providing a pooled cell or nucleus associated with or containing a nucleic acid having a first index array, wherein the first index array is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet, and the pooled cell or nucleus is provided by a protein binding reagent, such as an antibody, coupled to an oligonucleotide containing a barcode; (b) distributing a subset of the pooled cells or nuclei into a plurality of compartments having a second index array; and (c) incorporating the second index array into the nucleic acid within each compartment to generate a dual-indexed nucleic acid. In certain embodiments, the first index array is incorporated into the nucleic acid of the cell or nucleus in an emulsion droplet by a method that includes: (i) contacting a plurality of cells or nuclei with a plurality of particles having the first index array; (ii) partitioning the individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index array into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; and (iv) combining the cells or nuclei associated with or containing the indexed nucleic acid from the plurality of emulsion droplets to generate a pooled cell or nucleus associated with or containing a nucleic acid having the first index array. In certain embodiments, the pooled cells or nuclei are subjected to genomic screening, cell hashing techniques, lineage tracing methods, and / or treatment with therapeutic agents before incorporating the first index array into the nucleic acid of the pooled cells or nuclei, e.g., before step (a). 【0136】 The present disclosure further provides a method for generating a sequencing library. In certain embodiments, the sequencing library comprises nucleic acids indexed by the methods of the present disclosure. For example, without limitation, the sequencing libraries according to the present disclosure comprise nucleic acids from at least doubly-indexed single cells. In certain embodiments, the nucleic acids comprising the sequencing library are triply-indexed. In certain embodiments, the sequencing library represents, in part or in whole, the transcriptome of one or more single cells, e.g., all or a subset of the nucleic acids of a particular cell are labeled with an index sequence, e.g., a unique combination of index sequences. In certain embodiments, the sequencing library represents, in part or in whole, the genome of one or more single cells, e.g., all or a subset of the nucleic acids of a particular cell are labeled with an index sequence, e.g., a unique combination of index sequences. 【0137】 In certain embodiments, a method for generating a sequencing library involves combining multi-indexed nucleic acids that can be prepared as disclosed herein to generate a library from a plurality of single cells or nuclei. In certain embodiments, a sequencing library can be prepared by combining multi-indexed nucleic acids, such as dual-indexed nucleic acids, derived from a subset of whole cells or nuclei generated using the methods of the present disclosure. For example, without limitation, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the dual-indexed nucleic acids of cells or nuclei are combined to generate a sequencing library. In certain embodiments, at least about 20% of the dual-indexed nucleic acids of cells or nuclei are combined to generate a sequencing library. In certain embodiments, at least about 50% of the dual-indexed nucleic acids of cells or nuclei are combined to generate a sequencing library. In certain embodiments, at least about 90% of the dual-indexed nucleic acids of cells or nuclei are combined to generate a sequencing library. In certain embodiments, all multi-indexed nucleic acids, such as dual-indexed nucleic acids, can be combined to generate a sequencing library. 【0138】 In certain embodiments, nucleic acids with fewer than all multi - indexes, such as nucleic acids with dual indexes, can be combined to generate a sequencing library. For example, but not limited to, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 91%, less than about 92%, less than about 93%, less than about 94%, less than about 95%, less than about 96%, less than about 97%, less than about 98% or less than about 99% of the nucleic acids with dual indexes of cells or nuclei are combined to generate a sequencing library. In certain embodiments, less than about 50% of the nucleic acids with multi - indexes, such as nucleic acids with dual indexes, can be combined to generate a sequencing library. In certain embodiments, less than about 90% of the nucleic acids with multi - indexes, such as nucleic acids with dual indexes, can be combined to generate a sequencing library. 【0139】 In certain embodiments, about 10% to about 99% of the nucleic acids with dual indexes of cells or nuclei are combined to generate a sequencing library. In certain embodiments, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 99%, about 30% to about 99%, about 40% to about 99%, about 50% to about 99%, about 60% to about 99%, about 70% to about 99%, about 80% to about 99%, about 90% to about 99%, about 20% to about 80%, or about 30% to about 50% of the nucleic acids with dual indexes of cells or nuclei are combined to generate a sequencing library. 【0140】 In certain embodiments, a method for generating a sequencing library comprising nucleic acids from a plurality of single cells comprises: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles, such as microbeads, comprising a first index sequence; (b) partitioning individual particles, such as microbeads, and one or more of the cells or nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acids of the cells or nuclei to generate indexed nucleic acids; (d) combining cells or nuclei containing or associated with the indexed nucleic acids from the plurality of emulsion droplets to generate pooled indexed cells or nuclei; (e) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (f) incorporating the second index sequence into the indexed nucleic acids within each compartment to generate doubly indexed nucleic acids; and (g) combining all or a subset of the doubly indexed nucleic acids to generate a sequencing library from the plurality of cells or nuclei. In certain embodiments, the plurality of cells or nuclei are subjected to genomic screening, cell hashing techniques, lineage tracing methods, and / or treated with therapeutic agents prior to incorporating the first index sequence into the nucleic acids of the plurality of cells or nuclei, e.g., prior to step (a). 【0141】 In certain embodiments, the methods of the present disclosure can include cell hashing techniques. For example, without limitation, the methods of the present disclosure can include: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of protein binding reagents, such as a plurality of antibodies, coupled to oligonucleotides containing barcodes; (b) contacting the plurality of cells or nuclei isolated from the plurality of cells with a plurality of particles, such as microbeads, containing a first index sequence; (c) partitioning individual particles, such as microbeads, and one or more of the cells or nuclei into emulsion droplets; (d) incorporating the first index sequence into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; (e) combining the cells or nuclei associated with or containing the indexed nucleic acids from the plurality of emulsion droplets to generate pooled indexed cells or nuclei; (f) distributing a subset of the pooled cells or nuclei into a plurality of compartments containing a second index sequence; (g) incorporating the second index sequence into the indexed nucleic acid within each compartment to generate a doubly indexed nucleic acid; and (h) combining all or a subset of the doubly indexed nucleic acids to generate a sequencing library from the plurality of cells or nuclei. In certain embodiments, the plurality of cells or nuclei can be further subjected to genomic screening, subjected to lineage tracing methods, and / or treated with therapeutic agents before incorporating the first index sequence into the nucleic acid of the plurality of cells or nuclei and / or before contacting the plurality of cells or nuclei with the plurality of protein binding reagents, such as before step (a). 【0142】 In certain embodiments, a method for generating a sequencing library comprising nucleic acids from a plurality of single cells comprises: (a) providing pooled cells or nuclei associated with or comprising nucleic acids comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acids of the pooled cells or nuclei in emulsion droplets; (b) partitioning a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acids within each compartment to generate doubly-indexed nucleic acids; and (d) combining all or a subset of the doubly-indexed nucleic acids to generate a sequencing library from the plurality of cells or nuclei. In certain embodiments, the first index sequence is incorporated into the nucleic acids of the cells or nuclei in emulsion droplets by a method comprising: (i) contacting a plurality of cells or nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index sequence into the nucleic acids of the cells or nuclei to generate indexed nucleic acids; and (iv) combining the cells or nuclei associated with or comprising the indexed nucleic acids from the plurality of emulsion droplets to generate pooled cells or nuclei associated with or comprising nucleic acids comprising the first index sequence. In certain embodiments, the pooled cells or nuclei are subjected to genomic screening, subjected to cell hashing techniques, subjected to lineage tracing methods, and / or treated with a therapeutic agent prior to incorporating the first index sequence into the nucleic acids of the pooled cells or nuclei. 【0143】 In certain embodiments, the method comprises: (a) providing a pooled cell or nucleus associated with or containing a nucleic acid comprising a first index array, wherein the first index array is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet and the pooled cell or nucleus is provided with a protein-binding reagent, such as an antibody, coupled to an oligonucleotide comprising a barcode; (b) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index array; (c) incorporating the second index array into the nucleic acid within each compartment to generate a doubly-indexed nucleic acid; and (d) combining all or a subset of the doubly-indexed nucleic acids to generate a sequencing library from a plurality of cells or nuclei. In certain embodiments, the first index array is incorporated into the nucleic acid of the cell or nucleus in an emulsion droplet by a method comprising: (i) contacting a plurality of cells or nuclei with a plurality of particles comprising the first index array; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index array into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; and (iv) combining the cells or nuclei associated with or containing the indexed nucleic acid from the plurality of emulsion droplets to generate a pooled cell or nucleus associated with or containing a nucleic acid comprising the first index array. In certain embodiments, the pooled cell or nucleus is further subjected to genomic screening, subjected to lineage tracing methods, and / or treated with a therapeutic agent prior to incorporating the first index array into the nucleic acid of the pooled cell or nucleus. 【0144】 In another aspect, the present disclosure further provides a method for sequencing a library generated according to the methods disclosed herein. In certain embodiments, the sequencing method of the present disclosure includes sequencing a multi-indexed nucleic acid, e.g., a dual-indexed nucleic acid, from a single cell. In certain embodiments, the sequencing method of the present disclosure includes sequencing a dual-indexed nucleic acid that partially or fully represents the transcriptome of a single cell. In certain embodiments, the sequencing method of the present disclosure includes sequencing a dual-indexed nucleic acid that partially or fully represents the genome of a single cell. 【0145】 In certain embodiments, the sequencing method of the present disclosure involves sequencing all or a subset of the multi-indexed nucleic acids of the library, such as the dual-indexed nucleic acids. For example, without limitation, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the multi-indexed nucleic acids, such as the dual-indexed nucleic acids, contained in the library can be sequenced. In certain embodiments, about 10% to about 99% of the dual-indexed nucleic acids can be sequenced. In certain embodiments, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 99%, about 30% to about 99%, about 40% to about 99%, about 50% to about 99%, about 60% to about 99%, about 70% to about 99%, about 80% to about 99%, about 90% to about 99%, about 20% to about 80%, or about 30% to about 50% of the dual-indexed nucleic acids can be sequenced. 