Tagcentric single-cell ATAC+rna

Abstract

Improved tagmentation compositions and methods are provided. These include for example a method for efficient RNA-seq ATAC-seq multiomic approaches.

Description

PATENT Attorney DocketNo. 094868-1533629-122910PC Client Ref No. BRP01316-WO TAGCENTRIC SINGLE-CELL ATAC+RNACROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority to US Provisional Application No.63 / 754,806, filed February 6, 2025, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a sequence listing filed in electronic form as an xml file entitled 094868-1533629-122910PC_SL.xml, created on November 26. 2025, and having size of 6,598 bytes. The content of the sequence listing is incorporated herein in its entirety.BACKGROUND OF THE INVENTION

[0003] Single-cell multiomics is a technique that interrogates multiple molecular layers, such as DNA, RNA, proteins, and epigenetic modifications within individual cells. This method offers a detailed, high-resolution view of cellular states and functions, going far beyond the capabilities of traditional bulk analysis or single-modality approaches. By integrating data across multiple dimensions, single-cell multiomics reveals the cellular mechanisms underlying biological processes and diseases. It enables precise identification of cell types, uncovers disease pathways, and highlights variations in drug responses, paving the way for more effective treatments. To fully realize the potential of this technology, it can be useful to develop workflows that are cost-effective, high-throughput, and user-friendly, ensuring broader accessibility and application in both research and clinical settings.BRIEF SUMMARY OF THE INVENTION

[0004] In some embodiments, the disclosure provides a transposase comprising an amino acid sequence at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO:1 and comprising lysine (K) at position 54, arginine (R) at position 212, arginine (R) at position 214, arginine (R) at position 251, valine (V) at position 338, and proline (P) at position 372 as numbered with reference to SEQ ID NO:1. In some embodiments, position 56 is A. In some embodiments, the transposase comprises SEQ ID NO:3.

[0005] Also provided is a kit comprising the tagmentase as described above or elsewhere herein. In some embodiments, the kit further comprises oligonucleotide adaptors (e.g.. two identical or non-identical, partially double-stranded oligonucleotides) comprising a mosaic end (ME) sequence.

[0006] Also provided is transposome composed of the transposase as described above or elsewhere herein and oligonucleotide adaptors comprising a mosaic end (ME) sequence.

[0007] Also provided is a solution comprising polyethylene glycol (PEG) and the transposome as described above or elsewhere herein. In some embodiments, the transposase comprises SEQ ID NO:3. In some embodiments, the PEG has an average molecular weight between 200-10,000 g / mol, optionally 400-8000 g / mol. In some embodiments, the PEG is at a concentration of 0.5-10% w / v, optionally 4-8%. In some embodiments, the solution comprises one or more fixed and permeabilized cell.

[0008] Also provided is a nucleic acid encoding the tagmentase as described above or elsewhere herein.

[0009] Also provided is an expression cassette comprising a promoter operably linked to the nucleic acid as described above or elsewhere herein.

[0010] Also provided is a cell comprising the nucleic acid as described above or elsewhere herein or the expression cassette as described above or elsewhere herein.

[0011] Also provided is a method of producing the tagmentase as described above or elsewhere herein, the method comprising incubating the cell under conditions resulting in expression of the tagmentase in the cell, and optionally purifying the tagmentase from the cell.

[0012] Also provided is a method of tagmenting DNA / RNA duplex nucleic acids in the absence of dimethylformamide (DMF). In some embodiments, the method comprises contacting DNA / RNA duplex nucleic acids in a solution with a transposome composed of a transposase and oligonucleotide adaptors under conditions such that the transposase fragments the nucleic acids and introduces the oligonucleotide adapters into breakpoints in the fragmented nucleic acids, wherein the contacting is performed in the presence of polyethylene glycol (PEG) and the absence of DMF. In some embodiments, the transposase comprises an amino acid sequence at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO:1 and comprising K at position 54, R at position 212, R at position 214, R at position251, V at position 338, and P at position 372 as numbered with reference to SEQ ID NO:1. In some embodiments, the transposase comprises SEQ ID NO:3. In some embodiments, the PEG has an average molecular weight between 200-10,000 g / mol, optionally 400-8000 g / mol. In some embodiments, the PEG is at a concentration of 0.5-10% w / v, optionally 4-8%. In some embodiments, the solution comprises fixed and permeabilized cells.

[0013] Also provided is a method of preparing tagmented cDNA and gDNA from a cell. In some embodiments, the method comprisesproviding fixed and permeabilized cells comprising RNA and gDNA;diffusing into the cells a first transposome, composed of a first transposase and a first and second partially double-stranded oligonucleotide adaptor, that selectively fragments the gDNA while leaving the RNA intact, wherein the first transposase introduces the oligonucleotide adapters onto 5 ’ ends at breakpoints in the fragmented gDNA, wherein the first and second partially double-stranded oligonucleotide adaptors have different first and second single-stranded 5’ sequences, respectively;performing reverse transcription of the RNA in the cells by contacting the RNA with oligonucleotide primers and a reverse transcriptase, annealing the oligonucleotide primers to the RNA and extending the oligonucleotide primers with the reverse transcriptase to form first strand cDNA / RNA duplexes;optionally removing the oligonucleotide primers; andcontacting the first strand cDNA / RNA duplexes with a solution comprising a second transposome, composed of a second transposase and two copies of the first partially doublestranded oligonucleotide adaptor, that fragments cDNA / RNA duplexes, wherein the second transposase introduces the first oligonucleotide adapters onto 5' ends of breakpoints in the fragmented cDNA, thereby generating cells comprising tagmented cDNA and gDNA.

[0014] In some embodiments, the cells are in a bulk solution during the contacting with the first and second transposomes and the performing of reverse transcription. In some embodiments, following the contacting the cDNA / RNA duplexes with a second transposome, partitioning the cells to form partitions containing single cells. In some embodiments, partition-specific barcode oligonucleotides are attached to common sequences at the end of the tagmented cDNA / RNA duplexes and gDNA in the partitions. In some embodiments, the partition-specific barcode oligonucleotides are linked to a solid support and are combinedwith the cells comprising tagmented cDNA / RNA duplexes and gDNA and wherein the partitioning comprises partitioning the solid supports with the cells. In some embodiments, in the partitions, optionally raising the temperature of the tagmented gDNA and cDNA / RNA duplexes to displace the second tagmentases; gap-filling with a first polymerase the first and second single-stranded 5’ sequences of the tagmented gDNA and the first single-stranded 5’ sequences of the tagmented cDNA / RNA duplexes to form 3’ ends; annealing the partitionspecific barcode oligonucleotides to the 3’ ends formed from gap-filling the first singlestranded 5’ sequences on the gDNA and the cDNA / RNA duplexes and extending the partition-specific barcode oligonucleotides with the first polymerase or a second polymerase to form barcoded tagmented cDNA / RNA duplexes and barcoded tagmented gDNA; and generating copies of barcoded tagmented cDNA / RNA duplexes by extending the partitionspecific barcode oligonucleotide and a cDNA-specific reverse primer with a second polymerase and generating copies of barcoded tagmented gDNA by extending the partitionspecific barcode oligonucleotide and a gDNA-specific reverse primer with the second polymerase; and combining contents of the partitions, thereby forming a solution comprising copies of barcoded tagmented first strand cDNA and copies of barcoded tagmented gDNA.

[0015] In some embodiments, one of the cDNA-specific reverse primer or the gDNA-specific reverse primer is biotinylated such that one of the copies of barcoded tagmented cDNA / RNA duplexes or the copies of barcoded tagmented gDNA are biotinylated; and the method further comprises separating biotinylated copies from unbiotinylated copies, thereby forming a first solution comprising barcoded tagmented first strand cDNA and a second solution comprising barcoded tagmented first strand gDNA. In some embodiments, the gDNA-specific reverse primer is biotinylated such that the copies of barcoded tagmented gDNA are biotinylated and wherein the copies of barcoded tagmented cDNA / RNA duplexes are not biotinylated. In some embodiments, the cDNA-specific reverse primer is biotinylated such that the copies of barcoded tagmented cDNA / RNA duplexes are biotinylated and wherein the copies of barcoded tagmented gDNA are not biotinylated. In some embodiments, the separating comprises contacting the biotinylated and unbiotinylated copies with a solid support linked to streptavidin under conditions in which the biotinylated copies bind to the solid support and the unbiotinylated copies do not bind to the solid support, and removing unbiotinylated copies in a solution from the solid support and subsequently eluting the biotinylated copies from the solid support thereby forming separate solutions of biotinylatedand unbiotinylated copies. In some embodiments, the solid support is a magnetic or paramagnetic bead.

[0016] In some embodiments, the method further comprises aliquoting a first aliquot of the solution comprising copies of barcoded tagmented first strand cDNA and copies of barcoded tagmented gDNA into a first container and specifically amplifying the barcoded tagmented first strand cDNA using an oligonucleotide comprising the cDNA-specific reverse primer; and aliquoting a second aliquot of the solution comprising copies of barcoded tagmented first strand cDNA and copies of barcoded tagmented gDNA into a second container and specifically amplifying the barcoded tagmented first strand gDNA using an oligonucleotide comprising the gDNA-specific reverse primer, thereby forming a first solution comprising amplified barcoded tagmented first strand cDNA and a second solution comprising amplified barcoded tagmented first strand gDNA.

[0017] In some embodiments, the oligonucleotide primers in the reverse transcription comprise a selection of hexamer primers.

[0018] In some embodiments, the diffusing of the first transposome occurs in the absence of exogenous Mg++ and the method further comprises subsequently diffusing Mg++ into the cell, thereby activating the tagmentase.

[0019] In some embodiments, the removing of the oligonucleotides primers comprises diffusing a single-stranded exonuclease into the cells that digests non-annealed oligonucleotide primers following reverse transcription.

