Methods for producing singe-cell sequencing libraries of DNA originating from open chromatin

Abstract

The present disclosure is concerned with producing sequencing libraries of molecularly distinct polynucleotides. Provided are methods and compositions related to producing libraries, including single-cell libraries, that include DNA from regions of open chromatin. The methods and compositions can be optionally related to the simultaneous production of transcriptome libraries, including single-cell transcriptome libraries.

Description

METHODS FOR PRODUCING SINGE-CELL SEQUENCING LIBRARIES OF DNA ORIGINATING FROM OPEN CHROMATIN

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 738,876, filed December 26, 2024, the disclosure of which is incorporated by reference herein in its entirety.

[0003] FIELD

[0004] Embodiments of the present disclosure relate to sequencing nucleic acids. In particular, embodiments of the methods provided herein relate to producing single-cell sequencing libraries based on accessible genomic DNA and optionally cellular or nuclear RNA.

[0005] BACKGROUND

[0006] The growing movement toward personalized medicine is led by a fundamental shift from a one size fits all paradigm for patient treatment to one that embraces tailored therapies. One technology driving this reevaluation of treatment is the ability to focus on specific gene expression in individuals. An individual's transcriptome and spatial and temporal organization of genomic DNA can provide a window on gene expression, allowing medical personnel to assess the health of individual patients to detect diseases and identify effective treatments.

[0007] SUMMARY OF THE APPLICATION

[0008] Provided herein are methods for preparing libraries. In one embodiment, a method includes providing nuclei or cells that include fragmented double stranded genomic DNA, where the ends of each fragment include ligated exogenous DNA, and combining the nuclei or cells with template particles, where the template particles include a plurality of a nucleotide capture sequence attached by the 5’ end to the template particles. The method can further include generating a plurality of uniform partitions near-instantly thatencapsulate a single one of the template particles and a single one of the nuclei or cells to form pre-templated instant partitions (PIPs), and lysing the nuclei or cells to release in each PIP the fragmented double stranded genomic DNA. Optionally, the method can further include delivering a micellized lysis agent to the nuclei or cells in the PIPs. The method can optionally include forming cDNA, forming amplicons from the cDNA, and sequencing the amplicons to generate a chromatin accessibility profile.

[0009] The method can also include the use of template particles that further include a plurality of a second nucleotide capture sequence attached by the 5’ end to the template particles. In this embodiment, the lysing can further include releasing in each PIP RNA present in the nuclei. The method can optionally include forming cDNA, forming amplicons from the cDNA, and sequencing the amplicons to generate a chromatin accessibility profile and a transcriptome.

[0010] Also provided herein are compositions and kits. In one embodiment, a kit includes, in separate compartments, a transposome complex including an associated antigen binding molecule and a template particle. In one embodiment, the transposome complex includes an exogenous DNA, where the exogenous DNA includes a barcode.

[0011] BRIEF DESCRIPTION OF THE FIGURES

[0012] The following detailed description of illustrative embodiments of the present disclosure may be best understood when read in conjunction with the following drawings.

[0013] FIG. 1A-1C shows a schematic representation of closed and open chromatin (FIG. 1A), the insertion of transposon complexes into genomic DNA of open chromatin (FIG. IB), and the resulting fragments of genomic DNA after transposon-mediated fragmentation and ligation of exogenous DNA to the ends of the fragments (FIG. 1C).

[0014] FIG. 2A-2C shows examples of exogenous DNA of transposome complexes alone (FIG.2A-2B) or attached to a DNA fragment (FIG. 2C).

[0015] FIG. 3A-3B, 4A-4B, 5, and 6 show exemplary illustrations of embodiments that can occur during practice of the methods described herein.

[0016] The schematic drawings are not necessarily to scale. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar to other numbered components.

[0017] DETAILED DESCRIPTION

[0018] The present disclosure includes methods for evaluating gene expression in individual cells.In one embodiment, evaluating gene expression is accomplished by ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing). ATAC-seq is a method for sequencing DNA in single cells that is carried out on DNA containing native chromatin, which permits the conversion of DNA within open chromatin into library molecules. Open chromatin, also referred to herein as accessible DNA, corresponds to regions of DNA that include expressed coding regions and regulatory elements such as promotor, suppressor, and enhancer sequences. Closed chromatin is DNA that is closely associated with groups of histones (FIG. 1 ). Coding regions present in closed chromatin correspond to DNA that includes unexpressed coding regions. Thus, a library produced using an ATAC-seq method targets DNA present in open chromatin, and is predominately DNA that can be expressed and regulatory elements that can influence expression. In another embodiment, evaluating gene expression is accomplished by preparing libraries based on a cell’s RNA, such as mRNA. A library based on a cell’s mRNA can be referred to as a transcriptome library.

[0019] DNA Library Production Using ATAC-seq

[0020] Methods for making a library include providing nuclei or cells that include fragmented double stranded genomic DNA, where the fragmented DNA originates from open chromatin.

[0021] The method may further include preparing the nuclei. In one embodiment nuclei are from cells. Optionally, cells or nuclei are permeabilized. As used herein, “permeabilization”and “permeabilizing” refer to increasing the permeability of the cellular, nuclear, or both nuclear and cellular membrane without disrupting the membrane, where the permeability allows molecules such as transposome complexes to traverse across the permeabilized membrane. Alternatively, cells containing the nuclei can be permeabilized and the nuclei isolated later. Compounds and methods for permeabilizing nuclei, cells, or both nuclei and cells are known in the art (International Application Publication WO 2018 / 125982).Examples of compounds useful for permeabilizing nuclei or cells containing nuclei include, but are not limited to detergents and surfactants, such as Triton X-100 (IUPAC name 2-[4- (2,4,4-trimethylpentan-2-yl)phenoxy]ethano).

[0022] Nuclei or cells containing the nuclei may be fixed before permeabilizing. Without intending to be limited by theory, fixing nuclei or cells can help reduce the movement of cellular or nuclear molecules, such as fragmented DNA or mRNA, out of the cells or nuclei. However, fixation is optional. Methods for fixing nuclei and cells are known in the art and include, but are not limited to, treatment of nuclei with formaldehyde, DSP, gluteraldehyde, or one or more succinimide esters, or a combination thereof. In one embodiment, the fixation of the nuclei or cells can be reversed. Methods for reversing fixation of nuclei and cells are known in the art and include, but are not limited to, treatment of nuclei or cells with DTT, SDS, or proteinase K.

[0023] Nuclei that include fragmented double stranded genomic DNA, whether the nuclei are separated from cells or present in cells, can be produced. In one embodiment, fragmented double stranded genomic DNA inside nuclei can be produced enzymatically. Examples of enzymatic treatment include but are not limited to, DNAse I or restriction endonuclease digestion. Enzymatic fragmentation of nuclear DNA can be followed by attachment of exogenous DNA to both ends of the fragmented DNA. Methods for attaching exogenous DNA to the ends of fragments DNA are known and include, but are not limited to, ligation, hybridization of overhanging ends, or a combination thereof.

[0024] In one embodiment, fragmented double stranded genomic DNA inside nuclei can be produced by exposing permeabilized nuclei or cells to transposome complexes. A transposome complex is a transposase bound to exogenous DNA. The transposasetypically includes a transposase recognition site, which can insert the exogenous DNA into accessible DNA, e.g., open chromatin, within genomic DNA in a process sometimes referred to interchangeably as "tagmentation" and "transposition." Tagmentation combines fragmentation and ligation into a single step to add the exogenous DNA to both ends of the resulting fragmented DNA (Gunderson et al., WO 2016 / 130704). FIG. IB shows a representation of the association of transposome complexes with accessible DNA of open chromatin, and FIG. 1C shows the resulting fragments of genomic DNA after fragmentation and ligation to add the exogenous DNA to each end of each fragment.

[0025] In some embodiments transposome complexes are associated with an antigen binding molecule that can aid in recruiting transposome complexes to specific antigens, such as DNA-associated proteins. Examples of useful antigen binding molecules include, but are not limited to, antibodies or aptamers that bind DNA-associated proteins. Examples of DNA-associated proteins include, but are not limited to, transcription factors, histones, polymerases, zinc finger proteins, helix-turn-helix proteins, leucine zipper proteins, and DNA repair proteins. Use of an antigen binding molecule that binds a DNA-associated protein may aid in recruiting transposome complexes to regions of open chromatin. The antigen binding molecule can be covalently bound to a transposome complex, such as a transposase. Methods for conjugating an antigen binding molecule, such as an antibody or aptamer, to a protein are known in the art.

[0026] In some embodiments, use of transposome complexes associated with an antigen binding molecule that binds a DNA-associated protein can be used to identify locations in a genome where the targeted DNA-associated protein is bound. The antigen binding molecule recruits the associated transposome complexes to sites in the genome where the DNA-associated protein is bound. Localized insertion and ligation of the exogenous DNA can occur when the DNA-associated protein is bound to accessible DNA.

