Combination indexing for single cell nucleic acid sequencing

Abstract

The present disclosure provides methods for combinatorial indexing of nucleic acids of a single cell or nucleus. In particular, the methods of the present disclosure result in the incorporation of at least two index sequences within the nucleic acid of a single cell or cell nucleus. The disclosure further provides a method for producing libraries of such indexed nucleic acids and sequencing libraries of such indexed nucleic acids.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 349,184 filed on June 6, 2022, U.S. Provisional Application No. 63 / 441,744 filed on January 27, 2023, and U.S. Provisional Application No. 63 / 499,689 filed on May 2, 2023, the contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to methods for combinatorial indexing of nucleic acids of single cells or cell nuclei. The present disclosure further provides methods for generating libraries of such indexed nucleic acids and sequencing libraries of such indexed nucleic acids. Background Art

[0004] Nucleic acid sequencing of single cells has become a powerful tool for analyzing the genetic heterogeneity of a group of cells. For example, sequencing nucleic acids from single cells can be used to analyze the progression of cancer, map cellular heterogeneity in diseased and healthy tissues, understand the development of immune or autoimmune disorders, and better understand natural biological processes.

[0005] Single-cell sequencing typically involves adding a unique barcode to the nucleic acid of a single cell to allow for the profiling of many single cells in parallel. There are a variety of techniques for adding barcodes to the nucleic acid of a single cell, and typically involve the use of multi-well plates or microfluidic devices. However, such techniques are limited by the capacity of the plate or microfluidic device to avoid loading more than one cell in the wells of the multi-well plate or in the droplets produced by the microfluidic device, and to prevent labeling the nucleic acid of multiple cells with the same barcode. Therefore, there is a need in the art for improved high-throughput methods for indexing the nucleic acid of a single cell. Summary of the invention

[0006] The present disclosure provides methods for combinatorial indexing of nucleic acids of single cells as well as methods for generating libraries of such indexed nucleic acids and sequencing libraries of such indexed nucleic acids.

[0007] In one aspect, the present disclosure provides a method for combinatorial indexing of nucleic acids of a single cell. In certain embodiments, the method includes (a) providing a mixed (pooled) cell or cell nucleus associated with or containing a nucleic acid containing a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the mixed cell or cell nucleus in an emulsion droplet; (b) assigning a subset of the mixed cell or cell nucleus to a plurality of compartments including a second index sequence; and (c) merging the second index sequence into the nucleic acid in each compartment to produce a dual index nucleic acid. In certain embodiments, a first index sequence is incorporated into nucleic acid of cells or cell nuclei in emulsion droplets by a method comprising: (i) contacting a plurality of cells or cell nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning one or more of the individual particles and cells or cell nuclei into emulsion droplets; (iii) incorporating the first index sequence into nucleic acid of cells or cell nuclei to produce index nucleic acid; and (iv) combining cells or cell nuclei associated with or containing the index nucleic acid from a plurality of emulsion droplets to produce mixed cells or cell nuclei associated with or containing nucleic acid containing the first index sequence.

[0008] In certain embodiments, a method for combinatorial indexing of nucleic acids of a single cell comprises: (a) contacting a plurality of cells or cell nuclei separated from a plurality of cells with a plurality of particles comprising a first index sequence; (b) partitioning the individual particles and one or more of the cells or cell nuclei into emulsion droplets; and (c) incorporating the first index sequence into the nucleic acids of the cells or cell nuclei to produce indexed nucleic acids; (d) combining cells or cell nuclei containing or associated with indexed nucleic acids from a plurality of emulsion droplets to produce mixed cells or cell nuclei; (e) distributing subsets of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; and (f) incorporating the second index sequence into the indexed nucleic acid in each compartment to produce dual-indexed nucleic acids.

[0009] In another aspect, the present disclosure provides a method for generating a sequencing library comprising nucleic acids from multiple single cells or nuclei. For example, but not by way of limitation, a method for generating a sequencing library may include: (a) providing a mixed 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 mixed cell or nucleus in an emulsion droplet; (b) distributing a subset of the mixed cell or nucleus into a plurality of compartments comprising a second index sequence; (c) merging the second index sequence into the nucleic acid in each compartment to generate a dual index nucleic acid; and (d) combining all or a subset of the dual index nucleic acids to generate a sequencing library from a plurality of cells. In certain embodiments, a first index sequence is incorporated into nucleic acid of cells or cell nuclei in emulsion droplets by a method comprising: (i) contacting a plurality of cells or cell nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning one or more of the individual particles and cells or cell nuclei into emulsion droplets; (iii) incorporating the first index sequence into nucleic acid of cells or cell nuclei to produce index nucleic acid; and (iv) combining cells or cell nuclei associated with or containing the index nucleic acid from a plurality of emulsion droplets to produce mixed cells or cell nuclei associated with or containing nucleic acid containing the first index sequence.

[0010] In certain embodiments, a method for generating a sequencing library may include: (a) contacting a plurality of cells or cell nuclei isolated from a plurality of cells with a plurality of particles comprising a first index sequence; (b) partitioning the individual particles and one or more of the cells or cell nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cells or cell nuclei to generate an index nucleic acid; (d) combining cells or cell nuclei containing or associated with the index nucleic acid from a plurality of emulsion droplets to generate mixed cells or cell nuclei; (e) distributing a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; (f) incorporating the second index sequence into the index nucleic acid in each compartment to generate a dual-index nucleic acid; and (g) combining all or a subset of the dual-index nucleic acids to generate a sequencing library from a plurality of cells.

[0011] In another aspect, the present disclosure further provides a method for sequencing a library comprising nucleic acids from multiple single cells or cell nuclei. For example, but not by way of limitation, a method for sequencing a library comprising nucleic acids from multiple single cells or cell nuclei includes: (a) providing a mixed cell or cell 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 mixed cell or cell nucleus in an emulsion droplet; (b) distributing a subset of the mixed cell or cell nucleus into a plurality of compartments comprising a second index sequence; (c) merging the second index sequence into the nucleic acid in each compartment to produce a dual index nucleic acid; (d) combining all or a subset of the dual index nucleic acids to produce a sequencing library from multiple cells; and (e) sequencing all or a subset of the dual index nucleic acids. In certain embodiments, a first index sequence is incorporated into nucleic acid of cells or cell nuclei in emulsion droplets by a method comprising: (i) contacting a plurality of cells or cell nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning one or more of the individual particles and cells or cell nuclei into emulsion droplets; (iii) incorporating the first index sequence into nucleic acid of cells or cell nuclei to produce index nucleic acid; and (iv) combining cells or cell nuclei associated with or containing the index nucleic acid from a plurality of emulsion droplets to produce mixed cells or cell nuclei associated with or containing nucleic acid containing the first index sequence.

[0012] In certain embodiments, a method for sequencing a library comprising nucleic acids from multiple single cells or cell nuclei comprises: (a) contacting multiple cells or cell nuclei isolated from multiple cells with multiple particles comprising a first index sequence; (b) partitioning the individual particles and one or more of the cells or cell nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cells or cell nuclei to produce index nucleic acids; (d) combining cells or cell nuclei containing or associated with index nucleic acids from multiple emulsion droplets to produce mixed index cells or cell nuclei; (e) distributing subsets of the mixed cells or cell nuclei into multiple compartments comprising a second index sequence; (f) incorporating the second index sequence into the index nucleic acid in each compartment to produce dual index nucleic acids; (g) combining all or a subset of the dual index nucleic acids to produce a sequencing library from multiple cells; and (h) sequencing all or a subset of the dual index nucleic acids.

[0013] In certain embodiments, the methods of the present disclosure may further comprise incorporating a third index sequence into the dual-indexed nucleic acid to produce a triply-indexed nucleic acid.

[0014] In certain embodiments, the particles are beads, eg, gel microbeads.

[0015] In certain embodiments, emulsion droplets are generated in an emulsion droplet generating device or using an emulsion droplet generating technique.

[0016] In certain embodiments, the emulsion droplet comprises at least two or more cells or cell nuclei.

[0017] In some embodiments, the first index sequence of each particle or subset of particles is unique relative to the first index sequences of other particles. In some embodiments, the second index sequence in one compartment or subset of compartments is unique relative to the second index sequences of other compartments.

[0018] In certain embodiments, the first index sequence is incorporated into the nucleic acid by performing an amplification method, a reverse transcription method, or a ligation method. In certain embodiments, the second index sequence is incorporated into the index nucleic acid by performing an amplification method or a ligation method. In certain embodiments, the amplification method is polymerase chain reaction (PCR) or an isothermal amplification method.

[0019] In certain embodiments, the cells or nuclei are treated with a translocase, e.g., in a translocase complex, prior to or during the step of contacting a plurality of cells or cell nuclei isolated from a plurality of cells with a plurality of particles comprising a first index sequence. In certain embodiments, the translocase is a Tn5 translocase or a mutant thereof. In certain embodiments, the cells or nuclei are treated with a fixative prior to the step of contacting a plurality of cells or cell nuclei isolated from a plurality of cells with a plurality of particles comprising a first index sequence. In certain embodiments, the plurality of cells are permeabilized or lysed prior to the step of contacting a plurality of cells or cell nuclei isolated from a plurality of cells with a plurality of particles comprising a first index sequence. In certain embodiments, the cells or nuclei are treated with a multiple reagent prior to or during the step of contacting a plurality of cells or cell nuclei isolated from a plurality of cells with a plurality of particles comprising a first index sequence.

[0020] In certain embodiments, the plurality of compartments is a multiwell plate. In certain embodiments, the plurality of compartments is not an emulsion droplet.

[0021] In certain embodiments, the nucleic acids indexed using the disclosed methods comprise RNA, e.g., mRNA. In certain embodiments, the nucleic acids indexed using the disclosed methods comprise DNA, e.g., genomic DNA.

[0022] In certain embodiments, the plurality of cells comprises at least about 100,000 cells.

[0023] In certain embodiments, a plurality of cells include pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof. In certain embodiments, a plurality of cells include cells modified with a gene editing system, cells producing antibodies, cells in different developmental states, cells in different disease states, and / or cells treated with a pharmaceutical agent (e.g., a therapeutic agent).

[0024] 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 screen. For example, but not limited to, the genomic screen is a CRISPR-based screen, for example, a gene editing CRISPR screen, a CRISPRi screen, or a CRISPRa screen.

[0025] In certain embodiments, the method of the present disclosure further includes performing a cell hashing technique. In certain embodiments, a plurality of cells or cell nuclei separated from a plurality of cells are combined by a protein binding reagent coupled to an oligonucleotide comprising a barcode. For example, but not limited to, the method of the present disclosure may further include contacting a plurality of cells or cell nuclei separated from a plurality of cells with a plurality of protein binding reagents coupled to an oligonucleotide comprising a barcode before (a). In certain embodiments, a protein binding reagent binds to a protein on the surface of at least one cell located in the plurality of cells or a cell nucleus separated from a plurality of cells. In certain embodiments, a protein binding reagent is an antibody or a fragment thereof. In certain embodiments, the method of the present disclosure further includes determining the expression content of one or more proteins bound by the protein binding reagent.

[0026] In certain embodiments, the present disclosure further provides a kit for performing the methods of the present disclosure. For example, but not by way of limitation, the kit of the present disclosure may include a container comprising a plurality of particles comprising a first index sequence. In certain embodiments, the kit may further include a container, e.g., a microtiter plate, comprising a plurality of second index sequences. In certain embodiments, the kit of the present disclosure includes 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

[0027] Figure 1Schematic diagram showing an exemplary embodiment of an overloading and unpacking ("OAK") method for combinatorial indexing of nucleic acids of single cells or nuclei. The OAK method includes overloading cells or nuclei and gel beads in an emulsion droplet generation device to produce multicellular gel bead emulsification ("GEMS") (left) to incorporate a first index sequence into the nucleic acids of the cells or nuclei. The multicellular GEMS are then unpacked to produce mixed cells or nuclei with indexed nucleic acids (middle). The mixed indexed cells or nuclei are then distributed into different compartments to obtain a second index sequence (right) to produce dual indexed nucleic acids.

[0028] Figure 2 Images showing multicellular GEMs generated by loading a sample including 280,000 Jurkat cells into an emulsion droplet generation device are provided.

[0029] Figure 3 Images showing intact Jurkat cells recovered after unpacking of multicellular GEMs are provided.

[0030] Figure 4 Schematic diagrams of exemplary methods including single cell CRISPR screening and OAK methods of the present disclosure are provided.

[0031] Figure 5 Schematic diagrams of exemplary methods including the lineage tracing techniques and OAK methods of the present disclosure are provided.

[0032] Figure 6 A schematic diagram is provided showing loading different types of gel beads (e.g., 3' gel beads, 5' gel beads, or multiome gel beads) and cells into an emulsion droplet generation device to generate multicellular GEMS for performing an exemplary OAK multiome method or an exemplary OAK-scRNAseq method of the present disclosure.

[0033] FIG. 7A provides images showing multicellular GEMs generated by loading a sample including 150,000 cells into an emulsion droplet generating device.

[0034] Figure 7B Images showing multicellular GEMs produced by loading a sample including 450,000 cells into an emulsion droplet generation device are provided.

[0035] Figure 8 Performance metrics of exemplary OAK multi-omics methods of the present disclosure are provided.

[0036] Fig. 9 A schematic diagram of an exemplary method for performing the cell hashing technique in combination with the OAK method of the present disclosure is provided. Created by BioRender.com.

[0037] Fig.10 A graph showing the abundance of cells assigned 9 different labels in sequencing data obtained using the OAK method compared to a control method on the same cell sample is provided.

[0038] Fig.11 A ridge plot showing the label representation content of cells decentralized for each label identity is provided.

[0039] Fig.12 Provided by Fig. 9 Quality of transcriptome sequencing data from the experiments shown.

[0040] FIG. 13A to FIG. 13B A UMAP of scRNA-Seq is provided, showing label assignments ( Fig.13A ), showing that the label is evenly distributed across different cell types ( Fig. 13B ).

[0041] Fig. 13C A graph showing the frequency of cell types obtained using the OAK method compared to a control method on the same cell sample is provided.

[0042] Fig.14 A schematic diagram of an exemplary method for performing cell hashing techniques on two cell lines, Jurkat and K562, in combination with the OAK method of the present disclosure is provided. Created by BioRender.com.

[0043] Fig.15 Provided from Fig.14 Quality of transcriptome sequencing data obtained from the experiments shown.

[0044] Fig.16 A clustered UMAP showing scRNA-Seq data from Jurkat and K562 cell lines is provided.

[0045] Fig.17 Graphs showing signals derived from antibody labels of Jurkat and K562 cell lines are provided.

[0046] Fig.18 Shown are the frequencies of CDR3 clonotypes recovered from a homogenous population of Jurkat cells. DETAILED DESCRIPTION

[0047] The present disclosure relates to an improved method for combining indexes of nucleic acids from a single cell. In certain embodiments, the method of the present disclosure includes merging at least two index sequences into the nucleic acid of a single cell or a nucleus. By using at least two index sequences, the combined indexing method of the present disclosure allows the presence of a unique combination of two index sequences to identify nucleic acids from a single cell or a nucleus. In certain embodiments, the method of the present disclosure can be used to generate a library of index nucleic acids (e.g., dual index nucleic acids). In certain embodiments, the present disclosure further provides a method for sequencing a library generated from an index nucleic acid (e.g., dual index nucleic acids).

[0048] The present disclosure is based in part on the discovery that the throughput of an indexing method is increased more than 20-fold by overloading an emulsion droplet to contain at least two cells or nuclei for incorporating a first index sequence into the nucleic acid of the cell or nucleus. For example, by overloading an emulsion droplet with more than one cell or nucleus for incorporating a first index sequence, the disclosed method allows for combinatorial indexing of nucleic acids from more than 200,000 single cells. In addition, by using an emulsion droplet as the first step in adding an index sequence to the nucleic acid of a single cell, the disclosed combinatorial indexing method allows the cell to remain intact for adding a second index sequence to the index nucleic acid. Furthermore, using an emulsion droplet as the first step in the indexing method ensures compatibility with different emulsion-based kits, reagents, and methods, and allows subsets of cells to be sequenced independently of each other.

[0049] In certain embodiments, the nucleic acid indexed using the methods disclosed herein may be DNA or RNA. In certain embodiments, the methods disclosed herein may be used to index the RNA of a single cell. For example, but not limited to, the methods disclosed herein may be used to analyze all or part of the transcriptome of a single cell by indexing the mRNA of a single cell. In certain embodiments, the methods disclosed herein may be used to index the genomic DNA of a single cell or a cell nucleus. In certain embodiments, the methods disclosed herein may be used to analyze gene regulation in a single cell by indexing the genomic DNA of a single cell. In certain embodiments, the methods disclosed herein may be used to analyze chromatin accessibility in a single cell by indexing open chromatin sites in the genomic DNA of a single cell. In certain embodiments, the methods disclosed herein may be used to analyze multiple samples that have been mixed and indexed using sample-specific oligonucleotides or are genetically different before mixing. In certain embodiments, the methods disclosed herein may be used to quantify the presence of a protein after detecting the protein using an oligonucleotide-labeled antibody.

[0050] For purposes of clarity, and not limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0051] I. Definitions;

[0052] II. Cells;

[0053] III. How to use;

[0054] IV. Kits; and

[0055] V. Exemplary Embodiments.

[0056] I. Definitions

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the subject matter of the present disclosure belongs. The following references provide general definitions of many terms used by technicians in the present disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Glossary of Genetics (1991). As used herein, unless otherwise indicated, the following terms have the following meanings assigned to them.

[0058] As used herein, when used in conjunction with the term "comprising" in the claims and / or the specification, the use of the term "a / a" or "an" may mean "one / a / an", but is also consistent with the meaning of "one / a / one or more / multiple / multiple", "at least one / a / an", and "one / a / an or more than one / a / an".

[0059] 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, according to practice in the art, "about" can mean 3 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within a certain order of magnitude of a certain value, preferably within 5 times, more preferably within 2 times.

[0060] The term "amplification method" generally refers to any method in which a portion of a nucleic acid is copied or duplicated into at least one additional nucleic acid molecule.

[0061] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0062] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the 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 ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv) and multispecific antibodies formed from antibody fragments.

[0063] As used herein, the term "biological sample" refers to a sample of biological material obtained from a subject, including biological fluids, for example, blood, plasma, serum, urine, sputum, cerebrospinal fluid, pleural fluid, nipple aspirate, lymph fluid, fluids from the respiratory, intestinal and urogenital tracts, tears, saliva, milk, fluid from the lymphatic system, semen, cerebrospinal fluid, fluids within organ systems, ascites, tumor cyst fluid, amniotic fluid, bronchoalveolar fluid, biliary fluid, and combinations thereof.

[0064] The term "combinatorial indexing" refers to a method in which two or more index sequences are incorporated into a nucleic acid as an identification sequence.

[0065] As used herein, the term "compartment" refers to an area or volume that separates or isolates one or more components from other components. Non-limiting examples of compartments include vials, tubes, wells, drops, boluses, containers, or areas or volumes separated by physical forces such as fluid flow, magnetism, electric current, etc.

[0066] As used herein, the terms "comprises," "including," "having," "having," "may," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude additional actions or structures. The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements set forth herein, whether or not explicitly set forth.

[0067] The term "coupled" may refer to two or more components being connected or united by an interaction, bonding, connection, force or tie so as to hold the two or more components together. In certain embodiments, the term "coupled" encompasses direct or indirect binding, where, for example, a first component is directly bound to a second component, or one or more intermediate molecules are disposed between the first component and the second component. Exemplary bonds include covalent bonds, ionic bonds, van der Waals interactions, hydrogen bonds, and other bonds recognizable to the skilled artisan.