【0146】 In certain embodiments, the present disclosure provides a method for sequencing nucleic acids from a plurality of cells, comprising: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles, such as microbeads, comprising a first index sequence; (b) partitioning individual particles, such as microbeads, and one or more of the cells or nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acids of the cells or nuclei to generate indexed nucleic acids; (d) combining the cells or nuclei containing or associated with the indexed nucleic acids from the plurality of emulsion droplets to generate pooled indexed cells or nuclei; (e) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (f) incorporating the second index sequence into the indexed nucleic acids within each compartment to generate doubly indexed nucleic acids; (g) combining all or a subset of the doubly indexed nucleic acids to generate a sequencing library from the plurality of cells; and (h) sequencing all or a subset of the doubly indexed nucleic acids. In certain embodiments, the plurality of cells or nuclei are subjected to genomic screening, cell hashing techniques, lineage tracing methods, and / or treated with therapeutic agents prior to incorporating the first index sequence into the nucleic acids of the plurality of cells or nuclei. 【0147】 In certain embodiments, the method comprises: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of protein binding reagents, such as a plurality of antibodies, coupled to oligonucleotides comprising barcodes; (b) contacting the plurality of cells or nuclei isolated from the plurality of cells with a plurality of particles, such as microbeads, comprising a first index sequence; (c) partitioning individual particles, such as microbeads, and one or more of the cells or nuclei into emulsion droplets; (d) incorporating the first index sequence into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; (e) combining the cells or nuclei containing or associated with the indexed nucleic acids from the plurality of emulsion droplets to generate pooled indexed cells or nuclei; (f) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (g) incorporating the second index sequence into the indexed nucleic acid within each compartment to generate a dual-indexed nucleic acid; (h) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from the plurality of cells; and (i) sequencing all or a subset of the dual-indexed nucleic acids and / or sequencing the barcodes derived from the protein binding reagents. In certain embodiments, the plurality of cells or nuclei are further subjected to genomic screening, subjected to lineage tracing methods, and / or treated with therapeutic agents before incorporating the first index sequence into the nucleic acid of the plurality of cells or nuclei and / or before contacting the plurality of cells or nuclei with the plurality of protein binding reagents. 【0148】 In certain embodiments, the present disclosure provides a method for sequencing nucleic acids from a plurality of cells, comprising: (a) providing a pooled cell or nucleus associated with or containing a nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet; (b) partitioning a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acid within each compartment to generate a dual-indexed nucleic acid; (d) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from the plurality of cells; and (d) sequencing all or a subset of the dual-indexed nucleic acids. In certain embodiments, the first index sequence is incorporated into the nucleic acid of the cells or nuclei in an emulsion droplet by a method comprising: (i) contacting a plurality of cells or nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cells or nuclei to generate an indexed nucleic acid; and (iv) combining the cells or nuclei associated with or containing the indexed nucleic acid from the plurality of emulsion droplets to generate a pooled cell or nucleus associated with or containing a nucleic acid comprising the first index sequence. In certain embodiments, the pooled cells or nuclei are subjected to genomic screening, subjected to cell hashing technology, subjected to lineage tracing methods, and / or treated with a therapeutic agent prior to incorporating the first index sequence into the nucleic acid of the pooled cells or nuclei. 【0149】 In certain embodiments, the method comprises: (a) providing a pooled cell or nucleus associated with or containing a nucleic acid comprising a first index array, wherein the first index array is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet, and the pooled cell or nucleus is provided with a protein binding reagent, such as an antibody, coupled to an oligonucleotide comprising a barcode; (b) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index array; (c) incorporating the second index array into the nucleic acid within each compartment to generate a dual-indexed nucleic acid; (d) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from a plurality of cells; and (d) sequencing all or a subset of the dual-indexed nucleic acids and / or sequencing the barcode derived from the protein binding reagent. In certain embodiments, the first index array is incorporated into the nucleic acid of the cell or nucleus in an emulsion droplet by a method comprising: (i) contacting a plurality of cells or nuclei with a plurality of particles comprising the first index array; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index array into the nucleic acid of the cell or nucleus to generate an indexed nucleic acid; and (iv) combining cells or nuclei associated with or containing the indexed nucleic acid from a plurality of emulsion droplets to generate a pooled cell or nucleus associated with or containing a nucleic acid comprising the first index array. In certain embodiments, the pooled cell or nucleus is further subjected to genomic screening, a lineage tracing method, and / or treated with a therapeutic agent before incorporating the first index array into the nucleic acid of the pooled cell or nucleus and / or binding the protein binding reagent to the pooled cell or nucleus. 【0150】 Suitable sequencing methods can be used to sequence indexed nucleic acids. In certain embodiments, the nucleic acids can be sequenced by high-throughput sequencing methods. In certain embodiments, the nucleic acids can be sequenced by next-generation sequencing (NGS). For example, without limitation, sequencing can be performed using an Illumina NGS platform. In certain embodiments, the nucleic acids can be sequenced by pyrosequencing methods. In certain embodiments, the nucleic acids can be sequenced by Sanger sequencing. In certain embodiments, the nucleic acids can be sequenced using nanopore-based sequencing. In certain embodiments, the sequence reads obtained using the methods of the present disclosure can be attributable to specific cells based on a unique combination of two indexed sequences. 【0151】 IV. Kits The present disclosure provides kits for practicing the methods of the present disclosure. For example, without limitation, the present disclosure provides kits containing materials for practicing methods for combinatorial indexing of nucleic acids from multiple single cells. In certain embodiments, the present disclosure provides kits containing materials for practicing methods for generating a sequencing library containing nucleic acids from multiple single cells. In certain embodiments, the present disclosure provides kits containing materials for sequencing libraries containing nucleic acids from multiple single cells. 【0152】 In certain embodiments, the kits of the present disclosure include a container containing a plurality of particles comprising a first index array. In certain embodiments, the kit can further include a container containing a plurality of second index arrays. Non-limiting examples of suitable containers include bottles, test tubes, vials, and microtiter plates. The container can be formed from a variety of materials such as glass or plastic. For example, without limitation, the kits of the present disclosure can include a microtiter plate comprising a plurality of compartments comprising a second index array. 【0153】 In certain embodiments, the kits of the present disclosure can further include one or more protein binding reagents, such as antibodies. In certain embodiments, the kits of the present disclosure can include a container containing a plurality of protein binding reagents, such as antibodies. In certain embodiments, each protein binding reagent, such as an antibody, binds to an oligonucleotide comprising a barcode. 【0154】 In certain embodiments, the kit further includes an accompanying document providing instructions for using the components provided in the kit. For example, the kits of the present disclosure can include an accompanying document providing instructions for using a plurality of particles comprising a first index array in the disclosed methods. In certain embodiments, the kits of the present disclosure can include an accompanying document providing instructions for using a microtiter plate comprising a plurality of compartments comprising a second index array in the disclosed methods. 【0155】 In certain embodiments, the kits of the present disclosure can further include reagents for generating emulsion droplets. For example, without limitation, the kit can include one or more aqueous and / or non-aqueous fluids. In certain embodiments, the kit can include materials for performing emulsion droplet generation techniques, such as pipettes and / or vortex machines. Alternatively or additionally, the kits of the present disclosure can include an emulsion generation device, such as a microfluidic device. 【0156】 In certain embodiments, the kits of the present disclosure can further include reagents for performing amplification reactions. In certain embodiments, the reagents can include one or more of the following: polymerase, reverse transcriptase, nucleoside triphosphates or NTP analogs, primers, cofactors, ligation reaction reagents, endonucleases, lysis reagents, dyes, markers, and labels. 【0157】 In certain embodiments, the kits of the present disclosure can further include reagents for performing genomic screening, lineage tracing methods, and / or cell hashing techniques. In certain embodiments, the kits of the present disclosure can further include reagents for performing genomic screening. In certain embodiments, the kits of the present disclosure can further include reagents for performing lineage tracing methods. 【0158】 In certain embodiments, the kit can include other materials desirable from a commercial and user perspective, including other buffers and diluents. In certain embodiments, the kit can include materials or reagents for dissociating a sample into single cells or for isolating nuclei from single cells. In certain embodiments, the kit can include reagents for permeabilizing, fixing, and / or crosslinking cells or nuclei. In certain embodiments, the kits of the present disclosure can include a transposase, such as Tn5 transposase. 【0159】 In certain embodiments, the components of the kit are provided in a predetermined ratio by appropriately varying the relative amounts of the various reagents to obtain the desired sensitivity and throughput of the disclosed methods. 【0160】 V. Exemplary Embodiments A. The present disclosure provides a method for combinatorial indexing of nucleic acids from a plurality of single cells, comprising: (a) providing pooled cells or nuclei associated with or containing a nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the pooled cells or nuclei in emulsion droplets; (b) partitioning a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; and (c) incorporating the second index sequence into the nucleic acid within each compartment to generate a dual-indexed nucleic acid. 【0161】 A1. The method according to A, wherein the first index sequence is incorporated into the nucleic acid of the cells or nuclei in the emulsion droplets by the following: (i) contacting a plurality of cells or nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cells or nuclei to generate an indexed nucleic acid; and (iv) combining the cells or nuclei associated with or containing the indexed nucleic acid from the plurality of emulsion droplets to generate pooled cells or nuclei associated with or containing a nucleic acid comprising the first index sequence. 【0162】 A2. The method according to A1, wherein the cells or nuclei are treated with transposase before or during step (i). 【0163】 A3. The method according to A2, wherein the transposase is Tn5 transposase. 【0164】 A4. The method according to any one of A1 to A3, wherein the plurality of cells or nuclei are treated with a fixative before step (i). 【0165】 A5. The method according to any one of A1 to A4, wherein the plurality of cells are permeabilized or lysed before step (i). 【0166】 A6. The method according to any one of A1 to A5, wherein a plurality of cells or nuclei are treated with a multiplexing reagent before or during step (i). 【0167】 A7. The method according to any one of A1 to A6, wherein the first index array of each particle or subset of particles is unique to other particles. 