[0027] The exogenous DNA can include a double stranded region and a single stranded region (FIG. 2A-2B). The double stranded region typically includes the transposase recognition sequence, and the single stranded region typically includes a sequence that is complementary to the 3' region of a capture oligo. FIG. 2A shows an example of anexogenous DNA where the double stranded transposase recognition sequence includes a mosaic end 21, and the single stranded region includes a universal complementary sequence (CSU) 22. Essentially any sequence can be used as transposase recognition sequence, provided it functions with the transposase to insert the exogenous DNA into genomic DNA. Essentially any sequence can be used as a CSU, provided that it is complementary to a capture oligo. Without intending to be limiting, examples of CSUs include, but are not limited to, a Poly-A region. Typically, a combination of CSU and capture oligo will be selected that reduces the likelihood of non-specific hybridization between the CSU and other nucleotide sequences present, such as other sequences present on a capture oligo and genomic DNA. Optionally, the single stranded region includes two single strands, where the first includes a sequence that is complementary to a capture oligo and the second includes a sequence that can be used as an amplification handle. FIG. 2B shows an example of an exogenous DNA where the double stranded transposase recognition sequence includes a mosaic end 21, and the single stranded region includes a CSU 22 and an amplification handle 23. Following the fragmentation of genomic DNA and ligation to add exogenous DNA to the ends of each fragment, a structure 20 such as the one depicted in FIG. 2C can result. Nuclei containing fragmented DNA 24 with attached exogenous DNA 29 can be used in subsequent steps of the methods. Cells that include nuclei containing fragmented DNA with attached exogenous DNA can also be used in subsequent steps of the methods. Alternatively, nuclei can be isolated from the cells.

[0028] The single stranded region of the exogenous DNA of a transposome complex can include extra sequences in addition to a sequence that is complementary to the 3' region of a capture oligo. Typically, such an extra sequence is located between the transposase recognition site and the sequence that is complementary to the 3' region of a capture oligo. Examples of extra sequences include, but are not limited to, universal sequences that will hybridize to a primer for use in amplification or sequencing, and the like. In some embodiments, the single stranded region can include a transposase complex-specific barcode. Transposase complex-specific barcodes are useful in multiplexed approaches for identifying, in the same experiment, the locations of different DNA-associated proteins in a cell's genome. For instance, multiple populations of transposome complexes can be produced, where each population has an antigen binding molecule that binds to a specificDNA-associated protein and a unique barcode. Exposing permeabilized nuclei or cells to two or more populations of uniquely labeled transposome complexes, each targeting different DNA-associated proteins, allows the localization of areas in the genome where the different DNA-associated proteins are bound.

[0029] Partitioning into compartments

[0030] Nuclei or cells containing fragmented DNA with ligated exogenous DNA are used in a process that results in individual nuclei or cells present in pre-templated instant partitions (PIPs). Methods for forming PIPs are described in, for instance, U.S. Published Patent Application 2021 / 0340596 and International Patent Application WO 2022 / 245868.Briefly, template particles are combined in a first fluid with nuclei or cells that include fragmented DNA with ligated exogenous DNA. The first fluid is combined with a second fluid that is immiscible with the first fluid, and the resulting mixture is agitated such that a plurality of monodisperse droplets in the second fluid is formed. The monodisperse droplets include the first fluid, a single one of the template particles, and a single one of the nuclei or cells.

[0031] The template particles include a plurality of an attached capture oligo. In some embodiments described herein, template particles include a plurality of at least two distinct populations of attached capture oligos. Capture oligos are configured so the 3' end can hybridize with a CSU of a fragment that includes an exogenous DNA. Accordingly, the 3' end of a capture oligo is located distal to the point of attachment to the template particle. The capture oligo can include other useful nucleotide sequences including, but not limited to, a template particle-specific barcode, universal sequences that will hybridize to a primer for use in amplification or sequencing, and the like. In one embodiment, the capture oligos attached to any single template particle include the same barcode, and the barcode of each template particle is different from the barcode of other template particles. This use of a unique barcode results in all fragments within a droplet being tagged with the same barcode and permits identifying, after sequencing, which fragments originated from the same cell or nucleus. In one embodiment, the capture oligos attached to any single template particle do not include a universal molecular identifier (UMI). An example of a template particle 30and atached capture oligo 36 is shown in FTG. 3A. In this example the capture oligo 36 includes a complement of CSU, 32', a barcode 35, and universal primer site 38.

[0032] Nuclear or cellular lysis

[0033] The methods of the present disclosure further include lysing the nuclei or cells present in the droplets. Any method for lysing the nuclei or cells while maintaining the emulsion and the separation of the droplets can be used. Typically, nuclei or cells within the droplets are contacted with one or more lysis agents. In one embodiment, micelle-based methods are used to deliver a lysis agent to the droplets and then release the lysis agent to cause lysis of the nuclei or cells in the separate droplets. Methods for making micelles that include one or more lysis agents, referred to herein as a micellized lysis agent, and methods for delivering micelles to droplets are described in U.S. Patent Publication 2021 / 0214721. Methods for creating micellized lysis agents can include use of a fluorosurfactant. In one embodiment, template particles include compartments that contain one or more lysis agents. The compartments can be released by an external stimulus, such as heat, induced chemical cleavage of bound reagents, selective dissolution of hydrogel shells, or induced changes to porosity and diffusivity of the template particle.

[0034] Examples of lysis agents that can be used as a lysis agent include, but are not limited to, detergents, heat activated enzymes, enzymatic cofactors, and disulfide reducing agents. Examples of detergents include, but are not limited to, ionic type detergents, non-ionic type detergents, and zwitterionic type detergents, such as, but not limited to, sodium dodecyl sulfate (SDS), Sarkosyl, sodium deoxycholate, Capstone FS-61, CTAB, Triton X-100, Triton X-114, NP-40, Tween-80, Brij 35, Octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, ASB-14, ASB-16, SB-3-10, and SB-3-12.

[0035] Lysing the nuclei or cells releases the fragments, which can associate with the capture oligos present on each template particle. FIG. 3B shows an example of a structure that can result after a DNA fragment 37 hybridizes to a capture oligo 36. The DNA fragment includes an optional amplification handle 33, a fragment of accessible genomic DNA 34, a mosaic end 31, and a CSU 32. The CSU 32 is hybridized to the complement of CSU, 32' of capture oligo 36. The capture oligo 36 also includes a barcode 35. In one embodiment,a polymerase is introduced into the droplets and used to extend the 3' end of the nucleotide capture sequences using the annealed fragment as a template.

[0036] Breaking emulsion

[0037] Following the hybridization of fragments (e.g., DNA fragment 37 of FIG. 3B) to capture oligos within each droplet, the emulsion is disrupted, and the contents of the droplets are released into a bulk aqueous phase. The result is an aqueous phase that contains the template particles, where each template particle includes hybridized fragments representing a library originating from the single cell or nucleus originally present in the PIP with the template particle.

[0038] In some embodiments, the hybridized fragments can be treated to identify methylated nucleotides, including methylated cytosines. The treatment can occur while the droplets are present or after the bulk aqueous phase is produced. In one embodiment, an altered cytidine deaminase can be used to treat the hybridized fragments to selectively convert methylated cytosines, e.g., 5-methylcytosine (5mC), 5 -hydroxymethyl cytosine (5hmC), or both 5mC and 5hmC, to thymidine. Examples of altered cytidine deaminases and methods of using them to identify methylated cytosines are described in Internation Application Publication WO 2023 / 196572. Because amplification of DNA does not preserve the modification status of cytidine (e.g., the methylation status of 5mC and 5hmC is not retained), use of an altered cytidine deaminase typically occurs before extension of the 3' end of the capture oligos and before amplification of the hybridized fragments. Thus, the hybridized fragments can be contacted with an altered cytidine deaminase at essentially any time before hybridized fragments are used as a template by a polymerase.

[0039] Extension

[0040] Following the transfer of the contents of each droplet into a bulk aqueous phase, a polymerase can be used to extend the 3' end of the capture oligos using the annealed fragment as a template. Any suitable polymerase can be used, including but not limited to polymerases such as Phi29 and Klenow large fragment. In some embodiments, the polymerase is an isothermal polymerase. In some embodiments, the polymerase is a RNA-dependent DNA polymerase, e.g., a reverse transcriptase (RT). RT can be used with shorter DNA fragments. In one embodiment, a RT can be used for template switching. An illustration of an extension 40 of a capture oligo 36 by a polymerase is depicted in FIG. 4A. The result of extension is depicted in FIG. 4B, where the complement of CSU, 32', of capture oligo 36 has been extended to result in a complementary DNA (cDNA) 42. cDNA 42 includes the capture oligo 36 and the complement of fragment 37, and regions including universal primer site 38 and the complement of the amplification handle 33'. The result is an aqueous phase that contains the template particles, where each template particle includes attached cDNAs that represent a library of DNA fragments originating from a single cell.

[0041] Library preparation

[0042] Different approaches can be taken to prepare the single cell libraries in the aqueous phase for sequencing. In one embodiment, an amplification, such as a PCR, can be used to produce amplified libraries from each template particle, and optionally the PCR can be used to add an adapter to one or both ends of the amplified cDNAs. For instance, primers can be added to the aqueous phase. One primer can hybridize with the complement of the amplification handle (e.g., complement of the amplification handle 33' of FIG. 4B) and a second primer can hybridize with the complement of nucleotides present at the 5' end of the attached cDNA (e.g., the complement of a universal primer 38 of FIG. 4B). A primer that hybridizes with the complement of the amplification handle can further include adapter sequences at the 5' end. Incorporation of this primer during the PCR results in amplicons that include the adapter sequences at the 5' ends. An illustration of an amplification is depicted in FIG. 5. The in-solution primer 33 hybridizes to the 3' end of cDNA 42, and the PCR produces the extension product 41. The resulting polynucleotide 52 hybridizes with the in-solution universal primer 38, and the PCR produces the extension product 53. The remainder of the PCR continues with the resulting polynucleotide 54 hybridizing with insolution primer 33 and polynucleotide 52 hybridizing with in-solution universal primer 38.