[0068] As used herein, the term "decentralization" refers to the separation of different data obtained during a single experiment. In certain embodiments, the term "decentralization" refers to the separation of scRNA-SEQ data from tag sequencing and / or expression data.

[0069] As used herein, the term "detect" or "detection" indicates determining the presence and / or state of a target (e.g., a protein target or a nucleic acid target) in a limited portion of space, including but not limited to a sample. As used herein, the term "detect" or "detection" may include determining the chemical and / or biological properties of a target, including but not limited to the ability to interact, particularly the ability to bind to other compounds, the ability to activate another compound, and additional properties that a skilled person may identify after reading this disclosure. Detection may be quantitative or qualitative. Detection is "quantitative" when detection refers to, relates to, or is about the measurement of the quantity or amount of a target or signal (also referred to as quantification), including but not limited to any analysis designed to determine the amount or ratio of a target or signal. Detection is "qualitative" when detection refers to, relates to, or is about identifying the mass or type of a target or signal based on its relative abundance relative to another unquantified target or signal.

[0070] As used herein, the term "dual-indexed nucleic acid" refers to a nucleic acid having two index sequences.

[0071] As used herein, the term "emulsified droplets" refers to droplets formed by at least two immiscible phases (e.g., a continuous phase and a dispersed phase). In certain embodiments, the emulsified droplets can be formed in an emulsified droplet generating device by adding oil as a continuous phase to the emulsified droplet generating device. In certain embodiments, the emulsified droplets can be formed by a technique for generating emulsified droplets (such as, but not limited to, vortexing).

[0072] As used herein, the term "hybridization" refers to the process by which two single-stranded polynucleotides non-covalently associate to form a stable double-stranded polynucleotide.

[0073] As used herein, the term "index sequence" refers to a nucleotide sequence used to identify a single cell or cell nucleus associated with the index sequence.

[0074] As used herein, the term "individual" or "subject" refers to a vertebrate or invertebrate, such as a human or non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sports animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, e.g., mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, apes, and monkeys. In certain embodiments, the individual or subject is a human.

[0075] As used herein, the term "in vitro" refers to an artificial environment and a process or reaction that occurs in an artificial environment. In vitro environments are exemplified, but not limited to, test tubes and cell cultures.

[0076] As used herein, the term "in vivo" refers to the natural environment (eg, an animal or a cell) and processes or reactions that occur in the natural environment, such as embryonic development, cell differentiation, neural tube formation, and the like.

[0077] As used herein, the term "isolated cell nucleus" refers to a cell nucleus that has been separated from components of its natural environment.

[0078] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0079] 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 termini of two or more nucleic acid molecules.

[0080] As used herein, the term "ligation method" generally refers to a method in which two or more molecules are covalently linked together by an enzyme. For example, two or more nucleic acid molecules can be covalently linked together by a ligation method using a ligase.

[0081] As used herein, "marker" refers to an agent that allows for direct or indirect detection. Markers include, but are not limited to, fluorescent compositions, 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), incorporated herein by reference, 32P, 14C, 125I, 3H and 131I, fluorophores (such as rare earth chelates or lucifer yellow and its derivatives), rhodamine and its derivatives, dansyl, umbelliferone, luciferases (such as firefly luciferase and bacterial luciferase) (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, and enzymes that generate a detectable signal, such as horseradish peroxidase. peroxidase (HRP), alkaline phosphatase, 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).

[0082] As used herein, the term "multiple-indexed" refers to a nucleic acid having at least two index sequences.

[0083] The term "nucleic acid" or "polynucleotide" includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide consists of a base, particularly a purine or 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. Typically, nucleic acid molecules are described by a base sequence, wherein the base represents the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically expressed from 5' to 3'. The term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), such as messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid includes sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acids described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues.

[0084] As used herein, the term "partition" generally refers to the separation of one or more components from other components in a region or volume.

[0085] The term "plurality" refers to a number greater than one. In certain embodiments, the term "plurality of cells" refers to a number of cells greater than one. For example, but not limited to, a plurality of cells includes at least two cells. In certain embodiments, a plurality of cells includes 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 includes at least about 100,000 cells. In certain embodiments, the term "plurality of particles" refers to a number of particles greater than one.

[0086] The term "reverse transcription method" refers to a method of producing a complementary strand of DNA using an enzyme called reverse transcriptase.

[0087] As used herein, the term "specifically binds" refers to preferential binding to a target molecule (eg, a protein or nucleic acid) relative to other molecules (eg, proteins or nucleic acids) in a sample.

[0088] As used herein, "subset" refers to a small portion of a larger amount of material.

[0089] As used herein, the term "triply indexed" refers to a nucleic acid having three index sequences.

[0090] II. Cells

[0091] The present disclosure provides methods for indexing nucleic acids (e.g., dual-indexed nucleic acids) of a single cell or a cell nucleus obtained from a single cell. In certain embodiments, cells used in the methods of the present disclosure may include any type of cell. In certain embodiments, cell nuclei used in the methods of the present disclosure may be obtained (e.g., isolated) from any type of cell.

[0092] In certain embodiments, the cell is obtained from a subject. In certain embodiments, the subject can be a human, a non-human primate, e.g., an ape or monkey, livestock, a mouse, a rat, a hamster, a guinea pig, a rabbit, a dog, a cat, a sheep, a pig, a goat, a cow, or a horse. In certain embodiments, the subject is a human.

[0093] In certain embodiments, cells can be obtained from biological fluids. Non-limiting examples of biological fluids include whole blood, plasma, serum, sweat, urine, sputum, spinal fluid, pleural fluid, mucus, nipple aspirates, lymph fluid, fluids from the respiratory, intestinal and urogenital tracts, interstitial fluid, tears, saliva, breast milk, fluids from the lymphatic system, semen, vaginal secretions, cerebrospinal fluid, fluids within organ systems, ascites, tumor cyst fluid, amniotic fluid, bronchoalveolar fluid, bile fluid, and combinations thereof.

[0094] In certain embodiments, the cell can be obtained from a tissue (e.g., a tissue sample). Non-limiting examples of tissues include 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, thyroid, adrenal gland, or parathyroid tissue.

[0095] In certain embodiments, cells used in the present disclosure may be fetal cells, for example, obtained from fetal tissue and / or amniotic fluid. In certain embodiments, the methods of the present disclosure may be used to analyze fetal health and / or identify abnormalities in individual fetal cells.

[0096] In certain embodiments, the cells can be obtained from in vitro cell culture. For example, but not by way of limitation, the cells can include cell lines.

[0097] In certain embodiments, the cell may include a primary cell, a blood cell, a somatic cell, a cancer cell, a cell derived from an organoid or a xenograft, or a stem cell, such as an pluripotent stem cell (iPSC) or an embryonic stem cell. In certain embodiments, the cell used in the present disclosure may be derived from a stem cell, such as a reprogrammed or transdifferentiated stem cell that has undergone natural differentiation or artificial induction.

[0098] In certain embodiments, cells can be obtained from a preserved sample (eg, a fixed sample), from a frozen sample (eg, a frozen tissue sample), or from a fresh sample (eg, a fresh tissue sample).

[0099] In certain embodiments, cells for the present disclosure may be treated with an agent. For example, but not limited to, an agent may be a therapeutic agent. In certain embodiments, cells for the present disclosure may be contacted with an agent (e.g., a therapeutic agent) before undergoing the combined indexing method described herein. In certain embodiments, the methods of the present disclosure may be used for drug screening, for example, to determine the genome and / or transcriptional changes associated with a test therapeutic agent (e.g., a newly identified therapeutic agent). In certain embodiments, the methods of the present disclosure may be used to determine the genome and / or transcriptional changes associated with resistance to a therapeutic agent. Non-limiting examples of such therapeutic agents include polypeptide therapeutics, for example, antibody-based therapeutics, oligonucleotides, cell-based therapeutics, gene editing systems, and small molecule therapeutics. In certain embodiments, the therapeutic agent may 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 or antagonist antibody). In certain embodiments, the methods of the present disclosure include performing single cell therapeutic screening, followed by the combined indexing method described herein.

[0100] In certain embodiments, the cells have been genetically modified to express and / or secrete an agent, e.g., a therapeutic agent. Non-limiting examples of therapeutic agents are described herein. For example, but not by way of limitation, the cells used in the methods of the present disclosure express a polypeptide therapeutic agent, e.g., an antibody or antibody fragment.

[0101] In certain embodiments, the cell may be an immune cell. Non-limiting examples of immune cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells (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 cell may be a T cell. In certain embodiments, the cell may be a modified immune cell, such as a modified T cell, that is genetically engineered to express a chimeric antigen receptor (CAR) (e.g., CAR T cells and CAR NK cells).

[0102] In certain embodiments, cells can be obtained from malignant tumors of tissues or tumors. Non-limiting examples of such malignant tumors include carcinomas, adenocarcinomas, sarcomas, and fibroadenomas. In certain embodiments, 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, gastric 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 single cancer cells.

[0103] In certain embodiments, cells may undergo genetic regulation or regulation. For example, but not limited to, cells for the present disclosure may be genetically regulated or regulated by enzymes or fusion proteins including such enzymes, for example, by contacting cells with enzymes or fusion proteins. In certain embodiments, enzymes or fusion proteins including such enzymes can cut target genes, nick target genes, edit target genes, inhibit the expression of target genes, and / or activate the expression of target genes of cells. In certain embodiments, enzymes or fusion proteins including such enzymes are guided to the region of target genes by guide nucleic acids, for example, to regulate or regulate target genes. In certain embodiments, genetic regulation or regulation is achieved by nicking, cutting, editing, inhibiting, and / or activating target genes by enzymes or fusion proteins including such enzymes. Non-limiting examples of enzymes that can be used to modulate or regulate genes (e.g., for use in gene editing systems) include homing endonucleases or meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR enzymes, and fusion proteins comprising such enzymes. In certain embodiments, cells used in the present disclosure can be subjected to genomic screening, e.g., where target genes are modulated or regulated using enzymes disclosed herein or fusion proteins comprising such enzymes. In certain embodiments, the methods of the present disclosure can be used in combination with single-cell genomic screening, e.g., combined with single-cell CRISPR-based screening, such as Figure 4As shown. Non-limiting examples of CRISPR-based screening include gene editing CRISPR screening, CRISPR interference (CRISPRi) screening, and CRISPR activation (CRISPRa) screening. For example, but not limited to, single-cell genome screening can be performed, and then the combined indexing method of the present disclosure is performed. In certain embodiments, nucleic acids associated with single-cell genome screening (e.g., screening based on single-cell CRISPR) present in genetically modified cells) are processed by the combined indexing method described herein. In certain embodiments, the indexed guide nucleic acid molecules (e.g., gRNA) can be further subjected to a secondary enrichment method before sequencing. In certain embodiments, the identity of the guide nucleic acid molecule (e.g., gRNA) can be determined by direct sequencing of the guide nucleic acid molecule (e.g., gRNA) or by sequencing an alternative barcode sequence associated with the guide nucleic acid molecule (e.g., gRNA). In certain embodiments, methods comprising the combined indexing methods and single-cell genomic screening (e.g., single-cell CRISPR-based screening) described herein may further include modifying and / or manipulating cells before and / or during the single-cell genomic screening. In certain embodiments, methods comprising the combined indexing methods and single-cell genomic screening described herein may further include treating cells with one or more therapeutic agents (e.g., drugs of interest), e.g., prior to performing the single-cell genomic screening. For example, but not by way of limitation, Figure 4 As shown, the method including the combined indexing method described herein and the screening based on single cell CRISPR may further include treating cells with one or more drugs of interest, for example, before performing screening based on single cell CRISPR. In certain embodiments, the method including the combined indexing method described herein and the single cell genome screening (for example, screening based on single cell CRISPR) may further include subjecting the cells to development and / or differentiation. In certain embodiments, information, for example, the sequence of the guide nucleic acid molecule (for example, gRNA) and the associated cell results (for example, editing, inhibition or activation of genes and / or cell phenotypes) are obtained from the following methods: including the combined indexing method described herein, the single cell genome screening can be used to determine the function of the gene (for example, the gene targeted in the screening), the responsiveness to the drug and / or the drug resistance mechanism. In certain embodiments, the method disclosed herein includes performing single cell genome screening, and then performing the combined indexing method described herein.

[0104] In certain embodiments, cells used in the present disclosure can be genetically modified using 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 as a limitation, the methods of the present disclosure can be used to identify and / or quantify nucleic acid modifications produced by a gene editing system.

[0105] In some embodiments, cells can include one or more lineage barcodes. For example, but not by way of limitation, the combined indexing methods disclosed herein using cells with lineage barcodes can be combined with single-cell lineage tracing techniques. In some embodiments, single-cell lineage tracing techniques can be used to obtain information about lineage identity and to correlate cells across multiple time points during development, differentiation, and / or artificial manipulation (e.g., drug treatment). In some embodiments, the combined indexing methods described herein and the single-cell lineage tracing techniques described herein can be combined with single-cell lineage tracing techniques. Figure 5The method of the single-cell lineage tracing technology shown can be used to determine the cell type, the genome of the cell, or a specific gene mutation associated with the cell that causes the drug resistance of interest. A non-limiting example of a single-cell lineage tracing technology is TraCe-seq, as disclosed in WO 2021 / 188973 and Chang et al., Nature Biotechnology 40: 86-93 (2022), each of which is incorporated herein by reference in its entirety. In certain embodiments, the lineage barcode may be included in an exogenous nucleic acid construct introduced into the cell, for example, as described in Section III. Alternatively, in certain embodiments, the lineage barcode may be an endogenous genetic marker. In certain embodiments, the lineage barcode and other nucleic acids associated with the single-cell lineage tracing technology present in the cell are processed by the combined indexing method described herein. In certain embodiments, the lineage barcode may be further subjected to a secondary enrichment method before sequencing. In certain embodiments, the method of the present disclosure includes performing lineage tracing technology and performing the combined indexing method described herein at different time points during the treatment of the cell population with the drug of interest. In certain embodiments, the methods of the present disclosure comprise performing lineage tracing techniques and performing the combined indexing methods described herein at different time points during the development and / or differentiation of a cell population. In certain embodiments, information obtained from methods comprising the combined indexing methods and single-cell lineage tracing techniques described herein, such as information on the lineage identity and molecular phenotype of the cell contents, can be used to determine how different lineages undergo diverse developmental and / or differentiation processes. In certain embodiments, information obtained from methods comprising the combined indexing methods and single-cell lineage tracing techniques described herein can be used to determine how certain lineages share similar programs in development and / or differentiation. In certain embodiments, information obtained from methods comprising the combined indexing methods and single-cell lineage tracing techniques described herein can be used to determine how pre-existing genetic programs in each cell and lineage affect their response to external stimuli, including, but not limited to drug treatments, such as, for example. Figure 5 shown.

[0106] In certain embodiments, the cell may include one or more tags. For example, but not limited to, the combined indexing method of the present disclosure may use cells bound by protein binding reagents coupled to oligonucleotides comprising barcodes. In certain embodiments, protein binding reagents coupled to oligonucleotides comprising barcodes may be combined with cells for combined indexing methods of the present disclosure as described in Section III. In certain embodiments, the method of the present disclosure includes performing cell hashing technology and performing the combined indexing method described herein. A non-limiting example of cell hashing technology is CITE-seq, for example, as disclosed in Stoeckius et al., Nature Methods 14: 865-868 (2017), the contents of which are incorporated herein by reference in their entirety. In certain embodiments, barcodes coupled to protein binding reagents and other nucleic acids associated with performing cell hashing technology are processed by the combined indexing method described herein.

[0107] In certain embodiments, the cell may be a bacterial cell.For example, but not by way of limitation, the methods of the present disclosure may be used to analyze a subject's microbiome, for example, a subject's intestinal microbiome.

[0108] In certain embodiments, the cells may include cells at different developmental stages. In certain embodiments, the cells may include cells at different disease states. For example, but not limited to, the methods of the present disclosure may be used to analyze genetic regulation at different developmental stages. In certain embodiments, the methods of the present disclosure may be used to analyze molecular dynamics of different cell types during different developmental stages. In certain embodiments, the methods of the present disclosure may be used to identify rare and / or transient developmental stages and / or disease states.

[0109] In certain embodiments, the cells can be from any model organism. For example, but not limited to, the model organism can be Escherichia coli, yeast, Arabidopsis thaliana, African clawed frogs, zebrafish, fruit flies, sea squirts, nematodes, mice and monkeys.

[0110] In certain embodiments, the cell may be a cell that has been infected by an infectious agent. Non-limiting examples of infectious agents include viruses, bacteria, fungi, and protozoa.

[0111] In certain embodiments, cells may be enriched for cells of interest to produce an enriched cell sample, which may be subjected to the methods of the present disclosure. Any technique known in the art may be used to enrich for cells of interest.

[0112] In certain embodiments, the nucleus can be isolated from any cell type. For example, but not by way of limitation, the nucleus can be isolated from any cell disclosed herein. Methods for isolating the nucleus from a cell are known to those skilled in the art and can be used to isolate the nucleus for use in the present disclosure.

[0113] III. How to use

[0114] The present disclosure relates to improved methods for combinatorial indexing of nucleic acids from a single cell or cell nucleus. In certain embodiments, the methods of the present disclosure include incorporating at least two index sequences into the nucleic acid of a single cell or cell nucleus. Figure 1 A flow chart of an exemplary method of the present disclosure is provided.

[0115] In certain embodiments, the methods of the present disclosure may not include contacting the cell or a nucleus isolated from the cell with particles comprising the first index sequence. Figure 6 A schematic diagram showing exemplary particles (e.g., gel beads) that can be used in the present disclosure is provided. In certain embodiments, contacting cells or cell nuclei with particles can include contacting cells or cell nuclei with particles to produce a mixture of cells or cell nuclei and particles. For example, but not by way of limitation, cells or cell nuclei are present in an aqueous phase and particles are present in a second aqueous phase, and the two aqueous phases are combined to produce a single aqueous phase of cells or cell nuclei and particles.

[0116] In certain embodiments, a plurality of cells or a cell nucleus separated from a plurality of cells is contacted with a particle. In certain embodiments, a plurality of cells may include at least about 5,000 cells. For example, but not limited to, a plurality of cells 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 include at least about 100,000 cells. In certain embodiments, a plurality of cells include at least about 200,000 cells. In certain embodiments, the plurality of cells comprises about 5,000 to about 200,000 cells, e.g., about 10,000 to about 200,000 cells, about 50,000 to about 200,000 cells, about 100,000 to about 200,000 cells, about 150,000 to about 200,000 cells, about 5,000 to about 150,000 cells, about 5,000 to about 100,000 cells, about 5,000 to about 50,000 cells, about 5,000 to about 10,000 cells, about 10,000 to about 150,000 cells, or about 50,000 to about 100,000 cells.