【0168】 A8. The method according to any one of A to A7, wherein the second index array within one compartment or subset of compartments is unique to other second index arrays. 【0169】 A9. The method according to any one of A to A8, wherein the plurality of compartments are a multi-well plate. 【0170】 A10. The method according to any one of A to A9, wherein the emulsion droplets are generated within an emulsion droplet generation device or using an emulsion droplet generation technique. 【0171】 A11. The method according to any one of A to A10, further comprising incorporating a third index array into the dual-indexed nucleic acid to generate a triple-indexed nucleic acid. 【0172】 A12. The method according to any one of A to A11, wherein incorporating the first index array into the nucleic acid in the emulsion droplet is performed by an amplification process, a reverse transcription process, or a ligation process. 【0173】 A12.1. The method according to A12, wherein incorporating the first index array into the nucleic acid in the emulsion droplet is performed by a reverse transcription process. 【0174】 A13. The method according to any one of A to A12.1, wherein incorporating the second index array into the indexed nucleic acid is performed by an amplification process or a ligation process. 【0175】 A13.1. The method according to any one of A to A13, wherein incorporating the second index array into the indexed nucleic acid is performed by an amplification process. 【0176】 A14. The method according to any one of A12 to A13.1, wherein the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. 【0177】 A15. The method according to any one of A to A14, wherein the nucleic acid comprises RNA. 【0178】 A16. The method according to any one of A to A14, wherein the nucleic acid comprises DNA. 【0179】 A17. The method according to any one of A to A16, wherein the plurality of cells comprises at least about 100,000 cells. 【0180】 A18. The method according to any one of A to A17, wherein the plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof. 【0181】 A19. The method according to any one of A to A18, wherein the plurality of cells comprises any one of the following: (a) cells modified with a gene editing system; (b) antibody-producing cells; (c) cells in different developmental states; (d) cells in different disease states; (e) cells treated with a drug; and (f) cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide containing a barcode. 【0182】 A20. The method according to A19, wherein the plurality of cells comprises cells modified with a gene editing system. 【0183】 A21. The method according to A19, wherein the plurality of cells comprises antibody-producing cells. 【0184】 A22. The method according to A19, wherein the plurality of cells comprises cells in different developmental states. 【0185】 A23. The method according to A19, wherein the plurality of cells includes cells in different disease states. 【0186】 A24. The method according to A19, wherein the plurality of cells includes cells treated with a drug. 【0187】 A25. The method according to A24, wherein the drug is a therapeutic agent. 【0188】 A26. The method according to A25, wherein the therapeutic agent is selected from the group consisting of polypeptide therapeutics, oligonucleotides, cell-based therapeutics, gene editing systems, small molecule therapeutics, and combinations thereof. 【0189】 A27. The method according to A19, wherein the plurality of cells includes cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide containing a barcode. 【0190】 A28. The method according to any one of A to A27, wherein the emulsion droplet contains at least two cells or nuclei. 【0191】 A29. The method according to any one of A to A28, wherein the particles are gel beads. 【0192】 A30. The method according to any one of A to A29, further comprising performing a lineage tracing method. 【0193】 A31. The method according to any one of A to A30, further comprising performing a genomic screening. 【0194】 A32. The method according to A31, wherein the genomic screening is a CRISPR-based screening. 【0195】 A33. The method according to A32, wherein the CRISPR-based screening is selected from the group consisting of gene editing CRISPR screening, CRISPRi screening, and CRISPRa screening. 【0196】 A34. The method according to any one of A to A33, wherein a plurality of cells or nuclei isolated from a plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide containing a barcode. 【0197】 A35. The method according to any one of A to A33, further comprising contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of protein binding reagents coupled to an oligonucleotide containing a barcode, prior to (a). 【0198】 A36. The method according to A34 or A35, wherein the protein binding reagent binds to a protein located on the surface of at least one cell of a plurality of single cells. 【0199】 A37. The method according to A34 to A36, wherein the protein binding reagent is an antibody or a fragment thereof. 【0200】 A38. The method according to A36 or A37, further comprising determining the expression level of the protein. 【0201】 B. The present disclosure provides a method for generating a sequencing library comprising nucleic acids from a plurality of single cells, comprising: (a) providing a pooled cell or nucleus associated with or containing a nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet; (b) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acid within each compartment to generate a dual-indexed nucleic acid; and (d) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from a plurality of cells. 【0202】 The method according to B1.B, wherein the first index array is applied to the nucleic acid of cells or nuclei in an emulsion droplet by the following: (i) contacting a plurality of cells or nuclei with a plurality of particles comprising the first index array; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index array into the nucleic acid of the cells or nuclei to generate an indexed nucleic acid; and (iv) combining cells or nuclei associated with or containing the indexed nucleic acid from a plurality of emulsion droplets to generate a pooled cell or nucleus associated with or containing a nucleic acid comprising the first index array, the method being incorporated by the method. 【0203】 B2. The method according to B1, wherein the cells or nuclei are treated with transposase before or during step (i). 【0204】 B3. The method according to B2, wherein the transposase is Tn5 transposase. 【0205】 B4. The method according to any one of B1 to B3, wherein a plurality of cells or nuclei are treated with a fixative before step (i). 【0206】 B5. The method according to any one of B1 to B4, wherein a plurality of cells are permeabilized or lysed before step (i). 【0207】 B6. The method according to any one of B1 to B5, wherein a plurality of cells or nuclei are treated with a multiplexing reagent before or during step (i). 【0208】 B7. The method according to any one of B1 to B6, wherein the first index array of each particle or subset of particles is unique with respect to other particles. 【0209】 B8. The method according to any one of B to B7, wherein the second index array within one compartment or subset of compartments is unique with respect to other second index arrays. 【0210】 B9. The method according to any one of B to B8, wherein the plurality of compartments are multi-well plates. 【0211】 B10. The method according to any one of B to B9, wherein the emulsion droplets are generated within an emulsion droplet generation device or using an emulsion droplet generation technique. 【0212】 B11. The method according to any one of B to B10, further comprising incorporating a third index sequence into the dual-indexed nucleic acid to generate a triple-indexed nucleic acid. 【0213】 B12. The method according to any one of B to B11, wherein incorporating the first index sequence into the nucleic acid in the emulsion droplet is performed by an amplification process, a reverse transcription process, or a ligation process. 【0214】 B12.1. The method according to B12, wherein incorporating the first index sequence into the nucleic acid in the emulsion droplet is performed by a reverse transcription process. 【0215】 B13. The method according to any one of B to B12.1, wherein incorporating the second index sequence into the indexed nucleic acid is performed by an amplification process or a ligation process. 【0216】 B13.1. The method according to any one of B to B13, wherein incorporating the second index sequence into the indexed nucleic acid is performed by an amplification process. 【0217】 B14. The method according to any one of B12 to B13.1, wherein the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. 【0218】 B15. The method according to any one of B to B14, wherein the nucleic acid comprises RNA. 【0219】 The method according to any one of B to B14, wherein the nucleic acid comprises DNA. 【0220】 The method according to any one of B to B16, wherein the plurality of cells comprises at least about 100,000 cells. 【0221】 The method according to any one of B to B17, wherein the plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or combinations thereof. 【0222】 The method according to any one of B to B18, wherein the plurality of cells comprises any one of the following: (a) cells modified with a gene editing system; (b) antibody-producing cells; (c) cells in different developmental states; (d) cells in different disease states; (e) cells treated with a drug; and (f) cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide containing a barcode. 【0223】 The method according to B19, wherein the plurality of cells comprises cells modified with a gene editing system. 【0224】 The method according to B19, wherein the plurality of cells comprises antibody-producing cells. 【0225】 The method according to B19, wherein the plurality of cells comprises cells in different developmental states. 【0226】 The method according to B19, wherein the plurality of cells comprises cells in different disease states. 【0227】 The method according to B19, wherein the plurality of cells comprises cells treated with a drug. 【0228】 The method according to B24, wherein the drug is a therapeutic agent. 【0229】 The method according to B25, wherein the therapeutic agent is selected from the group consisting of polypeptide therapeutics, oligonucleotides, cell-based therapeutics, gene editing systems, small molecule therapeutics, and combinations thereof. 【0230】 The method according to B19, wherein a plurality of cells comprises cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide comprising a barcode. 【0231】 The method according to any one of B~B27, wherein the emulsion droplets contain at least two cells or nuclei. 【0232】 The method according to any one of B~B28, wherein the particles are gel beads. 【0233】 The method according to any one of B~B29, further comprising performing a lineage tracing method. 【0234】 The method according to any one of B~B30, further comprising performing a genome screening. 【0235】 The method according to B31, wherein the genome screening is a CRISPR-based screening. 【0236】 The method according to B32, wherein the CRISPR-based screening is selected from the group consisting of gene editing CRISPR screening, CRISPRi screening, and CRISPRa screening. 【0237】 The method according to any one of B~B33, wherein a plurality of cells or nuclei isolated from a plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide comprising a barcode. 【0238】 The method according to any one of B to B33, further comprising contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides containing barcodes, prior to B35.(a). 【0239】 The method according to B34 or B35, wherein the protein binding reagent binds to a protein located on the surface of at least one cell of a plurality of single cells. 【0240】 The method according to any one of B34 to B36, wherein the protein binding reagent is an antibody or a fragment thereof. 【0241】 The method according to B36 or B37, further comprising determining the expression level of a protein. 【0242】 C. The present disclosure provides a method for sequencing a library containing nucleic acids from a plurality of single cells, comprising: (a) providing a pooled cell or nucleus associated with or containing a nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet; (b) distributing a subset of the pooled cells or nuclei into a plurality of compartments containing a second index sequence; (c) incorporating the second index sequence into the nucleic acid within each compartment to generate a dual-indexed nucleic acid; (d) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from a plurality of cells; and (e) sequencing all or a subset of the dual-indexed nucleic acids. 【0243】 The method according to C1.C, wherein the first index array is incorporated into the nucleic acid of cells or nuclei in an emulsion droplet by the following method: (i) contacting a plurality of cells or nuclei with a plurality of particles containing the first index array; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index array into the nucleic acid of the cells or nuclei to generate an indexed nucleic acid; and (iv) combining cells or nuclei associated with or containing the indexed nucleic acid from a plurality of emulsion droplets to generate pooled cells or nuclei associated with or containing a nucleic acid containing the first index array. 