[0043] The PCR can be a limited cycle PCR, for instance, one that includes 1 to 5 cycles of amplification, or the PCR can be a complete PCR, for instance, one that includes at least 20 to 40 cycles of amplification.

[0044] Following amplification, the amplicons that include adapters at each end can be used immediately in the preparation of clusters and subsequent sequencing. Alternatively, the amplicons can be subjected to enzymatic digestion with, for instance, a restriction endonuclease that leaves a staggered end with non-hybridized nucleotides (e.g., sticky ends). Enzymatic digestion of fragments can be used for subsequent addition of adapters with non-hybridized complementary nucleotides for efficient addition of adapters. In one embodiment, an enzyme can be selected that will identify and cleave only nucleotide sequences that are present in the region originating from genomic DNA (e.g., fragment of accessible genomic DNA 44 of FIG. 4B). Optionally, the location of the enzymatic digestion can be used as a source of distinct molecular identity. Alternatively, the insertion site of exogenous DNA by the transposome complex can be used as a source of distinct molecular identity. Uniquely labeling fragments by enzymatic digestion site or exogenous DNA insertion site creates diversity within populations of identical molecules by producing fragments with ends at different cleavage locations (U.S. Patent Publication 2021 / 0214721). Accordingly, unique molecular identifiers (UMIs) are not required.

[0045] Preparation of transcriptome librariesThe methods described herein for producing a library using ATAC-seq can be modified to also produce a transcriptome library in addition to a library of genomic DNA fragments derived from regions of open chromatin. Examples of the use of template particles for transcriptome analysis are described in US 2022-0135966. The production of both DNA and transcriptome libraries simultaneously is referred to herein as a co-assay. In one embodiment, the fragmented double stranded DNA can be flanked by exogenous DNA that includes a poly-A region as a CSU, and RNAse free conditions can be used. After lysis, but fragmented double stranded DNA and mRNA can hybridize with capture oligos that include a nucleotide sequence that will hybridize to a Poly-A region. In one embodiment, a modification includes the use of a different template particle during production of PIPs. The template particles for producing a library using ATAC-seq include a plurality of an attached capture oligo. Template particles useful for a co-assay include a plurality of at least two distinct populations of attached capture oligos. One population is described herein for use with making an ATAC-seq library. The second population includes a 3' endhaving a nucleotide sequence designed to hybridize with RNA molecules, such as mRNA. Examples of template particles having two populations of capture oligo nucleotides are described in International Patent Publication WO 2022 / 245868. In one embodiment, suitable nucleotide sequences can be complementary to a universal complementary sequence (CSU) present on an RNA molecule, such as a Poly-A region on an mRNA molecule. Examples of suitable nucleotide sequences include, but are not limited to, a poly-T domain, a randomer domain, a target-specific domain, and a disrupted homopolymer.

[0046] A poly-T domain is useful in hybridizing to the poly-A region of mRNAs. A randomer domain includes a random sequence of nucleotides of 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length that anneals to a random location on a mRNA. As used herein, a “target-specific domain” when used in reference to a capture oligo refers to a 3' domain of a capture oligo that includes a nucleotide sequence complementary to a targeted polynucleotide. For instance, nucleotides of a target-specific domain will selectively anneal to a targeted polynucleotide, e.g., a mRNA encoded by a specific coding region. A surface can include capture oligos with a target-specific domain, and optionally a surface can include different populations of capture oligos, each population having a single target-specific domain that targets a different set of nucleotides of the same mRNA.

[0047] As used herein, a "disrupted homopolymer" refers to a hybridization region of a capture oligo that includes two or more nucleotides or two or more nucleotide sequences complementary to a mRNA poly-A region. In contrast to an uninterrupted series of consecutive thymidine nucleotides (e.g., a poly-T domain), a disrupted ymer includes at least two non-sequential nucleotides or non- sequent! al nucleotide sequences, where each of the non-sequential nucleotides or non-sequential nucleotide sequences is complementary to a portion of a mRNA poly-A region. Each of the non-sequential nucleotides or nonsequential nucleotide sequences are separated by an intervening nucleotide or intervening nucleotide sequence. For example, a disrupted homopolymer of a capture oligo can have a first series of three thymines, followed by a non-thymine nucleotide, and a second series of three thymines. Such patterns can be repeated multiple times within a hybridization region of a capture oligo. The presence of an intervening nucleotide or intervening nucleotidesequence between non-sequential nucleotides or non-sequential nucleotide sequences does not prevent hybridization between a mRNA poly-A region and complementary thymidine nucleotides present in a disrupted homopolymer.

[0048] In one embodiment, the mRNA-specific capture oligos do not include nucleotides that will hybridize with nucleotides on a genomic DNA-specific capture oligo. For instance, mRNA-specific capture oligos do not hybridize with a CSU of a DNA-specific capture oligo.

[0049] FIG. 6 shows an example of a template particle 30 that can be used in a co-assay. The attached capture oligo 36, includes a barcode 35, universal primer site 38, and a complement of CSU, 32' for use in capturing DNA fragments with attached exogenous DNA. The attached capture oligo 66, includes a barcode 65, universal primer site 68, and a poly-T region 62 for use in capturing mRNA molecules. Barcode 65 can be identical to barcode 35, and universal primer site 68 can be identical to universal primer 38.

[0050] Another modification for use of the method as a co-assay includes the use of a RNA- dependent DNA polymerase, e.g., reverse transcriptase, during the extension after transfer of the contents of each droplet into a bulk aqueous phase. After lysis of cells or nuclei and breaking the emulsion, the bulk aqueous phase includes DNA fragments with exogenous DNA at each end and mRNA. In one embodiment, the 3' ends of the DNA fragments with exogenous DNA hybridize to the 3' ends of one population of capture oligos (e.g., the complement of a CSU at the 3' end), and the 3' poly-A of mRNA hybridizes to the 3' ends of the other population of capture oligos (e.g., a poly-T domain, randomer domain, or target-specific domain at the 3' end). A RNA-dependent DNA polymerase is useful for extension of the mRNA, and in some embodiments can be used in conjunction with a DNA polymerase. In one embodiment, the RNA-dependent DNA polymerase, e g., reverse transcriptase, is one that will use both RNA and DNA as a template and produce a cDNA.

[0051] In some embodiments, the extension includes a method for template switching. Template switching typically includes the use of a reverse transcriptase that adds nucleotides to the end of the 3' end of the extended strand, and a template switching oligo (TSO) that acts as a primer for extension of a new strand that uses the newly synthesized cDNA as template.Examples of reverse transcriptases that add nucleotides to the extended strand (e.g., the first strand nucleotide sequence) include, but are not limited to, Moloney Murine Leukemia Virus Reverse Transcriptase. The number of nucleotides added, also referred to as nontemplate nucleotides, can be 2, 3, or more. The nucleotides added are typically the same, e.g., 2 or more cytosines, 2 or more adenines, 2 or more guanines, or 2 or more thymidines. The added nucleotides at the 3' end of the extended strand produce a DNA / RNA hybrid with a single stranded DNA overhang at the 3' end of the DNA strand (when mRNA is extended) or a DNA / DNA hybrid with a single stranded DNA overhang at the 3' end of the DNA strand (when a DNA fragment is extended).

[0052] The overhang can provide a nucleotide sequence to which complementary nucleotides can anneal and provide an additional template (e.g., a template switching oligo) for further extension of the cDNA. In some embodiments, the complementary nucleotides that anneal to the first strand nucleotide sequence overhang contain a template switching oligo, the complement of which is incorporated into the cDNA.

[0053] Following the initial extension, the attached polynucleotides can be amplified, for instance by PCR. The PCR can be a limited cycle PCR, for instance, one that includes 1 to 5 cycles of amplification, or the PCR can be a complete PCR, for instance, one that includes at least 20 to 40 cycles of amplification. In one embodiment, amplified products can be further fragmented after a limited amplification to increase diversity through the use of molecular diversity enhancers (MDEs) or intrinsic molecular identifiers (IMIs). The production of MDEs can include the addition of short random oligos to the fragmented ends. Methods for introducing MDEs to polynucleotide fragments is described at, for instance, US 2022 / 0135966.

[0054] Sequencing

[0055] A sequencing library, e.g., polynucleotides originating from accessible genomic DNA, RNA, or both accessible genomic DNA and RNA, having universal adapters at each end, can be prepared for sequencing. In one embodiment, amplicons are enriched using a plurality of universal capture oligonucleotides having specificity for the amplicons, and the universal capture oligonucleotides are immobilized on a surface of a solid substrate such asa flow cell or a bead. Methods for immobilizing amplicons to a surface and amplifying immobilized amplicons include, but are not limited to, bridge amplification and exclusion amplification (also referred to as kinetic exclusion amplification (KEA). Methods for immobilizing and amplifying prior to sequencing are described in, for instance, Bignell et al. (US 8,053,192), Gunderson et al. (W02016 / 130704), Shen et al. (US 8,895,249), and Pipenburg et al. (US 9,309,502). In some embodiments, a device available under the tradename cBOT (Illumina, San Deigo, CA) can be used to produce amplified immobilized amplicons.