[0117] In certain embodiments, a plurality of particles may be contacted with a cell or a cell nucleus (e.g., a plurality of cells or a cell nucleus isolated from a plurality of cells). In certain embodiments, a plurality of particles (e.g., beads) comprises about 5,000 to about 1,000,000 particles. For example, but not by way of limitation, the plurality of particles includes about 10,000 to about 1,000,000 particles, about 20,000 to about 1,000,000 particles, about 30,000 to about 1,000,000 particles, about 40,000 to about 1,000,000 particles, about 50,000 to about 1,000,000 particles, about 60,000 to about 1,000,000 particles, about 70,000 to about 1,000,000 particles, about 80,000 to about 1,000,000 particles, about 90,000 to about 1,000,000 particles, about 100,000 to about 1,000,000 particles, or about 150,000 to about 1,000,000 particles. 0 to about 1,000,000 particles, about 200,000 to about 1,000,000 particles, about 300,000 to about 1,000,000 particles, about 400,000 to about 1,000,000 particles, about 500,000 to about 1,000,000 particles, about 600,000 to about 1,000,000 particles, about 700,000 to about 1,000,000 particles, about 800,000 to about 1,000,000 particles, about 900,000 to about 1,000,000 particles, about 10,000 to about 500,000 particles, about 20,000 to about 500,000 particles, about 30,000 to about 500,000 particles, about 40,000 to about 500,000 particles, about 50,000 to about 500,000 particles, about 60,000 to about 500,000 particles, about 70,000 to about 500,000 particles, about 80,000 to about 500,000 particles, about 90,000 to about 500,000 particles, about 100,000 to about 500,000 particles, about 200,000 to about 500,000 particles, about 300,000 to about 500,000 particles , about 400,000 to about 500,000 particles, about 10,000 to about 400,000 particles, about 10,000 to about 300,000 particles, about 10,000 to about 200,000 particles, about 10,000 to about 100,000 particles, about 50,000 to about 200,000 particles, about 60,000 to about 190,000 particles, about 70,000 to about 180,000 particles, about 80,000 to about 170,000 particles, about 90,000 to about 150,000 particles, or about 90,000 to about 110,000 particles.In certain embodiments, a plurality of particles include about 50,000 to about 450,000 particles. In certain embodiments, a plurality of particles include about 400,000 to about 1,000,000 particles. In certain embodiments, a plurality of particles include about 200,000 to about 800,000 particles. In certain embodiments, a plurality of particles include about 300,000 to about 800,000 particles. In certain embodiments, a plurality of particles include about 400,000 to about 800,000 particles. In certain embodiments, a plurality of particles include about 50,000 particles. In certain embodiments, a plurality of particles include about 100,000 particles. In certain embodiments, a plurality of particles include about 400,000 particles.

[0118] In certain embodiments, the particles combined with the cells or cell nuclei can be glass, plastic, gel or metal particles. In certain embodiments, the particles can be beads, for example, microbeads. In certain embodiments, the particles are gel beads, for example, gel microbeads. In certain embodiments, the particles can be any shape, for example, the particles can have a spherical, non-spherical, elliptical, rectangular, non-amorphous, circular or cylindrical shape. In certain embodiments, the particles can have a diameter of about 0.5 μm 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.

[0119] In certain embodiments, the first index sequence is coupled to a particle, e.g., a bead. In certain embodiments, the first index sequence is coupled to a particle, e.g., a bead using a linker. In certain embodiments, the first index sequence is releasably attached to a particle, e.g., a bead. In certain embodiments, each particle can have a plurality of attached index sequences, e.g., about 10 index sequences, about 100 index sequences, about 1,000 index sequences, about 10,000 index sequences, or about 100,000 index sequences or more. In certain embodiments, each particle may have about 10 to about 100,000 linked index sequences, e.g., 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 linked index sequences. In certain embodiments, all index sequences linked to a particular particle (e.g., bead) may have the same nucleotide sequence. In certain embodiments, all index sequences attached to a particular particle (eg, bead) may have the same nucleotide sequence, and a large number of different index sequences may be represented among the multiple particles (eg, beads) used. For example, but not by way of limitation, a plurality of beads can comprise a library of different index sequences comprising at least about 100 different index sequences, at least about 1,000 different index sequences, at least about 10,000 different index sequences, at least about 100,000 index sequences, at least about 1,000,000 different index sequences, at least about 1,500,000 different index sequences, at least about 2,000,000 different index sequences, at least about 2,500,000 different index sequences, at least about 3,000,000 different index sequences, at least about 3,500,000 different index sequences, at least about 4,000,000 different index sequences, at least about 4,500,000 different index sequences, or at least about 5,000,000 different index sequences.In certain embodiments, the plurality of beads may include about 100 to about 5,000,000 different index sequences, e.g., about 100 to about 1,000,000, about 100 to about 500,000, about 100 to about 50,000, about 100 to about 10,000, about 100 to about 1,000, about 100 to about 500, about 500 to about 5,000,000, about 1,000 to about 5,000,000. In some embodiments, the present invention relates to a plurality of different index sequences. The present invention relates to a plurality of different index sequences. In some embodiments, the present invention relates to a plurality of different index sequences.

[0120] In certain embodiments, the first index sequence of each particle (e.g., bead) is unique relative to other particles in the plurality of particles. For example, but not by way of limitation, the first index sequence connected to a particle present in an emulsion droplet is unique compared to the sequence of the first index sequence connected to a particle present in a different emulsion droplet. Alternatively, no more than about 50% of the particle population in the plurality of particles, for example, no more than about 45%, no more than about 40%, no more than about 35%, no more than about 30%, no more than about 25%, no more than about 20%, no more than about 15%, no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, or no more than about 1% of the particle population is connected to an index sequence having the same nucleic acid sequence as a different particle. In certain embodiments, no more than about 10% of the particle population in the plurality of particles is connected to an index sequence having the same nucleic acid sequence as a different particle. In certain embodiments, no more than about 5% of the particle population in the plurality of particles is connected to an index sequence having the same nucleic acid sequence as a different particle. 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 a population of particles in a plurality of particles are linked to an index sequence having the same nucleic acid sequence as a different particle.

[0121] In certain embodiments, the index sequence used in the present disclosure, e.g., the first index sequence and / or the second index sequence, can be up to 200 nucleotides in length. In certain embodiments, the index sequence can be from about 5 to about 200 nucleotides in length, e.g., 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 sequence may be about 5 to about 50 nucleotides in length, for example, about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, about 10 to about 50 nucleotides in length, about 15 to about 50 nucleotides in length, about 20 to about 50 nucleotides in length, about 25 to about 50 nucleotides in length, about 30 to about 50 nucleotides in length, about 35 to about 50 nucleotides in length, about 40 to about 50 nucleotides in length, about 10 to about 40 nucleotides in length, or about 10 to about 30 nucleotides in length.

[0122] In certain embodiments, the first index sequence is provided in the first polynucleotide. For example, but not as a limitation, the first polypeptide comprising the first index sequence is coupled to a particle, for example, a bead, as described herein. In certain embodiments, the first polynucleotide may further comprise a universal sequence. For example, but not as a limitation, the universal sequence may be a sequence common to two or more first polynucleotides. In certain embodiments, the universal sequence may be a sequence common to all first polynucleotides. In certain embodiments, the universal sequence may be used as a primer binding site, for example, for binding to a primer to be used in an amplification method, or acting as a primer. In certain embodiments, the first polynucleotide may further comprise a sequence at least partially complementary to a nucleic acid sequence of a cell or a nucleus. In certain embodiments, the universal sequence may include a sequence that binds to an RNA poly A tail. For example, but not as a limitation, the universal sequence may include a poly T sequence that binds to a poly A tail of an mRNA. In certain embodiments, the first polynucleotide comprises a universal sequence and a first index sequence. In certain embodiments, the first polynucleotide comprises a first index sequence and a sequence complementary to a nucleic acid sequence of a cell or a nucleus. In certain embodiments, the first polynucleotide comprises a universal sequence, a first index sequence, and a sequence complementary to a nucleic acid sequence of a cell or a nucleus. In certain embodiments, the first polynucleotide comprises a universal sequence, a first index sequence for identifying a partition (eg, an emulsion droplet), and an additional sequence, such as, but not by way of limitation, a unique molecular identifier.

[0123] In certain embodiments, universal sequences may be designed to capture chromatin fragments and any other DNA fragments generated by transposase processing. Non-limiting examples of such capture mechanisms may include the binding and connection of complementary sequences. In certain embodiments, universal sequences may 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 connection of complementary sequences. In certain embodiments, the introduced nucleic acids may include, but are not limited to, guide RNA (gRNA), lineage barcodes, oligonucleotides bound to antibodies, cell hashing oligonucleotides, and other oligonucleotides for sample multiplexing.

[0124] In certain embodiments, the first polynucleotide can exist in multiple forms and use multiple forms of universal sequences to allow for simultaneous capture of multiple molecule types. For example, but not by way of limitation, the molecule types can be RNA, DNA, 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.

[0125] In certain embodiments, cells and / or cell nuclei may be fixed prior to use in the methods of the present disclosure. For example, but not by way of limitation, cells or cell nuclei are fixed prior to contacting the cells or cell nuclei with particles coupled to the first index sequence. Any technique known in the art for fixing cells and / or cell nuclei may be used in the present disclosure. For example, but not by way of limitation, cells and / or nucleic acids may be contacted with a fixative. In certain embodiments, the fixation method may comprise chemical crosslinking, for example, by using glutaraldehyde and / or formaldehyde. In certain embodiments, cells or cell nuclei may be fixed with alcohols (e.g., methanol and / or ethanol).

[0126] In certain embodiments, cells may be permeabilized prior to use in the methods of the present disclosure. For example, but not limited to, cells are permeabilized prior to contacting the cells or cell nuclei with particles coupled to the first index sequence. Any technique known in the art for permeabilizing cells may be used in the present disclosure. For example, but not limited to, cells may be permeabilized with alcohol (e.g., methanol) and / or detergents (e.g., such as Tween-20). In certain embodiments, cells may be fixed and permeabilized.

[0127] In certain embodiments, cells (e.g., cells of a tissue) may be dissociated prior to use in the methods of the present disclosure. For example, but not by way of limitation, cells may be dissociated to produce a single cell suspension for use in the present disclosure. In certain embodiments, cells (or nuclei obtained from such cells) may be dissociated prior to contacting the cells (or nuclei obtained from such cells) with particles coupled to a first index sequence. Methods for dissociating cells (e.g., cells of a tissue) are known in the art.

[0128] In certain embodiments, cells or cell nuclei may be cross-linked prior to use in the methods of the present disclosure. For example, but not limited to, cells or cell nuclei may be cross-linked prior to contacting the cells or cell nuclei with particles coupled to the first index sequence. Alternatively or additionally, cells or cell nuclei may be cross-linked in emulsion droplets. In certain embodiments, cell membranes used in the present disclosure may be cross-linked. Cross-linking of cells (e.g., cross-linking of cell membranes or cell nuclei) may be achieved by exposing cells to any cross-linking agent. In certain embodiments, cross-linking may be performed using a fixative as disclosed herein. In certain embodiments, cross-linking may be performed by exposing cells to dithiobis(succinimidyl propionate) (DSP).

[0129] In certain embodiments, the core used in the present disclosure may be nucleosome-free. In certain embodiments, the nucleus may be treated to deplete the nucleus of nucleosomes. In certain embodiments, before being used in any of the disclosed methods, the nucleus may be treated to deplete the nucleus of nucleosomes. For example, but not limited to, before contacting the nucleus with particles coupled to the first index sequence, the nucleus may be treated to deplete the nucleus of nucleosomes. Non-limiting examples of methods for depleting the nucleus of nucleosomes include contacting the isolated nucleus with a chaotropic agent or detergent that can destroy the interaction between nucleic acid and protein. In certain embodiments, nucleus-free or depleted nuclei can be used in the methods of the present disclosure to index genomic DNA.

[0130] In certain embodiments, the cell or nucleus used in the present disclosure may be contacted with a transposase. Non-limiting examples of transposases include Tn5, Mu and Tn7 or mutants thereof. For example, but not limited to, the transposase may be an overactive mutation of a transposase, for example, an overactive Tn5 transposase. Non-limiting examples of overactive Tn5 variants are disclosed in Picelli et al., Genome Res. 24 (12): 2033-2040 (2014), the contents of which are disclosed in full herein. In certain embodiments, before being used in the present disclosure, the nucleus may be treated with a transposase or a transposase (e.g., a transposase complexed with a DNA comprising a transposase recognition sequence). For example, but not limited to, before contacting the nucleus with particles coupled to a first index sequence, the nucleus may be treated with a transposase or a transposase. Alternatively or additionally, the transposase or transposase may be partitioned into an emulsion droplet with the particles and two or more nuclei. In certain embodiments, the transpososome is complexed with a DNA sequence of a transposon comprising a flanking transposase recognition sequence, wherein the transposon is inserted into the genomic DNA. In certain embodiments, the transposon may comprise a primer binding sequence and / or an adaptor. In certain embodiments, treatment of the nucleus with a transposase or a transpososome may be used in the methods of the present disclosure for determination of transposase accessible chromatin (ATAC) sequencing.

[0131] In certain embodiments, the method may further include partitioning 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 compartments are emulsions. For example, but not by way of limitation, the compartments are emulsion droplets.

[0132] In certain embodiments, the compartments are not wells of a multiwell plate, eg, wells of a microtiter plate.

[0133] In certain embodiments, individual beads and one or more cells or cell nuclei may be partitioned into emulsion droplets. In certain embodiments, partitioning individual particles and one or more cells or cell nuclei into emulsion droplets may include contacting a plurality of particles from a first aqueous phase and cells or cell nuclei from a second aqueous phase with another phase (e.g., a non-aqueous phase), i.e., immiscible with the first and / or second aqueous phases to form emulsion droplets. Alternatively, partitioning individual particles and one or more cells or cell nuclei into emulsion droplets may include contacting a first aqueous phase containing a plurality of particles and cells or cell nuclei with another phase (e.g., a non-aqueous phase), i.e., immiscible with the first aqueous phase to form emulsion droplets.

[0134] In certain embodiments, emulsion droplets can be produced by providing cells and / or nucleic acids in a first fluid (e.g., a first aqueous phase) and combining the first fluid with a second fluid (e.g., a second aqueous phase or a non-aqueous phase), and shearing the fluid to produce a plurality of emulsion droplets containing individual particles and one or more cells or cell nuclei. In certain embodiments, the fluid can be sheared using any known method, technique, or device for mixing solutions. For example, but not limitation, the shearing fluid can include vortexing, shaking, flicking, stirring, and / or pipetting. In certain embodiments, the shearing fluid includes swirling the fluid to produce the emulsion droplets.

[0135] In some embodiments, any emulsion droplet generating device can be used to generate emulsion droplets. In some embodiments, the emulsion droplet generating device can be a microfluidic device. For example, but not by way of limitation, the Chromium TM Droplets are generated on the platform. In certain embodiments, a first aqueous phase containing an index sequence carrying beads flows through a channel segment and a second aqueous phase containing cells or cell nuclei flows through a second channel segment toward a channel junction. A partitioning fluid (e.g., oil) is introduced from one or more side channels into the channel junction, and the combined stream flows into an outlet channel, such as Figure 1 As shown. Within the channel junction, the two water streams are combined with the partitioning oil and partitioned into droplets containing co-partitioned nucleic acids or cells, and the beads are partitioned into emulsion droplets (referred to herein as "GEMs") ( Figure 1). In certain embodiments, controlling the flow characteristics of each fluid combined at the channel junction and controlling the geometry of the channel junction can achieve a desired occupancy level of beads, cells (or nuclei), or both within the generated droplets. Additional disclosures regarding microfluidic devices for use in the present disclosure are provided in WO 2017 / 096158 and U.S. Patent No. 11,193,122, the contents of each of which are incorporated herein by reference in their entirety.

[0136] In certain embodiments, partitioning of individual particles and one or more cells or nuclei may result in the generation of a plurality of emulsified droplets. In certain embodiments, the methods of the present disclosure may comprise 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 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 5 In some embodiments, about 5,000 to about 200,000 droplets are produced.

[0137] In certain embodiments, each compartment (e.g., droplet) contains more than one cell or cell nucleus and a single bead. For example, but not by way of limitation, the number of cells or cell nuclei in each droplet can be from about 2 to about 20. In certain embodiments, the number of cells or cell nuclei in each droplet can be about 3 to about 20, about 4 to about 20, about 5 to about 20, about 6 to about 20, about 7 to about 20, about 8 to about 20, about 9 to about 20, about 10 to about 20, about 11 to about 20, about 12 to about 20, about 13 to about 20, about 14 to about 20, about 15 to about 20, about 16 to about 20, about 17 to about 20, about 18 to about 20, about 19 to about 20, about 2 to about 19, about 2 to about 18, about 2 to about 17, about 2 to about 16, about 2 to about 15, about 2 to about 14, about 2 to about 13, about 2 to about 12, about 2 to about 11, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, or about 3 to about 10 cells or cell 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 cell nuclei. In certain embodiments, the number of cells or cell nuclei in each droplet may be from about 2 to about 10. In certain embodiments, about 5,000 to about 200,000 droplets are generated and each droplet may include about 2 to about 10 cells or cell nuclei.

[0138] Incorporating more than one cell or cell nucleus and a single bead in each emulsion droplet allows for the use of the same index sequence to index nucleic acids of cells or cell nuclei present in the emulsion droplet. As described herein, incorporating more than one cell or cell nucleus in each emulsion droplet increases the throughput of the indexing technique and increases the capacity of an emulsion droplet generation device (e.g., a microfluidic device) used to generate the droplets by at least 20 times.

[0139] In certain embodiments, other reagents may be co-partitioned into the emulsion droplets. In certain embodiments, such reagents may be used to incorporate the first index sequence into the nucleic acid, for example, for an amplification method or a ligation method to incorporate the first index sequence into the nucleic acid. For example, but not by way of limitation, the reagent may include a polymerase, a reverse transcriptase, a nucleoside triphosphate or NTP analog, a primer, a cofactor, a ligation reaction reagent, an endonuclease, a dissolution reagent, a dye, a marker, or a label. In certain embodiments, additional reagents may include a protease to remove proteins bound to nucleic acids of a cell or nucleus and / or a translocase or transposase to fragment or insert a known sequence into nucleic acids of a cell or nucleus.

[0140] In certain embodiments, the method may further include merging the first index sequence disposed on the particle (e.g., a bead) into the nucleic acid of the cell or nucleus to produce an index nucleic acid. In certain embodiments, merging the first index sequence into the nucleic acid occurs in a droplet, for example, using a reagent that is commonly partitioned into the droplet. The first index sequence can be incorporated into the nucleic acid of the cell or nucleus using any suitable nucleic acid amplification method known in the art. Non-limiting examples of such amplification methods include polymerase chain reaction (PCR), reverse transcriptase PCR, real-time PCR, rolling circle amplification (RCA), self-sustaining 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 method is an isothermal amplification method, for example, 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 contents of each method are disclosed in their entirety herein. In certain embodiments, the amplification method uses a universal sequence, for example, a universal sequence present in a first polynucleotide, for binding to a primer used in the amplification method. Alternatively, a ligation method can be used to incorporate the first index sequence into the nucleic acid of a cell or nucleus. For example, but not by way of limitation, the index sequence can be ligated to the nucleic acid of a single cell or nucleus by a ligase.

[0141] In certain embodiments, reverse transcription is used to incorporate the first index sequence into the nucleic acid of the cell or nucleus. For example, but not limited to, reverse transcription is used to incorporate the first index sequence into the RNA of the cell (e.g., the mRNA of the cell). In certain embodiments, in a compartment (e.g., an emulsion droplet), a first polynucleotide providing a first index sequence is combined with the RNA (e.g., mRNA) of the cell. The reverse transcriptase binds to the RNA complexed with the first polynucleotide and initiates the synthesis of a complementary DNA (cDNA) chain. Any reverse transcriptase known in the art can be used in the method of the present disclosure. In certain embodiments, RNase H may be present in a partition (e.g., an emulsion droplet) to degrade the RNA of the cDNA: RNA complex. In certain embodiments, the reverse transcriptase may lack RNase H activity. In certain embodiments, the reverse transcriptase may have terminal transferase activity and template switching activity to incorporate a universal selection sequence at the end of the cDNA chain. In certain embodiments, the compartment (eg, emulsion droplet) may further comprise a DNA-dependent polymerase that synthesizes a complementary cDNA strand using the single-stranded cDNA as a template to form a double-stranded cDNA.