【0244】 The method according to C1, wherein the cells or nuclei are treated with transposase before or during step (i). 【0245】 The method according to C2, wherein the transposase is Tn5 transposase. 【0246】 The method according to any one of C1 to C3, wherein a plurality of cells or nuclei are treated with a fixative before step (i). 【0247】 The method according to any one of C1 to C4, wherein a plurality of cells are permeabilized or lysed before step (i). 【0248】 The method according to any one of C1 to C5, wherein a plurality of cells or nuclei are treated with a multiplexing reagent before or during step (i). 【0249】 The method according to any one of C1 to C6, wherein the first index array of each particle or subset of particles is unique to other particles. 【0250】 The method according to any one of C to C7, wherein the second index array within one compartment or subset of compartments is unique to other second index arrays. 【0251】 Method according to any one of C to C8, wherein the plurality of compartments are multi-well plates. 【0252】 Method according to any one of C to C9, wherein the emulsion droplets are generated within an emulsion droplet generation device or using an emulsion droplet generation technique. 【0253】 Method according to any one of C to C10, further comprising incorporating a third index sequence into the dual-indexed nucleic acid to generate a triple-indexed nucleic acid. 【0254】 Method according to any one of C to C11, wherein incorporation of the first index sequence into the nucleic acid in the emulsion droplet is performed by an amplification process, a reverse transcription process, or a ligation process. 【0255】 Method according to C12, wherein incorporation of the first index sequence into the nucleic acid in the emulsion droplet is performed by a reverse transcription process. 【0256】 Method according to any one of C to C12.1, wherein incorporation of the second index sequence into the indexed nucleic acid is performed by an amplification process or a ligation process. 【0257】 Method according to any one of C to C13, wherein incorporation of the second index sequence into the indexed nucleic acid is performed by an amplification process. 【0258】 Method according to any one of C12 to C13.1, wherein the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. 【0259】 Method according to any one of C to C14, wherein the nucleic acid comprises RNA. 【0260】 The method according to any one of C to C14, wherein the nucleic acid comprises DNA. 【0261】 The method according to any one of C to C16, wherein the plurality of cells comprises at least about 100,000 cells. 【0262】 The method according to any one of C to C17, wherein the plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof. 【0263】 The method according to any one of C to C18, wherein the plurality of cells comprises any one of the following: (a) cells modified with a gene editing system; (b) antibody-producing cells; (c) cells in different developmental states; (d) cells in different disease states; (e) cells treated with a drug; and (f) cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide containing a barcode. 【0264】 The method according to C19, wherein the plurality of cells comprises cells modified with a gene editing system. 【0265】 The method according to C19, wherein the plurality of cells comprises antibody-producing cells. 【0266】 The method according to C19, wherein the plurality of cells comprises cells in different developmental states. 【0267】 The method according to C19, wherein the plurality of cells comprises cells in different disease states. 【0268】 The method according to C19, wherein the plurality of cells comprises cells treated with a drug. 【0269】 The method according to C24, wherein the drug is a therapeutic agent. 【0270】 The method according to C25, wherein the therapeutic agent is selected from the group consisting of polypeptide therapeutics, oligonucleotides, cell-based therapeutics, gene editing systems, small molecule therapeutics, and combinations thereof. 【0271】 The method according to C19, wherein a plurality of cells comprise cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide comprising a barcode. 【0272】 The method according to any one of C~C27, wherein the emulsion droplets contain at least two cells or nuclei. 【0273】 The method according to any one of C~C28, wherein the particles are gel beads. 【0274】 The method according to any one of C~C29, further comprising performing a lineage tracing method. 【0275】 The method according to any one of C~C30, further comprising performing a genome screening. 【0276】 The method according to C31, wherein the genome screening is a CRISPR-based screening. 【0277】 The method according to C32, wherein the CRISPR-based screening is selected from the group consisting of gene editing CRISPR screening, CRISPRi screening, and CRISPRa screening. 【0278】 The method according to any one of C~C33, wherein a plurality of cells or nuclei isolated from a plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide comprising a barcode. 【0279】 The method according to any one of C33 to C35, further comprising contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides containing barcodes, prior to C35.(a). 【0280】 The method according to C34 or C35, wherein the protein binding reagent binds to a protein located on the surface of at least one cell of a plurality of single cells. 【0281】 The method according to any one of C34 to C36, wherein the protein binding reagent is an antibody or a fragment thereof. 【0282】 The method according to C36 or C37, further comprising determining the expression level of a protein. 【0283】 D. The present disclosure provides a method for combinatorial indexing of nucleic acids from a plurality of single cells, comprising: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles containing a first index sequence; (b) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cells or nuclei to generate indexed nucleic acids; (d) combining the cells or nuclei containing or associated with the indexed nucleic acids from the plurality of emulsion droplets to generate pooled cells or nuclei; (e) distributing a subset of the pooled cells or nuclei into a plurality of compartments containing a second index sequence; and (f) incorporating the second index sequence into the indexed nucleic acids within each compartment to generate doubly indexed nucleic acids. 【0284】 The method according to D, further comprising (g) incorporating a third index sequence into the doubly indexed nucleic acid to generate a triply indexed nucleic acid. 【0285】 The method according to D or D1, wherein the first index array of each particle or subset of particles is unique to other particles. 【0286】 The method according to any one of D to D2, wherein the second index array within one compartment or subset of compartments is unique to other second index arrays. 【0287】 The method according to any one of D to D3, wherein the emulsion droplets are generated within an emulsion droplet generation device or using an emulsion droplet generation technique. 【0288】 The method according to any one of D to D4, wherein the plurality of compartments are multi-well plates. 【0289】 The method according to any one of D to D5, wherein incorporating the first index array into a nucleic acid is performed by an amplification process, a reverse transcription process, or a ligation process. 【0290】 The method according to D6, wherein incorporating the first index array into a nucleic acid in an emulsion droplet is performed by a reverse transcription process. 【0291】 The method according to any one of D to D6, wherein incorporating the second index array into an indexed nucleic acid is performed by an amplification process or a ligation process. 【0292】 The method according to any one of D to D7, wherein incorporating the second index array into an indexed nucleic acid is performed by an amplification process. 【0293】 The method according to D6 to D7.1, wherein the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. 【0294】 The method according to any one of D to D8, wherein the cell or nucleus is treated with a transposase before or during step (a). 【0295】 The method according to D9, wherein the transposase is Tn5 transposase. 【0296】 The method according to any one of D to D10, wherein a plurality of cells or nuclei are treated with a fixative before step (a). 【0297】 The method according to any one of D to D11, wherein a plurality of cells are permeabilized or lysed before step (a). 【0298】 The method according to any one of D to D12, wherein a plurality of cells or nuclei are treated with a multiplexing reagent before or during step (a). 【0299】 The method according to any one of D to D13, wherein the nucleic acid comprises RNA. 【0300】 The method according to any one of D to D13, wherein the nucleic acid comprises DNA. 【0301】 The method according to any one of D to D15, wherein a plurality of cells comprise at least about 100,000 cells. 【0302】 The method according to any one of D to D16, wherein a plurality of cells comprise pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or combinations thereof. 【0303】 The method according to any one of D to D17, wherein a plurality of cells comprise somatic cells. 【0304】 A method according to any one of D19.D to D18, wherein a plurality of cells comprises any one of the following: (a) cells modified with a gene editing system; (b) antibody-producing cells; (c) cells in different developmental states; (d) cells in different disease states; (e) cells treated with a drug; and (f) cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide containing a barcode. 【0305】 D20. The method according to D19, wherein the plurality of cells comprises cells modified with a gene editing system. 【0306】 D21. The method according to D19, wherein the plurality of cells comprises antibody-producing cells. 【0307】 D22. The method according to D19, wherein the plurality of cells comprises cells in different developmental states. 【0308】 D23. The method according to D19, wherein the plurality of cells comprises cells in different disease states. 【0309】 D24. The method according to D19, wherein the plurality of cells comprises cells treated with a drug. 【0310】 D25. The method according to D24, wherein the drug is a therapeutic agent. 【0311】 D26. The method according to D25, wherein the therapeutic agent is selected from the group consisting of polypeptide therapeutics, oligonucleotides, cell-based therapeutics, gene editing systems, small molecule therapeutics, and combinations thereof. 【0312】 D27. The method according to D19, wherein the plurality of cells comprises cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide containing a barcode. 【0313】 D28. The method according to any one of D to D27, wherein the emulsion droplet contains at least two cells or nuclei. 【0314】 The method according to any one of D to D28, wherein the particles are gel beads. 【0315】 The method according to any one of D to D29, further comprising performing a lineage tracing method. 【0316】 The method according to any one of D to D30, further comprising performing a genome screening. 【0317】 The method according to D31, wherein the genome screening is a CRISPR-based screening. 【0318】 The method according to D32, wherein the CRISPR-based screening is selected from the group consisting of gene editing CRISPR screening, CRISPRi screening, and CRISPRa screening. 【0319】 The method according to any one of D to D33, wherein a plurality of cells or nuclei isolated from a plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide containing a barcode. 【0320】 The method according to any one of D to D33, further comprising contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of protein binding reagents coupled to an oligonucleotide containing a barcode prior to (a). 【0321】 The method according to D34 or D35, wherein the protein binding reagent binds to a protein located on the surface of at least one cell of a plurality of single cells. 【0322】 The method according to D34 to D36, wherein the protein binding reagent is an antibody or a fragment thereof. 【0323】 The method according to D36 or D37, further comprising determining the expression level of a protein. 【0324】 E. The present disclosure provides a method for generating a sequencing library comprising nucleic acids from a plurality of single cells, the method comprising: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles comprising a first index sequence; (b) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acids of the cells or nuclei to generate indexed nucleic acids; (d) combining the cells or nuclei containing or associated with the indexed nucleic acids from the plurality of emulsion droplets to generate pooled cells or nuclei; (e) distributing a subset of the pooled cells or nuclei into a plurality of compartments comprising a second index sequence; (f) incorporating the second index sequence into the indexed nucleic acids within each compartment to generate doubly indexed nucleic acids; and (g) combining all or a subset of the doubly indexed nucleic acids to generate a sequencing library from the plurality of cells. 