[0020] In some embodiments, the solution comprising the second transposome further comprises polyethylene glycol. In some embodiments, the PEG has an average molecular weight between 200-10,000 g / mol. optionally 400-8000 g / mol. In some embodiments, the PEG is at a concentration of 0.5-10% w / v, optionally 4-8%. In some embodiments, the second transposase comprises an amino acid sequence at least 95, 96, 97, 98 or 99% identical to SEQ ID NO:1 and comprising K at position 54, R at position 212, R at position 214, R at position 251, V at position 338, and P at position 372 as numbered with reference to SEQ ID NO:1. In some embodiments, the second transposase comprises SEQ ID NO:3.DEFINITIONS

[0021] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to whichthis disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., which is incorporated herein by reference), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, and organic synthetic described below are those well-known and commonly employed in the art.

[0022] The term "amplification reaction" refers to any in vitro means for multiplying the copies of a target sequence of nucleic acid in a linear or exponential manner. Such methods include but are not limited to polymerase chain reaction (PCR); DNA ligase chain reaction (see U. S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)) (LCR); QBeta RNA replicase and RNA transcriptionbased amplification reactions (e.g., amplification that involves T7, T3, or SP6 primed RNA polymerization), such as the transcription amplification system (TAS), nucleic acid sequence based amplification (NASBA), and self-sustained sequence replication (3SR); isothermal amplification reactions (e.g., single-primer isothermal amplification (SPIA)); as well as others known to those of skill in the art.

[0023] " Amplifying" refers to a step of submitting a solution to conditions sufficient to allow for amplification of a polynucleotide if all of the components of the reaction are intact. Components of an amplification reaction include, e.g., primers, a polynucleotide template, polymerase, nucleotides, and the like. The term "amplifying" typically refers to an "exponential" increase in target nucleic acid. However, "amplifying" as used herein can also refer to linear increases in the numbers of a select target sequence of nucleic acid, such as is obtained with cycle sequencing or linear amplification. In an exemplary embodiment, amplifying refers to PCR amplification using a first and a second amplification primer.

[0024] The term "amplification reaction mixture" refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid. These include enz mes, aqueous buffers, salts, amplification primers, target nucleic acid, and nucleoside triphosphates.Amplification reaction mixtures may also further include stabilizers and other additives to optimize efficiency and specificity. Depending upon the context, the mixture can be either a complete or incomplete amplification reaction mixture.

[0025] " Polymerase chain reaction" or " PCR" refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression. PCR is well known to those of skill in the art; see, e.g., U. S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds.1990. Exemplary PCR reaction conditions typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by a hybridization / elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.

[0026] A "primer" refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid and serves as a point of initiation of nucleic acid synthesis. Primers can be of a variety of lengths and are often less than 50 nucleotides in length, for example 12-30 nucleotides, in length. The length and sequences of primers for use in PCR can be designed based on principles known to those of skill in the art, see, e.g., Innis et al., supra. Primers can be DNA, RNA, or a chimera of DNA and RNA portions. In some cases, primers can include one or more modified or non-natural nucleotide bases. In some cases, primers are labeled.

[0027] ‘‘Primer extension” refers to any method in which a primer is extended in a template-specific manner. Examples of primer extension include, for example, methods in which a primer hybridizes to a template nucleic acid and a polymerase extends the primer in a template-specific manner. In some embodiments, the template is DNA and the polymerase is a DNA polymerase. In some embodiments, the template is RNA and the polymerase is a reverse-transcriptase. Primer extension can also include, for example, template switching (see, e.g.. Zhu YY, Machleder EM, et al. (2001) Biotechniques, 30(4): 892-897; Ramskold D, Luo S, et al. (2012) Nat Biotechnol, 30(8):777-78, and nick polymerization (also referred to as nick translation), the latter involving nicking one strand of a nucleic acid duplex and using the nicked strand as a primer that is extended using the other strand as a template (see, e.g., Leonard G. Davis Ph D., et al, in Basic Methods in Molecular Biology, 1986).

[0028] A nucleic acid, or a portion thereof, “hybridizes” or “anneals” to another nucleic acid under conditions such that non-specific hybridization is minimal at a definedtemperature in a physiological buffer (e.g.. pH 6-9, 25-150 mM chloride salt or in a PCR reaction mixture). In some cases, a nucleic acid, or portion thereof, hybridizes to a conserved sequence shared among a group of target nucleic acids. In some cases, a primer, or portion thereof, can hybridize to a primer binding site if there are at least about 6, 8, 10, 12, 14, 16, or 18 contiguous complementary nucleotides, including “universal” nucleotides that are complementary to more than one nucleotide partner. Alternatively, a primer, or portion thereof, can hybridize to a primer binding site if there are fewer than 1 or 2 complementarity mismatches over at least about 12, 14, 16, or 18 contiguous complementary nucleotides. In some embodiments, the defined temperature at which specific hybridization occurs is room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is higher than room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80 °C. In some embodiments, the defined temperature at which specific hybridization occurs is 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80 °C.

[0029] A "template" refers to a polynucleotide sequence that comprises the polynucleotide to be amplified, flanked by or a pair of primer hybridization sites. Thus, a "target template" comprises the target polynucleotide sequence adjacent to at least one hybridization site for a primer. In some cases, a "target template" comprises the target polynucleotide sequence flanked by a hybridization site for a “forward” primer and a “reverse” primer.

[0030] As used herein, "nucleic acid" means DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, points of attachment and functionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiester group modifications (e.g.. phosphorothioates, methylphosph onates), 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases, isocytidine and isoguanidine and the like. Nucleic acids can also include non-natural bases, such as, for example, nitroindole. Modifications can also include 3' and 5' modifications including but not limited to capping with a fluorophore (e.g., quantum dot) or another moiety.

[0031] A "polymerase" refers to an enzyme that performs template-directed synthesis of polynucleotides, e.g. DNA and / or RNA. The term encompasses both the full-length polypeptide and a domain that has polymerase activity. DNA polymerases are well-known to those skilled in the art, including but not limited to DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. Additional examples of commercially available polymerase enzymes include, but are not limited to: Klenow fragment (New England Biolabs® Inc.), Taq DNA polymerase (QIAGEN), 9 °N™ DNA polymerase (New England Biolabs® Inc ), Deep Vent™ DNA polymerase (New England Biolabs® Inc.), Manta DNA polymerase (Enzymatics®), Bst DNA polymerase (New England Biolabs® Inc.), and phi29 DNA polymerase (New England Biolabs® Inc.).

[0032] Polymerases include both DNA-dependent polymerases and RNA-dependent polymerases such as reverse transcriptase. At least five families of DNA-dependent DNA polymerases are known, although most fall into families A, B and C. Other types of DNA polymerases include phage polymerases. Similarly, RNA polymerases typically include eukaryotic RNA polymerases I, II, and III. and bacterial RNA polymerases as well as phage and viral polymerases. RNA polymerases can be DNA-dependent and RNA-dependent.

[0033] As used herein, the term "partitioning" or "partitioned" refers to separating a sample into a plurality of portions, or "partitions." Partitions are generally physical, such that a sample in one partition does not, or does not substantially, mix with a sample in an adjacent partition. Partitions can be solid or fluid. In some embodiments, a partition is a solid partition, e.g., a microchannel. In some embodiments, a partition is a fluid partition, e.g., a droplet. In some embodiments, a fluid partition (e.g., a droplet) is a mixture of immiscible fluids (e.g., water and oil). In some embodiments, a fluid partition (e.g., a droplet) is an aqueous droplet that is surrounded by an immiscible carrier fluid (e.g., oil). Other partitions can include, but are not limited to wells (e.g., microwells) and capsules, including capsules that can later be degraded or semi-permeable capsules, that retain larger molecules such as polynucleotides but allow for diffusion of reagents such as enzy mes. Exemplary array of wells and well descriptions can be found for example in U. S. Patent No. 9,103,754 and 10,391.493. The array of wells (set of nanowells, microwells, wells) can function to capture the solid supports, optionally in addressable, know n locations. As such, the array of wells can be configured to facilitate bead capture in at least one of a single-solid support format or optionally in small groups of solid supports. Exemplary microwell arrays and methods ofdelivery of beads to the microwells and analysis thereof is described in, e.g., PCT / US2021 / 034152.

[0034] As used herein a “barcode” is a short nucleotide sequence (e.g., at least about 4, 6, 8, 10, 12, 14, 16, 18, 20 or more nucleotides long) that identifies a molecule to which it is conjugated. Barcodes can be used, e.g., to identify molecules in a partition. Such a partitionspecific barcode should be unique for that partition as compared to barcodes present in other partitions. For example, partitions containing target RNA from single-cells can subject to reverse transcription conditions using primers that contain a different partition-specific barcode sequence in each partition, thus incorporating a copy of a unique “cellular barcode” into the reverse transcribed nucleic acids of each partition. Thus, nucleic acid from each cell can be distinguished from nucleic acid of other cells due to the unique “cellular barcode.” In some cases, the cellular barcode is provided by a “bead barcode” that is present on oligonucleotides conjugated to a bead, wherein the bead barcode is shared by (e.g., identical or substantially identical amongst) all, or substantially all, of the oligonucleotides conjugated to that bead but is different from most or substantially all oligonucleotides conjugated to other beads. Thus, cellular and bead barcodes can be present in a partition, attached to a bead, or bound to cellular nucleic acid as multiple copies of the same barcode sequence. Cellular or bead barcodes of the same sequence can be identified as deriving from the same cell, partition, or bead. Such partition-specific, cellular, or bead barcodes can be generated using a variety of methods, which methods result in the barcode conjugated to or incorporated into a solid or hydrogel support (e.g., a solid bead or particle or hydrogel bead or particle). In some cases, the partition-specific, cellular, or bead barcode is generated using a split and mix (also referred to as split and pool) synthetic scheme as described herein. A partition-specific barcode can be a cellular barcode and / or a bead barcode (for example when associated with a cell or partition or both). Similarly, a cellular barcode can be a partition specific barcode (when provided in a partition) and / or a bead barcode (when delivered by a bead). Additionally, a bead barcode can be a cellular barcode and / or a partition-specific barcode.