[0056] Following attachment of amplicons to a surface, the sequence of the immobilized and amplified amplicons can be determined. Sequencing can be carried out using any suitable sequencing technique, and methods for determining the sequence of immobilized and amplified modified target nucleic acids, including strand re-synthesis, are known in the art and are described in, for instance, Bignell et al. (US 8,053,192), Gunderson et al.(W02016 / 130704), Shen et al. (US 8,895,249), and Pipenburg et al. (US 9,309,502).

[0057] Sequencing data is subsequently processed using readily available computer algorithms.Processing can include aligning reads to an annotated reference genome and quantifying expression of coding regions.

[0058] Compositions

[0059] The present disclosure includes template particles and compositions that can result during the practice of the methods described herein. For instance, template particles disclosed herein include those having one population of attached capture oligos, where the 3' ends include a universal complementary sequence (CSU) or a complement of a CSU.Optionally, the attached capture oligos further include a barcode located at the 5' end of the CSU or complement of the CSU. In one embodiment, the population of attached capture oligos have a 3' end that is not a poly-T domain, a randomer domain, or a target-specific domain. Another template particle provided herein includes one having two populations of attached capture oligos. One population of attached capture oligos have a 3' end that is a poly-T domain, a randomer domain, a target-specific domain, or a disrupted homopolymer. The second population of attached capture oligos has 3' ends that include a universalIP-2927-PCT / 0531.002927WQ01complementary sequence (CSU) or a complement of a CSU. Optionally, the second population of attached capture oligos further include a barcode located at the 5' end of the CSU or complement of the CSU. Also provided herein are compositions that include a template particle and fragmented DNA with ligated exogenous DNA, RNA, a template particle and RNA, and a template particle and fragmented DNA with ligated exogenous DNA and RNA.

[0060] Kits

[0061] The present disclosure also provides kits for producing sequencing libraries. A kit can include at least one transposome complex. The transposome complex can include an associated antigen binding molecule, such as an antibody or aptamer that binds to a DNA- associated protein. The kit can further include an exogenous DNA that includes a transposase recognition site, a barcode, and a CSU. Optionally the exogenous DNA can be associated with the transposome complex. In one embodiment, a population of transposome complexes having a specific antigen binding molecule also have the same barcode. In one embodiment, a kit can include multiple populations of transposome complexes, where each population has a unique combination of antigen binding molecule and a barcode, where the antigen binding molecule is different from the antigen binding molecule of the other populations and the barcode is different from the barcodes of the other populations.

[0062] A kit can also include template particles. The template particles can include one population of attached capture oligos, where the 3' end of the capture oligos include a nucleotide sequence that is complementary to the CSU of the exogenous DNA. In another embodiment, the template particles can include two populations of attached capture oligos. One population of attached capture oligo has a 3' end that includes a nucleotide sequence that is complementary to the CSU of the exogenous DNA. The other population of attached capture oligo has a 3' end that includes a nucleotide sequence that is complementary to RNA molecules, e.g., a poly-T domain, a randomer domain, a targetspecific domain, or a disrupted homopolymer. The template particles can be present in an amount suitable for analysis of at least 1,000 cells, at least 100,000 cells, or at least1,000,000 cells. Examples of other components include positive control polynucleotides and / or negative control polynucleotides. Optionally, other reagents such as buffers and solutions needed to use the transposome complexes and template particles are also included. Instructions for use of the packaged components are also typically included.

[0063] As used herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit. The packaging material is constructed by known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the components can be used for producing sequencing libraries. In addition, the packaging material contains instructions indicating how the materials within the kit are employed to practice reaction with a transposome complex and how to use template particles to form pre-templated instant partitions. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the components. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent / sample admixtures, temperature, buffer conditions, and the like.

[0064] Definitions

[0065] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below.

[0066] As used herein, the term "partition" is intended to mean a compartment, such as an area or volume, that separates or isolates something from other things.

[0067] As used herein, the term “amplicon,” when used in reference to a nucleic acid, means the product of copying the nucleic acid, where the product has a nucleotide sequence that is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. An amplicon can be produced by any of a variety of amplification methods that use the nucleic acid, e.g., a target polynucleotide or an amplicon thereof, as a templateincluding, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation extension, or ligation chain reaction. An amplicon can be a nucleic acid molecule having a single copy of a particular nucleotide sequence (e.g., a polymerase extension product) or multiple copies of the nucleotide sequence (e.g., a concatemeric product of RCA). A first amplicon of a target polynucleotide is typically a complementary copy. Subsequent amplicons are copies that are created, after generation of the first amplicon, from the target polynucleotide or from the first amplicon. A subsequent amplicon can have a sequence that is substantially complementary to the target polynucleotide or substantially identical to the target polynucleotide.

[0068] As used herein, the terms “polynucleotide” and “nucleic acid” are used interchangeably and are intended to be consistent with their use in the art and includes naturally occurring polynucleotides and functional analogs thereof. Particularly useful functional analogs are capable of hybridizing to a nucleic acid in a sequence specific fashion or capable of being used as a template for replication of a particular nucleotide sequence. Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety of those known in the art. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)). A nucleic acid can contain any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include native or non-native bases. In this regard, a native deoxyribonucleic acid can have one or more bases selected from adenine, thymine, cytosine or guanine and a ribonucleic acid can have one or more bases selected from uracil, adenine, cytosine or guanine. Useful non-native bases that can be included in a nucleic acid are known in the art. The term “target,” when used in reference to a polynucleotide, is intended as a semantic identifier for the polynucleotide in the context of a method or composition set forth herein and does not necessarily limit the structure or function of the polynucleotide beyond what is otherwise explicitly indicated. A target polynucleotide having an exogenous DNA at each end can be referred to as a modified targetpolynucleotide.

[0069] As used herein, the term “polymerase” is intended to be consistent with its use in the art and includes, for example, an enzyme that produces a complementary replicate of a nucleic acid molecule using the nucleic acid as a template strand. Typically, polymerases bind to the template strand and then move down the template strand sequentially adding nucleotides to the free hydroxyl group at the 3' end of a growing strand of nucleic acid. DNA polymerases typically synthesize complementary DNA molecules from DNA templates and RNA-dependent DNA polymerases (e.g., reverse transcriptases) typically synthesize DNA molecules from RNA templates. Polymerases can use a short DNA strand, called a primer, to begin strand growth. Some polymerases can displace the strand upstream of the site where they are adding bases to a chain. Such polymerases are said to be strand displacing, meaning they have an activity that removes a complementary strand from a template strand being read by the polymerase. Exemplary polymerases having strand displacing activity include, without limitation, the large fragment of Bsu (Bacillus subtilis), Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase or sequencing grade T7 exo-polymerase. Some polymerases degrade the strand in front of them, effectively replacing it with the growing chain behind (5' exonuclease activity). Some polymerases have an activity that degrades the strand behind them (3' exonuclease activity). Some useful polymerases have been modified, either by mutation or otherwise, to reduce or eliminate 3' and / or 5' exonuclease activity.

[0070] As used herein, a “transcriptome” refers to RNA transcripts a cell or nucleus at a specific time. In some embodiments, a transcriptome can refer to the mRNA transcripts in a cell or nucleus at a specific time.

[0071] As used herein, the term "primer" and its derivatives refer generally to any polynucleotide that can hybridize to a target sequence of interest. Typically, the primer functions as a substrate onto which nucleotides can be polymerized by a polymerase or to which a polynucleotide can be ligated; in some embodiments, however, the primer can become incorporated into the synthesized nucleic acid strand and provide a site to which another primer can hybridize to prime synthesis of a new strand that is complementary to the synthesized nucleic acid molecule. In some embodiments, a primer includes a sequence present in a guide RNA used with a CRISPR-based system to hybridize to a predeterminedsequence. In some embodiments, a primer includes an additional sequence at the 5' end. The primer can include any combination of nucleotides or analogs thereof. In some embodiments, the primer is a single- stranded polynucleotide.

[0072] As used herein, an "exogenous DNA" is a double stranded DNA that includes a transposase recognition site. An example of a mosaic site is a mosaic end or mosaic element. In some embodiments, two exogenous DNAs can be associated with a transposase dimer to form a transposase complex. An exogenous DNA can further include an additional single stranded DNA attached to the 3' end of one strand of the transposase.

[0073] As used herein, "amplify", "amplifying" or "amplification reaction" and their derivatives, refer generally to any action or process whereby at least a portion of a nucleic acid molecule is replicated or copied into at least one additional nucleic acid molecule. The additional nucleic acid molecule optionally includes sequence that is substantially identical or substantially complementary to at least some portion of the template nucleic acid molecule. The template nucleic acid molecule can be single-stranded or double-stranded and the additional nucleic acid molecule can independently be single-stranded or doublestranded. Amplification is the linear or exponential replication of a nucleic acid molecule. In some embodiments, such amplification can be performed using isothermal conditions; in other embodiments, such amplification can include thermocycling. In some embodiments, the amplification is a multiplex amplification that includes the simultaneous amplification of a plurality of target sequences in a single amplification reaction. In some embodiments, "amplification" includes amplification of at least some portion of DNA and RNA based nucleic acids alone, or in combination. The amplification reaction can include any of the amplification processes known to one of ordinary skill in the art. In some embodiments, the amplification reaction includes polymerase chain reaction (PCR).