【0325】 E1. The method according to E, wherein the first index sequence of each particle or subset of particles is unique relative to other particles. 【0326】 E2. The method according to E or E1, wherein the second index sequence within one compartment or subset of compartments is unique relative to other second index sequences. 【0327】 E3. The method according to any one of E to E2, wherein the emulsion droplets are generated within an emulsion droplet generation device or using an emulsion droplet generation technique. 【0328】 E4. The method according to any one of E to E3, wherein the plurality of compartments is a multi-well plate. 【0329】 E5. The method according to any one of E to E4, wherein incorporating the first index array into the nucleic acid is performed by an amplification process, a reverse transcription process, or a ligation process. 【0330】 E5.1. The method according to E5, wherein incorporating the first index array into the nucleic acid in an emulsion droplet is performed by a reverse transcription process. 【0331】 E6. The method according to any one of E to E5.1, wherein incorporating the second index array into the indexed nucleic acid is performed by an amplification process or a ligation process. 【0332】 E6.1. The method according to any one of E to E6, wherein incorporating the second index array into the indexed nucleic acid is performed by an amplification process. 【0333】 E7. The method according to E5 to E6.1, wherein the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. 【0334】 E8. The method according to any one of E to E7, wherein the cell or nucleus is treated with a transposase before or during step (a). 【0335】 E9. The method according to E8, wherein the transposase is Tn5 transposase. 【0336】 E10. The method according to any one of E to E9, wherein a plurality of cells or nuclei are treated with a fixative before step (a). 【0337】 E11. The method according to any one of E to E10, wherein a plurality of cells are permeabilized or lysed before step (a). 【0338】 E12. The method according to any one of E to E11, wherein a plurality of cells or nuclei are treated with a multiplexing reagent before or during step (a). 【0339】 The method according to any one of E to E12, wherein the nucleic acid comprises RNA. 【0340】 The method according to any one of E to E12, wherein the nucleic acid comprises DNA. 【0341】 The method according to any one of E to E14, wherein the plurality of cells comprises at least about 100,000 cells. 【0342】 The method according to any one of E to E15, wherein the plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof. 【0343】 The method according to any one of E to E17, wherein the plurality of cells comprises somatic cells. 【0344】 The method according to any one of E to E18, wherein the plurality of cells comprises any one of the following: (a) cells modified with a gene editing system; (b) antibody-producing cells; (c) cells in different developmental states; (d) cells in different disease states; (e) cells treated with a drug; and (f) cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide containing a barcode. 【0345】 The method according to E19, wherein the plurality of cells comprises cells modified with a gene editing system. 【0346】 The method according to E19, wherein the plurality of cells comprises antibody-producing cells. 【0347】 The method according to E19, wherein the plurality of cells comprises cells in different developmental states. 【0348】 The method according to E19, wherein the plurality of cells comprises cells in different disease states. 【0349】 The method according to E19, wherein the plurality of cells comprises cells treated with a drug. 【0350】 The method according to E24, wherein the agent is a therapeutic agent. 【0351】 The method according to E25, wherein the therapeutic agent is selected from the group consisting of polypeptide therapeutics, oligonucleotides, cell-based therapeutics, gene editing systems, small molecule therapeutics, and combinations thereof. 【0352】 The method according to E19, wherein a plurality of cells comprise cells bound by a protein binding reagent coupled to an oligonucleotide comprising a barcode. 【0353】 The method according to any one of E~E27, wherein the emulsion droplet contains at least two cells or nuclei. 【0354】 The method according to any one of E~E28, wherein the particle is a gel bead. 【0355】 The method according to any one of E~E29, further comprising performing a lineage tracing method. 【0356】 The method according to any one of E~E30, further comprising performing a genomic screening. 【0357】 The method according to E31, wherein the genomic screening is a CRISPR-based screening. 【0358】 The method according to E32, wherein the CRISPR-based screening is selected from the group consisting of gene editing CRISPR screening, CRISPRi screening, and CRISPRa screening. 【0359】 The method according to any one of E~E33, further comprising contacting a plurality of cells or nuclei isolated from the plurality of cells with a plurality of protein binding reagents coupled to an oligonucleotide comprising a barcode prior to (a). 【0360】 The method according to E27 or E35, wherein the protein-binding reagent binds to a protein located on the surface of at least one cell of a plurality of single cells. 【0361】 The method according to any one of E27 and E35 to E36, wherein the protein-binding reagent is an antibody or a fragment thereof. 【0362】 The method according to E36 or E37, further comprising determining the expression level of the protein. 【0363】 F. The present disclosure provides a method for sequencing a library containing nucleic acids from a plurality of single cells, comprising: (a) contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of particles containing a first index sequence; (b) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cells or nuclei to generate indexed nucleic acids; (d) combining the cells or nuclei containing or associated with the indexed nucleic acids from the plurality of emulsion droplets to generate pooled cells or nuclei; (e) distributing a subset of the pooled cells or nuclei into a plurality of compartments containing a second index sequence; (f) incorporating the second index sequence into the indexed nucleic acids within each compartment to generate doubly indexed nucleic acids; (g) combining all or a subset of the doubly indexed nucleic acids to generate a sequencing library from the plurality of cells; and (h) sequencing all or a subset of the doubly indexed nucleic acids. 【0364】 The method according to F, wherein the first index sequence of each particle or subset of particles is unique to other microbeads. 【0365】 The method according to F or F1, wherein a second index array within one compartment or a subset of compartments is unique with respect to other second index arrays. 【0366】 The method according to F to F2, wherein the microfluidic droplets are generated within an emulsion droplet generation device or using an emulsion droplet generation technique. 【0367】 The method according to F to F3, wherein the plurality of compartments are multi-well plates. 【0368】 The method according to F to F4, wherein incorporating the first index array into a nucleic acid is performed by an amplification process, a reverse transcription process, or a ligation process. 【0369】 The method according to F5, wherein incorporating the first index array into a nucleic acid in an emulsion droplet is performed by a reverse transcription process. 【0370】 The method according to F to F5.1, wherein incorporating the second index array into an indexed nucleic acid is performed by an amplification process or a ligation process. 【0371】 The method according to any one of F to F6, wherein incorporating the second index array into an indexed nucleic acid is performed by an amplification process. 【0372】 The method according to F5 to F6.1, wherein the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. 【0373】 The method according to F to F2, wherein a plurality of cells or nuclei are treated with a transposase before or during step (a). 【0374】 The method according to F8, wherein the transposase is Tn5 transposase. 【0375】 F10. The method according to F to F9, wherein a plurality of cells or nuclei are treated with a fixative before step (a). 【0376】 F11. The method according to F to F10, wherein a plurality of cells are permeabilized or lysed before step (a). 【0377】 F12. The method according to F to F11, wherein a plurality of cells or nuclei are treated with a multiplexing reagent before or during step (a). 【0378】 F13. The method according to F to F12, wherein the nucleic acid comprises RNA. 【0379】 F14. The method according to F to F12, wherein the nucleic acid comprises DNA. 【0380】 F15. The method according to F to F14, wherein a plurality of cells comprises at least about 100,000 cells. 【0381】 F16. The method according to F to F15, wherein a plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or combinations thereof. 【0382】 F17. The method according to F to F16, wherein a plurality of cells comprises immune cells. 【0383】 F18. The method according to F to F17, wherein a plurality of cells comprises somatic cells. 【0384】 F19. The method according to F to F18, wherein a plurality of cells comprises any one of the following: (a) cells modified with a gene editing system; (b) antibody-producing cells; (c) cells in different developmental states; (d) cells in different disease states; (e) cells treated with a drug; and (f) cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide containing a barcode. 【0385】 F20. The method according to F19, wherein a plurality of cells comprises cells modified with a gene editing system. 【0386】 F21. The method according to F19, wherein the plurality of cells include antibody-producing cells. 【0387】 F22. The method according to F19, wherein the plurality of cells include cells in different developmental states. 【0388】 F23. The method according to F19, wherein the plurality of cells include cells in different disease states. 【0389】 F24. The method according to F19, wherein the plurality of cells include cells treated with a drug. 【0390】 F25. The method according to F24, wherein the drug is a therapeutic agent. 【0391】 F26. The method according to F25, wherein the therapeutic agent is selected from the group consisting of polypeptide therapeutics, oligonucleotides, cell-based therapeutics, gene editing systems, small molecule therapeutics, and combinations thereof. 【0392】 F27. The method according to F19, wherein the plurality of cells include cells bound by a protein binding reagent, such as an antibody, coupled to an oligonucleotide containing a barcode. 【0393】 F28. The method according to any one of F to F27, wherein the emulsion droplet contains at least two cells or nuclei. 【0394】 F29. The method according to any one of F to F28, wherein the particles are gel beads. 【0395】 F30. The method according to any one of F to F29, further comprising performing a lineage tracing method. 【0396】 F31. The method according to any one of F to F30, further comprising performing a genome screening. 【0397】 F32. The method according to F31, wherein the genome screening is a CRISPR-based screening. 【0398】 F33. A method according to F32, wherein the CRISPR-based screening is selected from the group consisting of gene editing CRISPR screening, CRISPRi screening, and CRISPRa screening. 【0399】 F34. A method according to any one of F33, wherein a plurality of cells or nuclei isolated from a plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide containing a barcode. 【0400】 F35. A method according to any one of F33, further comprising contacting a plurality of cells or nuclei isolated from a plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides containing barcodes, prior to (a). 【0401】 F36. A method according to F34 or F35, wherein the protein binding reagent binds to a protein located on the surface of at least one cell of a plurality of single cells. 【0402】 F37. A method according to any one of F34 to F36, wherein the protein binding reagent is an antibody or a fragment thereof. 【0403】 F38. A method according to F36 or F37, further comprising determining the expression level of a protein. 【0404】 G. The present disclosure provides a kit for carrying out any one of the methods of A to F28. 【0405】 G1. A kit according to G, comprising one or more of the following: (a) at least one container containing a plurality of particles comprising a first index sequence; and (b) at least one container containing a plurality of second index sequences. 【0406】 G2. The kit according to G1, further comprising a container containing one or more protein binding reagents coupled to an oligonucleotide containing a barcode. 【0407】 The kit according to G2, wherein the protein binding reagent is an antibody or a fragment thereof. 