[0035] In other cases, barcodes uniquely identify the molecule to which it is conjugated and are referred to as a unique molecular identifier (UMI). The number of nucleotides of the UMI, which can be continuous, or discontinuous, will depend on the number of UMI sequences required. In some embodiments, the number of UMIs available are many times (e.g., 2X, 10X, 100X, etc.) higher than possible conjugation partners, thereby reducing thechance of rare duplicates being linked to different molecules. In some embodiments, pools of different UMIs are present in a partition and the composition of the pool acts as an identifiers for the partition, with some UMIs being in common with some other partitions but the total pool of UMIs being unique or substantially unique between partitions. UMI sequences can be generated for example as random sequences of a set length, and in some embodiments is identified by a flanking known sequence.

[0036] The length of the barcode sequence determines how many unique samples can be differentiated. For example, a 1 nucleotide barcode can differentiate 4, or fewer, different samples or molecules; a 4-nucleotide barcode can differentiate 44or 256 samples or less; a 6 nucleotide barcode can differentiate 4096 different samples or less; and an 8 nucleotide barcode can index 65,536 different samples or less. Additionally, barcodes can be attached to both strands either through barcoded primers for both first and second strand synthesis, through ligation, or in a tagmentation reaction.

[0037] Barcodes are typically synthesized and / or polymerized (e.g., amplified) using processes that are inherently inexact. Thus, barcodes that are meant to be uniform (e.g., a cellular, particle, or partition-specific barcode shared amongst all barcoded nucleic acid of a single partition, cell, or bead) can contain various N-l deletions or other mutations from the canonical barcode sequence. Thus, barcodes that are referred to as “identical” or “substantially identical” copies refer to barcodes that differ due to one or more errors in, e.g., synthesis, polymerization, or purification errors, and thus contain various N-l deletions or other mutations from the canonical barcode sequence. Moreover, the random conjugation of barcode nucleotides during synthesis using e.g., a split and pool approach and / or an equal mixture of nucleotide precursor molecules as described herein, can lead to low probability events in which a barcode is not absolutely unique (e.g., different from all other barcodes of a population or different from barcodes of a different partition, cell, or bead). However, such minor variations from theoretically ideal barcodes do not interfere with the high-throughput sequencing analysis methods, compositions, and kits described herein. Therefore, as used herein, the term “unique” in the context of a particle, cellular, partition-specific, or molecular barcode encompasses various inadvertent N-l deletions and mutations from the ideal barcode sequence. In some cases, issues due to the inexact nature of barcode synthesis, polymerization, and / or amplification, are overcome by oversampling of possible barcode sequences as compared to the number of barcode sequences to be distinguished (e.g., at least about 2-, 5-, 10-fold or more possible barcode sequences). For example, 10,000 cells can beanalyzed using a cellular barcode having 9 barcode nucleotides, representing 262,144 possible barcode sequences. The use of barcode technology is well known in the art. see for example Katsuyuki Shiroguchi, et al. Proc Natl Acad Sci U S A., 2012 Jan 24;109(4):1347-52; and Smith, AM et al., Nucleic Acids Research Can 11, (2010). Further methods and compositions for using barcode technology include those described in U. S. 2016 / 0060621.

[0038] A "transposase" or “tagmentase’' means an enzyme that is capable of forming a functional complex with a transposon end-containing composition and catalyzing insertion or transposition of the transposon end-containing composition into the double-stranded target DNA with which it is incubated in an in vitro transposition reaction.

[0039] The term “transposon end” means a double-stranded DNA that exhibits the nucleotide sequences (the “transposon end sequences”) that are necessary to form the complex with the transposase that is functional in an in vitro transposition reaction. A transposon end forms a “complex” or a “synaptic complex” or a “transposome complex” or a “transposome composition” with a transposase or integrase that recognizes and binds to the transposon end, and which complex is capable of inserting or transposing the transposon end into target DNA with which it is incubated in an in vitro transposition reaction. A transposon end exhibits two complementary sequences consisting of a “transferred transposon end sequence” or “transferred strand” and a “non-transferred transposon end sequence,” or “non transferred strand” For example, one transposon end that forms a complex with a hyperactive Tn5 transposase (e.g., EZ-Tn5™ Transposase, EPICENTRE Biotechnologies, Madison, Wis., USA) that is active in an in vitro transposition reaction comprises a transferred strand that exhibits a “transferred transposon end sequence” as follows:5' AGATGTGTATAAGAGACAG 3' (SEQ ID NO: 4),and a non-transferred strand that exhibits a “non-transferred transposon end sequence” (also known as a “mosaic end” or “ME” sequence) as follows:5' CTGTCTCTTATACACATCT 3' (SEQ ID NO: 5).

[0040] The 3'-end of a transferred strand is joined or transferred to target DNA in an in vitro transposition reaction. The non-transferred strand, which exhibits a transposon end sequence that is complementary to the transferred transposon end sequence, is not joined or transferred to the target DNA in an in vitro transposition reaction.

[0041] The term “solid support” refers to the surface of a bead, microtiter well or other surface that is useful for attaching a nucleic acid, such as an oligonucleotide or polynucleotide. The surface of the solid support can be treated to facilitate attachment of a nucleic acid, such as a single stranded nucleic acid.

[0042] The term “bead” refers to any solid support that can be in a partition, e.g., a small particle or other solid support. In some embodiments, the beads comprise an alginate matrix, i.e., calcium alginate. In some embodiments, the beads comprise polyacrylamide. For example, in some embodiments, the beads incorporate barcode oligonucleotides into the gel matrix through an acrydite chemical modification attached to each oligonucleotide.Exemplary beads can include hydrogel beads. In some cases, the hydrogel is in sol form. In some cases, the hydrogel is in gel form. An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in. e.g., U. S. Patent Nos. 4,438,258; 6,534,083; 8,008,476; 8,329,763; U. S. Patent Appl. Nos. 2002 / 0,009,591;2013 / 0,022,569; 2013 / 0,034,592; and International Patent Publication Nos.WO / 1997 / 030092; and WO / 2001 / 049240.

[0043] It will be understood that any range of numerical values disclosed herein can include the endpoints of the range, and any values or subranges in between the endpoints. For example, the range 1 to 10 includes the endpoints 1 and 10, and any value between 1 and 10. The values typically include one significant digit.

[0044] The term “sample” refers to a biological composition, such as a cell, comprising a target nucleic acid.

[0045] The term “about” refers to the usual error range for the respective value that is known by a person of ordinary skill in the art for this technical field, for example, a range of ± 10%, ± 5%, or ± 1% can encompass the recited value, even if the recited value is not modified by the term “about.”

[0046] All ranges described herein can include the end point values of the range, and any sub-range of values included between the endpoints of the range, where the values include the first significant digit. For example, a range of 1 to 10 includes a range from 2 to 9, 3 to 8, 4 to 7, 5 to 6, 1 to 5, 2 to 5, 2 to 10, 3 to 10, and so on.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 shows the effect of PEG buffer on cDNA / RNA hybrid tagmentation using transposase mutant. The figure shows size distributions of tagmented products of SEQ ID NO: 3 in the presence of PEG.

[0048] FIGs. 2A-B show size distributions of tagmented products of SEQ ID NO:3 in the presence of the indicated concentration of PEG.

[0049] FIG. 3A-C illustrates the effect of DMF buffer on AT AC profile generation of cells using transposase mutant (SEQ ID NO: 3).

[0050] FIG. 4A-D illustrates the differential effect of DMF & PEG buffer on tagmentation of various substrates using the transposase mutant (SEQ ID NO:3).

[0051] FIG. 5 illustrates the performance comparison of two distinct tagmentation strategies, assessing their efficacy in producing high-quality sequencing libraries.

[0052] FIG. 6 illustrates an exemplary workflow for RNA-seq and ATAC-seq.

[0053] FIG. 7 illustrates single-cell ATACseq and RNAseq multi omic sequencing profiles generated according to the workflow in FIG. 6.

[0054] FIG. 8 illustrates a barnyard plot of the AT AC library derived from the single-cell multiomic (ATAC+RNA) workflow of FIG. 6.

[0055] FIG. 9 shows the size profile of a AT AC library derived from the current single-cell multiomic methodology of FIG. 6.

[0056] FIG. 10 illustrates a barnyard plot of the cDNA library derived from the single-cell multiomic (ATAC+RNA) workflow of FIG. 6.

[0057] FIG. 11 illustrates a transcript body coverage plot of the reads of cDNA library derived from the single-cell multiomic (ATAC+RNA) workflow of FIG. 6.DETAILED DESCRIPTION OF THE INVENTION

[0058] Methods of tagmenting both RNA and DNA in cells are provided. The methods can comprise for example, providing fixed and permeabilized cells, tagmenting genomic DNA in the cell, generating cDNAs in the cell from RNA in the cell, and subsequently tagmenting RNA / first strand cDNA duplexes in the cell, thereby generating tagmented gDNA and cDNAs in the permeabilized cell. In some embodiments, the cells can be partitioned, forexample after tagmentation, and tagmented nucleic acids from the cells can be barcoded and amplified, allowing for single-cell sequencing. In some embodiments, the methods can be combined with oligonucleotide-tagged antibodies to further immunolabel proteins on and / or in cells.

[0059] Also provided herein are new ways to tagment RNA / DNA duplexes in situ in cells. In some embodiments, these methods can involve using certain transposase mutants, optionally in combination with polyethylene glycol, allowing for efficient tagmentation of first strand cDNA / RNA duplexes in the cytosol of cells.