[0074] As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202), which describe a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of DNA without cloning or purification. In one embodiment, this process for amplifying the polynucleotide of interest includes introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired polynucleotide of interest, followed by a series of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded polynucleotide of interest. The mixture is denatured at a higher temperature first and the primers are then annealed to complementary sequences within the polynucleotide of interest molecule. Following annealing, the primers are extended with a polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (referred to as thermocycling) to obtain a high concentration of an amplified segment of the desired polynucleotide of interest. The length of the amplified segment of the desired polynucleotide of interest (amplicon) is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of repeating the process, the method is referred to as PCR. Because the desired amplified segments of the polynucleotide of interest become the predominant nucleic acid sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified". In a modification to the method discussed above, the target nucleic acid molecules can be PCR amplified using a plurality of different primer pairs, in some cases, one or more primer pairs per target nucleic acid molecule of interest, thereby forming a multiplex PCR reaction.

[0075] As used herein, the term "adapter" and its derivatives, e g., universal adapter, refers generally to any linear polynucleotide that can be attached to a target nucleic acid. An adapter can be single- stranded or double-stranded DNA, or can include both doublestranded and single-stranded regions. An adapter can include a universal sequence that is substantially identical, or substantially complementary, to at least a portion of a primer, for example a universal primer; a barcode to assist with downstream error correction, identification, or sequencing; and / or a unique molecular identifier. In some embodiments, the adapter is substantially non-complementary to the 3' end or the 5' end of any target sequence present in the sample. The terms "adaptor" and "adapter" are used interchangeably.

[0076] As used herein, the term "universal," when used to describe a nucleotide sequence, refers to a region of sequence that is common to two or more nucleic acid molecules where themolecules also have regions of sequence that differ from each other. A universal sequence that is present in different members of a collection of nucleic acids can be used as, for instance, a "landing pad" in a subsequent step to anneal a nucleotide sequence that can be used as a primer for addition of another nucleotide sequence, such as a universal sequence, to a target nucleic acid such as a fragmented double stranded genomic DNA or a cDNA. A universal sequence that is present in different members of a collection of nucleic acids can allow capture of multiple different nucleic acids using a population of capture nucleic acids, e.g., capture oligonucleotides, also referred to as capture oligos and nucleotide capture sequences, that are complementary to a portion of the universal sequence, e.g., a universal capture sequence. Non-limiting examples of universal capture sequences include sequences that are identical to or complementary to P5 and P7 primers and sequences that are identical to or complementary to a CSU. Similarly, a universal sequence present in different members of a collection of molecules can allow the replication (e.g., sequencing) or amplification of multiple different nucleic acids using a population of universal primers that are complementary to a portion of the universal sequence, e.g., a universal anchor sequence. In one embodiment universal anchor sequences are used as a site to which a universal primer (e.g., a sequencing primer for read 1 or read 2) anneals for sequencing. A capture oligo or a universal primer can therefore include a sequence that can hybridize specifically to a universal sequence.

[0077] As used herein, a “barcode” (also referred to as an “index” or a “tag”) refers to a unique nucleic acid that can be used to identify a sample or source of a particular fragment of genomic DNA or a RNA, or a compartment in which a particular fragment of genomic DNA or a RNA was present. The barcode can be present in solution or on a template particle, or attached to or associated with a template particle and released in solution or compartment. When samples are derived from multiple sources such as multiple cells, the nucleic acids in each nucleic acid sample can be tagged with different nucleic acid barcodes such that the source of the sample can be identified. Any suitable barcode or set of barcodes can be used, as known in the art and as exemplified by the disclosures of U.S. Pat. No. 8,053,192, PCT Publication No. WO 05 / 068656, and U.S. Pat. Publication No.2013 / 0274117.

[0078] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0079] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. The use of "and / or" in some instances does not imply that the use of "or" in other instances may not mean "and / or."

[0080] The words "preferred" and "preferably" refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.

[0081] As used herein, "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" or the like are used in their open ended inclusive sense, and generally mean "include, but not limited to," "includes, but not limited to," or "including, but not limited to."

[0082] It is understood that wherever embodiments are described herein with the language "have,""has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" and the like, otherwise analogous embodiments described in terms of "consisting of' and / or "consisting essentially of are also provided. The term "consisting of means including, and limited to, whatever follows the phrase "consisting of." That is, "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0083] As used herein, "providing" in the context of a cell, a nucleus, or a composition means making the cell, the nucleus, or the composition, purchasing the cell, the nucleus, or the composition, or otherwise obtaining the cell, the nucleus, or the composition.

[0084] Reference throughout this specification to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0085] Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0086] In the description herein particular embodiments may be described in isolation for clarity.Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.

[0087] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0088] In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0089] The invention is defined in the claims. However, a non-exhaustive listing of non-limiting exemplary aspects is provided below. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0090] Exemplary Aspects

[0091] Aspect 1. A library preparation method, the method including: providing isolated nuclei including fragmented double stranded genomic DNA, wherein the ends of each fragment include ligated exogenous DNA; combining the isolated nuclei with template particles, wherein the template particles include a plurality of a nucleotide capture sequence attached by the 5’ end to the template particles; generating a plurality of uniform partitions near- instantly that encapsulate a single one of the template particles and a single one of the isolated nuclei to form pre-templated instant partitions (PIPs); delivering a micellized lysis agent to the isolated nuclei in the PIPs; lysing the isolated nuclei to release in each PIP the fragmented double stranded genomic DNA.

[0092] Aspect 2. The method of any of aspects 1 or 3-37, wherein the template particles further include a plurality of a second nucleotide capture sequence attached by the 5’ end to the template particles, and wherein the lysing further includes releasing in each PIP RNA present in the isolated nuclei.

[0093] Aspect 3. The method of any of aspects 1-2 or 4-37, wherein the providing includes fixing isolated nuclei or cells including nuclei.

[0094] Aspect 4. The method of any of aspects 1-3 or 5-37, wherein the fixing includes exposing the isolated nuclei or cells including nuclei to DSP, formaldehyde, gluteraldehyde, one or more succinimide ester, or a combination thereof.

[0095] Aspect 5. The method of any of aspects 1-4 or 6-37, wherein the providing includes permeabilizing cells including nuclei.

[0096] Aspect 6. The method of any of aspects 1-5 or 7-37, wherein the providing includes permeabilizing isolated nuclei.

[0097] Aspect 7. The method of any of aspects 1-6 or 8-37, wherein the permeabilizing includes treating the cells or the isolated nuclei with a detergent, a surfactant, or a combination thereof.

[0098] Aspect 8. The method of any of aspects 1-7 or 9-37, wherein the surfactant includes Triton X-100.

[0099] Aspect 9. The method of any of aspects 1-8 or 10-37, wherein the providing includes isolating nuclei from cells.

[0100] Aspect 10. The method of any of aspects 1-9 or 11-37, wherein the providing includes exposing permeabilized cells or permeabilized nuclei to transposome complexes, wherein the transposome complexes include exogenous DNA, and wherein the transposome complexes (i) fragment accessible double stranded genomic DNA and (ii) ligate the exogenous DNA to result in the fragments including the ligated exogenous DNA.

[0101] Aspect 11. The method of any of aspects 1-10 or 12-37, wherein the fragments are double stranded and the exogenous DNA include a double stranded region and a single stranded region, and wherein the double stranded region of the exogenous DNA is ligated to each end of the fragments.

[0102] Aspect 12. The method of any of aspects 1-11 or 13-37, wherein the transposome complexes further include an associated antigen binding molecule.

[0103] Aspect 13. The method of any of aspects 1-12 or 14-37, wherein the antigen binding molecule includes an antibody or aptamer that binds to a DNA-associated protein.

[0104] Aspect 14. The method of any of aspects 1-13 or 15-37, wherein the providing includes exposing the permeabilized cells or the permeabilized nuclei to two or more populations of transposome complexes, wherein each population of transposome complexes include anassociated antigen binding molecule that binds to a different DNA-associated protein, and wherein the exogenous DNA of each population of transposome complexes includes a barcode that is unique compared to the barcodes of the other populations of transposome complexes.

[0105] Aspect 15. The method of any of aspects 1-14 or 16-37, wherein the combining includes:combining the isolated nuclei with the template particles in a first fluid and adding a second fluid to the first fluid; and wherein the generating includes: agitating the fluids to generate the PIPs that contain a single one of the isolated nuclei and a single one of the template particles.

[0106] Aspect 16. The method of any of aspects 1-15 or 17-37, wherein the agitating includes vortexing.

[0107] Aspect 17. The method of any of aspects 1-16 or 18-37, wherein the first fluid and the second fluid are immiscible.

[0108] Aspect 18. The method of any of aspects 1-17 or 19-37, wherein the combining and the generating are performed in a tube, wherein the tube is a centrifuge, microcentrifuge, or polymerase chain reaction (PCR) tube.

[0109] Aspect 19. The method of any of aspects 1-18 or 20-37, wherein the PIPs are monodisperse aqueous droplets.

[0110] Aspect 20. The method of any of aspects 1-19 or 21-37, further including prior to the delivering step, creating the micellized lysis agent using a fluorosurfactant.

[0111] Aspect 21. The method of any of aspects 1-20 or 22-37, wherein the micellized lysis agent includes a detergent or a heat activated enzyme.

[0112] Aspect 22 The method of any of aspects 1-21 or 23-37, wherein the micellized lysis agent is ionic type detergent, non-ionic type detergent, or zwitterionic type detergent.