【Example】 【0408】 The subject matter disclosed herein will be better understood by reference to the following examples, which are provided as illustrations of the subject matter disclosed herein, and not as limitations. 【0409】 Example 1: Combinatorial indexing for OAK-scRNAseq This example describes a combinatorial indexing method for sequencing single-cell RNA as shown in FIG. 1. In particular, this example describes the addition of two index sequences to single-cell RNA. 【0410】 Cell preparation: The combinatorial indexing method begins with the preparation of a single-cell suspension for fixation. When an adherent cell line or solid tissue was used, cell dissociation was performed prior to fixation. The cells were harvested in medium and then washed with phosphate-buffered saline (PBS). The washed cells were gently resuspended in 200 μl of cold PBS per 1×106 cells to obtain a single-cell suspension. 【0411】 Subsequently, the single cells were fixed by adding 800 μl of cold pure methanol dropwise per 1×106 cells while gently stirring with a pipette tip to prevent cell aggregation. The sample was then stored at -20°C for 40 minutes. The fixed sample was placed on ice for 5 minutes and then centrifuged at 1000 g for 5 minutes at 4°C. The supernatant was removed, and the cells were resuspended in a buffer containing 3×SSC, 1% BSA, 1 mM DTT, and 0.2 U / μl RNase inhibitor. The volume was adjusted with the buffer to achieve a concentration of 50,000 cells / μl. 【0412】 Integration of the primary barcode: The master mix was prepared on ice based on the user guide of Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (referred to as the “Chromium Next GEM Single Cell User Guide” in this specification). 31.8 μl of the master mix was aliquoted for each sample to be loaded. The following formula was used to calculate the volume of fixed cells to be loaded into each sample: Volume_cell stock (μl) = Normalized cell number * 2.3 / Concentration_cell stock. For example, when the cell stock concentration was 50,000 cells / μl and the target was 100,000 cells, 100k * 2.3 / 50k = 4.6 μl of cell stock was used as input to load 230,000 cells. For each channel, based on the calculations of the previous step, 43.3 μl of nuclease-free water was added to the tube containing 31.8 μl of the master mix. Second, the corresponding volume of fixed cells was added. The cell-reagent mixture was gently mixed with a pipette, and using the same pipette tip, 70 μl of the mixture was dispensed into the wells of the column labeled 1 on the chip. The previous step was repeated for each of the samples to be loaded. Each sample was placed in a well. The remaining wells were loaded according to the Chromium Next GEM Single Cell User Guide. The GEM gasket was attached, and the Chromium controller was operated based on the Chromium Next GEM Single Cell User Guide. After the run was completed, the GEM was transferred according to the Chromium Next GEM Single Cell User Guide. As shown in Figure 2, this process was successful in generating multi-cell GEMs. Figure 7A shows the generation of multi-cells by loading 150,000 cells per channel (resulting in the recovery of 85,000 cells and the sequencing of approximately 2,000 - 4,000 genes per cell), and Figure 7B shows the generation of multi-cell GEMs by loading 450,000 cells per channel (resulting in the recovery of 273,000 cells and the sequencing of approximately 600 genes per cell). Representative droplets containing 0 - 8 cells are shown.Next, the migrating GEMs were incubated in a thermal cycler at 53 °C for 45 minutes to enable primary barcoding during the reverse transcription process, and subsequently held at 4 °C for a short time as needed. At the end of the incubation, the GEMs were unpacked as follows. 【0413】 GEM unpacking: 125 μl of recovery agent was added to each sample at room temperature. After incubation for 5 - 10 minutes, two-phase separation formed and stabilized. The aqueous phase was slowly transferred from the top of each sample tube to a round-bottom 2.0 mL Eppendorf tube. 500 μl of 3×SSC was slowly added to each sample. The tubes were centrifuged at 650 g for 5 minutes at 4 °C in a swinging bucket centrifuge. The supernatant was removed without disrupting the cell pellet. 500 μl of 3×SSC was slowly added to each sample without disrupting the cell pellet. The tubes were centrifuged at 650 g for 5 minutes at 4 °C in a swinging bucket centrifuge, and the supernatant was removed without disrupting the cell pellet. The cells were resuspended in 3×SSC using a regular bore P200 pipette tip. An appropriate resuspension volume should be used such that each 10 μl contains 1,000 - 10,000 cells based on the multiplex tolerance of each project. As shown in Figure 3, after unpacking the GEMs, cell recovery was successful. Each sample was aliquoted into multiple individual PCR tubes such that each PCR tube contained 10 μl of resuspended cells. Each 10 μl contains 1,000 - 10,000 cells. The aliquots were frozen at -80 °C until the next step. In a thermal cycler, the aliquots were heated at 95 °C for 5 minutes. 90 μl of EB was added to each aliquot and purification was performed using Dynabeads MyOne Silane. Heating at 95 °C and purification with Dynabeads MyOne Silane resulted in cell lysis. 【0414】 Integration of the secondary barcode: For each aliquot, cDNA amplification was performed using uniquely barcoded primers that hybridize to a common sequence located upstream of the primary cell barcode and UMI in the first-strand cDNA. The other primers in the PCR reaction cover the TSO side of the first-strand cDNA and are thus shared across all aliquots. After the PCR reaction was completed, the products from different aliquots were pooled. The PCR products were purified with 0.6× SPRIselect beads. 1 μl of the resulting cDNA was run on a D5000 tape to evaluate the amount and length distribution of the cDNA. 【0415】 Subsequently, the cDNA library was sequenced by using the Illumina sequencing library construction process followed by sequencing. Table 1 shows the performance metrics of the OAK-scRNAseq method. As shown in Table 1, this method enables the analysis of many cells with a low multiplexing rate. 【Table 1】 【0416】 Example 2: Combinatorial indexing for OAK-multiome (ATAC&RNA co-assay) This example describes a combinatorial indexing method for sequencing single-cell RNA and genomic DNA as shown in FIG. 1. In particular, this example describes the addition of two index sequences to the genomic DNA of a single cell. 【0417】 Nuclear preparation: The method begins with the preparation of a single nuclear suspension for fixation. Cells were harvested into medium. When using adherent cell lines or solid tissues, cell dissociation can be performed prior to fixation. The cells were then washed with phosphate-buffered saline (PBS) and gently resuspended in 200 μl of cold PBS per 1×106 cells to obtain a single-cell suspension. Subsequently, nuclei were isolated from the single-cell suspension. 【0418】 Subsequently, single nuclei were fixed by adding an appropriate volume of formaldehyde and PBS to the single nucleus suspension such that 0.3% formaldehyde was used for fixation. The samples were stored on ice for 15 minutes. The fixed nuclei were then washed with a buffer containing PBS, 1% BSA, and 0.2 U / μl RNase inhibitor. The fixed nuclei were gently resuspended with 10× Genomics in cooled dilution nuclear buffer. The volume was adjusted to achieve a desired concentration such as 4,000 nuclei / μl. Nuclei translocation was performed according to Chromium Next GEM Single Cell Multiome ATAC+Gene Expression (the "Chromium Next GEM Single Cell Multiome User Guide"). A maximum of 20,000 nuclei were used for each translocation reaction with a reaction volume of 15 μl. To obtain sufficient samples for overloading the microfluidic device, multiple translocation reactions were prepared in parallel. Ten such reactions were prepared in parallel for each sample, and a total of 200,000 nuclei were translocated for each sample. If additional ATAC enzyme is required, TDE1 enzyme from Illumina Tagment DNA TDE1 Enzyme and Buffer Kits can be used. Alternatively, a self-cultured Tn5 enzyme loaded with appropriate adapters can be used. After the translocation reaction, for each sample, the nuclei were collected from all reaction tubes into a round-bottom 2.0 mL Eppendorf tube. The tubes were centrifuged at 500 g for 5 minutes at 4°C in a swinging bucket centrifuge. Most of the supernatant was removed without disrupting the nuclear pellet. For each sample, the last 15 μl of supernatant was left in the tube. The translocated nuclei were gently resuspended with the remaining buffer. 【0419】 Integration of primary barcode: The master mix was prepared on ice based on the Chromium Next GEM Single Cell Multiome User Guide. 60 μl of the master mix was added to the sample tube containing 15 μl of translocated concentrated nuclei. 【0420】 The nuclear-reagent mixture was gently mixed with a pipette and, using the same pipette tip, 70 μl of the mixture was dispensed into the wells of the column labeled 1 on the chip. The previous step was repeated for each of the samples to be loaded. Each sample was placed in a well. The remaining wells were loaded according to the Chromium Next GEM Single Cell Multiome User Guide. The GEM gasket was attached and the Chromium Controller was operated based on the user guide. After the run was completed, the GEM was transferred according to the Chromium Next GEM Single Cell Multiome User Guide. 1 μl of the GEM could be examined under a microscope. The transferred GEM was then incubated in a thermal cycler at 37 °C for 45 minutes, followed by 30 minutes at 25 °C to enable primary barcoding and subsequently held at 4 °C for a short time if necessary. At the end of the incubation, 5 μl of quenching agent was added to each sample according to the user guide and the GEM was unpacked as follows. 【0421】 GEM unpacking: 125 μl of the recovery agent was added to each sample at room temperature. After incubation for 5 - 10 minutes, two-phase separation was formed and stabilized. The aqueous phase was slowly transferred from the top of each sample tube to a round-bottom 2.0 mL Eppendorf tube. 500 μl of 3×SSC was slowly added to each sample. The tubes were centrifuged at 650 g for 5 minutes at 4°C in a swinging bucket centrifuge. The supernatant was removed without disrupting the cell pellet. 500 μl of 3×SSC was slowly added to each sample without disrupting the nuclear pellet. The tubes were centrifuged at 650 g for 5 minutes at 4°C in a swinging bucket centrifuge, and the supernatant was removed without disrupting the nuclear pellet. The nuclei were resuspended in 3×SSC using a regular bore P200 pipette tip. Based on the multiplex resistance of each project, an appropriate resuspension volume should be used so that each 10 μl contains 1,000 - 10,000 cells. Each sample was aliquoted into multiple individual PCR tubes so that each PCR tube contained 10 μl of the resuspended nuclei. Each 10 μl contains 1,000 - 10,000 nuclei. The aliquots were frozen at -80°C until the next step. The aliquots were heated at 80°C for 10 minutes in a thermal cycler. 90 μl of EB was added to each aliquot and purification was performed using Dynabeads MyOne Silane. 【0422】 Integration of secondary barcodes: For each aliquot, pre-amplification was performed on both the cDNA fragment and the ATAC fragment using a mixture of primers that amplify both the cDNA fragment and the ATAC fragment. The PCR products of each aliquot were washed with 1.6× SPRI Select beads. 25% of the product of each aliquot was used from the previous step for cDNA amplification using uniquely barcoded primers that hybridize to a common sequence located upstream of the primary cell barcode and UMI in the first-strand cDNA. The other primers in the PCR reaction cover the TSO side of the first-strand cDNA and are thus shared across all aliquots. After the PCR reaction is complete, the products from different aliquots can be pooled. The cDNA amplification products were purified with 0.6× SPRIselect beads. 1 μl of the resulting cDNA was run on a D5000 tape to evaluate the amount and length distribution of the cDNA. 【0423】 Subsequently, the cDNA library was sequenced by using the Illumina sequencing library construction process followed by sequencing. Tables 2 and 8 show the performance metrics of the OAK-multiome method. As shown in Table 2, this method enables the analysis of many cells with a low multiplexing rate. 【Table 2】 【0424】 Example 3: OAK combinatorial indexing using cell hashing This example discloses a combinatorial indexing method for sequencing single-cell RNA labeled with an antibody coupled to a barcoded oligonucleotide (referred to herein as a "hash tag"). 