[0142] In certain embodiments, PCR is used to incorporate the first index sequence into the nucleic acid of the cell or nucleus. For example, but not by way of limitation, if the nucleic acid to be indexed is DNA (e.g., genomic DNA), PCR is used.

[0143] In certain embodiments, the first index sequence is released from a particle (e.g., a gel bead). In certain embodiments, the index sequence is released from the particle before the index sequence is incorporated into the nucleic acid. Alternatively or additionally, the index sequence is released from the particle after the index sequence is incorporated into the nucleic acid. In certain embodiments, the first index sequence is released from the particle by dissolving the particle (e.g., a gel bead). In certain embodiments, a reducing agent may be used to dissolve or degrade the particle, change the temperature (e.g., heat the droplet), change the pH, and / or expose to light. In certain embodiments, the first index sequence is released from the particle by cutting a linker that couples the index sequence to the particle. In certain embodiments, the first index sequence is released from the particle in the droplet.

[0144] In certain embodiments, the method may further include combining index nucleic acids from multiple compartments (e.g., emulsion droplets) to produce mixed index nucleic acids. For example, but not by way of limitation, the method includes combining cells and / or nuclei that contain index nucleic acids from multiple compartments (e.g., emulsion droplets) or are associated with the index nucleic acids to produce mixed cells and / or nuclei. In certain embodiments, the nucleic acid containing the first index sequence is removed from the compartment (e.g., emulsion droplets). For example, but not by way of limitation, the nucleic acid containing the first index sequence can be separated from the emulsion droplets by an unpacking method, wherein the index nucleic acid present in the emulsion droplets is released. Any technique known in the art for demulsification can be used for the unpacking method. Non-limiting examples of techniques for demulsification include acidification, centrifugation, filtration, and salting. In certain embodiments, the cells containing the index nucleic acids remain intact during the merging of the first index sequence, and the unpacking method releases intact cells from the emulsion droplets.

[0145] In certain embodiments, subsets of released index nucleic acids are mixed together. In certain embodiments, subsets of cells or cell nuclei comprising index nucleic acids released from compartments (eg, emulsion droplets) are mixed together. For example, but not by way of 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 index nucleic acids (or cells comprising or associated with the index nucleic acids) released from a compartment (e.g., an emulsion droplet) are mixed together. In certain embodiments, at least about 1% of the index nucleic acids (or cells comprising or associated with the index nucleic acids) released from a compartment (e.g., an emulsion droplet) are mixed together. In certain embodiments, at least about 2% of the index nucleic acids (or cells comprising or associated with the index nucleic acids) released from a compartment (e.g., an emulsion droplet) are mixed together. In certain embodiments, at least about 10% of the index nucleic acids (or cells comprising or associated with the index nucleic acids) released from a compartment (e.g., an emulsion droplet) are mixed together. In certain embodiments, at least about 50% of the index nucleic acids (or cells comprising or associated with the index nucleic acids) released from a compartment (e.g., an emulsion droplet) are mixed together. In certain embodiments, about 1% to about 5% of the index nucleic acids (or cells comprising or associated with the index nucleic acids) released from a compartment (e.g., an emulsion droplet) are mixed together. In certain embodiments, about 1% to about 10% of the index nucleic acids (or cells comprising or associated with the index nucleic acids) released from a compartment (e.g., an emulsion droplet) are mixed together. In certain embodiments, all index nucleic acids released from a compartment (e.g., an emulsion droplet) are mixed together. In certain embodiments, all cells comprising the index nucleic acids released from a compartment (e.g., an emulsion droplet) are mixed together.

[0146] In certain embodiments, the method may further include distributing a subset of the mixed index nucleic acids into a plurality of compartments including a second index sequence. The index cells or nuclei may be distributed 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 compartments may be wells of a multi-well plate (e.g., a 48-well, 96-well, 384-well, or 1536-well plate), e.g., wells of a microtiter plate. In certain embodiments, the compartments in which the second index sequence is obtained by the index nucleic acids are not emulsion droplets.

[0147] In certain embodiments, each compartment (e.g., well) contains more than one cell or cell nucleus comprising an index nucleic acid. For example, but not by way of limitation, the number of cells or cell nuclei in each compartment may be about 2 to about 20,000. In certain embodiments, the number of cells or cell nuclei in each compartment (e.g., well) may be about 10 to about 20,000, about 100 to about 20,000, about 1,000 to about 20,000, about 2,000 to about 20,000, about 3,000 to about 20,000, about 4,000 to about 20,000, about 5,000 to about 20,000, about 6,000 to about 20,000, or about 7,000 to about 20,000. 000, about 7,000 to about 20,000, about 8,000 to about 20,000, about 9,000 to about 20,000, from about 10,000 to about 20,000, about 11,000 to about 20,000, about 12,000 to about 20,000, about 13,000 to about 20,000, about 14,000 to about 20,000, about 15,000 to about 20 ,000, about 16,000 to about 20,000, about 17,000 to about 20,000, about 18,000 to about 20,000, about 19,000 to about 20,000, about 2 to about 19,000, about 2 to about 18,000, about 2 to about 17,000, about 2 to about 16,000, about 2 to about 15,000, about 2 to about 14,000, about 2 to about 13, In some embodiments, the present invention relates to a plurality of cells or cell nuclei. In some embodiments, the present invention relates to a plurality of cells or cell nuclei. In some embodiments, the present invention relates to a plurality of cells or cell nuclei. In some embodiments, the present invention relates to a plurality of cells or cell nuclei. In certain embodiments, the number of cells or cell nuclei in each compartment (e.g., well) can be about 100 to about 15,000, about 5,000 to about 15,000, about 1,000 to about 15,000, about 2,000 to about 15,000, about 3,000 to about 15,000, about 4,000 to about 15,000, about 5,000 to about 15,000, about 6,000 to about 15,000, about 7,000 to about 15,000, about 8,000 to about 15,000, or about 9,000 to about 10,000. 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 cell nuclei. In certain embodiments, the mixed index nucleic acids (e.g., mixed cells or nuclei comprising index nucleic acids) are sufficiently mixed so that cells or nuclei having nucleic acids indexed with the same first index sequence are not partitioned into the same compartment. As disclosed herein, the use of two index sequences in the methods of the present disclosure allows for labeling of nucleic acids from a single cell with a unique combination of the two index sequences.

[0148] In certain embodiments, the method may further include incorporating a second index sequence into the index nucleic acid in each compartment to produce a dual index nucleic acid. The second index sequence may be incorporated into the index nucleic acid of the cell or nucleus using any suitable nucleic acid sequence amplification method known in the art. Non-limiting examples of such amplification methods are provided herein. For example, but not limited to, the amplification method is an isothermal amplification method. In certain embodiments, a ligation method may be used to incorporate the second index sequence into the index nucleic acid. In certain embodiments, a polymerase chain reaction may be used to incorporate the second index sequence into the index nucleic acid.

[0149] In certain embodiments, each compartment contains a second index sequence that is unique relative to the index sequences in other compartments. Alternatively, no more than about 50% of the compartments, for example, no more than about 45%, no more than about 40%, no more than about 35%, no more than about 30%, no more than about 25%, no more than about 20%, no more than about 15%, no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, or no more than about 1% 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, no more than 10% 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, no more than 5% of the compartments contain a second index sequence having the same nucleic acid sequence as the second index sequence of other compartments.

[0150] In certain embodiments, the second index sequence is provided in a second polynucleotide. In certain embodiments, the second polynucleotide further comprises a sequence that is reverse complementary to the sequence of the index 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, reverse complementation 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 connection to the index nucleic acid. In certain embodiments, the second polynucleotide may comprise a sequence that can bind to a primer (e.g., a primer binding site), or a sequence that acts as a primer. For example, but not limited to, the second polynucleotide comprises a sequence that can bind to a primer for sequencing the index nucleic acid and / or for an amplification method.

[0151] In certain embodiments, each compartment contains reagents for merging the second index sequence into the index nucleic acid. For example, but not by way of limitation, the reagents may include reagents for performing an amplification or ligation method in 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 may further include a dissolution reagent.

[0152] In certain embodiments, the method may further include lysing the cells. For example, but not by way of limitation, the cells may be lysed using sodium hydroxide, potassium hydroxide, sodium dodecyl sulfate, nonionic surfactants, saponins, proteases, lytic enzymes, freeze-thaw methods, ultraviolet light, and / or heat. In certain embodiments, the cells are lysed after incorporating the second index sequence into the index nucleic acid to produce a dual index nucleic acid. Alternatively, but before incorporating the second index sequence into the index nucleic acid, the cells are lysed in a compartment containing the second index sequence. In certain embodiments, the cells are lysed after the second round of compartmentalization but before adding the reagent for incorporating the second index sequence.

[0153] In certain embodiments, the method may further include merging one or more additional index sequences into the dual index nucleic acid. In certain embodiments, one or more additional index sequences (e.g., two or more, three or more or four or more additional index sequences) may be incorporated into the dual index nucleic acid. For example, but not limited to, the method of the present disclosure may further include merging the third index sequence into the dual index nucleic acid to produce a triple index nucleic acid. In certain embodiments, merging the third index sequence into the dual index nucleic acid may be carried out in another plurality of compartments containing the third index sequence as described for the second index sequence. In certain embodiments, the method of the present disclosure may further include merging the fourth index sequence into the triple index nucleic acid to produce a quadruple index nucleic acid. In certain embodiments, merging one or more additional index sequences into the dual index nucleic acid may include providing an additional index sequence (e.g., a triple index sequence), which may be coupled to the first and / or second index sequence to produce a triple index nucleic acid. In certain embodiments, an additional index sequence (e.g., a triple index sequence) may be connected to the first and / or second index sequence to produce a triple index nucleic acid. In certain embodiments, a fourth index sequence can be coupled to the first, second, and / or third index sequence, for example, a fourth index sequence can be linked to the third index sequence to generate a quadruple-indexed nucleic acid.

[0154] In certain embodiments, the method of the present disclosure may further include performing a cell hashing technique. For example, but not by way of limitation, the method of the present disclosure may further include contacting the cells and / or cell nuclei with a reagent that binds to a target protein (referred to herein as a "protein binding reagent"). In certain embodiments, a protein binding reagent is a reagent that specifically binds to a target protein (e.g., specifically binds to a target protein of cells and / or cell 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, a protein binding reagent is an antibody or an antibody binding fragment thereof.

[0155] In certain embodiments, the protein bound by the protein binding reagent can be any protein present in a cell or on the surface of a cell. For example, but not limited to, 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 the protein or a wild-type form of the 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., allowing differentiation of two or more cell types).

[0156] In certain embodiments, the protein binding reagent may be coupled to an oligonucleotide. In certain embodiments, the oligonucleotide bound to the protein binding reagent may include a barcode (referred to herein as a "tag"). In certain embodiments, the protein binding reagent may be an antibody (or fragment thereof) coupled to an oligonucleotide comprising a barcode. In certain embodiments, the barcode allows identification of the protein binding reagent (e.g., antibody) and the corresponding protein to which it binds. In certain embodiments, the protein binding reagent may be a TotalSeq TM Antibodies (BioLegend, San Diego, CA). In certain embodiments, the use of protein binding reagents (e.g., antibodies) allows for the detection of proteins in a single sample (e.g., proteins in a single cell in a sample). In certain embodiments, the use of protein binding reagents (e.g., antibodies) allows for the quantification of proteins in a single sample (e.g., proteins in a single cell in a sample).

[0157] In certain embodiments, the methods of the present disclosure may include contacting cells and / or cell nuclei with a variety of protein binding reagents. For example, but not limited to, each protein binding reagent in a variety 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 variety of protein binding reagents can be used to bind to a variety of different target proteins in a sample. In certain embodiments, the use of a variety of protein binding reagents (e.g., antibodies) allows a variety of proteins in a single sample (e.g., proteins in a single cell in a sample) to be detected. In certain embodiments, the use of a variety of protein binding reagents (e.g., antibodies) allows a variety of proteins in a single sample (e.g., proteins in a single cell in a sample) to be quantified.

[0158] In certain embodiments, before contacting the cell and / or nucleus with a plurality of particles, the cell and / or nucleus may be contacted with a protein binding reagent (e.g., to perform the combined indexing method of the present disclosure). In certain embodiments, before fixing the cell and / or nucleus with a plurality of particles (e.g., methanol fixation), the cell and / or nucleus may be contacted with a protein binding reagent. In certain embodiments, a subset of cells and / or nuclei to be contacted with a plurality of particles may be contacted with a protein binding reagent (e.g., a protein binding reagent coupled to an oligonucleotide comprising a barcode), and different subsets of cells and / or nuclei to be contacted with a plurality of particles may be contacted with different protein binding reagents (e.g., different protein binding reagents coupled to oligonucleotides comprising different barcodes). Alternatively or additionally, cells and / or nuclei to be contacted with a plurality of particles may be contacted with a plurality of protein binding reagents. In certain embodiments, at least two or more protein binding agents, at least three or more protein binding agents, at least four or more protein binding agents, at least five or more protein binding agents, at least six or more protein binding agents, at least seven or more protein binding agents, at least eight or more protein binding agents, at least nine or more protein binding agents, at least ten or more protein binding agents can be used in the present disclosure, at least 50 or more protein binding agents, at least 100 or more protein binding agents, at least 150 or more protein binding agents, at least 200 or more protein binding agents, at least 250 or more protein binding agents, or at least 300 or more protein binding agents, wherein each protein binding agent specifically binds to a single target protein, and each protein binding agent is coupled to a unique barcode. In certain embodiments, a sample (e.g., a plurality of cells and / or cell nuclei) can be contacted with a protein binding agent (e.g., an antibody specific for a target protein coupled to a barcode) for a period of time and under conditions that support specific binding of the protein binding agent to the target protein.

[0159] In certain embodiments, the method of the present disclosure may further include treating cells and / or cell nuclei with a medicament (e.g., a therapeutic agent). For example, but not limited to, the method of the present disclosure may include contacting cells and / or cell nuclei with a medicament (e.g., a therapeutic agent), for example, before the combined indexing method described herein. In certain embodiments, before performing the combined indexing method described herein, the method of the present disclosure may include contacting cells and / or cell nuclei with a medicament (e.g., 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. In certain embodiments, the methods of the present disclosure may include contacting the cell and / or cell nucleus with an agent (e.g., 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 may include polypeptide therapeutics, e.g., antibody-based therapeutics, oligonucleotides, cell-based therapeutics, gene editing systems, and small molecule therapeutics.

[0160] In certain embodiments, the method of the present disclosure may further include subjecting cells and / or cell nuclei to gene regulation or control. In certain embodiments, the method of the present disclosure may further include subjecting cells and / or cell nuclei to genome screening, for example, before performing the combined indexing method described herein. For example, but not limited to, the method of the present disclosure may further include contacting cells and / or cell nuclei with a gene editing system, for example, before performing the combined indexing method. Non-limiting examples of gene editing systems include homing endonucleases or large-range nucleases, ZFNs, TALENs, and CRISPR gene editing systems. In certain embodiments, the method of the present disclosure may further include subjecting cells and / or cell nuclei to CRISPR-based screening, for example, before performing the combined indexing method described herein.

[0161] In certain embodiments, an exemplary method for combinatorial indexing of nucleic acids of a single cell or cell nucleus may include: (a) contacting a plurality of cells or cell nuclei separated from a plurality of cells with a plurality of particles (e.g., microbeads) comprising a first index sequence; (b) partitioning the individual particles and one or more of the cells or cell nuclei into an emulsion (e.g., emulsion droplets); and (c) incorporating the first index sequence into the nucleic acids of the cells or cell nuclei to produce indexed nucleic acids; (d) combining cells or cell nuclei containing or associated with indexed nucleic acids from a plurality of emulsions (e.g., emulsion droplets) to produce mixed cells or cell nuclei; (e) distributing subsets of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; and (f) incorporating the second index sequence into the indexed nucleic acid in each compartment to produce a dual-indexed nucleic acid. In certain embodiments, the method may include: (a) contacting a plurality of cells or cell nuclei isolated from a plurality of cells with a plurality of protein binding agents (e.g., a plurality of antibodies) coupled to an oligonucleotide comprising a barcode; (b) contacting a plurality of cells or cell nuclei isolated from a plurality of cells with a plurality of particles (e.g., microbeads) comprising a first index sequence; (c) partitioning one or more of the individual particles and cells or cell nuclei into an emulsion (e.g., an emulsion droplet); and (d) merging the first index sequence into the nucleic acid of the cell or cell nucleus to produce an index nucleic acid; (e) combining cells or cell nuclei containing or associated with an index nucleic acid from a plurality of emulsions (e.g., an emulsion droplet) to produce mixed cells or cell nuclei; (f) distributing a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; and (g) merging the second index sequence into the index nucleic acid in each compartment to produce a dual index nucleic acid. In certain embodiments, the method may further include merging a third index sequence into the dual index nucleic acid to produce a triple index nucleic acid. In certain embodiments, prior to incorporating the first index sequence into the nucleic acids of the plurality of cells or cell nuclei, the plurality of cells or cell nuclei have been subjected to genomic screening, subjected to cell hashing techniques, subjected to lineage tracing methods, and / or treated with a therapeutic agent. In certain embodiments, prior to contacting the cells or cell nuclei with a protein binding agent, the plurality of cells or cell nuclei have been subjected to genomic screening, subjected to cell hashing techniques, subjected to lineage tracing methods, and / or treated with a therapeutic agent.

[0162] In certain embodiments, an exemplary method for combinatorial indexing of nucleic acids of a single cell or cell nucleus may include: (a) providing mixed cells or cell nuclei associated with or containing nucleic acids comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acids of the mixed cells or cell nuclei in emulsion droplets; (b) distributing subsets of the mixed cells or cell nuclei into multiple compartments comprising a second index sequence; and (c) incorporating the second index sequence into the nucleic acid in each compartment to produce a dual-indexed nucleic acid. In certain embodiments, the first index sequence is incorporated into the nucleic acid of the cells or nuclei in the 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 one or more of the individual particles and cells or nuclei into the emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cells or nuclei to produce the index nucleic acid; and (iv) combining the cells or nuclei associated with or comprising the index nucleic acid from the plurality of emulsion droplets to produce a mixed cell or nucleus associated with or comprising the nucleic acid comprising the first index sequence. In certain embodiments, the mixed cells or nuclei have been subjected to genomic screening, cell hashing techniques, lineage tracing methods, and / or treated with a therapeutic agent prior to incorporating the first index sequence into the nucleic acid of the mixed cells or nuclei.

[0163] In certain embodiments, the method may include: (a) providing a mixture of cells or cell nuclei 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 mixed cells or cell nuclei in emulsion droplets, wherein the mixed cells or cell nuclei are bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode; (b) distributing a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; and (c) incorporating the second index sequence into the nucleic acid in each compartment to produce a dual-indexed nucleic acid. In certain embodiments, the first index sequence is incorporated into the nucleic acid of the cells or nuclei in the 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 one or more of the individual particles and cells or nuclei into the emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cells or nuclei to produce the index nucleic acid; and (iv) combining the cells or nuclei associated with or comprising the index nucleic acid from the plurality of emulsion droplets to produce a mixed cell or nucleus associated with or comprising the nucleic acid comprising the first index sequence. In certain embodiments, prior to incorporating the first index sequence into the nucleic acid of the mixed cells or nuclei, e.g., prior to step (a), the mixed cells or nuclei have been subjected to genomic screening, subjected to cell hashing techniques, subjected to lineage tracing methods, and / or treated with a therapeutic agent.