[0007] FIG. 6 depicts a non-limiting example workflow. FIG. 6 illustrates an exemplary process workflow of ATAC+RNA multiomic assay. The process begins with chemical crosslinking of cells or nuclei to preserve cellular structure and chromatin integrity. After permeabilization. chromatin tagmentation (the first tagmentation step) is performed using a first tagmentase (e.g., SEQ ID NO:2) in a two-step tagmentation reaction by using DMF-based tagmentation reaction buffer. During the two-step chromatin tagmentation reaction the tagmentase binds to genomic DNA of open chromatin regions without activating its cleavage or tagging functions. This process ensures the tagmentase is evenly distributed throughout the cell, reaching equilibrium with the chromatin binding. Following binding step, cleavage and tagging functions of the tagmentase are then activated by a quick buffer exchange process, enabling the tagmentase to label the open chromatin regions with preloaded adapters.

[0007] FIG. 6 illustrates that after removing excess tagmentase through the first washing step, a transcriptome-specific not-so-random " NSR" primer panel and optionally adapter-linked oligo dT primers are added. These primers bind to RNA molecules and then initiates cDNA synthesis via in-situ reverse transcription (RT) reaction. Following the in-situ RT step, any leftover primers are removed with exonuclease digestion, which selectively degrades single-stranded DNA. After exonuclease removal by the second washing step. The cells are then resuspended in the PEG-based tagmentation reaction buffer for the second cDNA / RNA hybrid tagmentation step by using a mutant tagmentase that selectively targets RNA / DNA duplexes (e.g., SEQ ID NO:3). In contrast to the first chromatin tagmentation step, the mutant tagmentase is fully active and targets cDNA / RNA hybrids in the cytosol while diffusing through the cells. Meanwhile, the mutant tagmentase demonstrates preference to tagmenting cDNA / RNA hybrid molecules, and poor tagmentation efficiency towards chromatin structure within nuclei. Furthermore, the use of PEG-based reaction buffer reduces the mutanttagmentase’s activity in chromatin tagmentation. As a result, the combined effects of mutant usage, reaction buffer, and reaction process minimizing unintended tagmentation and crosscontamination between cDNA and ATAC libraries in the ATAC+RNA multiomic assay.

[0007] As shown in FIG. 6, after this second cDNA / RNA tagmentation, cells are partitioned into droplets as single-cell compartment by using a microfluidic water-in-oil system. Each droplet contains one cell, barcode beads, and necessary reagents. Within the droplets, reactions for gap-filling, barcode attachment, and PCR amplification are performed. After the in-droplet reaction, droplets are disrupted and then the pooled barcoded nucleic acids content is separated into two portions for cDNA and ATAC library amplification by using library-specific primers forNGS library-preparation. Following sequencing, chromatin accessibility’ and transcriptomic profile are analyzed together to enable comprehensive insights into gene regulation, epigenetics, and cellular heterogeneity at single-cell resolution.

[0007] In some embodiments, the methods further comprise immunolabeling antibody targets (e.g., proteins) on or in cells using oligonucleotide-barcoded antibodies. In brief, antibodies specific to cell surface proteins can be conjugated to unique DNA oligonucleotides such that the specificity of the antibodies correspond to a sequence in the attached oligonucleotides. Upon binding to the cell surface proteins, the antibody-oligonucleotide conjugates label specific cell populations. The oligonucleotides serve as molecular barcodes that uniquely identify the antibody, thus the antibody’s corresponding target protein and the distinctive cell types.

[0060] In these embodiments, by combining traditional immunolabeling with sequencing techniques, the resolution of cell profiling is enhanced through next-generation sequencing for determining cell surface expression markers, enabling the precise identification and distinction of subpopulations within heterogeneous cell ty pes. The oligonucleotides attached to the antibodies can comprise one or more PCR handle sequence allowing for example a partition-specific barcoding oligonucleotide to be annealed in later partition steps and sequenced, allowing one to align RNA and ATACseq data as described herein with immunoprofiling data on a per cell (e.g., per-partition) basis.

[0061] By pairing antibody-oligonucleotide conjugate-based immunolabeling with the RNA sequencing described herein, one can link the surface protein expression to the RNA profile of each individual cell, allowing researchers to gain a comprehensive understanding of cellular identity’, function and state simultaneously. This dual-modality approach can providea comprehensive view of cellular function, facilitating more precise cell type identification and a deeper understanding of cellular heterogeneity.

[0062] For the cell type classification application, in some embodiments, antibody-oligonucleotide conjugates are used to distinguish subpopulations of cells that may express similar transcriptomes but differ significantly in their surface protein markers. For example, in immunology, this method can separate subsets of T cells, like naive and memory T cells, that have distinct functions but overlapping RNA profiles.

[0063] The application of combining transcriptomic and proteomic data can improve the resolution of cell type classification. Traditional single-cell RNAseq may not distinguish cell types with similar transcriptional profiles, but the addition of surface protein information enables more accurate differentiation. For example, immune cell subsets that are transcriptionally similar can be effectively distinguished based on their surface protein expression patterns.

[0007] The methods described herein can comprise permeabilized cells, which for example can be fixed or encapsulated in a hydrogel bead, for example such that the RNA of the cells are compartmentalized from each other in a bulk mixture (e.g., by the structure of the fixed or encapsulated cells) and the cells can subsequently be introduced into separate partitions.

[0064] Thus, in some embodiments, permeabilized cells are provided. The cells can be permeabilized to allow for entry of reagents while the cells themselves remain substantially intact. Permeabilization can remove cellular membrane lipids to allow molecules such as enzymes to enter the cell while substantially retaining RNA (e.g., >100 or 500 nucleotides long). In some embodiments, a detergent is used for permeabilization. Exemplary detergents can include, for example, Tween-20, Triton X-100, and NP-40 are used for permeabilization (for example, at 0.1-0.5% (v / v, in PBS). In some embodiments, a steroidal saponin (or saraponin) is used to solubilize lipid, resulting in permeabilization. An exemplary' saraponin is Digitonin. The appropriate permeabilization reagent can be selected to be compatible with the integrity of a downstream partition, if used. In some embodiments, the permeabilization and fixation do not use dimethylformamide (DMF) such that no exogenous DMF is present in the cells.

[0065] In some embodiments, the permeabilized cells are fixed cells. For example, in some embodiments, the cells are formalin-fixed, paraffin-embedded (FFPE) samples. In embodiments here the cells are permeabilized and fixed, the cells themselves can act aspartitions. In other embodiments, the permeabilized cells are not fixed. In these embodiments, the permeabilized cells can be provided encapsulated in a hydrogel bead, allowing for containment of the cell and its contents while allowing for diffusion of reagents into the cell. Examples of methods for hydrogel bead-encapsulation of cells are described in, for example, Utrech et al., Advanced Healthcare Materials, Volume 4, Issue 11, August 5, 2015, pages 1628-1633. Alternatively, in some embodiments, the cells, permeabilized and / or fixed or not, can be encompassed in a semi-permeable capsule (SPC).

[0066] Any type of cells can be used according to the methods and compositions described herein. In some embodiments, the cells are mammalian, for example human cells. In some embodiments, the cells are from a biological sample. Biological samples can be obtained from any biological organism, e.g., an animal, plant, fungus, pathogen (e.g., bacteria or virus), or any other organism. In some embodiments, the biological sample is from an animal, e.g., a mammal (e.g., a human or anon-human primate, a cow, horse, pig, sheep, cat, dog, mouse, or rat), a bird (e.g., chicken), or a fish. A biological sample can be any tissue or bodily fluid obtained from the biological organism, e.g., blood, a blood fraction, or a blood product (e.g.. serum, plasma, platelets, red blood cells, and the like), sputum or saliva, tissue (e.g., kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue); cultured cells, e.g., primary cultures, explants, and transformed cells, stem cells, or cells found in stool, urine, etc.

[0067] As noted above, reagents can be added to the bulk solution comprising the permeabilized cells allowing diffusion of reagents such as enzymes, nucleotides and smaller reagents into the cells for molecular activities, and because the nucleus will also be permeabilized, reagents can reach the interior of the nucleus, e.g., to genomic DNA and chromatin. Thus, an appropriate concentration of reagents is added to the bulk solution and allowed to diffuse into the cells, optionally with stirring or other types of mixing.

[0068] As noted above, permeabilized and fixed cells will retain genomic DNA as well as cytosolic RNA. As an initial tagmentation, a transposase carrying oligonucleotide adaptors is diffused into the cell and the nucleus such that the genomic DNA in the nucleus is tagmented, i.e., the transposase fragments the nucleic acids and introduces the oligonucleotide adapters into breakpoints in the fragmented nucleic acids. A transposase carrying oligonucleotide adaptors is referred to as a "transposome ’ In some embodiments, the transposome is diffused into the nuclei of the permeablized cells in the absence of exogenous magnesium. Thisallows the transposase to localize in genomic DNA without cleaving the DNA.Subsequently, magnesium (e.g., MgCl2) can be diffused into the cells, enabling the tagmentases to cleave at the positions in the DNA to which they have located. Commercial tagmentases, for example comprising SEQ ID NO:2 or similar, can be used in this initial tagmentation of genomic DNA. Examples of tagmentases can also be found in, e.g., U. S. Patent No. 9790476.