【0425】 As shown in FIG. 9, differentiated human bronchial epithelial cells were tagged with antibodies coupled to oligonucleotides containing barcodes (i.e., hashtags) and further processed using combinatorial indexing methods or control methods. In particular, primary lung cells were grown in plates, stained with commercially available hashtag antibodies specific for beta2-microglobulin B2M and CD298 expressed on all cells, and coupled to oligonucleotides containing known barcodes. A total of nine different barcodes were used. After antibody binding, the cells were washed, then pooled and sorted by cytometry for viability. 100,000 cells were further processed using the OAK method described in Example 1, and 7,000 cells were used for droplet sequencing using the 10× Chromium Single Cell 3′ Reagent Kit and the Chromium platform as a control. 【0426】 For the OAK method, a first index array was added to cDNA and antibody-derived oligonucleotides in droplets. By tagging the antibody-derived oligonucleotides and RNA-derived cDNA with the same first index array in droplets, the RNA and antibody-derived sequences were linked in the data. After unpacking the emulsion, the cells were placed into different aliquots for secondary barcode integration. Secondary barcode integration was performed as follows: for each aliquot, the cDNA and antibody (protein or hashtag)-derived oligonucleotides were amplified with a mixture of primers that amplify both the cDNA and antibody-derived oligonucleotides. During this reaction, a secondary index was added to the cDNA. The PCR products of each aliquot were separated with SPRI Select beads. 0.6× SPRI Select beads were used to collect the cDNA of the gene expression library similar to Example 1. The supernatant was maintained from the first separation from the cDNA for further purification using 2.1× SPRI Select beads to obtain the antibody-derived oligonucleotides. Amplification of the antibody-derived oligonucleotides used uniquely barcoded primers that hybridize to a common sequence located upstream of the primary cell barcode, and the other primers in the PCR reaction covered the common sequence within the antibody-derived oligonucleotides and were thus shared across all aliquots. 【0427】 The sequencing libraries from the transcriptome and hashtags were then depleted for analysis. 【0428】 The abundances of cells assigned nine different hashtag were analyzed in sequencing data obtained using the OAK method and the control method. As shown in Fig. 10, a high correlation was observed between the sequencing data obtained from the OAK method and the sequencing data of the control method. These data confirm that the OAK method functions well using cell hashing. Next, ridge plots were generated to show the hashtag expression levels of depleted cells for each hashtag identity. As shown in Fig. 11, the ridge plots showed a high signal-to-noise ratio for each cell assigned using the hashtag. Fig. 12 shows that the quality of the transcriptome sequencing data obtained from the OAK method using cell hashing is comparable to the quality of the transcriptome sequencing data obtained from the control method. UMAP analysis of scRNA-Seq data obtained using the OAK method with hashtags showed that the hashtags were evenly distributed among different cell types as expected for unbiased cell indexing (Figs. 13A–13B), and that the frequencies of cell types were equivalent between the OAK method and the control method (Fig. 13C). 【0429】 To confirm whether protein levels can be determined using the OAK methodology with cell hashing, additional experiments were conducted. Figure 14 provides a schematic of the methodology used in this experiment. As shown in Figure 14, two human cell lines (Jurkat cells and K562 cells) were independently stained with antibodies against proteins known to be differentially expressed between the two cell lines (CD45, CD3, and CD4 conjugate antibodies) and antibodies against proteins known not to be expressed by the two cell lines (RatIgG2b isotype control and CD8 conjugate antibodies), along with two different hashing tag antibodies. After staining, the cells were combined at a 1:1 ratio, fixed with methanol, and then processed using the OAK method described in Example 1 with the 10x Chromium Single Cell 3’ Reagent Kit and Chromium platform. Secondary indices were added to both the antibody hash library and the gene expression library along with the primers. The addition of the index to the antibody fraction was either during direct amplification from cDNA or after a second PCR to amplify antibody-derived sequences (described in Example 3). Two OAK aliquots were sequenced for two different temperature conditions (37°C and 53°C). A non-fixed control using the 10x Genomics protocol was also performed on 12,000 cells for comparison. 【0430】 As shown in Figure 15, the OAK methodology enables the processing of a large number of cells at once. The UMAP analysis shown in Figure 16 indicates how clustering separates the two cell lines based on scRNA-Seq data and that the clustering matched the assigned hash tags. The expression of proteins bound by the antibodies was further analyzed and compared to the signals from the antibody hash tags (to identify cell types). As shown in Figure 17, the signals derived from the antibody hash tags were consistent with the predictions of protein expression in the two cell lines tested. 【0431】 As shown in this example, cell hashing is compatible with the OAK combinatorial indexing method. In particular, the binding of antibodies to specific target proteins was maintained during the fixation process, during exposure to the lysing and reducing agents in the droplets, and during the reverse transcription process of the OAK combinatorial indexing method. This example further confirms that cell hashing can be used in combination with the OAK combinatorial indexing and sequencing methods to simultaneously analyze the protein expression and transcriptome of single cells. The use of cell hashing in combination with the OAK method can simplify the upstream processes of the cell array, increase the number of cells, and can be used to improve the accuracy of multiplet calling in the analysis. Cell hashing can also be used to provide a reference abundance for normalizing protein expression data. 【0432】 Example 4: OAK Combinatorial Indexing for Detecting V(D)J Recombination This example shows the use of the OAK method to identify V(D)J recombination patterns in single cells using a combinatorial indexing method. 【0433】 The OAK method described in Example 1 was performed on Jurkat cells using the 10x Chromium Single Cell 5' Reagent Kit. cDNA and TCR amplicons were indexed with a second index sequence by aliquoting to generate a sequencing library. 683 cells from a homogeneous population of Jurkat cells were sequenced, and as shown in Figure 18, 422 (61.8%) cells had the expected captured CDR3 sequence. This example confirms that the OAK method enables the sequencing of V(D)J transcripts obtained from single cells. 【0434】 Although the subject matter and its advantages disclosed in this specification have been described in detail, it should be understood that various changes, substitutions, and modifications can be made to this specification without departing from the spirit and scope of the present disclosure. Furthermore, the scope of this application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, and compositions of matter, means, methods, and steps described herein. Accordingly, the appended claims are intended to include such processes, machines, manufactures, compositions of matter, means, methods, or steps within their scope. 【0435】 Throughout this application, various patents, patent applications, publications, product descriptions, and protocols have been cited, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

Claims

[Claim 1] A method for combinatorial indexing of nucleic acids from multiple single cells, (a) Providing a pooled cell or nucleus relating to or containing a nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet; (b) Distributing the pooled subset of cells or nuclei into a plurality of compartments containing a second index sequence; and (c) Incorporating the second index sequence into the nucleic acid in each compartment to generate a dual-indexed nucleic acid. Methods that include... [Claim 2] A method for generating a sequencing library containing nucleic acids from multiple single cells, (a) Providing a pooled cell or nucleus relating to or containing a nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet; (b) Distributing the pooled subset of cells or nuclei into a number of compartments containing a second index sequence; (c) Incorporating the second index sequence into the nucleic acid in each compartment to produce a dual-indexed nucleic acid; and (d) Combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from the plurality of cells. Methods that include... [Claim 3] A method for sequencing a library containing nucleic acids from multiple single cells, (a) Providing a pooled cell or nucleus relating to or containing a nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the pooled cell or nucleus in an emulsion droplet; (b) Distributing the pooled subset of cells or nuclei into a number of compartments containing a second index sequence; (c) Incorporating the second index sequence into the nucleic acid in each compartment to produce a dual-indexed nucleic acid; (d) Combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from the plurality of cells; and (e) Sequencing all or a subset of the dual-indexed nucleic acids. Methods that include... [Claim 4] A method according to any one of claims 1 to 3, wherein the first index sequence is the nucleic acid of the cell or nucleus in the emulsion droplet, as follows: (i) bringing a plurality of cells or nuclei into contact with a plurality of particles containing the first index sequence; (ii) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (iii) incorporating the first index sequence into a cellular or nuclear nucleic acid to produce an indexed nucleic acid; and (iv) Combining the cells or nuclei associated with or containing the indexed nucleic acid from a plurality of emulsion droplets to generate the pooled cells or nuclei associated with or containing the nucleic acid containing the first index sequence. A method incorporated by a method that includes [Claim 5] The method according to claim 4, wherein the cells or nuclei are treated with a transposase before or during step (i). [Claim 6] The method according to claim 5, wherein the transposase is Tn5 transposase. [Claim 7] The method according to claim 4, wherein the plurality of cells or nuclei are treated with a fixative before step (i). [Claim 8] The method according to claim 4, wherein the plurality of cells are permeabilized or lysed before step (i). [Claim 9] The method according to claim 4, wherein the plurality of cells or nuclei are treated with a multiplexing reagent before or during step (i). [Claim 10] The method according to claim 4, wherein the first index sequence of each particle or subset of particles is unique to other particles. [Claim 11] The method according to any one of claims 1 to 3, wherein the second index sequence in one compartment or a subset of compartments is unique to other second index sequences. [Claim 12] The method according to any one of claims 1 to 3, wherein the plurality of compartments are a multiwell plate. [Claim 13] The method according to any one of claims 1 to 3, wherein the emulsion droplets are generated in an emulsion droplet generation device or by using an emulsion droplet generation technique. [Claim 14] The method according to any one of claims 1 to 3, further comprising incorporating a third index sequence into the double-indexed nucleic acid to produce a triple-indexed nucleic acid. [Claim 15] The method according to any one of claims 1 to 3, wherein the incorporation of the first index sequence into the nucleic acid in the emulsion droplet is performed by an amplification process, a reverse transcription process, or a ligation process. [Claim 16] The method according to any one of claims 1 to 3, wherein the incorporation of the second index sequence into the indexed nucleic acid is performed by an amplification process or a ligation process. [Claim 17] The method according to claim 15, wherein the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. [Claim 18] The method according to any one of claims 1 to 3, wherein the nucleic acid comprises RNA. [Claim 19] The method according to any one of claims 1 to 3, wherein the nucleic acid comprises DNA. [Claim 20] The method according to any one of claims 1 to 3, wherein the plurality of cells comprises at least about 100,000 cells. [Claim 21] The method according to any one of claims 1 to 3, wherein the plurality of cells include pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof. [Claim 22] A method according to any one of claims 1 to 3, wherein the plurality of cells are: (a) Cells modified by gene editing systems; (b) antibody-producing cells; (c) Cells in different developmental states; (d) Cells in different disease states; and (e) Cells treated with drugs A method that includes one of the following. [Claim 23] A method for combinatorial indexing of nucleic acids from multiple