[0164] The present disclosure further provides a method for producing a sequencing library. In certain embodiments, the sequencing library comprises nucleic acids indexed by the method of the present disclosure. For example, but not limited to, the sequencing library according to the present disclosure comprises nucleic acids from a single cell of at least a dual index. In certain embodiments, the nucleic acids comprising the sequencing library are triple indexed. In certain embodiments, the sequencing library partially or completely represents the transcriptome of one or more single cells, for example, wherein all or a subset of the nucleic acids of a particular cell have been marked with an indexed sequence (e.g., a unique combination of the sequences of the index). In certain embodiments, the sequencing library partially or completely represents the genome of one or more single cells, for example, wherein all or a subset of the nucleic acids of a particular cell have been marked with an indexed sequence (e.g., a unique combination of the sequences of the index).

[0165] In certain embodiments, methods of generating sequencing libraries include combining multiple index nucleic acids, which can be prepared as disclosed herein, to generate libraries from multiple single cells or nuclei. In certain embodiments, multiple index nucleic acids (e.g., dual index nucleic acids) derived from subsets of total cells or nuclei generated using the methods of the present disclosure can be combined to prepare sequencing libraries. For example, but not by way of 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 the cells or nuclei are combined to generate a sequencing library. In certain embodiments, at least about 20% of the dual-indexed nucleic acids of the cells or nuclei are combined to generate a sequencing library. In certain embodiments, at least about 50% of the dual-indexed nucleic acids of the cells or nuclei are combined to generate a sequencing library. In certain embodiments, dual-indexed nucleic acids from at least about 90% of cells or nuclei are combined to generate a sequencing library. In certain embodiments, all multiply-indexed nucleic acids (eg, dual-indexed nucleic acids) can be combined to generate a sequencing library.

[0166] In certain embodiments, less than all of the multiply-indexed nucleic acids (e.g., dual-indexed nucleic acids) may be combined to generate a sequencing library. For example, but not by way of limitation, 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 dual-indexed nucleic acids of a cell or nucleus may be combined to generate a sequencing library. In certain embodiments, less than about 50% of the multiply-indexed nucleic acids (e.g., dual-indexed nucleic acids) may be combined to generate a sequencing library. In certain embodiments, less than about 90% of the multiply-indexed nucleic acids (eg, dually-indexed nucleic acids) can be combined to generate a sequencing library.

[0167] In certain embodiments, about 10% to about 99% of the dual indexed nucleic acids of a cell or cell nucleus 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 dual indexed nucleic acids of a cell or cell nucleus are combined to generate a sequencing library.

[0168] In certain embodiments, a method for generating a sequencing library comprising nucleic acids from multiple single cells comprises: (a) contacting multiple cells or cell nuclei isolated from multiple cells with multiple particles (e.g., microbeads) comprising a first index sequence; (b) partitioning the individual particles (e.g., microbeads) and one or more of the cells or cell nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cells or cell nuclei to produce index nucleic acids; (d) combining cells or cell nuclei containing or associated with index nucleic acids from multiple emulsion droplets to produce mixed index cells or cell nuclei; (e) distributing subsets of the mixed cells or cell nuclei into multiple compartments comprising a second index sequence; (f) incorporating the second index sequence into the index nucleic acid in each compartment to produce dual-index nucleic acids; and (g) combining all or a subset of the dual-index nucleic acids to generate a sequencing library from multiple cells or cell nuclei. In certain embodiments, prior to incorporating the first index sequence into the nucleic acids of the plurality of cells or cell nuclei, e.g., prior to step (a), the plurality of cells or cell nuclei have been subjected to genomic screening, subjected to cell hashing techniques, subjected to lineage tracing methods, and / or treated with a therapeutic agent.

[0169] In certain embodiments, the methods of the present disclosure may include cell hashing techniques. For example, but not by way of limitation, the methods of the present disclosure include: (a) contacting a plurality of cells or cell nuclei separated from a plurality of cells with a plurality of protein binding agents (e.g., a plurality of antibodies) coupled to an oligonucleotide comprising a barcode; (b) contacting a plurality of cells or cell nuclei separated from a plurality of cells with a plurality of particles (e.g., microbeads) comprising a first index sequence; (c) partitioning the individual particles (e.g., microbeads) and one or more of the cells or cell nuclei into emulsion droplets; (d) merging the first index sequence into the nucleic acid of the cell or cell nucleus to produce an index nucleic acid; (e) combining cells or cell nuclei containing or associated with an index nucleic acid from a plurality of emulsion droplets to produce mixed index cells or cell nuclei; (f) distributing a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; (g) merging the second index sequence into the index nucleic acid in each compartment to produce a dual index nucleic acid; and (h) combining all or a subset of the dual index nucleic acids to produce a sequencing library from a plurality of cells or cell nuclei. In certain embodiments, prior to incorporating the first index sequence into the nucleic acids of the plurality of cells or nuclei and / or prior to contacting the plurality of cells or nuclei with the plurality of protein binding agents, e.g., prior to step (a), the plurality of cells or nuclei may be further subjected to genomic screening, subjected to lineage tracing methods, and / or treated with a therapeutic agent.

[0170] In certain embodiments, a method for generating a sequencing library comprising nucleic acids from multiple single cells comprises: (a) providing mixed cells or cell 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 mixed cells or cell nuclei in emulsion droplets; (b) partitioning subsets of the mixed cells or cell nuclei into multiple compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acids in each compartment to generate dual-index nucleic acids; and (d) combining all or a subset of the dual-index nucleic acids to generate a sequencing library from multiple cells or cell nuclei. In certain embodiments, the first index sequence is incorporated into the nucleic acid of the cells or nuclei in the 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 one or more of the individual particles and cells or nuclei into the emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cells or nuclei to produce the index nucleic acid; and (iv) combining the cells or nuclei associated with or comprising the index nucleic acid from the plurality of emulsion droplets to produce a mixed cell or nucleus associated with or comprising the nucleic acid comprising the first index sequence. In certain embodiments, the mixed cell or nucleus has been 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 acid of the mixed cell or nucleus.

[0171] In certain embodiments, the method comprises: (a) providing mixed cells or cell nuclei associated with or comprising nucleic acid comprising a first index sequence, wherein the first index sequence is incorporated into the nucleic acid of the mixed cells or cell nuclei in emulsion droplets, wherein the mixed cells or cell nuclei are bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode; (b) partitioning a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acid in each compartment to produce dual-index nucleic acids; and (d) combining all or a subset of the dual-index nucleic acids to produce a sequencing library from a plurality of cells or cell nuclei. In certain embodiments, the first index sequence is incorporated into the nucleic acid of the cells or nuclei in the 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 one or more of the individual particles and cells or nuclei into the emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cells or nuclei to produce the index nucleic acid; and (iv) combining the cells or nuclei associated with or comprising the index nucleic acid from the plurality of emulsion droplets to produce a mixed cell or nucleus associated with or comprising the nucleic acid comprising the first index sequence. In certain embodiments, the mixed cell or nucleus has been further subjected to genomic screening, subjected to a lineage tracing method, and / or treated with a therapeutic agent prior to incorporating the first index sequence into the nucleic acid of the mixed cell or nucleus.

[0172] On the other hand, the present disclosure further provides methods for sequencing libraries generated according to the methods disclosed herein. In certain embodiments, the sequencing methods of the present disclosure include sequencing multiple index nucleic acids (e.g., dual index nucleic acids) from a single cell. In certain embodiments, the sequencing methods of the present disclosure include sequencing dual index nucleic acids that partially or completely represent the transcriptome of a single cell. In certain embodiments, the sequencing methods of the present disclosure include sequencing dual index nucleic acids that partially or completely represent the genome of a single cell.

[0173] In certain embodiments, the sequencing methods disclosed herein include sequencing all or a subset of a multi-indexed nucleic acid (e.g., a dual-indexed nucleic acid) of a library. For example, but not limited to, 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 (e.g., dual-indexed nucleic acids) included in the library may be sequenced. In certain embodiments, about 10% to about 99% of the dual-indexed nucleic acids may 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 may be sequenced.

[0174] In certain embodiments, the present disclosure provides a method for sequencing nucleic acids from multiple cells, the method comprising: (a) contacting multiple cells or cell nuclei separated from multiple cells with multiple particles (e.g., microbeads) comprising a first index sequence; (b) partitioning the individual particles (e.g., microbeads) and one or more of the cells or cell nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cells or cell nuclei to produce index nucleic acids; (d) combining cells or cell nuclei containing or associated with index nucleic acids from multiple emulsion droplets to produce mixed index cells or cell nuclei; (e) distributing a subset of the mixed cells or cell nuclei into multiple compartments comprising a second index sequence; (f) incorporating the second index sequence into the index nucleic acid in each compartment to produce dual-index nucleic acids; (g) combining all or a subset of the dual-index nucleic acids to produce a sequencing library from multiple cells; and (h) sequencing all or a subset of the dual-index nucleic acids. In certain embodiments, prior to incorporating the first index sequence into the nucleic acid of the plurality of cells or cell nuclei, the plurality of cells or cell nuclei have been subjected to genomic screening, subjected to cell hashing techniques, subjected to lineage tracing methods, and / or treated with a therapeutic agent.

[0175] In certain embodiments, the method comprises: (a) contacting a plurality of cells or cell nuclei separated from a plurality of cells with a plurality of protein binding agents (e.g., a plurality of antibodies) coupled to oligonucleotides comprising a barcode; (b) contacting a plurality of cells or cell nuclei separated from a plurality of cells with a plurality of particles (e.g., microbeads) comprising a first index sequence; (c) partitioning the individual particles (e.g., microbeads) and one or more of the cells or cell nuclei into emulsion droplets; (d) incorporating the first index sequence into nucleic acids of the cells or cell nuclei to generate index nucleic acids; (e) Cells or cell nuclei containing or associated with index nucleic acids from a plurality of emulsion droplets are combined to produce mixed index cells or cell nuclei; (f) subsets of the mixed cells or cell nuclei are distributed into a plurality of compartments including a second index sequence; (g) the second index sequence is incorporated into the index nucleic acid in each compartment to produce a dual index nucleic acid; (h) all or a subset of the dual index nucleic acids are combined to produce a sequencing library from a plurality of cells; and (i) all or a subset of the dual index nucleic acids are sequenced and / or barcodes derived from protein binding agents are sequenced. In certain embodiments, the plurality of cells or cell nuclei have been further subjected to genomic screening, lineage tracing methods, and / or treated with a therapeutic agent prior to incorporating the first index sequence into the nucleic acids of the plurality of cells or cell nuclei and / or prior to contacting the plurality of cells or cell nuclei with a plurality of protein binding agents.

[0176] In certain embodiments, the present disclosure provides a method for sequencing nucleic acids from multiple cells, the method comprising: (a) providing mixed cells or cell 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 mixed cells or cell nuclei in emulsion droplets; (b) distributing subsets of the mixed cells or cell nuclei into multiple compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acids in each compartment to produce dual-index nucleic acids; (d) combining all or a subset of the dual-index nucleic acids to produce a sequencing library from multiple cells; and (d) sequencing all or a subset of the dual-index nucleic acids. In certain embodiments, the first index sequence is incorporated into the nucleic acid of the cells or nuclei in the 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 one or more of the individual particles and cells or nuclei into the emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cells or nuclei to produce the index nucleic acid; and (iv) combining the cells or nuclei associated with or comprising the index nucleic acid from the plurality of emulsion droplets to produce a mixed cell or nucleus associated with or comprising the nucleic acid comprising the first index sequence. In certain embodiments, the mixed cell or nucleus has been 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 acid of the mixed cell or nucleus.

[0177] In certain embodiments, the method comprises: (a) providing mixed cells or cell 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 mixed cells or cell nuclei in emulsion droplets, wherein the mixed cells or cell nuclei are bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode; (b) partitioning a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acids in each compartment to produce dual-indexed nucleic acids; (d) combining all or a subset of the dual-indexed nucleic acids to produce 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 barcodes derived from the protein binding reagent. In certain embodiments, the first index sequence is incorporated into the nucleic acid of the cells or nuclei in the 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 one or more of the individual particles and cells or nuclei into the emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cells or nuclei to produce the index nucleic acid; and (iv) combining the cells or nuclei associated with or comprising the index nucleic acid from the plurality of emulsion droplets to produce a mixed cell or nucleus associated with or comprising the nucleic acid comprising the first index sequence. In certain embodiments, the mixed cells or nuclei have been further subjected to genomic screening, subjected to a lineage tracing method, and / or treated with a therapeutic agent prior to incorporating the first index sequence into the nucleic acid of the mixed cells or nuclei and / or prior to binding the protein binding agent to the mixed cells or nuclei.

[0178] The index nucleic acid can be sequenced using an appropriate sequencing method. In certain embodiments, the nucleic acid can be sequenced by a high-throughput sequencing method. In certain embodiments, the nucleic acid can be sequenced by next generation sequencing (NGS). For example, but not limited to, the Illumina NGS platform can be used for sequencing. In certain embodiments, the nucleic acid can be sequenced by a pyrophosphate sequencing method. In certain embodiments, the nucleic acid can be sequenced by Sanger sequencing. In certain embodiments, the nucleic acid can be sequenced using nanopore-based sequencing. In certain embodiments, the sequence reads obtained using the method disclosed herein can be attributed to specific cells based on a unique combination of sequences of two indexes.

[0179] IV. Kit

[0180] The present disclosure further provides kits for performing the methods of the present disclosure. For example, but not by way of limitation, the present disclosure provides kits containing materials for performing methods for combinatorial indexing of nucleic acids from multiple single cells. In certain embodiments, the present disclosure provides kits containing materials for performing methods for generating sequencing libraries comprising nucleic acids from multiple single cells. In certain embodiments, the present disclosure provides kits containing materials for sequencing libraries comprising nucleic acids from multiple single cells.

[0181] In certain embodiments, the kit of the present disclosure includes a container containing a plurality of particles comprising a first index sequence. In certain embodiments, the kit may further include a container containing a plurality of second index sequences. Non-limiting examples of suitable containers include bottles, tubes, vials, and microplates. The container may be formed of various materials such as glass or plastic materials. For example, but not by way of limitation, the kit of the present disclosure may include a microtiter plate comprising a plurality of compartments comprising a second index sequence.

[0182] In certain embodiments, the kit of the present disclosure may further include one or more protein binding reagents, such as antibodies. In certain embodiments, the kit of the present disclosure may include a container containing multiple protein binding reagents (e.g., antibodies). In certain embodiments, each protein binding reagent (e.g., antibody) is bound to an oligonucleotide comprising a barcode.

[0183] In certain embodiments, the kit further comprises a package insert providing instructions for using the components provided in the kit. For example, a kit of the present disclosure may include a package insert providing instructions for using a plurality of particles comprising a first index sequence in the disclosed methods. In certain embodiments, a kit of the present disclosure may include a package insert providing instructions for using a microtiter plate comprising a plurality of compartments comprising a second index sequence in the disclosed methods.

[0184] In certain embodiments, the kit of the present disclosure may further include reagents for generating emulsion droplets. For example, but not by way of limitation, the kit may include one or more aqueous and / or non-aqueous fluids. In certain embodiments, the kit may include materials for performing emulsion droplet generation techniques (e.g., a pipette and / or a vortexer). Alternatively or additionally, the kit of the present disclosure may include an emulsion generation device, e.g., a microfluidic device.

[0185] In certain embodiments, the kit of the present disclosure may further include reagents for performing an amplification reaction. In certain embodiments, the reagents may include one or more of the following: a polymerase, a reverse transcriptase, a nucleoside triphosphate or NTP analog, a primer, a cofactor, a ligation reaction reagent, an endonuclease, a dissolution reagent, a dye, a marker, or a label.

[0186] In certain embodiments, the kits of the present disclosure may further include reagents for performing genomic screening, lineage tracing methods, and / or cell hashing techniques. In certain embodiments, the kits of the present disclosure may further include reagents for performing genomic screening. In certain embodiments, the kits of the present disclosure may further include reagents for performing lineage tracing methods.

[0187] In certain embodiments, the kit may include other materials desired from a commercial and user perspective, including other buffers and diluents. In certain embodiments, the kit may include materials or reagents for dissociating a sample into a single cell or for separating a nucleus from a single cell. In certain embodiments, the kit may include reagents for permeabilizing, fixing and / or cross-linking cells or nuclei. In certain embodiments, the kit of the present disclosure may include a translocase, for example, a Tn5 translocase.

[0188] In certain embodiments, the components of the kit are provided in predetermined ratios, wherein the relative amounts of the various reagents are varied appropriately to obtain the desired sensitivity and throughput of the disclosed methods.

[0189] V. Exemplary Embodiments

[0190] A. The present disclosure provides a method for combinatorial indexing of nucleic acids of single cells, the method comprising: (a) providing mixed cells or cell nuclei associated with or containing nucleic acids containing a first index sequence, wherein the first index sequence is incorporated into the nucleic acids of the mixed cells or cell nuclei in emulsion droplets; (b) distributing subsets of the mixed cells or cell nuclei into multiple compartments including a second index sequence; and (c) incorporating the second index sequence into the nucleic acid in each compartment to produce a dual-indexed nucleic acid.

[0191] A1. A method according to A, wherein the first index sequence is incorporated into the nucleic acid of the cells or cell nuclei in the emulsion droplets by a method comprising: (i) contacting a plurality of cells or cell nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning individual particles and one or more of the cells or cell nuclei into emulsion droplets; (iii) merging the first index sequence into the nucleic acid of the cells or cell nuclei to produce an index nucleic acid; and (iv) combining the cells or cell nuclei associated with or containing the index nucleic acid from a plurality of the emulsion droplets to produce the mixed cells or cell nuclei associated with or containing the nucleic acid containing the first index sequence.

[0192] A2. The method according to A1, wherein before or during step (i), the cell or cell nucleus is treated with a translocase.

[0193] A3. The method according to A2, wherein the translocase is Tn5 translocase.

[0194] A4. The method according to any one of A1 to A3, wherein prior to step (i), the plurality of cells or cell nuclei are treated with a fixative.

[0195] A5. The method according to any one of A1 to A4, wherein prior to step (i), the plurality of cells are permeabilized or lysed.

[0196] A6. The method according to any one of A1 to A5, wherein before or during step (i), the multiple cells or cell nuclei are treated with multiple reagents.

[0197] A7. A method according to any one of A1 to A6, wherein the first index sequence of each particle or subset of particles is unique relative to other particles.

[0198] A8. A method according to any one of A to A7, wherein the second index sequence in a compartment or a subset of compartments is unique relative to other second index sequences.

[0199] A9. The method according to any one of claims A to A8, wherein the plurality of compartments is a multi-well plate.

[0200] A10. The method according to any one of A to A9, wherein the emulsified droplets are generated in an emulsified droplet generating device or using an emulsified droplet generating technology.

[0201] A11. The method of any one of claims A to A10, further comprising incorporating a third index sequence into the dual-indexed nucleic acid to produce a triply-indexed nucleic acid.

[0202] A12. The method according to any one of A to A11, wherein incorporating the first index sequence into the nucleic acid in the emulsion droplet is performed by an amplification method, a reverse transcription method or a ligation method.

[0203] A12.1. A method according to A12, wherein the first index sequence is incorporated into the nucleic acid in the emulsion droplets by a reverse transcription method.