[0069] A tagmentase is an enzyme that is capable of forming a functional complex with a transposon end-containing composition and catalyzing insertion or transposition of the transposon end-containing composition into the double-stranded target nucleic acid (e.g., DNA or a DNA / RNA duplex) with which it is incubated in an in vitro transposition reaction. Exemplary transposases include but are not limited to modified Tn5 transposases that are hyperactive compared to wildtype Tn5, for example can have one or more mutations selected from E54K, M56A, or L372P. Wild-type Tn5 transposon (e.g., SEQ ID NO: 1) is a composite transposon in which two near-identical insertion sequences (IS50L and IS50R) are flanking three antibiotic resistance genes (Reznikoff WS. Annu Rev Genet 42: 269-286 (2008)). Each IS50 contains two inverted 19-bp end sequences (ESs). an outside end (OE) and an inside end (IE). However, wild-type ESs have a relatively low activity and were replaced in vitro by hyperactive mosaic end (ME) sequences. A complex of the transposase with the 19-bp ME is thus all that is necessary for transposition to occur, provided that the intervening DNA is long enough to bring two of these sequences close together to form an active Tn5 transposase homodimer (ReznikoffMol Microbiol 47: 1199-1206 (2003)). Transposition is a very infrequent event in vivo, and hyperactive mutants were historically derived by introducing three missense mutations in the 476 residues of the Tn5 protein (E54K, M56A, L372P), which is encoded by IS50R (Goryshin IY, Reznikoff WS. 1998. J Biol Chem 273: 7367-7374 (1998)). Transposition works through a ■'cut-and-paste” mechanism, where the Tn5 excises itself from the donor DNA and inserts into a target sequence, creating a 9-bp duplication of the target (Schaller H. Cold Spring Harb Symp Quant Biol 43: 401-408 (1979); Reznikoff S., Annu Rev Genet 42: 269-286 (2008)). In current commercial solutions (Nextera™ DNA kits, Illumina), free synthetic ME adaptors are end-joined to the 5'-end of the target DNA by the transposase (tagmentase).

[0070] In this initial tagmentation of the genomic DNA, the transposase will carry heteroadaptors, i.e., a first and second partially double-stranded oligonucleotide adaptor, wherein the first and second partially double-stranded oligonucleotide adaptors have differentsequences. For example, the double-stranded portion can comprise identical mosaic end (ME) sequences and the single-stranded 5’ portions of the first and second partially doublestranded oligonucleotide adaptors are different. Following tagmentation, genomic DNA fragments will thus have different 5 ’ single-stranded end sequences that can be filled in with a polymerase at a later step (see gap-filling below). The reverse complement of the first 5’ single-stranded end sequence can be used to barcode the resulting molecule in a downstream step as detailed below.

[0071] Following tagmentation, the cells can be washed, optionally more than once, to remove the tagmentase and / or reagents used in the tagmentation, from the bulk solution comprising the cells. Washing can include for example, centrifugation and removal of the supernatant and addition of new buffer, optionally repeated at least one additional time. In other embodiments, addition of new buffer alone allows for dilution of the tagmentation reagents without their removal.

[0072] Following tagmentation of the genomic DNA, and optional washing, reverse transcription can be initiated to reverse transcribe RNA (for example, but not limited to, mRNA) in the cytosol of the cell. Reverse transcription reagents can be diffused into the cells. For example, transcriptome-specific primers can be added to the solution and diffused into the cytosol of the cells along with a reverse transcriptase (RT) and buffers and dNTPs required for RT activity. The transcriptome-specific primers anneal to RNA and cDNA synthesis is initiated during reverse transcription (i.e. as an in-situ RT reaction).

[0073] Reverse transcription in the permeabilized will result in generation of first strand cDNAs. Reverse transcription (RT) is an amplification method that copies RNA into DNA. The disclosure provides for reverse transcribing one or more RNA in the permeabilized cells under conditions to allow for reverse transcription and generation of a first strand cDNA. First strand cDNAs and the RNA sequence template can anneal to each other, forming a cDNA / RNA double-stranded duplex in which one strand is DNA and one strand is RNA. The RT reaction can be primed with primers to prime an RT reaction from at least one target RNA molecule. The primers can have a 3’ end sequence that anneals to target RNA. For example, the 3’ end sequence of the primers can be one that is random, an oligo dT (also referred to herein as a ‘“poly T ’) sequence, or an RNA-specific sequence. Oligo dT sequences are single stranded sequences of deoxythymine (dT). The length of the oligo dT sequence can vary, for example, from 6 bases to 30 bases, and may be a mixture of oligo dTsequences with different lengths. In some embodiments, in addition to random, oligo dT, or an RNA-specific sequences, or instead of oligo dT, or an RNA-specific sequences, “not so random’’ primers can be included. Not so random primers are described in, for example, Armour, et al., Nature Methods volume 6, pages 647-649 (2009) and US Patent Publication No. 20100029511. Not so random primers can be selected from possible oligonucleotides, e.g., having a length of from 6 to 9 nucleotides, that hybridize under defined conditions to non-redundant target population of nucleic acid molecules, and do not hybridize under defined conditions to the non-target redundant population of nucleic acid molecules in the sample. Selection of the RT primer will affect the distribution of cDNAs. If a polyT primer is used, most cDNAs will be from the polyA tail of the RNA, whereas a selection of primers, either targeted to specific genes, or random, will generate cDNAs from different locations of RNAs. Components and conditions for RT reactions are generally known. The components for the RT reaction, such as for example the RTase and nucleotides, and buffers can be applied to a solution comprising the permeabilized cells and then passively diffused into the cells such that the RNA in the cells are reverse transcribed. Nucleotides used can be deoxyribonucleotides (dNTPs) or can be ribonucleotides or modified non-natural nucleotides or mixtures thereof. Depending on the nucleotide types used, specific RT enzymes may need to be selected that can incorporate the nucleotides into a cDNA or otherwise complementary polynucleotide.

[0074] Suitable reverse transcriptases can include but are not limited to Maxima RNAse+ (Thermo), Maxima RNAse- (Thermo), murine leukemia virus (MLV) reverse transcriptase (Gerard and Grandgenett, Journal of Virology 15:785-797, 1975; Verma, Journal of Virology 15:843-854, 1975) or feline leukemia virus (FLV) reverse transcriptase (Rho and Gallo, Cancer Lett., 10:207-221, 1980 or SEQ ID NO:1, bovine leukemia virus (BLV) (Demirhan et al., Anticancer Res., 16:2501-5, 1996; Drescher et al.. Arch Geschwulstforsch.. 49:569-79.1979), Avian Myeloblastosis Virus (AMV) reverse transcriptase, Respiratory Syncytial Virus (RSV) reverse transcriptase, Equine Infectious Anemia Virus (EIAV) reverse transcriptase, Rous-associated Virus-2 (RAV2) reverse transcriptase, SUPERSCRIPT II reverse transcriptase, SUPERSCRIPT III reverse transcriptase (US8541219, US7056716, US7078208), THERMOSCRIPT reverse transcriptase and MMLV RNase H- reverse transcriptase and Sensiscript (Qiagen).

[0075] Optionally, following reverse transcription, remaining transcriptome-specific primers can be removed from the cells. In some embodiments, the transcriptome-specificprimers are digested with an exonuclease, which is diffused into the cells. The exonuclease can be for example an exonuclease that selectively digests single stranded nucleic acids, thereby targeting transcriptome-specific primers that are not annealed to RNA. An exemplary nuclease is Exonuclease I.

[0076] A second tagmentation can be performed targeting the cDNA / RNA duplexes in the cytosol. The tagmentase and reagents can be diffused into the cells. In some embodiments, to optimize tagmentase targeting of cDNA / RNA duplexes in the cytosol, the tagmentase can be reacted with the cDNA / RNA duplexes in the cytosol in the presence of polyethylene glycol (PEG). The PEG can be added before, or after, the tagmentase is added to the cells or the PEG can be mixed with the tagmentase and then contacted to the cells as a mixture. Optionally, the tagmentation occurs in the presence of PEG and in the absence of exogenous DMF. It is believed any PEG can be used in conjunction with the tagmentase. In some embodiments, the PEG is selected from, e.g., PEG 400 to PEG 8000. In some embodiments, the concentration of PEG in the mixture contacted to the cells is 1-10% (w / v), e.g., 4-8%. Any transposase that targets and cleaves DNA / RNA duplexes can be used (e.g., in combination with PEG as described herein). In some embodiments, the transposase comprises K54 and P372, previously described to cause hyperactivity, and amino acid substitutions K212R, P214R, G251R and A338V corresponding to positions in SEQ ID NO: 1. In some embodiments, the transposase having these substitutions is at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1. An exemplary tagmentase comprises SEQ ID NO:3.

[0077] Table 1 below shows the position of amino acid substitution of the mutant transpoase described herein compared to wildtype Tn5 and a commercially-available tagmentase. SEQ ID NO:1 is the wild-type Tn5 transposase. SEQ ID NO:2 is a commercially-available tagmentase. SEQ ID NO:3 is an exemplary mutant as described in the examples. Bolded are the previously-known mutations of hyperactive Tn5 transposes (e.g., in SEQ ID NO:2). The underlined positions are the new changes in the mutant transposase (SEQ ID NO: 3) described herein.Table 1Amino acid positionSEQ ID NO:1 E54 M56 K212 P214 G251 A338 L372SEQ ID NO:2 K54 M56 K212 P214 G251 A338 P372SEQ ID NO:3 K54 A56 R212 R214 R251 V338 P372

[0078] In this tagmentation of the cDNA / RNA duplexes, the transposase will carry homoadaptors, i.e., a two identical partially double-stranded oligonucleotide adaptors. As described herein, optimally these partially double-stranded oligonucleotide adaptors have an identical 5’ single-stranded sequence as the first partially double-stranded oligonucleotide adaptor used in the first tagmentation (of gDNA), allowing for a later downstream step to barcode the same reverse complement of this 5’ single-stranded sequence in a later barcoding step.