[0113] Aspect 23. The method of any of aspects 1-22 or 24-37, wherein the micellized lysis agent includes sodium dodecyl sulfate (SDS), Sarkosyl, sodium deoxycholate, Capstone FS-61, or CT AB.

[0114] Aspect 24. The method of any of aspects 1-23 or 25-37, wherein the micellized lysis agent includes Triton X-100, Triton X-114, NP-40, Tween-80, Brij 35, Octyl glucoside, or octyl thioglucoside.

[0115] Aspect 25. The method of any of aspects 1-24 or 26-37, wherein the micellized lysis agent includes CHAPS, CHAPSO, ASB-14, ASB-16, SB-3-10, or SB-3-12.

[0116] Aspect 26. The method of any of aspects 1-25 or 27-37, wherein the ligated exogenous DNA includes a 3 ’ end that is complementary to a 3 ’ end of the nucleotide capture sequence, further including hybridizing the 3 ’ end of the ligated exogenous DNA to the 3 ’ end of the nucleotide capture sequence within each PIP.

[0117] Aspect 27. The method of any of aspects 1-26 or 28-37, wherein the ligated exogenous DNA includes a 5’ end that includes an amplification handle.

[0118] Aspect 28. The method of any of aspects 1-27 or 29-37, wherein the ligated exogenous DNA includes a 5’ end that includes a transposome complex-specific barcode.

[0119] Aspect 29. The method of any of aspects 1-28 or 30-37, wherein the fragments are double stranded and the exogenous DNA include a double stranded region and a single stranded region including two strands, wherein the double stranded region of the ligated exogenous DNA is ligated to each end of the fragments, wherein one strand of the single stranded region includes a 5’ end including an amplification handle, and the other strand of the single stranded region includes a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence.

[0120] Aspect 30. The method of any of aspects 1-29 or 31-37, wherein the 3’ end of the second nucleotide capture sequence includes a Poly-T domain, a randomer domain, or a targetspecific domain, further including hybridizing the 3’ end of the second nucleotide capture sequence to the RNA within each PIP.

[0121] Aspect 31. The method of any of aspects 1-30 or 32-37, wherein the 3’ end of the second nucleotide capture sequence includes the Poly-T domain, and wherein mRNA is hybridized to the 3’ of the second nucleotide capture sequence.

[0122] Aspect 32. The method of any of aspects 1- 31 or 33-37, further including extending the 3’ end of the nucleotide capture sequences using the hybridized fragment including the ligated exogenous DNA as template to produce a complementary DNA (cDNA).

[0123] Aspect 33. The method of any of aspects 1-32 or 34-37, wherein the extending includes an enzyme selected from a reverse transcriptase or a DNA polymerase.

[0124] Aspect 34. The method of any of aspects 1-33 or 35-37, wherein the DNA polymerase includes phi29 or Klenow large fragment.

[0125] Aspect 35. The method of any of aspects 1-34 or 36-37, wherein the ligated exogenous DNA includes a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence, wherein the hybridizing further including hybridizing the 3’ end of the ligated exogenous DNA to the 3’ end of the nucleotide capture sequence within each PIP.

[0126] Aspect 36. The method of any of aspects 1-35 or 37, further including forming cDNA;forming amplicons from the cDNA; and sequencing the amplicons to generate a chromatin accessibility profile.

[0127] Aspect 37. The method of any of aspects 1-36, further including forming cDNA; forming amplicons from the cDNA; and sequencing the amplicons to generate a chromatin accessibility profile and a transcriptome.

[0128] Aspect 38. A kit including in separate compartments: a transposome complex including an associated antigen binding molecule; and a template particle.

[0129] Aspect 39. The kit of aspect 38, wherein the transposome complex includes an exogenous DNA, wherein the exogenous DNA includes a barcode.

[0130] Aspect 40. A library preparation method, the method including: providing nuclei or cells including fragmented double stranded genomic DNA, wherein the ends of each fragmentinclude attached exogenous DNA; combining the nuclei or cells with template particles, wherein the template particles include a plurality of a nucleotide capture sequence attached by the 5’ end to the template particles; generating a plurality of uniform partitions near- instantly that encapsulate a single one of the template particles and a single one of the nuclei or cells to form pre-templated instant partitions (PIPs); lysing the nuclei or cells to release in each PIP the fragmented double stranded genomic DNA.

[0131] Aspect 41. The method of any of aspects 39-40 or 42-86, wherein the template particles further include a plurality of a second nucleotide capture sequence attached by the 5’ end to the template particles, and wherein the lysing further includes releasing in each PIP RNA present in the nuclei or cells.

[0132] Aspect 42. The method of any of aspects 39-41 or 43-86, wherein the providing includes fixing nuclei or cells.

[0133] Aspect 43. The method of any of aspects 39-42 or 44-86, wherein the fixing includes exposing the nuclei or cells to DSP, formaldehyde, gluteraldehyde, one or more succinimide ester, or a combination thereof.

[0134] Aspect 44. The method of any of aspects 39-43 or 45-86, wherein the providing includes permeabilizing cells.

[0135] Aspect 45. The method of any of aspects 39-44 or 46-86, wherein the providing includes permeabilizing nuclei.

[0136] Aspect 46. The method of any of aspects 39-45 or 47-86, wherein the permeabilizing includes treating the cells or the nuclei with a detergent, a surfactant, or a combination thereof.

[0137] Aspect 47. The method of any of aspects 39-46 or 48-86, wherein the surfactant includes Triton X-100.

[0138] Aspect 48. The method of any of aspects 39-47 or 49-86, wherein the providing includes isolating nuclei from cells.

[0139] Aspect 49. The method of any of aspects 39-48 or 50-86, wherein the providing includes exposing permeabilized cells or permeabilized nuclei to transposome complexes, wherein the transposome complexes include exogenous DNA, and wherein the transposome complexes (i) fragment accessible double stranded genomic DNA and (ii) ligate the exogenous DNA to result in the fragments including the attached exogenous DNA.

[0140] Aspect 50. The method of any of aspects 39-49 or 51-86, wherein the fragments are double stranded and the exogenous DNA include a double stranded region and a single stranded region, and wherein the double stranded region of the exogenous DNA is ligated to each end of the fragments.

[0141] Aspect 51. The method of any of aspects 39-50 or 52-86, wherein the attached exogenous DNA includes a universal complementary sequence (CSU), and wherein the nucleotide capture sequence includes at the 3’ end a nucleotide sequence that is complementary to at least a portion of the CSU.

[0142] Aspect 52. The method of any of aspects 39-51 or 53-86, wherein the attached exogenous DNA includes a Poly-A region, and wherein the nucleotide capture sequence includes at the 3’ end a Poly-T domain, a randomer domain, a target-specific domain, or a disrupted homopolymer.

[0143] Aspect 53. The method of any of aspects 39-52 or 54-86, wherein the transposome complexes further include an associated antigen binding molecule.

[0144] Aspect 54. The method of any of aspects 39-53 or 55-86, wherein the antigen binding molecule includes an antibody or aptamer that binds to a DNA-associated protein.

[0145] Aspect 55. The method of any of aspects 39-54 or 56-86, wherein the DNA-associated protein is a transcription factor, a histone, a polymerase, a zinc finger protein, a helix-turn- helix protein, a leucine zipper protein, or a DNA repair protein.

[0146] Aspect 56. The method of any of aspects 39-55 or 57-86, wherein the providing includes exposing the permeabilized cells or the permeabilized nuclei to two or more populations of transposome complexes, wherein each population of transposome complexes include anassociated antigen binding molecule that binds to a different DNA-associated protein, and wherein the exogenous DNA of each population of transposome complexes includes a barcode that is unique compared to the barcodes of the other populations of transposome complexes.

[0147] Aspect 57. The method of any of aspects 39-56 or 58-86, wherein the combining includes:combining the nuclei or cells with the template particles in a first fluid and adding a second fluid to the first fluid; and wherein the generating includes: agitating the fluids to generate the PIPs that contain a single one of the nuclei and a single one of the template particles.

[0148] Aspect 58. The method of any of aspects 39-57 or 59-86, wherein the agitating includes vortexing.

[0149] Aspect 59. The method of any of aspects 39-58 or 60-86, wherein the first fluid and the second fluid are immiscible.

[0150] Aspect 60. The method of any of aspects 39-59 or 61-86, wherein the combining and the generating are performed in a tube, wherein the tube is a centrifuge, microcentrifuge, or polymerase chain reaction (PCR) tube.

[0151] Aspect 61. The method of any of aspects 39-60 or 62-86, wherein the PIPs are monodisperse aqueous droplets.

[0152] Aspect 62. The method of any of aspects 39-61 or 63-86, further including contacting the nuclei or cells within the PIPs with a lysis agent.

[0153] Aspect 63. The method of any of aspects 39-62 or 64-86, wherein the template particles include the lysis agent, and wherein the contacting includes release of the lysis agent from the template particles.

[0154] Aspect 64. The method of any of aspects 39-63 or 65-86, wherein the contacting includes delivery of the lysis agent by micelle.

[0155] Aspect 65. The method of any of aspects 39-64 or 66-86, further including prior to the lysing, creating the micellized lysis agent using a fluorosurfactant.

[0156] Aspect 66. The method of any of aspects 39-65 or 67-86, wherein the lysis agent includes a detergent or a heat activated enzyme.

[0157] Aspect 67. The method of any of aspects 39-66 or 68-86, wherein the lysis agent is ionic type detergent, non-ionic type detergent, or zwitterionic type detergent.