[0204] A13. A method according to any one of A to A12.1, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0205] A13.1. The method according to any one of A to A13, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0206] A14. The method according to any one of A12 to A13.1, wherein the amplification method is polymerase chain reaction (PCR) or an isothermal amplification method.

[0207] A15. A method according to any one of A to A14, wherein the nucleic acid comprises RNA.

[0208] A16. A method according to any one of A to A14, wherein the nucleic acid comprises DNA.

[0209] A17. The method of any one of A to A16, wherein the plurality of cells comprises at least about 100,000 cells.

[0210] A18. A 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.

[0211] A19. A method according to any one of A to A18, wherein the multiple cells include any one of the following: (a) cells modified with a gene editing system; (b) cells that produce antibodies; (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 (e.g., an antibody) coupled to an oligonucleotide containing a barcode.

[0212] A20. A method according to A19, wherein the plurality of cells comprises cells modified with a gene editing system.

[0213] A21. A method according to A19, wherein the plurality of cells comprises antibody-producing cells.

[0214] A22. A method according to A19, wherein the plurality of cells comprises cells in different developmental states.

[0215] A23. A method according to A19, wherein the plurality of cells comprises cells in different disease states.

[0216] A24. A method according to A19, wherein the plurality of cells comprises cells treated with an agent.

[0217] A25. The method of A24, wherein the agent is a therapeutic agent.

[0218] A26. A 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.

[0219] A27. A method according to A19, wherein the plurality of cells comprises cells bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode.

[0220] A28. A method according to any one of A to A27, wherein the emulsion droplets contain at least two cells or cell nuclei.

[0221] A29. The method of any one of A to A28, wherein the particles are gel beads.

[0222] A30. A method according to any one of A to A29, further comprising performing a lineage tracing method.

[0223] A31. A method according to any one of A to A30, further comprising performing genomic screening.

[0224] A32. A method according to A31, wherein the genomic screening is a CRISPR-based screening.

[0225] A33. A 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.

[0226] A34. A method according to any one of A to A33, wherein the plurality of cells or cell nuclei isolated from the plurality of cells are bound by coupling to a protein binding reagent coupled to an oligonucleotide comprising a barcode.

[0227] A35. The method according to any one of A to A33, further comprising, before (a), contacting the plurality of cells or cell nuclei isolated from the plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides comprising a barcode.

[0228] A36. A method according to A34 or A35, wherein the protein binding reagent binds to a protein located on the surface of at least one cell among the plurality of single cells.

[0229] A37. A kit according to A34 to A36, wherein the protein binding reagent is an antibody or a fragment thereof.

[0230] A38. The method according to A36 or A37, further comprising determining the expression level of the protein.

[0231] B. A method for sequencing a library comprising nucleic acids from multiple single cells, the method comprising: (a) providing mixed cells or cell 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 mixed cells or cell nuclei in emulsion droplets; (b) distributing subsets of the mixed cells or cell nuclei into multiple compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acids in each compartment to produce dual-index nucleic acids; and (d) combining all or a subset of the dual-index nucleic acids to produce a sequencing library from multiple cells.

[0232] B1. A method according to B, wherein the first index sequence is incorporated into the nucleic acid of the cells or cell nuclei in the emulsion droplets by a method comprising: (i) contacting a plurality of cells or cell nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning separate particles and one or more of the cells or cell nuclei into emulsion droplets; (iii) merging the first index sequence into the nucleic acid of the cells or cell nuclei to produce an index nucleic acid; and (iv) combining the cells or cell nuclei associated with or containing the index nucleic acid from a plurality of the emulsion droplets to produce the mixed cells or cell nuclei associated with or containing the nucleic acid containing the first index sequence.

[0233] B2. The method according to B1, wherein the cell or cell nucleus is treated with a translocase before or during step (i).

[0234] B3. The method according to B2, wherein the translocase is Tn5 translocase.

[0235] B4. The method according to any one of B1 to B3, wherein prior to step (i), the plurality of cells or cell nuclei are treated with a fixative.

[0236] B5. The method according to any one of B1 to B4, wherein prior to step (i), the plurality of cells are permeabilized or lysed.

[0237] B6. A method according to any one of B1 to B5, wherein before or during step (i), the multiple cells or cell nuclei are treated with multiple reagents.

[0238] B7. A method according to any one of B1 to B6, wherein the first index sequence of each particle or subset of particles is unique relative to other particles.

[0239] B8. The method of any one of roots B to B7, wherein the second index sequence in a compartment or a subset of compartments is unique relative to other second index sequences.

[0240] B9. A method according to any one of B to B8, wherein the plurality of compartments is a multi-well plate.

[0241] B10. The method according to any one of B to B9, wherein the emulsified droplets are generated in an emulsified droplet generating device or using an emulsified droplet generating technology.

[0242] B11. The method of any one of B to B10, further comprising incorporating a third index sequence into the dual-indexed nucleic acid to produce a triply-indexed nucleic acid.

[0243] 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 method, a reverse transcription method or a ligation method.

[0244] B12.1. A method according to B12, wherein the first index sequence is incorporated into the nucleic acid in the emulsion droplets by a reverse transcription method.

[0245] B13. A method according to any one of B to B12.1, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0246] B13.1. A method according to any one of B to B13, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0247] B14. The method according to B12 to B13.1, wherein the amplification method is polymerase chain reaction (PCR) or an isothermal amplification method.

[0248] B15. A method according to any one of B to B14, wherein the nucleic acid comprises RNA.

[0249] B16. A method according to any one of B to B14, wherein the nucleic acid comprises DNA.

[0250] B17. A method according to any one of B to B16, wherein the plurality of cells comprises at least about 100,000 cells.

[0251] B18. A 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 a combination thereof.

[0252] B19. A 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) cells producing antibodies; (c) cells in different developmental states; (d) cells in different disease states; (e) cells treated with an agent; and (f) cells bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode.

[0253] B20. A method according to B19, wherein the plurality of cells comprises cells modified with a gene editing system.

[0254] B21. A method according to B19, wherein the plurality of cells comprises antibody-producing cells.

[0255] B22. A method according to B19, wherein the plurality of cells comprises cells in different developmental states.

[0256] B23. A method according to B19, wherein the plurality of cells comprise cells in different disease states.

[0257] B24. A method according to B19, wherein the plurality of cells comprises cells treated with an agent.

[0258] B25. A method according to B24, wherein the agent is a therapeutic agent.

[0259] B26. A 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.

[0260] B27. A method according to B19, wherein the plurality of cells comprises cells bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode.

[0261] B28. A method according to any one of B to B27, wherein the emulsion droplets contain at least two cells or cell nuclei.

[0262] B29. A method according to any one of B to B28, wherein the particles are gel beads.

[0263] B30. A method according to any one of B to B29, further comprising performing a lineage tracing method.

[0264] B31. A method according to any one of B to B30, further comprising performing genomic screening.

[0265] B32. A method according to B31, wherein the genomic screening is a CRISPR-based screening.

[0266] B33. A 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.

[0267] B34. A method according to any one of B to B33, wherein the plurality of cells or cell nuclei isolated from the plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide comprising a barcode.

[0268] B35. The method according to any one of B to B33, further comprising, before (a), contacting the plurality of cells or cell nuclei isolated from the plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides comprising a barcode.

[0269] B36. A method according to B34 or B35, wherein the protein binding reagent binds to a protein located on the surface of at least one cell among the plurality of single cells.

[0270] B37. A kit according to B34 to B36, wherein the protein binding reagent is an antibody or a fragment thereof.

[0271] B38. The method according to B36 or B37, further comprising determining the expression level of the protein.

[0272] C. The present disclosure provides a method for sequencing a library comprising nucleic acids from multiple single cells, the method comprising: (a) providing mixed cells or cell 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 mixed cells or cell nuclei in emulsion droplets; (b) distributing subsets of the mixed cells or cell nuclei into multiple compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acids in each compartment to produce dual-index nucleic acids; (d) combining all or a subset of the dual-index nucleic acids to produce a sequencing library from multiple cells; and (e) sequencing all or a subset of the dual-index nucleic acids.

[0273] C1. A method according to C, wherein the first index sequence is incorporated into the nucleic acid of the cells or cell nuclei in the emulsion droplets by a method comprising: (i) contacting a plurality of cells or cell nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning separate particles and one or more of the cells or cell nuclei into emulsion droplets; (iii) merging the first index sequence into the nucleic acid of the cells or cell nuclei to produce an index nucleic acid; and (iv) combining the cells or cell nuclei associated with or containing the index nucleic acid from a plurality of the emulsion droplets to produce the mixed cells or cell nuclei associated with or containing the nucleic acid containing the first index sequence.

[0274] C2. The method according to C1, wherein the cell or cell nucleus is treated with a translocase before or during step (i).

[0275] C3. The method according to C2, wherein the translocase is Tn5 translocase.

[0276] C4. The method according to any one of C1 to C3, wherein prior to step (i), the plurality of cells or cell nuclei are treated with a fixative.

[0277] C5. The method according to any one of C1 to C4, wherein prior to step (i), the plurality of cells are permeabilized or lysed.

[0278] C6. A method according to any one of C1 to C5, wherein before or during step (i), the multiple cells or cell nuclei are treated with multiple reagents.

[0279] C7. A method according to any one of C1 to C6, wherein the first index sequence of each particle or subset of particles is unique relative to other particles.

[0280] C8. A method according to any one of C to C7, wherein the second index sequence in a compartment or a subset of compartments is unique relative to other second index sequences.

[0281] C9. A method according to any one of claims C to C8, wherein the multiple compartments are porous plates.

[0282] C10. A method according to any one of C to C9, wherein the emulsified droplets are generated in an emulsified droplet generating device or using an emulsified droplet generating technology.

[0283] C11. The method of any one of claims C to C10, further comprising incorporating a third index sequence into the dual-indexed nucleic acid to produce a triply-indexed nucleic acid.

[0284] C12. The method according to any one of C to C11, wherein incorporating the first index sequence into the nucleic acid in the emulsion droplet is performed by an amplification method, a reverse transcription method, or a ligation method.

[0285] C12.1. A method according to C12, wherein the first index sequence is incorporated into the nucleic acid in the emulsion droplets by a reverse transcription method.

[0286] C13. A method according to any one of C to C12.1, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0287] C13.1. A method according to any one of C to C13, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0288] C14. A method according to C12 to C13.1, wherein the amplification method is a polymerase chain reaction (PCR) or an isothermal amplification method.

[0289] C15. A method according to any one of C to C14, wherein the nucleic acid comprises RNA.

[0290] C16. A method according to any one of C to C14, wherein the nucleic acid comprises DNA.

[0291] C17. A method according to any one of C to C16, wherein the plurality of cells comprises at least about 100,000 cells.

[0292] C18. A 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.

[0293] C19. A 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) cells producing antibodies; (c) cells in different developmental states; (d) cells in different disease states; (e) cells treated with an agent; and (f) cells bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode.

[0294] C20. A method according to C19, wherein the plurality of cells comprises cells modified with a gene editing system.

[0295] C21. A method according to C19, wherein the plurality of cells comprises antibody-producing cells.

[0296] C22. A method according to C19, wherein the plurality of cells comprises cells in different developmental states.

[0297] C23. A method according to C19, wherein the plurality of cells comprises cells in different disease states.

[0298] C24. A method according to C19, wherein the plurality of cells comprises cells treated with an agent.

[0299] C25. A method according to C24, wherein the agent is a therapeutic agent.

[0300] C26. A 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.

[0301] C27. A method according to C19, wherein the plurality of cells comprises cells bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode.

[0302] C28. A method according to any one of C to C27, wherein the emulsion droplets contain at least two cells or cell nuclei.

[0303] C29. A method according to any one of C to C28, wherein the particles are gel beads.

[0304] C30. A method according to any one of C to C29, further comprising performing a lineage tracing method.

[0305] C31. A method according to any one of C to C30, further comprising performing genomic screening.

[0306] C32. A method according to C31, wherein the genomic screening is a CRISPR-based screening.

[0307] C33. A 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.

[0308] C34. A method according to any one of C to C33, wherein the plurality of cells or cell nuclei isolated from the plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide comprising a barcode.

[0309] C35. A method according to any one of C to C33, further comprising, before (a), contacting the plurality of cells or cell nuclei isolated from the plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides comprising a barcode.

[0310] C36. A method according to C34 or C35, wherein the protein binding reagent binds to a protein located on the surface of at least one cell among the plurality of single cells.

[0311] C37. A kit according to C34 to C36, wherein the protein binding reagent is an antibody or a fragment thereof.

[0312] C38. The method according to C36 or C37, further comprising determining the expression level of the protein.

[0313] D. The present disclosure provides a method for combinatorial indexing of nucleic acids of a single cell, the method comprising: (a) contacting a plurality of cells or cell nuclei separated from a plurality of cells with a plurality of particles comprising a first index sequence; (b) partitioning the individual particles and one or more of the cells or cell nuclei into emulsion droplets; (c) merging the first index sequence into the nucleic acids of the cells or cell nuclei to produce indexed nucleic acids; (d) combining the cells or cell nuclei containing the indexed nucleic acids or associated with the indexed nucleic acids from a plurality of the emulsion droplets to produce mixed cells or cell nuclei; (e) distributing a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; and (f) merging the second index sequence into the indexed nucleic acids in each compartment to produce dual-indexed nucleic acids.

[0314] D1. The method of D, further comprising (g) incorporating a third index sequence into the dual-indexed nucleic acid to generate a triply-indexed nucleic acid.

[0315] D2. A method according to D or D1, wherein the first index sequence of each particle or subset of particles is unique relative to other particles.

[0316] D3. A method according to any one of D to D2, wherein the second index sequence in a compartment or a subset of compartments is unique relative to other second index sequences.

[0317] D4. The method according to any one of D to D3, wherein the emulsified droplets are generated in an emulsified droplet generating device or using an emulsified droplet generating technology.

[0318] D5. The method according to any one of claims D to D4, wherein the plurality of compartments are multi-well plates.

[0319] D6. The method according to any one of D to D5, wherein incorporating the first index sequence into the nucleic acid is performed by an amplification method, a reverse transcription method or a ligation method.

[0320] D6.1. A method according to D6, wherein the first index sequence is incorporated into the nucleic acid in the emulsion droplets by a reverse transcription method.

[0321] D7. A method according to any one of D to D6, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0322] D7.1. The method according to any one of D to D7, wherein the incorporation of the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0323] D8. The method according to D6 to D7.1, wherein the amplification method is polymerase chain reaction (PCR) or an isothermal amplification method.

[0324] D9. The method of any one of D to D8, wherein the cell or cell nucleus is treated with a translocase before or during step (a).

[0325] D10. The method according to D9, wherein the translocase is Tn5 translocase.

[0326] D11. The method according to any one of D to D10, wherein prior to step (a), the plurality of cells or cell nuclei are treated with a fixative.

[0327] D12. The method according to any one of D to D11, wherein prior to step (a), the plurality of cells are permeabilized or lysed.

[0328] D13. The method of any one of D to D12, wherein the plurality of cells or cell nuclei are treated with a plurality of reagents before or during step (a).

[0329] D14. A method according to any one of D to D13, wherein the nucleic acid comprises RNA.

[0330] D15. A method according to any one of D to D13, wherein the nucleic acid comprises DNA.

[0331] D16. The method of any one of D to D15, wherein the plurality of cells comprises at least about 100,000 cells.

[0332] D17. A method according to any one of D to D16, wherein the plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof.

[0333] D18. A method according to any one of D to D17, wherein the plurality of cells comprises somatic cells.

[0334] D19. A method according to any one of D to D18, wherein the plurality of cells comprises any one of the following: (a) cells modified with a gene editing system; (b) cells producing antibodies; (c) cells in different developmental states; (d) cells in different disease states; (e) cells treated with an agent; and (f) cells bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode.

[0335] D20. A method according to D19, wherein the plurality of cells comprises cells modified with a gene editing system.

[0336] D21. A method according to D19, wherein the plurality of cells comprises antibody-producing cells.

[0337] D22. A method according to D19, wherein the multiple cells comprise cells in different developmental states.

[0338] D23. A method according to D19, wherein the plurality of cells comprise cells in different disease states.

[0339] D24. A method according to D19, wherein the plurality of cells comprises cells treated with an agent.

[0340] D25. The method of D24, wherein the agent is a therapeutic agent.

[0341] D26. A 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.

[0342] D27. A method according to D19, wherein the plurality of cells comprises cells bound by a protein binding agent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode.

[0343] D28. A method according to any one of D to D27, wherein the emulsion droplets contain at least two cells or cell nuclei.

[0344] D29. A method according to any one of D to D28, wherein the particles are gel beads.

[0345] D30. A method according to any one of D to D29, further comprising performing a lineage tracing method.

[0346] D31. A method according to any one of D to D30, further comprising performing genomic screening.

[0347] D32. A method according to D31, wherein the genomic screening is a CRISPR-based screening.

[0348] D33. The method of D32, wherein the CRISPR-based screening is selected from the group consisting of: gene editing CRISPR screening, CRISPRi screening, and CRISPRa screening.

[0349] D34. A method according to any one of D to D33, wherein the plurality of cells or cell nuclei isolated from the plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide comprising a barcode.

[0350] D35. The method according to any one of D to D33, further comprising, before (a), contacting the plurality of cells or cell nuclei isolated from the plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides comprising a barcode.

[0351] D36. A method according to D34 or D35, wherein the protein binding reagent binds to a protein located on the surface of at least one cell among the plurality of single cells.

[0352] D37. A kit according to D34 to D36, wherein the protein binding reagent is an antibody or a fragment thereof.

[0353] D38. The method according to D36 or D37, further comprising determining the expression level of the protein.

[0354] E. The present disclosure provides a method for sequencing a library comprising nucleic acids from multiple single cells, the method comprising: (a) contacting multiple cells or cell nuclei separated from multiple cells with multiple particles comprising a first index sequence; (b) partitioning the individual particles and one or more of the cells or cell nuclei into emulsion droplets; (c) merging the first index sequence into the nucleic acid of the cells or cell nuclei to produce index nucleic acids; (d) combining the cells or cell nuclei containing or associated with the index nucleic acids from multiple emulsion droplets to produce mixed cells or cell nuclei; (e) assigning subsets of the mixed cells or cell nuclei to multiple compartments comprising a second index sequence; (f) merging the second index sequence into the index nucleic acid in each compartment to produce dual-index nucleic acids; and (g) combining all or a subset of the dual-index nucleic acids to produce a sequencing library from multiple cells.

[0355] E1. The method according to E, wherein the first index sequence of each particle or subset of particles is unique relative to other particles.

[0356] E2. A method according to E or E1, wherein the second index sequence in a compartment or a subset of compartments is unique relative to other second index sequences.

[0357] E3. A method according to any one of E to E2, wherein the emulsified droplets are generated in an emulsified droplet generating device or using an emulsified droplet generating technology.

[0358] E4. The method according to any one of claims E to E3, wherein the plurality of compartments is a multi-well plate.

[0359] E5. The method according to any one of E to E4, wherein incorporating the first index sequence into the nucleic acid is performed by an amplification method, a reverse transcription method or a ligation method.

[0360] E5.1. A method according to E5, wherein the first index sequence is incorporated into the nucleic acid in the emulsion droplets by a reverse transcription method.

[0361] E6. The method according to any one of E to E5.1., wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0362] E6.1. The method according to any one of E to E6, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0363] E7. The method according to E5 to E6.1, wherein the amplification method is polymerase chain reaction (PCR) or an isothermal amplification method.