[0079] The inventors have discovered that efficient tagmentation in permeabilized cells can be achieved, optionally in the presence of PEG, optionally in the absence of DMF, and using a transposase comprising K54 and P372. previously described to cause hyperactivity, and further amino acid substitutions K212R, P214R, G251R and A338V corresponding to positions in SEQ ID NO: 1. In some embodiments, the transposase having these substitutions is at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1. An exemplary tagmentase comprises SEQ ID NO: 3. Accordingly the disclosure provides a reaction mixture comprising (i) permeabilized and optionally fixed cells, optionally in a bulk solution, (ii) a transposase as described herein (e g., this paragraph) and (iii) PEG. The PEG can be the type and concentration as described directly above. Also provided is a method of tagmenting nucleic acids, e g., including but not limited to RNA / DNA duplexes, in permeabilized and optionally fixed cells, regardless of whether the initial steps described herein (e.g.. an earlier tagmentation) has been performed.

[0080] In some embodiments, following the above steps, including both tagmentation steps, a bulk solution comprising permeabilized cells have both tagmented gDNA and tagmented cDNA / RNA duplexes is generated. As noted above, in some embodiments, the tagmented gDNA comprises first and second (different) end 5’ overhang sequences and the tagmented RNA / cDNA duplexes comprise either the same first end 5’ overhang sequences on both ends, or for ends of the RNA and thus cDNA, some RNA / cDNA duplexes will only have the first end 5’ overhang sequence on one end and the poly A or 5’ end of the RNA at the other and the corresponding cDNA sequence for these ends.

[0081] The bulk solution can then be partitioned to form partitions that have single cells. Partitioning can be performed to introduce additional agents in the partitions, for examplebarcoding oligonucleotides, optionally linked to a solid support, e.g., a hydrogel bead. The tagmented products and the permeabilized cells that contain them can be partitioned to form partitions containing single cells and one or more bead comprising a plurality of clonal barcoding oligonucleotides having free 3’ ends that will be used subsequently to add a barcode sequence specific for the bead, allowing for partition-specific barcoding.

[0082] In some embodiments, the permeabilized cells can be partitioned such that individual cells are the only cell within a particular partition. Exemplary partitions can include but are not limited to droplets within an emulsion, microwells, capsules, including but not limited to semi-permeable capsules (SPCs). Methods and compositions for partitioning are described, for example, in published patent applications WO 2010 / 036352, US 2010 / 0173394, US 2011 / 0092373, and US 2011 / 0092376.

[0083] In some embodiments, one or more reagents are added during droplet formation or to the droplets after the droplets are formed. Methods and compositions for delivering reagents to one or more partitions include microfluidic methods as known in the art; droplet or microcapsule combining, coalescing, fusing, bursting, or degrading (e.g., as described in U. S. 2015 / 0027,892; US 2014 / 0227,684; WO 2012 / 149,042; and WO 2014 / 028,537); droplet injection methods (e.g, as described in WO 2010 / 151,776); and combinations thereof. In some embodiments, the droplets described herein are relatively stable and have minimal coalescence between two or more droplets. In some embodiments, less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. or 10% of droplets generated from a sample coalesce with other droplets. The emulsions can also have limited flocculation, a process by which the dispersed phase comes out of suspension in flakes. In some cases, such stability or minimal coalescence is maintained for up to 4, 6, 8, 10, 12, 24, or 48 hours or more (e.g, at room temperature, or at about 0, 2, 4, 6, 8, 10, or 12 °C). In some embodiments, the droplet is formed by flowing an oil phase through an aqueous sample or reagents.

[0084] The oil phase of an emulsion can comprise a fluorinated base oil which can additionally be stabilized by combination with a fluorinated surfactant such as a perfluorinated poly ether. Exemplary oil phase compositions along these lines are described in, e.g., PCT WO 2020 / 247950 and US Patent Publication No. 2017 / 0175179.

[0085] In some embodiments, the sample is partitioned into, or into at least, 500 partitions.1000 partitions, 2000 partitions, 3000 partitions, 4000 partitions, 5000 partitions, 6000partitions, 7000 partitions, 8000 partitions, 10,000 partitions, 15,000 partitions, 20,000 partitions, 30,000 partitions, 40,000 partitions. 50.000 partitions, 60,000 partitions, 70,000 partitions, 80,000 partitions, 90,000 partitions, 100,000 partitions, 200,000 partitions, 300,000 partitions, 400,000 partitions, 500,000 partitions, 600,000 partitions, 700,000 partitions, 800,000 partitions, 900,000 partitions, 1,000,000 partitions, 2,000,000 partitions, 3,000,000 partitions, 4,000,000 partitions, 5,000,000 partitions, 10,000,000 partitions, 20,000,000 partitions, 30,000,000 partitions, 40,000,000 partitions, 50,000,000 partitions, 60,000,000 partitions, 70,000,000 partitions, 80,000,000 partitions, 90,000,000 partitions, 100,000,000 partitions, 150,000,000 partitions, or 200,000,000 partitions.

[0086] A plurality of copies of barcoding oligonucleotides linked to a bead (e.g., a hydrogel bead) can be delivered to the partitions (including for example forming the partitions with the beads and the cells), wherein different partitions receive different beads and accompanying barcoding oligonucleotides. The bead can be attached to multiple copies of the same oligonucleotide, for example, at least about 10, 50, 100, 500, 1000, 5000, 10,000, 50,000, 100,000, 500.000, 1,000,000, 5,000,000, 10,000,000, 108, 109, 1010or more copies of the same or substantially identical oligonucleotide can be attached to one (e.g., the same) bead. The barcoding oligonucleotides can be covalently or non-covalently linked to the solid support(s). The barcoding oligonucleotides will comprise at least a bead-specific barcode sequence and a 3’ capture sequence for annealing to a target sequence (e.g., the reverse complement of the 5' end of the first adaptor oligonucleotide introduced by the tagmentases). The length of the barcode can depend on the number of different barcodes desired, and in some embodiments, can be between 4-20 nucleotides, which can be contiguous or noncontiguous. In some embodiments, the barcoding oligonucleotides further comprise a 5’ PCR handle sequence, allowing for a common 5’ sequence between sequences comprising different partition (e.g.. cell)-specific barcodes, allowing them all to be amplified with a universal primer that anneals to the PCR handle sequence. Optionally, once the bead is present in the partition, and before linkage to target fragments, the oligonucleotides can be cleaved from the bead prior to linking the oligonucleotides to the target nucleic acid fragments. In some embodiments, the cleavable linker linking the oligonucleotide to the solid support comprises a uridine incorporated site in a portion of a nucleotide sequence. A uridine incorporated site can be cleaved, for example, using a uracil glycosylase enzyme (e.g., a uracil N-glycosylase enzyme or uracil DNA glycosylase (UDG) enzyme). In some embodiments, the cleavable linker comprises a photocleavable nucleotide. Photocleavablenucleotides include, for example, photocleavable fluorescent nucleotides and photocleavable biotinylated nucleotides. See, e.g., Li et al., PNAS. 2003, 100:414-419; Luo et al., Methods Enzymol, 2014, 549:115-131. In some cases, the oligonucleotides are attached to bead through a disulfide linkage (e.g., through a disulfide bond between a sulfide of the solid support and a sulfide covalently attached to the 5’ or 3’ end, or an intervening nucleic acid, of the oligonucleotide). In such cases, the oligonucleotide can be cleaved from the solid support by contacting the solid support with a reducing agent such as a thiol or phosphine reagent, including but not limited to a beta mercaptoethanol, dithiothreitol (DTT), or tris(2-carboxyethyl)phosphine (TCEP).

[0087] Any bead of useful size and composition for delivery to partitions can be used. The particle or bead can be any particle or bead having a solid support surface. Solid supports suitable for particles include controlled pore glass (CPG)(available from Glen Research, Sterling, Va.), oxalyl-controlled pore glass (See, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527), TentaGel Support— an aminopolyethyleneglycol derivatized support (See, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373), polystyrene, Poros (a copolymer of polystyrene / divinylbenzene). or reversibly cross-linked acrylamide. Many other solid supports are commercially available and amenable to the present methods. In some embodiments, the bead material is a polystyrene resin or poly(methyl methacrylate) (PMMA). The bead material can be metal. In some embodiments, the particle or bead comprises hydrogel or another similar composition. In some cases, the hydrogel is in sol form. In some cases, the hydrogel is in gel form. An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in, e.g., U. S. Patent Nos. 4,438,258; 6,534,083; 8,008,476; 8,329,763; U. S. Patent Appl. Nos. 20020009591; 20130022569; 20130034592; and International Patent Publication Nos. W01997030092; and WO2001049240. Additional compositions and methods for making and using hydrogels, such as barcoded hydrogels, include those described in, e.g., Klein et al., Cell, 2015 May 21;161(5):1187-201.

[0088] Following formation of partitions, any tagmentases still associated with nucleic acids can be disassociated from the nucleic acids, for example by raising the temperature. In some embodiments, the temperature is raised above 65, e.g., 72 but no higher than 85 C such that tagmentases are disassociated from the nucleic acids without substantially harming the viability of the partitions.

[0089] Gap-filling can occur following tagmentation to produce blunt ends, allowing for example, for primer annealing sites for barcoding, and can occur before or after partitioning. In some embodiments, in the partitions, a gap-filling step can be performed to fill in singlestranded nucleic acids in the tagmented nucleic acids in the cells within the partitions. Gapfilling fills for example, any 5’ single-stranded ends in the tagmented nucleic acids. As indicated above, in some embodiments, the first tagmentation of the genomic DNA as well as the second tagmentation of the RNA / cDNA duplexes will include the same 5’ single-stranded end sequence in the oligonucleotide adaptors introduced by the tagmentases (e.g., referred to herein as “the first single-stranded 5 ' sequence of the first partially double-stranded oligonucleotide adaptors'’, see also FIG. 6), and these will be filled in by the gap-filling step such that the opposing strand includes a 3’ end that is the reverse complement of the first single-stranded 5’ sequence of the first partially double-stranded oligonucleotide adaptors. Thus both tagmented gDNA and tagmented cDNA / RNA duplexes will have a common 3’ end that is the reverse complement of the first single-stranded 5 ’ sequence of the first partially double-stranded oligonucleotide adaptors.