[0158] Aspect 68. The method of any of aspects 39-67 or 69-86, wherein the lysis agent includes sodium dodecyl sulfate (SDS), Sarkosyl, sodium deoxycholate, Capstone FS-61, or CTAB.

[0159] Aspect 69. The method of any of aspects 39-68 or 70-86, wherein the lysis agent includes Triton X-100, Triton X-114, NP-40, Tween-80, Brij 35, Octyl glucoside, or octyl thioglucoside.

[0160] Aspect 70. The method of any of aspects 39-69 or 71-86, wherein the lysis agent includes CHAPS, CHAPSO, ASB-14, ASB-16, SB-3-10, or SB-3-12.

[0161] Aspect 71. The method of any of aspects 39-70 or 72-86, wherein the attached exogenous DNA includes a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence, further including hybridizing the 3’ end of the attached exogenous DNA to the 3’ end of the nucleotide capture sequence within each PIP.

[0162] Aspect 72. The method of any of aspects 39-71 or 73-86, wherein the 3’ end of the attached exogenous DNA includes a CSU.

[0163] Aspect 73. The method of any of aspects 39-72 or 74-86, wherein the attached exogenous DNA includes a 5’ end that includes an amplification handle.

[0164] Aspect 74. The method of any of aspects 39-73 or 75-86, wherein the attached exogenous DNA includes a transposome complex-specific barcode.

[0165] Aspect 75. The method of any of aspects 39-74 or 76-86, wherein the fragments are double stranded and the attached exogenous DNA include a double stranded region and a single stranded region including two strands, wherein the double stranded region of the attached exogenous DNA is ligated to each end of the fragments, wherein one strand of the single stranded region includes a 5’ end including an amplification handle, and the other strand ofthe single stranded region includes a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence.

[0166] Aspect 76. The method of any of aspects 39-75 or 77-86, wherein the 3’ end of the attached exogenous DNA includes a CSU.

[0167] Aspect 77. The method of any of aspects 39-76 or 78-86, wherein the fragments are double stranded and the attached exogenous DNA include a double stranded region and a single stranded region including one strand, wherein the double stranded region of the attached exogenous DNA is ligated to each end of the fragments, wherein the strand of the single stranded region includes a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence.

[0168] Aspect 78. The method of any of aspects 39-77 or 79-86, wherein the 3’ end of the attached exogenous DNA includes a CSU.

[0169] Aspect 79. The method of any of aspects 39-78 or 80-86, wherein the 3’ end of the second nucleotide capture sequence includes a Poly-T domain, a randomer domain, a target-specific domain, or a disrupted homopolymer, further including hybridizing the 3’ end of the second nucleotide capture sequence to the RNA within each PIP.

[0170] Aspect 80. The method of any of aspects 39-79 or 81-86, wherein the 3’ end of the second nucleotide capture sequence includes the Poly-T domain, and wherein mRNA is hybridized to the 3’ of the second nucleotide capture sequence.

[0171] Aspect 81. The method of any of aspects 39-80 or 82-86, further including extending the 3’ end of the nucleotide capture sequences using the hybridized fragment including the attached exogenous DNA as template to produce a complementary DNA (cDNA).

[0172] Aspect 82. The method of any of aspects 39-81 or 83-86, wherein the extending includes an enzyme selected from a reverse transcriptase or a DNA polymerase.

[0173] Aspect 83. The method of any of aspects 39-82 or 84-86, wherein the DNA polymerase includes phi29 or Klenow large fragment.

[0174] Aspect 84. The method of any of aspects 39-83 or 85-86, wherein the attached exogenous DNA includes a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence, wherein the hybridizing further including hybridizing the 3’ end of the attached exogenous DNA to the 3’ end of the nucleotide capture sequence within each PIP.

[0175] Aspect 85. The method of any of aspects 39-84 or 86, further including forming cDNA;forming amplicons from the cDNA; and sequencing the amplicons to generate a chromatin accessibility profde.

[0176] Aspect 86. The method of any of aspects 39-85, further including forming cDNA; forming amplicons from the cDNA; and sequencing the amplicons to generate a chromatin accessibility profde and a transcriptome.

[0177] Incorporation by Reference

[0178] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and / or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.

[0179] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated tothe contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0180] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0181] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

CLAIMS1. A library preparation method, the method comprising:providing isolated nuclei comprising fragmented double stranded genomic DNA, wherein the ends of each fragment comprise ligated exogenous DNA;combining the isolated nuclei with template particles, wherein the template particles comprise a plurality of a nucleotide capture sequence attached by the 5’ end to the template particles;generating a plurality of uniform partitions near-instantly that encapsulate a single one of the template particles and a single one of the isolated nuclei to form pre-templated instant partitions (PIPs);delivering a micellized lysis agent to the isolated nuclei in the PIPs;lysing the isolated nuclei to release in each PIP the fragmented double stranded genomic DNA.

2. The method of claim 1, wherein the template particles further comprise a plurality of a second nucleotide capture sequence attached by the 5’ end to the template particles, and wherein the lysing further comprises releasing in each PIP RNA present in the isolated nuclei.

3. The method of claim 1, wherein the providing comprises fixing isolated nuclei or cells comprising nuclei.

4. The method of claim 3, wherein the fixing comprises exposing the isolated nuclei or cells comprising nuclei to DSP, formaldehyde, gluteraldehyde, one or more succinimide ester, or a combination thereof.

5. The method of claim 1, wherein the providing comprises permeabilizing cells comprising nuclei.

6. The method of claim 1, wherein the providing comprises permeabilizing isolated nuclei.

7. The method of claim 5 or 6, wherein the permeabilizing comprises treating the cells or the isolated nuclei with a detergent, a surfactant, or a combination thereof.

8. The method of claim 7, wherein the surfactant comprises Triton X-100.

9. The method of claim 1, wherein the providing comprises isolating nuclei from cells.

10. The method of claim 1, wherein the providing comprises exposing permeabilized cells or permeabilized nuclei to transposome complexes, wherein the transposome complexes comprise exogenous DNA, and wherein the transposome complexes (i) fragment accessible double stranded genomic DNA and (ii) ligate the exogenous DNA to result in the fragments comprising the ligated exogenous DNA.

11. The method of claim 10, wherein the fragments are double stranded and the exogenous DNA comprise a double stranded region and a single stranded region, and wherein the double stranded region of the exogenous DNA is ligated to each end of the fragments.

12. The method of claim 10, wherein the transposome complexes further comprise an associated antigen binding molecule.

13. The method of claim 12, wherein the antigen binding molecule comprises an antibody or aptamer that binds to a DNA-associated protein.

14. The method of claim 10, wherein the providing comprises exposing the permeabilized cells or the permeabilized nuclei to two or more populations of transposome complexes, wherein each population of transposome complexes comprise an associated antigen binding molecule that binds to a different DNA-associated protein, and wherein the exogenous DNA of each population of transposome complexes comprises a barcode that is unique compared to the barcodes of the other populations of transposome complexes.

15. The method of claim 1, wherein the combining comprises:combining the isolated nuclei with the template particles in a first fluid and adding a second fluid to the first fluid; andwherein the generating comprises:agitating the fluids to generate the PIPs that contain a single one of the isolated nuclei and a single one of the template particles.

16. The method of claim 15, wherein the agitating comprises vortexing.

17. The method of claim 15, wherein the first fluid and the second fluid are immiscible.

18. The method of claim 1, wherein the combining and the generating are performed in a tube, wherein the tube is a centrifuge, microcentrifuge, or polymerase chain reaction (PCR) tube.

19. The method of claim 1, wherein the PIPs are monodisperse aqueous droplets.

20. The method of claim 1, further comprising prior to the delivering step, creating the micellized lysis agent using a fluorosurfactant.

21. The method of claim 1, wherein the micellized lysis agent comprises a detergent or a heat activated enzyme.22 The method of claim 1, wherein the micellized lysis agent is ionic type detergent, non-ionic type detergent, or zwitterionic type detergent.

23. The method of claim 22, wherein the micellized lysis agent comprises sodium dodecyl sulfate (SDS), Sarkosyl, sodium deoxycholate, Capstone FS-61, or CTAB.

24. The method of claim 22, wherein the micellized lysis agent comprises Triton X-100, Triton X-l 14, NP-40, Tween-80, Brij 35, Octyl glucoside, or octyl thioglucoside.

25. The method of claim 22, wherein the micellized lysis agent comprises CHAPS, CHAPSO, ASB-14, ASB-16, SB-3-10, or SB-3-12.

26. The method of claim 1, wherein the ligated exogenous DNA comprises a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence, further comprising hybridizing the 3’ end of the ligated exogenous DNA to the 3’ end of the nucleotide capture sequence within each PIP.

27. The method of claim 26, wherein the ligated exogenous DNA comprises a 5’ end that comprises an amplification handle.

28. The method of claim 26, wherein the ligated exogenous DNA comprises a 5’ end that comprises a transposome complex-specific barcode.

29. The method of claim 1, wherein the fragments are double stranded and the exogenous DNA comprise a double stranded region and a single stranded region comprising two strands, wherein the double stranded region of the ligated exogenous DNA is ligated to each end of the fragments, wherein one strand of the single stranded region comprises a 5’ end comprising an amplification handle, and the other strand of the single stranded region comprises a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence.