[0364] E8. The method according to any one of E to E7, wherein the cell or cell nucleus is treated with a translocase before or during step (a).

[0365] E9. The method according to E8, wherein the translocase is Tn5 translocase.

[0366] E10. The method according to any one of E to E9, wherein prior to step (a), the plurality of cells or cell nuclei are treated with a fixative.

[0367] E11. The method according to any one of E to E10, wherein prior to step (a), the plurality of cells are permeabilized or lysed.

[0368] E12. The method according to any one of E to E11, wherein before or during step (a), the plurality of cells or cell nuclei are treated with a plurality of reagents.

[0369] E13. A method according to any one of E to E12, wherein the nucleic acid comprises RNA.

[0370] E14. A method according to any one of E to E12, wherein the nucleic acid comprises DNA.

[0371] E15. The method according to any one of E to E14, wherein the plurality of cells comprises at least about 100,000 cells.

[0372] E16. 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.

[0373] E18. A method according to any one of E to E17, wherein the plurality of cells comprises somatic cells.

[0374] E19. A 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) cells producing antibodies; (c) cells in different developmental states; (d) cells in different disease states; (e) cells treated with an agent; and (f) cells bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode.

[0375] E20. A method according to E19, wherein the plurality of cells comprises cells modified with a gene editing system.

[0376] E21. The method according to E19, wherein the plurality of cells comprises antibody-producing cells.

[0377] E22. The method of E19, wherein the plurality of cells comprises cells in different developmental states.

[0378] E23. The method according to E19, wherein the plurality of cells comprises cells in different disease states.

[0379] E24. The method of E19, wherein the plurality of cells comprises cells treated with an agent.

[0380] E25. The method according to E24, wherein the agent is a therapeutic agent.

[0381] E26. A 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.

[0382] E27. A method according to E19, wherein the plurality of cells comprises cells bound by a protein binding reagent coupled to an oligonucleotide comprising a barcode.

[0383] E28. A method according to any one of E to E27, wherein the emulsion droplets contain at least two cells or cell nuclei.

[0384] E29. A method according to any one of E to E28, wherein the particles are gel beads.

[0385] E30. A method according to any one of E to E29, further comprising performing a lineage tracing method.

[0386] E31. A method according to any one of E to E30, further comprising performing genomic screening.

[0387] E32. A method according to E31, wherein the genomic screening is a CRISPR-based screening.

[0388] E33. The method of E32, wherein the CRISPR-based screening is selected from the group consisting of: gene editing CRISPR screening, CRISPRi screening, and CRISPRa screening.

[0389] E35. The method according to any one of E to E33, further comprising, before (a), contacting the plurality of cells or cell nuclei isolated from the plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides comprising a barcode.

[0390] E36. A method according to E27 or E35, wherein the protein binding reagent binds to a protein located on the surface of at least one cell among the plurality of single cells.

[0391] E37. The method according to any one of E27 and E35 to E36, wherein the protein binding agent is an antibody or a fragment thereof.

[0392] E38. The method according to E36 or E37, further comprising determining the expression level of the protein.

[0393] F. The present disclosure provides a method for sequencing a library comprising nucleic acids from multiple single cells, the method comprising: (a) contacting multiple cells or cell nuclei separated from multiple cells with multiple particles comprising a first index sequence; (b) partitioning the individual particles and one or more of the cells or cell nuclei into emulsion droplets; (c) merging the first index sequence into the nucleic acid of the cells or cell nuclei to produce index nucleic acids; (d) combining the cells or cell nuclei containing or associated with the index nucleic acids from multiple emulsion droplets to produce mixed cells or cell nuclei; (e) distributing subsets of the mixed cells or cell nuclei into multiple compartments comprising a second index sequence; (f) merging the second index sequence into the index nucleic acid in each compartment to produce dual-index nucleic acids; (g) combining all or a subset of the dual-index nucleic acids to produce a sequencing library from multiple cells; and (h) sequencing all or a subset of the dual-index nucleic acids.

[0394] F1. A method according to F, wherein the first index sequence of each particle or subset of particles is unique relative to other microbeads.

[0395] F2. A method according to F or F1, wherein the second index sequence in a compartment or a subset of compartments is unique relative to other second index sequences.

[0396] F3. A method according to F to F2, wherein the microfluidic droplets are generated in an emulsion droplet generation device or using an emulsion droplet generation technology.

[0397] F4. A method according to claims F to F3, wherein the multiple compartments are multi-well plates.

[0398] F5. The method according to F to F4, wherein incorporating the first index sequence into the nucleic acid is performed by an amplification method, a reverse transcription method or a ligation method.

[0399] F5.1. A method according to F5, wherein the first index sequence is incorporated into the nucleic acid in the emulsion droplets by a reverse transcription method.

[0400] F6. The method according to F to F5.1., wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0401] F6.1. A method according to any one of F to F6, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

[0402] F7. A method according to F5 to F6.1, wherein the amplification method is polymerase chain reaction (PCR) or an isothermal amplification method.

[0403] F8. A method according to F to F2, wherein before or during step (a), the cell or cell nucleus is treated with a translocase.

[0404] F9. The method according to F8, wherein the translocase is Tn5 translocase.

[0405] F10. A method according to F to F9, wherein prior to step (a), the plurality of cells or cell nuclei are treated with a fixative.

[0406] F11. A method according to F to F10, wherein before step (a), the plurality of cells are permeabilized or lysed.

[0407] F12. A method according to F to F11, wherein before or during step (a), the multiple cells or cell nuclei are treated with multiple reagents.

[0408] F13. A method according to F to F12, wherein the nucleic acid comprises RNA.

[0409] F14. A method according to F to F12, wherein the nucleic acid comprises DNA.

[0410] F15. A method according to F to F14, wherein the plurality of cells comprises at least about 100,000 cells.

[0411] F16. A method according to F to F15, wherein the plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells or a combination thereof.

[0412] F17. A method according to F to F16, wherein the plurality of cells comprises immune cells.

[0413] F18. A method according to any one of F to F17, wherein the plurality of cells comprises somatic cells.

[0414] F19. A method according to F to F18, wherein the multiple cells include any one of the following: (a) cells modified with a gene editing system; (b) cells producing antibodies; (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 (e.g., an antibody) coupled to an oligonucleotide containing a barcode.

[0415] F20. A method according to F19, wherein the plurality of cells comprises cells modified with a gene editing system.

[0416] F21. A method according to F19, wherein the plurality of cells comprises antibody-producing cells.

[0417] F22. A method according to F19, wherein the plurality of cells comprises cells in different developmental states.

[0418] F23. A method according to F19, wherein the plurality of cells comprises cells in different disease states.

[0419] F24. A method according to F19, wherein the plurality of cells comprises cells treated with an agent.

[0420] F25. The method of F24, wherein the agent is a therapeutic agent.

[0421] F26. A 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.

[0422] F27. A method according to F19, wherein the plurality of cells comprises cells bound by a protein binding reagent (e.g., an antibody) coupled to an oligonucleotide comprising a barcode.

[0423] F28. A method according to any one of F to F27, wherein the emulsion droplets contain at least two cells or cell nuclei.

[0424] F29. A method according to any one of F to F28, wherein the particles are gel beads.

[0425] F30. A method according to any one of F to F29, further comprising performing a lineage tracing method.

[0426] F31. A method according to any one of F to F30, further comprising performing genomic screening.

[0427] F32. A method according to F31, wherein the genomic screening is a CRISPR-based screening.

[0428] 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.

[0429] F34. A method according to any one of F to F33, wherein the plurality of cells or cell nuclei isolated from the plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide comprising a barcode.

[0430] F35. The method according to any one of F to F33, further comprising, before (a), contacting the plurality of cells or cell nuclei isolated from the plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides comprising a barcode.

[0431] 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 among the plurality of single cells.

[0432] F37. A method according to F34 to F36, wherein the protein binding agent is an antibody or a fragment thereof.

[0433] F38. The method according to F36 or F37, further comprising determining the expression level of the protein.

[0434] G. A kit for performing the method according to any one of A to F28.

[0435] G1. A kit according to G, wherein the kit comprises one or more of the following: (a) at least one container comprising a plurality of particles comprising the first index sequence; and (b) at least one container comprising a plurality of second index sequences.

[0436] G2. The kit according to G1, further comprising a container comprising one or more protein binding reagents coupled to the oligonucleotide comprising the barcode.

[0437] G3. A kit according to G2, wherein the protein binding reagent is an antibody or a fragment thereof.

[0438] Examples

[0439] The objects of the present disclosure will be better understood by reference to the following examples, which are provided by way of illustration and not by way of limitation of the objects of the present disclosure.

[0440] Example 1: Combined index for OAK-scRNAseq

[0441] This example describes a combined indexing method for Figure 1 The RNA of the single cell shown is sequenced. In particular, this example describes adding two index sequences to the RNA of the single cell.

[0442] Cell preparation:

[0443] The combined indexing method begins by preparing a single cell suspension for fixation. If using adherent cell lines or solid tissues, dissociate the cells before fixation. Collect the cells in culture medium and then wash with phosphate-buffered saline (PBS). 6 Gently resuspend the washed cells with 200 μl of chilled PBS to obtain a single-cell suspension.

[0444] Then, by adding 1x10 6 800 μl of frozen pure methanol was added dropwise to each cell for fixation while gently stirring with a pipette tip to prevent cell condensation. The samples were then stored at -20°C for 40 min. The fixed samples were placed on ice for 5 minutes and then centrifuged at 1000 g for 5 min at 4°C. The supernatant was removed and the cells were resuspended in a buffer including 3X SSC, 1% BSA, 1 mM DTT and 0.2 U / ul RNase inhibitor. The volume was adjusted with buffer to achieve a concentration of 50,000 cells / μl.

[0445] Primary barcode integration:

[0446] Prepare a master mix on ice based on the Chromium Next GEM Single Cell 3'Reagent Kits v3.1 User Guide (referred to herein as the "Chromium Next GEM Single Cell User Guide"). Divide 31.8 μl of the master mix into equal portions for each sample to be loaded. Use the following formula for the volume of fixed cells loaded for each sample: Volume_CellStock(μl)=TargetedCellNumber*2.3 / Concentration_CellStock. For example, if the cell stock concentration is 50,000 cells / μl to target 100,000 cells, 230,000 cells are loaded by using 100k*2.3 / 50k=4.6μl cell stock as input. For each channel, 43.3μl of nuclease-free water was added to the tube containing 31.8μl of the master mix based on the calculation in the previous step. Secondly, add the corresponding volume of fixed cells. Gently mix the cell-reagent mixture with a pipette and use the same pipette tip to dispense 70 μl of the mixture into the well in the row labeled 1 on the chip. Repeat the previous step for each of the samples to be loaded. Each sample is placed in a well. Load the remaining wells according to the ChromiumNext GEM Single Cell User Guide. Connect the GEM spacer and run the Chromium controller according to the Chromium Next GEM Single Cell User Guide. After the run is complete, transfer the GEMs according to the Chromium Next GEM SingleCell User Guide. Figure 2 As shown, this method resulted in the successful generation of multicellular GEMs. FIG7A shows the generation of multicellular GEMs by loading 150,000 cells per channel (resulting in the recovery of 85,000 cells and the sequencing of approximately 2,000 to 4,000 genes per cell), and Figure 7B The generation of multicellular GEMs by loading 450,000 cells per channel (resulting in the recovery of 273,000 cells and sequencing of approximately 600 genes per cell) is shown. Representative droplets containing 0 to 8 cells are shown. The transferred GEMs were then incubated in a thermal cycler at 53°C for 45 minutes to enable primary barcoding during the reverse transcription process, and then kept at 4°C for a short period of time if necessary. At the end of the incubation, the GEMs were unpacked as follows.

[0447] GEM unpacking:

[0448] 125 μl of recovery agent was added to each sample at room temperature, and after incubation for 5 to 10 minutes, a 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 centrifuge tube. 500 μl of 3X SSC was slowly added to each sample. The tubes were centrifuged at 650 g for 5 min at 4°C in a swinging bucket centrifuge. The supernatant was removed without disrupting the cell pellet. 500 μl of 3X SSC was slowly added to each sample without disrupting the cell pellet. The tubes were centrifuged at 650 g for 5 min at 4°C in a swinging bucket centrifuge, and the supernatant was removed without disrupting the cell pellet. The cells were resuspended in 3X SSC using a regular caliber P200 pipette tip. An appropriate resuspension volume should be used so that each 10 μl will contain 1,000 to 10,000 cells based on the multiple tolerance of each project. As Figure 3 As shown, cells were successfully recovered after GEM unpacking. Each sample was aliquoted into multiple individual PCR tubes so that each PCR tube contained 10 μl of resuspended cells. Each 10 μl contained 1,000 to 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 cleaned up using Dynabeads MyOne Silane. Heating at 95 °C and cleanup using Dynabeads MyOne Silane resulted in cell lysis.

[0449] Integration of secondary barcodes:

[0450] Each aliquot was subjected to cDNA amplification using a unique barcode primer that hybridizes to a common sequence located upstream of the primary cell barcode and the UMI in the first strand cDNA. The other primers in the PCR reaction covered the TSO side of the first strand cDNA and were therefore shared among all aliquots. After the PCR reaction was complete, the products from the different aliquots were mixed. The PCR products were cleaned up with 0.6XSPRIselect beads. 1 μl of the resulting cDNA was run in a D5000 tape to assess cDNA quantity and length distribution.

[0451] The cDNA library was then sequenced by using the Illumina sequencing library construction step followed by sequencing. Table 1 shows the performance metrics of the OAK-scRNAseq method. As shown in Table 1, this method allows the analysis of many cells with low multiplet rates.

[0452] Table 1

[0453] Median number of genes per cell (K562) 1608 Median number of genes per cell (NIH / 3T3) 628 Multiplet rate 4.4％ Fractional readout in cells 80.7％

[0454] Example 2: OAK multi-omics combined index (ATAC and RNA co-assay)

[0455] This example describes a combined indexing approach for sequencing RNA and genomic DNA from single cells. Figure 1 Specifically, this example describes the addition of two index sequences to the genomic DNA of a single cell.

[0456] Nuclei preparation:

[0457] The method begins by preparing a mononuclear suspension for fixation. The cells are collected in culture medium. If an adherent cell line or solid tissue is used, the cells can be dissociated before fixation. The cells are then washed with phosphate-buffered saline (PBS) and 1x10 6 The cells were gently resuspended in 200 μl of chilled PBS to obtain a single cell suspension. The nuclei were then isolated from the single cell suspension.

[0458] The single nucleus was then fixed by adding an appropriate volume of formaldehyde and PBS to the single nucleus suspension so as to be fixed using 0.3% formaldehyde. The sample was stored on ice for 15 minutes. The fixed nucleus was then washed with a buffer including PBS, 1% BSA and 0.2U / μl RNase inhibitor. The fixed nucleus was gently resuspended with frozen diluted nucleus buffer by 10x Genomics. The volume was adjusted to achieve the desired concentration, such as 4,000 nuclei / μl. Nuclear translocation was performed according to Chromium Next GEM single cell multi-omics ATAC+ gene expression ("Chromium Next GEM single cell multi-omics user guide"). Up to 20,000 nuclei were used in each of the translocation reactions of 15 μl reaction volumes. In order to have enough samples for the overload of the microfluidic device, multiple translocation reactions were established in parallel. Ten such reactions were established in parallel for each sample so that a total of 200,000 nuclei were translocated for each sample. If additional ATAC enzyme is needed, the TDE1 enzyme from the Illumina Tagment DNA TDE1 Enzyme and Buffer Kit can be used. Alternatively, a homemade Tn5 enzyme loaded with the appropriate adapter can be used. After the transposition reaction, for each sample, collect the nuclei from all reaction tubes into a round-bottom 2.0 mL centrifuge tube. Centrifuge the tubes at 500 g for 5 min at 4°C in a swinging bucket centrifuge. Remove most of the supernatant without disrupting the nuclei pellet. The last 15 μl of supernatant from each sample remains in the tube. Gently resuspend the transposed nuclei with the remaining buffer.

[0459] Primary barcode integration:

[0460] The master mix was prepared on ice based on the Chromium Next GEM Single Cell Multi-Omics User Guide. 60 μl of the master mix was added to the sample tube containing 15 μl of translocated and enriched nuclei.

[0461] Gently mix the nuclei-reagent mixture with a pipette and use the same pipette tip to dispense 70 μl of the mixture into the wells in the row marked 1 on the chip. Repeat the previous step for each of the samples to be loaded. Each sample is placed in a well. Load the remaining wells according to the Chromium Next GEM Single Cell Multi-omics User Guide. Connect the GEM gasket and run the Chromium controller based on the user guide. After the run is complete, transfer the GEM according to the Chromium Next GEM Single Cell Multi-omics User Guide. 1 μl GEM can be examined under a microscope. The transferred GEM is then incubated at 37 ° C for 45 min in a thermal cycler, then at 25 ° C for 30 minutes to enable the primary barcode, and then kept at 4 ° C for a short period of time if necessary. At the end of the incubation, 5 μl of quencher is added to each sample according to the user guide and the GEM is unpacked as follows.

[0462] GEM unpacking:

[0463] 125 μl of recovery agent was added to each sample at room temperature, and after incubation for 5 to 10 minutes, a 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 centrifuge tube. 500 μl of 3X SSC was slowly added to each sample. The tubes were centrifuged at 650 g for 5 min at 4°C in a swinging bucket centrifuge. The supernatant was removed without disrupting the cell pellet. 500 μl of 3X SSC was slowly added to each sample without disrupting the nuclear pellet. The tubes were centrifuged at 650 g for 5 min at 4°C in a swinging bucket centrifuge, and the supernatant was removed without disrupting the nuclear pellet. The nuclei were resuspended in 3X SSC using a regular-bore P200 pipette tip. The appropriate resuspension volume should be used so that each 10 μl will contain 1,000 to 10,000 cells based on the multiple tolerance of each project. Aliquot each sample into multiple individual PCR tubes so that each PCR tube contains 10 μl of resuspended nuclei. Each 10 μl contains 1,000 to 10,000 nuclei. Freeze the aliquots at -80 °C until the next step. Heat the aliquots at 80 °C for 10 min in a thermal cycler. Add 90 μl of EB to each aliquot and clean up using Dynabeads MyOne Silane.

[0464] Integration of secondary barcodes:

[0465] For each aliquot, cDNA and ATAC fragments were pre-amplified using a mixture of primers that can amplify both cDNA and ATAC fragments. The PCR products of each aliquot were cleaned up with 1.6X SPRI Select beads. 25% of the products of each aliquot were used in the previous step of cDNA amplification using unique barcode 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 therefore shared among all aliquots. After the PCR reaction is completed, the products from different aliquots can be mixed. The cDNA amplification products were cleaned up with 0.6X SPRIselect beads. 1 μl of the resulting cDNA was run in a D5000 tape to assess the cDNA quantity and length distribution.

[0466] The cDNA library was then sequenced by using the Illumina sequencing library construction steps and then sequenced. Figure 8 Performance metrics of the OAK multi-omics approach are shown. As shown in Table 2, this approach allows analysis of many cells with low multiplet rates.

[0467] Table 2

[0468] Fractional readout in cells 97.1％ Multiplet rate 5.5％ Median number of genes per cell (K562) 3348 Median number of genes per cell (NIH / 3T3) 2607 TSS enrichment score 11.62 Median fragment / cell (K-562) 12821 Median fragments / cell (NIH / 3T3) 10016

[0469] Example 3: OAK Composite Index Using Cell Hashing

[0470] This example discloses a combinatorial indexing method for sequencing single-cell RNA that has been labeled with antibodies coupled to barcode oligonucleotides (referred to herein as "tags").