[0090] Because of the nature of the tagmentation of a cDNA / RNA duplex, the gap-filling is performed with RNA- and DNA-dependent polymerase activity, which can be provided in a single enzyme or a cocktail of different enzymes. Exemplary polymerases having RNA- and DNA-dependent DNA polymerase activity can include, for example, Bst 2.0, Bst 3.0, Superscript II, Superscript III and polymerases described in for example US Patent Publication No. 20230287364.

[0091] Following gap-filling, a first PCR cycle allows for barcoding oligonucleotides to anneal to reverse complementary ends of the tagmented cDNA or gDNA (i.e., the 3’ ends having reverse complement of the first single-stranded 5’ sequence of the first partially double-stranded oligonucleotide adaptors) and extended, either by the same polymerase that gap-filled or by a second DNA-dependent DNA polymerase also added to the partitions, in which case the second polymerase can be any thermostable polymerase that can be used in PCR. See, e.g., FIG. 6.

[0092] Further rounds of PCR in the presence of the second polymerase and reverse primers results in amplification of barcoded tagmented cDNA and barcoded tagmented gDNA in the partitions. See, e.g., FIG. 6. For example, in some embodiments, copies of barcoded tagmented cDNA / RNA duplexes are generated by extending a cDNA-specificreverse primer with a second polymerase and copies of barcoded tagmented gDNA are generated by extending a gDNA-specific reverse primer with the second polymerase. In some embodiments, the cDNA-specific reverse primer comprises a 3’ poly-T sequence that can anneal to a poly-A sequence on the barcoded tagmented second strand cDNA.Optionally the cDNA-specific reverse primer can also include, for example, a PCR handle sequence and / or a unique molecular identifier (UMI) sequence that allows for counting of unique copies of nucleic acids. The gDNA-specific reverse primer 3’ sequence can target sequences introduced in tagmentation such as a sequence from the second partially doublestranded oligonucleotide adaptor, or a reverse complement thereof, that is therefore unique to the tagmented gDNA in that it includes an adaptor not added in the second tagmentation. See, e.g., FIG. 6.

[0093] Following barcoding in the partitions and amplification, the contents of the partitions can be combined to form a new bulk solution comprising copies of the barcoded tagmented cDNAs and copies of the barcoded tagmented gDNA. Combining of contents of partitions will depend on the type of partitions used. Examples of combining contents of partitions can include but is not limited to. mixing of different microwell contents, disruption of droplets or SPCs, e.g., with heat and / or detergents, etc. Optionally the permeabilized cells can be disrupted and / or cellular debris in the new bulk solution can be removed, e.g., by pelleting the debris and removing the supernatant containing the copies of the barcoded tagmented products.

[0094] In some embodiments, separate libraries of the barcoded tagmented cDNAs and gDNAs can be formed by forming different aliquots from the bulk solution and then selectively amplifying the cDNAs or the gDNA. For example, in one aliquot, the cDNAs can be selectively amplified and in a second aliquot the gDNA can be selectively amplified. Selective amplification can be achieved because ends of the cDNAs and gDNAs are different in view of the above workflow, allowing for selective amplification. For example, a primer comprising the barcoding oligonucleotide can be used as a forw ard primer and a reverse comprise comprising the oligonucleotide comprising the cDNA-specific reverse primer, and optionally having further 5’ sequences such as a PCR handle, sequencing index or other sequences, can be used to amplify the cDNA library. In some embodiments, a primer comprising the barcoding oligonucleotide can be used as a forward primer and a reverse comprise comprising the oligonucleotide comprising the gDNA-specific reverse primer, andoptionally having further 5’ sequences such as a PCR handle, sequencing index or other sequences, can be used to amplify the gDNA library.

[0095] In alternative embodiments, separate libraries of the barcoded tagmented cDNAs and gDNAs can be formed by physical separation of cDNA copies from gDNA copies. In some embodiments, this can involve using one or more biotinylated primer in amplification of the gDNA but not the cDNA, or alternatively one or more biotinylated primer in amplification of the cDNA but not the gDNA. In either option, a solution is formed with biotinylated copies and unbiotinylated copies. Any method for separating biotinylated copies from unbiotinylated copies can then be used to form separate solutions of gDNA and cDNA. For example, one or more solid support linked to streptavidin can be contacted to the solution having biotinylated and unbiotinylated copies under conditions to allow binding of the biotinylated copies to the streptavidin. The remaining solution comprising unbiotinylated copies can be removed as a first solution and the biotinylated copies can then be eluted from the solid support(s) to form a second solution comprising biotinylated copies. The solid support can be selected as desired and in some embodiments can be beads, optionally that are magnetic or paramagnetic and then a magnetic field can be applied to immobilize the beads while the first solution is separated from the beads.

[0096] The resulting aliquots, following selective amplification, will contain barcoded cDNA or gDNA libraries that can be subsequently analyzed, e.g., by nucleotide sequencing. Because the cDNAs and gDNAs from the same cell is barcoded by the same barcode sequence, sequencing results from the cDNAs and the gDNAs can be compiled per cell, allowing for analysis of both DNA and cDNA on a single-cell basis. Because in some embodiments the initial tagmentation is performed in genomic DNA comprising chromatin, Assay for Transposase- Accessible Chromatin with Sequencing (ATAC-seq) analysis can be performed based on the gDNAs generated. The cDNA library allows for RNA-seq analysis for example.

[0097] Sequencing of the DNA molecules can be performed as desired. Methods for high throughput sequencing and genotyping are known in the art. For example, such sequencing technologies include, but are not limited to, pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), massive parallel clonal, massive parallel single molecule SBS. massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology, etc. Morozova and Marra provide a review of somesuch technologies in Genomics, 92: 255 (2008), herein incorporated by reference in its entirety.

[0098] Exemplary DNA sequencing techniques include fluorescence-based sequencing methodologies (See, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N. Y.; herein incorporated by reference in its entirety). In some embodiments, automated sequencing techniques understood in that art are utilized. In some embodiments, the present technology provides parallel sequencing of partitioned amplicons (PCT Publication No. WO 2006 / 0841,32, herein incorporated by reference in its entirety). In some embodiments, DNA sequencing is achieved by parallel oligonucleotide extension (See, e.g., U. S. Pat. Nos. 5,750,341; and 6,306,597, both of which are herein incorporated by reference in their entireties). Additional examples of sequencing techniques include the Church polony technology (Mitra et al., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732; and U. S. Pat. Nos. 6,432,360; 6,485,944; 6,511,803; herein incorporated by reference in their entireties), the 454 picotiter pyrosequencing technology (Margulies et al., 2005 Nature 437, 376-380; U. S. Publication No. 2005 / 0130173; herein incorporated by reference in their entireties), the Solexa single base addition technology (Bennett etal., 2005, Pharmacogenomics, 6, 373-382; U. S. Pat. Nos. 6,787,308; and 6,833,246; herein incorporated by reference in their entireties), the Lynx massively parallel signature sequencing technology (Brenner et al. (2000). Nat. Biotechnol. 18:630-634; U. S. Pat. Nos. 5.695,934; 5,714.330; herein incorporated by reference in their entireties), and the Adessi PCR colony technology (Adessi etal. (2000). Nucleic Acid Res. 28. E87; WO 2000 / 018957; herein incorporated by reference in its entirety).

[0099] Typically, high throughput sequencing methods share the common feature of massively parallel, high-throughput strategies, with the goal of lower costs in comparison to older sequencing methods (See, e.g., Voelkerding et al., Clinical Chem, 55: 641-658, 2009; MacLean et al.. Nature Rev. Microbiol., 7:287-296; each herein incorporated by reference in their entirety). Such methods can be broadly divided into those that typically use template amplification and those that do not. Amplification-requiring methods include pyrosequencing commercialized by Roche as the 454 technology platforms (e.g., GS 20 and GS FLX), the Solexa platform commercialized by Illumina, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems. Non-amplification approaches, also known as single-molecule sequencing, are exemplified by the HeliScope platform commercialized by Helicos BioSciences, andplatforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., Life Technologies / Ion Torrent, and Pacific Biosciences, respectively.

[0100] Also provided are tagmentases comprising an amino acid sequence comprising K at position 54, R at position 212, R at position 214, R at position 251, V at position 338, and P at position 372 as numbered with reference to SEQ ID NO: 1. In some embodiments, position 56 is A. In some embodiments, the transposase having these substitutions is at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1. An exemplary tagmentase comprises SEQ ID NO:3.

[0101] Also provided is a nucleic acid (e.g., DNA or RNA) encoding a tagmentase comprising an amino acid sequence comprising K at position 54, R at position 212, R at position 214, R at position 251, V at position 338, and P at position 372 as numbered with reference to SEQ ID NO: 1. In some embodiments, position 56 is A. In some embodiments, the nucleic acid encodes the transposase having these substitutions is at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1. In some embodiments, the nucleic acid encodes a tagmentase comprising SEQ ID NO: 3. In some embodiments, an expression cassette comprising a promoter operably linked to the nucleic acid such that the tagmentase can be expressed in a cell. In some embodiments, the expression cassette is provided in a vector, e.g., an expression vector. Also provided is a cell comprising the nucleic acid encoding the tagmentase, the expression cassette or the vector described above. Exemplary cells can be prokaryotic (e.g., E. coli or other bacterial species used to produce proteins) or eukaryotic cells.

[0102] Also provided are methods of producing a tagmentase as described above. In some embodiments, the cell comprising the nucleic acid encoding the tagmentase, the expression cassette or the vector is incubated such that the tagmentase is produced by the cell.Optionally the tagmentase is subsequently purified from the cell to produce a purified tagmentase.