single cells, (a) Contacting multiple cells or nuclei isolated from multiple cells with multiple particles containing a first index sequence; (b) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (c) Incorporating the first index sequence into a nucleic acid of a cell or nucleus to produce an indexed nucleic acid; (d) Combining the cells or nuclei containing or associated with the indexed nucleic acids from a plurality of emulsion droplets to generate pooled cells or nuclei; (e) Distributing the pooled subset of cells or nuclei into a plurality of compartments containing a second index sequence; and (f) Incorporating the second index sequence into the indexed nucleic acid in each compartment to generate a double-indexed nucleic acid. Methods that include... [Claim 24] (g) The method according to claim 23, further comprising incorporating a third index sequence into the double-indexed nucleic acid to produce a triple-indexed nucleic acid. [Claim 25] The method according to claim 23 or 24, wherein the first index sequence of each particle or subset of particles is unique to other particles. [Claim 26] The method according to claim 23 or 24, wherein the second index sequence in one compartment or a subset of compartments is unique to other second index sequences. [Claim 27] The method according to claim 23 or 24, wherein the emulsion droplets are generated in an emulsion droplet generation device or by using an emulsion droplet generation technique. [Claim 28] The method according to claim 23 or 24, wherein the plurality of compartments are a multiwell plate. [Claim 29] The method according to claim 23 or 24, wherein the incorporation of the first index sequence into the nucleic acid is performed by an amplification process, a reverse transcription process, or a ligation process. [Claim 30] The method according to claim 23 or 24, wherein the incorporation of the second index sequence into the indexed nucleic acid is performed by an amplification process or a ligation process. [Claim 31] The method according to claim 29, wherein the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. [Claim 32] The method according to claim 23 or 24, wherein the cells or nuclei are treated with a transposase before or during step (a). [Claim 33] The method according to claim 32, wherein the transposase is Tn5 transposase. [Claim 34] The method according to claim 23 or 24, wherein the plurality of cells or nuclei are treated with a fixative before step (a). [Claim 35] The method according to claim 23 or 24, wherein the plurality of cells are permeabilized or lysed before step (a). [Claim 36] The method according to claim 23 or 24, wherein the plurality of cells or nuclei are treated with a multiplexing reagent before or during step (a). [Claim 37] The method according to claim 23 or 24, wherein the nucleic acid comprises RNA. [Claim 38] The method according to claim 23 or 24, wherein the nucleic acid comprises DNA. [Claim 39] The method according to claim 23 or 24, wherein the plurality of cells comprises at least about 100,000 cells. [Claim 40] The method according to claim 23 or 24, wherein the plurality of cells include pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof. [Claim 41] A method according to claim 23 or 24, wherein the plurality of cells are: (a) Cells modified by gene editing systems; (b) antibody-producing cells; (c) Cells in different developmental states; (d) Cells in different disease states; and (e) Cells treated with drugs A method that includes one of the following. [Claim 42] A method for generating a sequencing library containing nucleic acids from multiple single cells, (a) Contacting multiple cells or nuclei isolated from multiple cells with multiple particles containing a first index sequence; (b) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (c) Incorporating the first index sequence into a nucleic acid of a cell or nucleus to produce an indexed nucleic acid; (d) Combining the cells or nuclei containing or associated with the indexed nucleic acids from a plurality of emulsion droplets to generate pooled cells or nuclei; (e) Distributing the pooled subset of cells or nuclei into a number of compartments containing a second index sequence; (f) Incorporating the second index sequence into the indexed nucleic acid in each compartment to generate a double-indexed nucleic acid; and (g) Combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from the plurality of cells. Methods that include... [Claim 43] The method according to claim 42, wherein the first index sequence of each particle or subset of particles is unique to other particles. [Claim 44] The method according to claim 42 or 43, wherein the second index sequence in one compartment or a subset of compartments is unique to other second index sequences. [Claim 45] The method according to claim 42 or 43, wherein the emulsion droplets are generated in an emulsion droplet generation device or by using an emulsion droplet generation technique. [Claim 46] The method according to claim 42 or 43, wherein the plurality of compartments are a multiwell plate. [Claim 47] The method according to claim 42 or 43, wherein the incorporation of the first index sequence into the nucleic acid is performed by an amplification process, a reverse transcription process, or a ligation process. [Claim 48] The method according to claim 42 or 43, wherein the incorporation of the second index sequence into the indexed nucleic acid is performed by an amplification process or a ligation process. [Claim 49] The method according to claim 47, wherein the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. [Claim 50] The method according to claim 42 or 43, wherein the cells or nuclei are treated with a transposase before or during step (a). [Claim 51] The method according to claim 50, wherein the transposase is Tn5 transposase. [Claim 52] The method according to claim 42 or 43, wherein the plurality of cells or nuclei are treated with a fixative before step (a). [Claim 53] The method according to claim 42 or 43, wherein the plurality of cells are permeabilized or lysed before step (a). [Claim 54] The method according to claim 42 or 43, wherein the plurality of cells or nuclei are treated with a multiplexing reagent before or during step (a). [Claim 55] The method according to claim 42 or 43, wherein the nucleic acid comprises RNA. [Claim 56] The method according to claim 42 or 43, wherein the nucleic acid comprises DNA. [Claim 57] The method according to claim 42 or 43, wherein the plurality of cells comprises at least about 100,000 cells. [Claim 58] The method according to claim 42 or 43, wherein the plurality of cells include pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof. [Claim 59] The method according to claim 42 or 43, wherein the plurality of cells are: (a) Cells modified by gene editing systems; (b) antibody-producing cells; (c) Cells in different developmental states; (d) Cells in different disease states; and (e) Cells treated with drugs A method that includes one of the following. [Claim 60] A method for sequencing a library containing nucleic acids from multiple single cells, (a) Contacting multiple cells or nuclei isolated from multiple cells with multiple particles containing a first index sequence; (b) partitioning individual particles and one or more of the cells or nuclei into emulsion droplets; (c) Incorporating the first index sequence into the nucleic acid of the cell or nucleus to produce an indexed nucleic acid; (d) Combining the cells or nuclei containing or associated with the indexed nucleic acids from a plurality of emulsion droplets to generate pooled cells or nuclei; (e) Distributing the pooled subset of cells or nuclei into a number of compartments containing a second index sequence; (f) Incorporating the second index sequence into the indexed nucleic acid in each compartment to generate a double-indexed nucleic acid; (g) Combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from the plurality of cells; and (h) Sequencing all or a subset of the dual-indexed nucleic acids. Methods that include... [Claim 61] The method according to claim 60, wherein the first index sequence of each particle or subset of particles is unique to other microbeads. [Claim 62] The method according to claim 60 or 61, wherein the second index sequence in one compartment or a subset of compartments is unique to other second index sequences. [Claim 63] The method according to claim 60 or 61, wherein microfluidic droplets are generated in an emulsion droplet generation device or by using an emulsion droplet generation technique. [Claim 64] The method according to claim 60 or 61, wherein the plurality of compartments are a multiwell plate. [Claim 65] The method according to claim 60 or 61, wherein the incorporation of the first index sequence into the nucleic acid is performed by an amplification process, a reverse transcription process, or a ligation process. [Claim 66] The method according to claim 60 or 61, wherein the incorporation of the second index sequence into the indexed nucleic acid is performed by an amplification process or a ligation process. [Claim 67] The method according to claim 65, wherein the amplification process is a polymerase chain reaction (PCR) or an isothermal amplification process. [Claim 68] The method according to claim 60 or 61, wherein the plurality of cells or nuclei are treated with a transposase before or during step (a). [Claim 69] The method according to claim 68, wherein the transposase is Tn5 transposase. [Claim 70] The method according to claim 60 or 61, wherein the plurality of cells or nuclei are treated with a fixative before step (a). [Claim 71] The method according to claim 60 or 61, wherein the plurality of cells are permeabilized or lysed before step (a). [Claim 72] The method according to claim 60 or 61, wherein the plurality of cells or nuclei are treated with a multiplexing reagent before or during step (a). [Claim 73] The method according to claim 60 or 61, wherein the nucleic acid comprises RNA. [Claim 74] The method according to claim 60 or 61, wherein the nucleic acid comprises DNA. [Claim 75] The method according to claim 60 or 61, wherein the plurality of cells comprises at least about 100,000 cells. [Claim 76] The method according to claim 60 or 61, wherein the plurality of cells include pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof. [Claim 77] A method according to claim 60 or 61, wherein the plurality of cells are: (a) Cells modified by gene editing systems; (b) antibody-producing cells; (c) Cells in different developmental states; (d) Cells in different disease states; and (e) Cells treated with drugs A method that includes one of the following. [Claim 78] The method according to claim 60 or 61, wherein the emulsion droplet comprises at least two cells or nuclei. [Claim 79] The method according to claim 60 or 61, wherein the particles are gel beads. [Claim 80] The method according to any one of claims 1, 23, 42, and 60, further comprising performing a lineage tracing method. [Claim 81] The method according to any one of claims 1, 23, 42, and 60, further comprising performing genome screening. [Claim 82] The method according to claim 81, wherein the genome screening is a CRISPR-based screening. [Claim 83] The method according to claim 82, wherein the CRISPR-based screening is selected from the group consisting of gene editing CRISPR screening, CRISPRi screening, and CRISPRRa screening. [Claim 84] The method according to any one of claims 1, 23, 42, and 60, wherein the plurality of cells or nuclei isolated from the plurality of cells are bound by a protein-binding reagent coupled to an oligonucleotide containing a barcode. [Claim 85] The method according to any one of claims 1, 23, 42, and 60, further comprising contacting the plurality of cells or nuclei isolated from the plurality of cells with a plurality of protein-binding reagents coupled to oligonucleotides containing barcodes, prior to (a). [Claim 86] The method according to claim 84, wherein the protein-binding reagent binds to a protein located on the surface of at least one of the plurality of single cells. [Claim 87] The method according to claim 84, wherein the protein-binding reagent is an antibody or a fragment thereof. [Claim 88] The method according to claim 86, further comprising determining the expression level of the protein. [Claim 89] A kit for carrying out the method according to any one of claims 1, 23, 42, and 60. [Claim 90] The kit according to claim 89, wherein: (a) at least one container containing a plurality of particles including the first index sequence; and (b) At least one container containing a plurality of second index arrays A kit containing one or more of the following. [Claim 91] The kit according to claim 90, further comprising a container containing one or more protein-binding reagents coupled to an oligonucleotide containing a barcode. [Claim 92] The kit according to claim 91, wherein the protein-binding reagent is an antibody or a fragment thereof.

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