[0471] like Fig. 9 As shown, differentiated human bronchial epithelial cells were hash-tagged using antibodies coupled to oligonucleotides containing barcodes (i.e., tags) and further processed using a combined indexing method or a control method. Specifically, primary lung cells were grown in a dish and stained with commercially available tag antibodies that were specific to β2-microglobulin B2M and CD298 expressed on all cells and coupled to oligonucleotides containing known barcodes. A total of 9 different barcodes were used. After antibody binding, the cells were washed, then mixed and cell viability was sorted by cell analysis technology. 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 10x Chromium single cell 3' reagent kit and the Chromium platform as a control.

[0472] For the OAK method, a first index sequence is added to the cDNA and antibody-derived oligonucleotides in the droplets. The antibody-derived oligonucleotides and cDNA from RNA are labeled with the same first index sequence in the droplets, allowing the RNA and antibody-derived sequences to be connected in the data. After emulsification and lysis, the cells are placed in different aliquots to integrate the secondary barcodes. The integration of the secondary barcodes is performed as follows: For each aliquot, the cDNA and antibody (protein or tag) derived oligonucleotides are amplified using a mixture of primers that amplify both the cDNA and antibody-derived oligonucleotides. During this reaction, a secondary index is added to the cDNA. The PCR products of each aliquot are separated using SPRI Select beads. 0.6XSPRI Select beads are used to collect cDNA of a gene expression library similar to Example 1. The supernatant is retained from the initial separation of the cDNA for further cleaning using 2.1X SPRI Select beads to obtain the antibody-derived oligonucleotides. Amplification of the antibody-derived oligonucleotides used a unique barcode primer that hybridized to a common sequence located upstream of the primary cell barcode, while the other primers in the PCR reaction covered the common sequence in the antibody-derived oligonucleotides and were therefore shared among all aliquots.

[0473] Sequencing libraries from the transcriptome and tags were then decentralized for analysis.

[0474] The cellular abundance of nine different tags in sequencing data obtained using the OAK method and control methods were analyzed. Fig.10 As shown, a high correlation was observed between the sequencing data obtained from the OAK method and the sequencing data from the control method. These data confirm that the OAK method works well in cell hashing. A ridge plot was then generated to show the tag expression content of the cells decentralized for each tag identity. Fig.11 As shown, the ridge plots demonstrate a high signal / noise ratio for each cell using label assignments. Fig.12 The results show that the quality of transcriptome sequencing data obtained from the OAK method with cell hashing is comparable to that obtained from the control method. UMAP analysis of scRNA-Seq data obtained using the OAK method with tags shows that tags are evenly distributed across different cell types ( FIG. 13A to FIG. 13B ), and the frequencies of cell types were comparable between the OAK method and the control method ( Fig. 13C ).

[0475] Further experiments were performed to confirm whether the OAK method with cell hashing could be used to determine protein content. Fig.14 A schematic diagram of the method used for this experiment is provided. Fig.14As shown, two human cell lines (Jurkat cells and K562 cells) were stained independently with two different tag antibodies and antibodies to proteins known to be differentially expressed between the two cell lines (CD45, CD3 and CD4 binding antibodies) and antibodies to known proteins not expressed by the two cell lines (RatIgG2b isotype control and CD8 binding antibodies). After staining, the cells were combined at a ratio of 1: 1, fixed with methanol, and then processed using the OAK method described in Example 1 using the 10xChromium single cell 3' reagent kit and the Chromium platform. The secondary index was added to both the antibody hash library and the gene expression library together with the primers. The addition of the index to the antibody part was during direct amplification from cDNA, or after the second PCR to amplify the antibody-derived sequence (as described in Example 3). Two OAK aliquots were sequenced for two different temperature conditions (37°C and 53°C). An unfixed control using the 10x genomics protocol was also performed on 12,000 cells for comparison.

[0476] like Fig.15 As shown, the OAK method allows processing of large numbers of cells at one time. Fig.16 The UMAP analysis shown illustrates how clustering separates two cell lines based on scRNA-Seq data, and the clusters match the assigned labels. The representation of proteins bound by antibodies was further analyzed and compared to the signal from the antibody label (which identifies the cell type). Fig.17 As shown, the signal derived from the antibody tag matches the expectation for protein expression in both cell lines tested.

[0477] As shown in this example, cell hashing is compatible with the OAK combined indexing method. In particular, the binding of the antibody to the specific target protein is maintained during the fixation method, during exposure to the solvent and reducing agent in the droplet, and during the reverse transcription method of the OAK combined indexing method. This example further confirms that cell hashing can be used in combination with the OAK combined indexing and sequencing method to simultaneously analyze the protein expression and transcriptome of a single cell. Combining cell hashing with the OAK method can be used to simplify the upstream method of the cell array, to increase the number of cells and to improve the accuracy of calling multiple states in the analysis. Cell hashing can also be used to provide reference abundance for standardized protein expression data.

[0478] Example 4: OAK combined index detection of V(D)J recombination

[0479] This example demonstrates the use of the OAK method to identify V(D)J recombination patterns in single cells using a combinatorial indexing approach.

[0480] Jurkat cells were subjected to the OAK method described in Example 1 using the 10x Chromium Single Cell 5' Kit. The cDNA and TCR amplicons were indexed with a second index sequence by aliquoting and a sequencing library was generated. 683 cells from a homogenous population of Jurkat cells were sequenced, and 422 (61.8%) cells had the same captured CDR3 sequence as expected, as shown in Table 1. Fig.18 This example confirms that the OAK method allows sequencing of V(D)J transcripts obtained from single cells.

[0481] ********

[0482] Although the objects and advantages of the present disclosure have been described in detail, it should be understood that various changes, substitutions and modifications may be made herein without departing from the spirit and scope of the present disclosure. In addition, the scope of the present case is not intended to be limited to the specific embodiments of the processes, machines, manufactures and material compositions, means, methods and steps described in the specification. Therefore, the attached claims are intended to include such processes, machines, manufactures, material compositions, means, methods or steps within the scope of the claims.

[0483] Throughout this application, various patents, patent applications, publications, product descriptions, and protocols are cited, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method for combined indexing of nucleic acids from multiple single cells, the method comprising: (a) providing a mixed cell or cell 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 mixed cell or cell nucleus in an emulsion droplet; (b) assigning a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; as well as (c) incorporating the second index sequence into the nucleic acid in each compartment to produce a dual-indexed nucleic acid.

2. A method for generating a sequencing library comprising nucleic acids from a plurality of single cells, the method comprising: (a) providing a mixed cell or cell 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 mixed cell or cell nucleus in an emulsion droplet; (b) assigning a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; (c) incorporating the second index sequence into the nucleic acid in each compartment to produce a dual-indexed nucleic acid; as well as (d) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from multiple cells.

3. A method for sequencing a library comprising nucleic acids from a plurality of single cells, the method comprising: (a) providing a mixed cell or cell 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 mixed cell or cell nucleus in an emulsion droplet; (b) assigning a subset of the mixed cells or cell nuclei into a plurality of compartments comprising 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 a plurality of cells; as well as (e) sequencing all or a subset of the dual-indexed nucleic acids.

4. The method of any one of claims 1 to 3, wherein the first index sequence is incorporated into the nucleic acid of the cell or cell nucleus in the emulsion droplet by a method comprising: (i) contacting a plurality of cells or cell nuclei with a plurality of particles comprising the first index sequence; (ii) partitioning individual particles and one or more of the cells or cell nuclei into emulsion droplets; (iii) incorporating the first index sequence into the nucleic acid of the cell or cell nucleus to produce an index nucleic acid; as well as (iv) combining the cells or cell nuclei associated with or containing the index nucleic acid from a plurality of the emulsion droplets to produce the mixed cells or cell nuclei associated with or containing the nucleic acid comprising the first index sequence.

5. The method of claim 4, wherein before or during step (i), the cell or cell nucleus is treated with a transposase. The method according to claim 5 , wherein the transposase is Tn5 transposase.

7. The method according to any one of claims 4 to 6, wherein prior to step (i), the plurality of cells or cell nuclei are treated with a fixative.

8. The method according to any one of claims 4 to 7, wherein prior to step (i), the plurality of cells are permeabilized or lysed.

9. The method according to any one of claims 4 to 8, wherein before or during step (i), the plurality of cells or cell nuclei are treated with a plurality of reagents.

10. The method according to any one of claims 4 to 9, wherein the first index sequence of each particle or subset of particles is unique relative to the other particles.

11. The method according to any one of claims 1 to 10, wherein the second index sequence in one compartment or a subset of compartments is unique relative to other second index sequences.

12. The method according to any one of claims 1 to 11, wherein the plurality of compartments is a multi-well plate.

13. The method according to any one of claims 1 to 12, wherein the emulsion droplets are generated in an emulsion droplet generating device or using an emulsion droplet generating technology.

14. The method of any one of claims 1 to 13, further comprising incorporating a third index sequence into the dual-indexed nucleic acid to generate a triply-indexed nucleic acid.

15. The method according to any one of claims 1 to 14, wherein incorporating the first index sequence into the nucleic acid in the emulsion droplets is performed by an amplification method, a reverse transcription method or a ligation method.

16. The method according to any one of claims 1 to 15, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

17. The method according to claim 15 or 16, wherein the amplification method is polymerase chain reaction (PCR) or an isothermal amplification method.

18. The method of any one of claims 1 to 17, wherein the nucleic acid comprises RNA.

19. The method of any one of claims 1 to 17, wherein the nucleic acid comprises DNA.

20. The method of any one of claims 1 to 19, wherein the plurality of cells comprises at least about 100,000 cells.

21. The method of any one of claims 1 to 20, wherein the plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof.

22. The method of any one of claims 1 to 21, wherein the plurality of cells comprises any of: (a) Cells modified with gene editing systems; (b) antibody-producing cells; (c) cells in different developmental states; (d) cells in different disease states; and (e) Cells treated with drug.

23. A method for combinatorial indexing of nucleic acids from a plurality of single cells, the method comprising: (a) contacting a plurality of cells or cell nuclei separated 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 cell nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cell or cell nucleus to produce an index nucleic acid; (d) combining the cells or cell nuclei containing or associated with the index nucleic acid from a plurality of the emulsion droplets to produce mixed cells or cell nuclei; (e) assigning a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; as well as (f) incorporating the second index sequence into the index nucleic acid in each compartment to produce a dual index nucleic acid.

24. The method of claim 23, further comprising (g) incorporating a third index sequence into the dual-indexed nucleic acid to generate a triply-indexed nucleic acid.

25. The method of claim 23 or 24, wherein the first index sequence of each particle or subset of particles is unique relative to the other particles.

26. The method of any one of claims 23 to 25, wherein the second index sequence in one compartment or a subset of compartments is unique relative to other second index sequences.

27. The method according to any one of claims 23 to 26, wherein the emulsion droplets are generated in an emulsion droplet generating device or using an emulsion droplet generating technology.

28. The method of any one of claims 23 to 27, wherein the plurality of compartments is a multiwell plate.

29. The method according to any one of claims 23 to 28, wherein incorporating the first index sequence into the nucleic acid is performed by an amplification method, a reverse transcription method or a ligation method.

30. The method of any one of claims 23 to 29, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

31. The method of claim 29 or 30, wherein the amplification method is polymerase chain reaction (PCR) or an isothermal amplification method.

32. The method of any one of claims 23 to 31 , wherein before or during step (a), the cell or cell nucleus is treated with a transposase.

33. The method of claim 32, wherein the transposase is Tn5 transposase.

34. The method of any one of claims 23 to 33, wherein prior to step (a), the plurality of cells or cell nuclei are treated with a fixative.

35. The method of any one of claims 23 to 34, wherein prior to step (a), the plurality of cells are permeabilized or lysed.

36. The method of any one of claims 23 to 35, wherein prior to or during step (a), the plurality of cells or cell nuclei are treated with a multiplex reagent.

37. The method of any one of claims 23 to 36, wherein the nucleic acid comprises RNA.

38. The method of any one of claims 23 to 36, wherein the nucleic acid comprises DNA.

39. The method of any one of claims 23 to 38, wherein the plurality of cells comprises at least about 100,000 cells.

40. The method of any one of claims 23 to 39, wherein the plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof.

41. The method of any one of claims 23 to 40, wherein the plurality of cells comprises any of: (a) Cells modified with gene editing systems; (b) antibody-producing cells; (c) cells in different developmental states; (d) cells in different disease states; and (e) Cells treated with drug.

42. 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 cell nuclei separated 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 cell nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cell or cell nucleus to produce an index nucleic acid; (d) combining the cells or cell nuclei containing or associated with the index nucleic acid from a plurality of the emulsion droplets to produce mixed cells or cell nuclei; (e) assigning a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; (f) incorporating the second index sequence into the index nucleic acid in each compartment to produce a dual index nucleic acid; as well as (g) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from multiple cells.

43. The method of claim 42, wherein the first index sequence of each particle or subset of particles is unique relative to other particles.

44. A method according to claim 42 or 43, wherein the second index sequence in a compartment or a subset of compartments is unique relative to other second index sequences.

45. The method according to any one of claims 42 to 44, wherein the emulsion droplets are generated in an emulsion droplet generation device or using an emulsion droplet generation technology.

46. ​​The method of any one of claims 42 to 45, wherein the plurality of compartments is a multiwell plate.

47. The method of any one of claims 42 to 46, wherein incorporating the first index sequence into the nucleic acid is performed by an amplification method, a reverse transcription method, or a ligation method.

48. The method of any one of claims 42 to 47, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

49. The method of claim 47 or 48, wherein the amplification method is polymerase chain reaction (PCR) or an isothermal amplification method.

50. The method of any one of claims 42 to 49, wherein the cell or cell nucleus is treated with a transposase before or during step (a).

51. The method of claim 50, wherein the transposase is Tn5 transposase.

52. The method of any one of claims 42 to 51, wherein prior to step (a), the plurality of cells or cell nuclei are treated with a fixative.

53. The method of any one of claims 42 to 52, wherein prior to step (a), the plurality of cells are permeabilized or lysed.

54. The method of any one of claims 42 to 53, wherein prior to or during step (a), the plurality of cells or cell nuclei are treated with a multiplex reagent.

55. The method of any one of claims 42 to 54, wherein the nucleic acid comprises RNA.

56. The method of any one of claims 42 to 54, wherein the nucleic acid comprises DNA.

57. The method of any one of claims 42 to 56, wherein the plurality of cells comprises at least about 100,000 cells.

58. The method of any one of claims 42 to 57, wherein the plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof.

59. The method of any one of claims 42 to 58, wherein the plurality of cells comprises any of: (a) Cells modified with gene editing systems; (b) antibody-producing cells; (c) cells in different developmental states; (d) cells in different disease states; and (e) Cells treated with drug.

60. A method for sequencing a library comprising nucleic acids from a plurality of single cells, the method comprising: (a) contacting a plurality of cells or cell nuclei separated 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 cell nuclei into emulsion droplets; (c) incorporating the first index sequence into the nucleic acid of the cell or cell nucleus to produce an index nucleic acid; (d) combining the cells or cell nuclei containing or associated with the index nucleic acid from a plurality of the emulsion droplets to produce mixed cells or cell nuclei; (e) assigning a subset of the mixed cells or cell nuclei into a plurality of compartments comprising a second index sequence; (f) incorporating the second index sequence into the index nucleic acid in each compartment to produce a dual index nucleic acid; (g) combining all or a subset of the dual-indexed nucleic acids to generate a sequencing library from a plurality of cells; as well as (h) sequencing all or a subset of the dual-indexed nucleic acids.

61. The method of claim 60, wherein the first index sequence of each particle or subset of particles is unique relative to other microbeads.

62. A method according to claim 60 or 61, wherein the second index sequence in a compartment or a subset of compartments is unique relative to other second index sequences.

63. The method according to any one of claims 60 to 62, wherein the microfluidic droplets are generated in an emulsion droplet generation device or using an emulsion droplet generation technique.

64. The method of any one of claims 60 to 63, wherein the plurality of compartments is a multiwell plate.

65. The method of any one of claims 60 to 64, wherein incorporating the first index sequence into the nucleic acid is performed by an amplification method, a reverse transcription method, or a ligation method.

66. The method of any one of claims 60 to 65, wherein incorporating the second index sequence into the index nucleic acid is performed by an amplification method or a ligation method.

67. The method of claim 65 or 66, wherein the amplification method is polymerase chain reaction (PCR) or an isothermal amplification method.

68. The method of any one of claims 60 to 67, wherein before or during step (a), the plurality of cells or cell nuclei are treated with a transposase.

69. The method of claim 68, wherein the transposase is Tn5 transposase.

70. The method of any one of claims 60 to 69, wherein prior to step (a), the plurality of cells or cell nuclei are treated with a fixative.

71. The method of any one of claims 60 to 70, wherein prior to step (a), the plurality of cells are permeabilized or lysed.

72. The method of any one of claims 60 to 71, wherein the plurality of cells or cell nuclei are treated with a plurality of reagents prior to or during step (a).

73. The method of any one of claims 60 to 72, wherein the nucleic acid comprises RNA.

74. The method of any one of claims 60 to 72, wherein the nucleic acid comprises DNA.

75. The method of any one of claims 60 to 74, wherein the plurality of cells comprises at least about 100,000 cells.

76. The method of any one of claims 60 to 75, wherein the plurality of cells comprises pluripotent stem cells, embryonic stem cells, somatic cells, immune cells, cancer cells, or a combination thereof.

77. The method of any one of claims 60 to 76, wherein the plurality of cells comprises any of: (a) Cells modified with gene editing systems; (b) antibody-producing cells; (c) cells in different developmental states; (d) cells in different disease states; and (e) Cells treated with drug.

78. The method of any one of claims 60 to 77, wherein the emulsion droplet comprises at least two cells or cell nuclei.

79. The method of any one of claims 60 to 78, wherein the particles are gel beads.

80. The method of any one of claims 1 to 79, further comprising performing a lineage tracing method.

81. The method of any one of claims 1 to 79, further comprising performing a genomic screen.

82. The method of claim 81, wherein the genomic screen is a CRISPR-based screen.

83. The method of claim 82, wherein the CRISPR-based screen is selected from the group consisting of a gene editing CRISPR screen, a CRISPRi screen, and a CRISPRa screen.

84. The method of any one of claims 1 to 83, wherein the plurality of cells or cell nuclei isolated from the plurality of cells are bound by a protein binding reagent coupled to an oligonucleotide comprising a barcode.

85. The method of any one of claims 1 to 83, further comprising, prior to (a), contacting the plurality of cells or cell nuclei isolated from the plurality of cells with a plurality of protein binding reagents coupled to oligonucleotides comprising a barcode.

86. The method of claim 84 or 85, wherein the protein binding reagent binds to a protein located on the surface of at least one cell in the plurality of single cells.

87. The method of any one of claims 84 to 86, wherein the protein binding agent is an antibody or fragment thereof.

88. The method of claim 86 or 87, further comprising determining the expression level of the protein.

89. A kit for performing the method according to any one of claims 1 to 88.

90. The kit of claim 89, wherein the kit comprises one or more of: (a) at least one container comprising a plurality of particles comprising said first index sequence; and (b) at least one container comprising a plurality of second index sequences.

91. The kit of claim 90, further comprising a container comprising one or more protein binding reagents coupled to an oligonucleotide comprising a barcode.

92. The kit of claim 91, wherein the protein binding agent is an antibody or fragment thereof.

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