30. The method of claim 2, wherein the 3’ end of the second nucleotide capture sequence comprises aPoly-T domain, a randomer domain, or a target-specific domain, further comprising hybridizing the 3’ end of the second nucleotide capture sequence to the RNA within each PIP.

31. The method of claim 30, wherein the 3’ end of the second nucleotide capture sequence comprises the Poly-T domain, and wherein mRNA is hybridized to the 3’ of the second nucleotide capture sequence.

32. The method of claim 26, further comprising extending the 3’ end of the nucleotide capture sequences using the hybridized fragment comprising the ligated exogenous DNA as template to produce a complementary DNA (cDNA).

33. The method of claim 32, wherein the extending comprises an enzyme selected from a reverse transcriptase or a DNA polymerase.

34. The method of claim 33, wherein the DNA polymerase comprises phi29 or Klenow large fragment.

35. The method of claim 31, wherein the ligated exogenous DNA comprises a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence, wherein the hybridizing further comprising hybridizing the 3’ end of the ligated exogenous DNA to the 3’ end of the nucleotide capture sequence within each PIP.

36. The method of claim 1, further comprisingforming cDNA;forming amplicons from the cDNA;sequencing the amplicons to generate a chromatin accessibility profile.

37. The method of claim 2, further comprisingforming cDNA;forming amplicons from the cDNA;sequencing the amplicons to generate a chromatin accessibility profile and a transcriptome.

38. A kit comprising in separate compartments:a transposome complex comprising an associated antigen binding molecule; and a template particle.

39. The kit of claim 38, wherein the transposome complex comprises an exogenous DNA, wherein the exogenous DNA comprises a barcode.

40. A library preparation method, the method comprising:providing nuclei or cells comprising fragmented double stranded genomic DNA, wherein the ends of each fragment comprise attached exogenous DNA;combining the nuclei or cells with template particles, wherein the template particles comprise a plurality of a nucleotide capture sequence attached by the 5’ end to the template particles;generating a plurality of uniform partitions near-instantly that encapsulate a single one of the template particles and a single one of the nuclei or cells to form pre-templated instant partitions (PIPs);lysing the nuclei or cells to release in each PIP the fragmented double stranded genomic DNA.

41. The method of claim 40, wherein the template particles further comprise a plurality of a second nucleotide capture sequence attached by the 5’ end to the template particles, and wherein the lysing further comprises releasing in each PIP RNA present in the nuclei or cells.

42. The method of claim 40, wherein the providing comprises fixing nuclei or cells.

43. The method of claim 42, wherein the fixing comprises exposing the nuclei or cells to DSP, formaldehyde, gluteraldehyde, one or more succinimide ester, or a combination thereof.

44. The method of claim 40, wherein the providing comprises permeabilizing cells.

45. The method of claim 40, wherein the providing comprises permeabilizing nuclei .

46. The method of claim 44 or 45, wherein the permeabilizing comprises treating the cells or the nuclei with a detergent, a surfactant, or a combination thereof.

47. The method of claim 46, wherein the surfactant comprises Triton X-100.

48. The method of claim 40, wherein the providing comprises isolating nuclei from cells.

49. The method of claim 40, wherein the providing comprises exposing permeabilized cells or permeabilized nuclei to transposome complexes, wherein the transposome complexes comprise exogenous DNA, and wherein the transposome complexes (i) fragment accessible double stranded genomic DNA and (ii) ligate the exogenous DNA to result in the fragments comprising the attached exogenous DNA.

50. The method of claim 49, wherein the fragments are double stranded and the exogenous DNA comprise a double stranded region and a single stranded region, and wherein the double stranded region of the exogenous DNA is ligated to each end of the fragments.

51. The method of claim 40, wherein the attached exogenous DNA comprises a universal complementary sequence (CSU), and wherein the nucleotide capture sequence comprises at the 3’ end a nucleotide sequence that is complementary to at least a portion of the CSU.

52. The method of claim 40, wherein the attached exogenous DNA comprises a Poly-A region, and wherein the nucleotide capture sequence comprises at the 3’ end a Poly-T domain, a randomer domain, a target-specific domain, or a disrupted homopolymer.

53. The method of claim 49, wherein the transposome complexes further comprise an associated antigen binding molecule.

54. The method of claim 53, wherein the antigen binding molecule comprises an antibody or aptamer that binds to a DNA-associated protein.

55. The method of claim 53, wherein the DNA-associated protein is a transcription factor, a histone, a polymerase, a zinc finger protein, a helix-tum-helix protein, a leucine zipper protein, or a DNA repair protein.

56. The method of claim 49, wherein the providing comprises exposing the permeabilized cells or the permeabilized nuclei to two or more populations of transposome complexes, wherein each population of transposome complexes comprise an associated antigen binding molecule that binds to a different DNA-associated protein, and wherein the exogenous DNA of each population of transposome complexes comprises a barcode that is unique compared to the barcodes of the other populations of transposome complexes.

57. The method of claim 40, wherein the combining comprises:combining the nuclei or cells with the template particles in a first fluid and adding a second fluid to the first fluid; andwherein the generating comprises:agitating the fluids to generate the PIPs that contain a single one of the nuclei and a single one of the template particles.

58. The method of claim 57, wherein the agitating comprises vortexing.

59. The method of claim 57, wherein the first fluid and the second fluid are immiscible.

60. The method of claim 40, wherein the combining and the generating are performed in a tube, wherein the tube is a centrifuge, microcentrifuge, or polymerase chain reaction (PCR) tube.

61. The method of claim 40, wherein the PIPs are monodisperse aqueous droplets.

62. The method of claim 40, further comprising contacting the nuclei or cells within the PIPs with a lysis agent.

63. The method of claim 62, wherein the template particles comprise the lysis agent, and wherein the contacting comprises release of the lysis agent from the template particles.

64. The method of claim 62, wherein the contacting comprises delivery of the lysis agent by micelle.

65. The method of claim 64, further comprising prior to the lysing, creating the micellized lysis agent using a fluorosurfactant.

66. The method of claim 62, wherein the lysis agent comprises a detergent or a heat activated enzyme.

67. The method of claim 62, wherein the lysis agent is ionic type detergent, non-ionic type detergent, or zwitterionic type detergent.

68. The method of claim 67, wherein the lysis agent comprises sodium dodecyl sulfate (SDS), Sarkosyl, sodium deoxy cholate, Capstone FS-61, or CTAB.

69. The method of claim 67, wherein the lysis agent comprises Triton X-100, Triton X-114, NP-40, Tween-80, Brij 35, Octyl glucoside, or octyl thioglucoside.

70. The method of claim 67, wherein the lysis agent comprises CHAPS, CHAPSO, ASB-14, ASB-16, SB-3-10, or SB-3-12.

71. The method of claim 40, wherein the attached exogenous DNA comprises a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence, further comprising hybridizing the 3’ end of the attached exogenous DNA to the 3’ end of the nucleotide capture sequence within each PIP.

72. The method of claim 71, wherein the 3’ end of the attached exogenous DNA comprises a CSU.

73. The method of claim 72, wherein the attached exogenous DNA comprises a 5’ end that comprises an amplification handle.

74. The method of claim 72, wherein the attached exogenous DNA comprises a transposome complex-specific barcode.

75. The method of claim 40, wherein the fragments are double stranded and the attached exogenous DNA comprise a double stranded region and a single stranded region comprising two strands, wherein the double stranded region of the attached exogenous DNA is ligated to each end of the fragments, wherein one strand of the single stranded region comprises a 5’ end comprising an amplification handle, and the other strand of the single stranded region comprises a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence.

76. The method of claim 75, wherein the 3’ end of the attached exogenous DNA comprises a CSU.

77. The method of claim 40, wherein the fragments are double stranded and the attached exogenous DNA comprise a double stranded region and a single stranded region comprising one strand, wherein the double stranded region of the attached exogenous DNA is ligated to each end of the fragments, wherein the strand of the single stranded region comprises a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence.

78. The method of claim 77, wherein the 3’ end of the attached exogenous DNA comprises a CSU.

79. The method of claim 41, wherein the 3’ end of the second nucleotide capture sequence comprises aPoly-T domain, a randomer domain, a target-specific domain, or a disrupted homopolymer, further comprising hybridizing the 3’ end of the second nucleotide capture sequence to the RNA within each PIP.

80. The method of claim 79, wherein the 3’ end of the second nucleotide capture sequence comprises the Poly-T domain, and wherein mRNA is hybridized to the 3’ of the second nucleotide capture sequence.

81. The method of claim 72, further comprising extending the 3 ’ end of the nucleotide capture sequences using the hybridized fragment comprising the attached exogenous DNA as template to produce a complementary DNA (cDNA).

82. The method of claim 81, wherein the extending comprises an enzyme selected from a reverse transcriptase or a DNA polymerase.

83. The method of claim 82, wherein the DNA polymerase comprises phi29 or Klenow large fragment.

84. The method of claim 80, wherein the attached exogenous DNA comprises a 3’ end that is complementary to a 3’ end of the nucleotide capture sequence, wherein the hybridizing further comprising hybridizing the 3’ end of the attached exogenous DNA to the 3’ end of the nucleotide capture sequence within each PIP.

85. The method of claim 40, further comprisingforming cDNA;forming amplicons from the cDNA;sequencing the amplicons to generate a chromatin accessibility profile.

86. The method of claim 41, further comprisingforming cDNA;forming amplicons from the cDNA;sequencing the amplicons to generate a chromatin accessibility profile and a transcriptome.

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