[0103] Also provided are kits that can be used for performing part or all of the methods described herein. In some embodiments, the kit will comprise one or more container for holding various reagents, and optionally instructions. Exemplary components of a kit, which can be provided separately or in a mixture, as the methods described herein allow, can include, for example, one or more of cell fixatives, cell permeation reagents, adaptor-loaded transposases (optionally more than one as described herein), one or more polymerase, whichcan include for example a reverse transcriptase and a DNA polymerase, one or more exonuclease, primers for primer extension and / or amplification as described herein, and reagents for performing droplet-based amplification (dPCR). In some embodiments, the kit comprises a tagmentase that selectively tagments RNA / DNA duplexes optionally in a solution comprising PEG. In some embodiments, the PEG is selected from, e.g., PEG 400 to PEG 8000 g / mol. In some embodiments, the concentration of PEG in the mixture contacted to the cells is 1-10% (w / v), e.g., 4-8%. Any transposase that targets and cleaves DNA / RNA duplexes can be used (e.g., in combination with PEG as described herein). In some embodiments, the transposase comprises comprising K at position 54, R at position 212, R at position 214, R at position 251, V at position 338, and P at position 372 as numbered with reference to SEQ ID NO: 1. In some embodiments, the transposase having these substitutions is at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1. An exemplary tagmentase comprises SEQ ID NO: 3. Other reagents as described herein or as useful for performing the steps described herein can also be included in the kit.EXAMPLENovel activity of mutant transposase including for RNA / DNA duplexes

[0104] DNA size distribution profiles of libraries created by using commercially available Tn5 transposase (SEQ ID NO:2) and a mutant Tn5 transposase (SEQ ID NO: 3) at low and high enzyme concentrations across different sample types were measured. The resulting electropherogram provides information on the size distribution of samples through electrophoresis-based separation. Nucleic acid samples, including naked double-stranded DNA (dsDNA), naked double-stranded RNA:cDNA hybrid molecule, and nucleosomal double-stranded genomic DNA (gDNA) of nuclei, were subjected to tagmentation reaction by using the corresponding Tn5 transposase. Tagmentation is a single-step process that fragments and tags DNA. For double-stranded DNA (dsDNA) samples, increasing the concentration (from 4 nM [A-2] to 17nM [A-l]) of Tn5 transposase leads to more frequent tagmentation, producing smaller DNA fragments and a higher overall yield. For RNA:cDNA hybrid samples, the double-stranded helical structure is not an ideal substrate for Tn5 transposase. The commercially available Tn5 transposase (SEQ ID NO:2) had low tagmentation efficiency on RNA:cDNA hybrids, resulting in lower yield regardless of enzyme concentration. In contrast, the mutant Tn5 transposase (SEQ ID NO:3) had improvedtolerance to the helical structure of RNA:cDNA hybrid and recognized it as a substrate of the tagmentation, leading to a significant improvement in reaction efficiency and yield.

[0105] Tagmentation is a useful step in the ATAC (Assay for Transposase-Accessible Chromatin) reaction, where Tn5 transposase is used to fragment and tag DNA regions accessible within chromatin. This reaction takes advantage of the enzyme’s ability to preferentially target nucleosome-free or loosely packed dsDNA regions, leading to a unique fingerprint pattern of chromatin structure, including sub-, mono-, di-, tri-nucleosome peaks as shown in [A-5] and [A-6], Unlike the commercial Tn5 transposase (SEQ ID NO:2), the mutant Tn5 transposase (SEQ ID NO:3) does not perform well with nuclei samples, despite showing compatibility with naked double-stranded DNA in earlier experiments. This indicates that the mutant Tn5 transposase has distinct properties and behaves differently in various biological sample types during in vitro reactions.Activity of mutant transposase in presence of PEG

[0106] Bulk cDNA / RNA hybrid was synthesized from RNA by using a commercial first-strand cDNA synthesis product. The same amount of RNA / cDNA hybrid was subjected to tagmentation reaction with the same amount of Tn5 under different tagmentation buffers. The tagmented products were further amplified by PCR and purified before the size distribution was measured on a bioanalyzer automated electrophoresis to generate FIG.1 image. The sizes and amounts of tagmentation products under various tagmentation buffers containing 6% polyethylene glycol (PEG) of molecular weight of 400, 1000, 1500, 3000, 4000, 6000, or 8000 g / mol. FIG.1 indicates tagmentation of cDNA / RNA hybrid inside individual cells can be performed under DMF-free. PEG-based tagmentation buffer.

[0107] Single-cell cDNA / RNA hybrid w as synthesized by in-situ reverse transcription inside individual fixed and permeabilized cells. The same number of cells was subjected to in-situ tagmentation reaction by diffusing the same amount of Tn5 along with different tagmentation buffers into these cells. The tagmented products were further amplified by PCR and purified before the size distribution was measured on a bioanalyzer automated electrophoresis to generate FIG. 2A-B images. FIG. 2A shows the sizes and amounts of tagmentation products under tagmentation buffers containing 4% polyethylene glycol (PEG) with molecular weight of 400, 1500, or 6000 g / mol. FIG. 2B shows the sizes and amounts of tagmentation products under tagmentation buffers containing 1%. 2%, or 4% polyethyleneglycol (PEG) with molecular weight of 400 g / mol. FIG. 2A-B indicates tagmentation of cDNA / RNA hybrid can be performed under DMF-free, PEG-based tagmentation buffer.

[0108] FIG. 3 shows the DNA profiling of transposase-accessible chromatin with sequencing (ATAC-Seq) in the presence of various concentration of DMF: 16% (A), 10% (B), and 0% (C) DMF using the workflow of FIG. 6. In brief, following fixation and permeation, cells were tagmented by using the current invention workflow to introduce sequencing adapters into open chromatin regions. Subsequently, the tagged DNA is amplified by PCR to generate a sequencing library. The quality of the tagmentation process was assessed by using Bioanalyzer to assess the size distribution of libraries generated under tested conditions. In the presence of effective amount of DMF, the size profile of atypical ATACseq assay reflects the distribution of DNA fragments of a typical characteristic pattern, with distinct peaks corresponding to nucleosome-free DNA (i), mono-nucleosomal (ii), di-nucleosomal (iii), and tri-nucleosomal (iv) DNA peaks. These peaks arise because of the transposase preferentially accessing open regions of chromatin and fragmenting DNA at intervals determined by nucleosome spacing.

[0109] FIG. 4 shows Tn5-Gamma transposome activity on cDNA-RNA hybrid and doublestranded DNA substrates. Briefly, rRNA-depleted RNA was reverse transcribed into a cDNA / RNA hybrid using a commercial first-strand cDNA synthesis kit. The resulting hybrid or dsDNA substrate was tagmented by the Tn5 transposome in the presence of tagmentation buffers containing Polyethylene Glycol (Fig. 4A, 4C) and Dimethylformamide (DMF) (Fig.4B, 4D). The tagmentation reaction was incubated at 37°C for 30 minutes. PCR amplification with index primers was then performed, and the resulting library was purified. The size distribution of the purified tagmented library was measured using an Agilent 2100 Bioanalyzer as shown.

[0110] FIG. 5 illustrates the performance comparison of two distinct tagmentation strategies, assessing their efficacy in producing high-quality sequencing libraries. The cDNA / RNA hybrids were synthesized in situ via reverse transcription of fixed and permeabilized cells. Equal numbers of single cells underw ent two distinct tagmentation strategies - One and Two-step tagmentation. During One-step tagmentation, Tn5 transposase and tagmentation buffer (containing Mg2+) were simultaneously diffused into cells, allowing diffusion and tagmentation to occur concurrently. While during the two-step tagmentation, Tn5 transposase and tagmentation buffer (lacking Mg2+) were first diffused into cells,allowing the enzyme bind to the target substate only, but not tagmenting the substrate.Subsequently, Mg2+was added to initiate the tagmentation reaction after diffusion / binding. All tagmented products were then amplified by polymerase chain reaction, purified, and analyzed by using Bioanalyzer to assess the size distribution of libraries generated under tested conditions. The one-step tagmentation approach (entitled “red” trace with a solid line) yielded small-size libraries with a uniform distribution of fragment sizes. In contrast, the two-step tagmentation (entitled “blue” trace with dashed lines) produced additional periodic peaks corresponding to chromatin structure, as typically observed in ATAC-seq libraries (inset, right). These results suggest that the delayed activation of tagmentation (two-step strategy)) facilitates redistribution and enhanced deep penetration of enzyme into the nucleus tagmenting the genomic DNA of open chromatin regions. Moreover, this observation demonstrates that the switching between one and two-step tagmentation strategies can provides a significant advantage in controlling and decoupling of the diffusion / binding and tagmentation processes, enabling versatile application potential in single-cell application.

[0111] FIG. 7A-B shows the multiome, RNA (FIG. 7A) and ATAC (FIG. 7B) libraries of single-cells generated by using the workflow as described in the current invention. The distribution of fragment size of the corresponding libraries was assessed by using Bioanalyzer automated electrophoresis. The fragments in the ATAC library (FIG. 7B) exhibited atypical characteristic of nucleosomal patterns, while the RNA library (FIG. 7A) showed a broader fragments size distribution peak, demonstrating the application feasibility of the FIG. 6 workflow in multiome sequencing library generation.

[0112] FIG. 8 shows the barnyard plot of ATAC library derived from the FIG. 6 single-cell multiomic methodology. Single-cell library-prep of the current invention is performed on a mixed species experiment to validate the purity and percentage crosstalk of the current experimental workflow. The designated clusters provide a graphical representation of the sequencing fragment origin, distinguishing between species of sources in the mixed-species experiments. Cells expressing only human ATAC fragments cluster along the X-axis. Cells expressing only mouse ATAC fragments cluster along the Y-axis. Cells carry ATAC fragments that map to both species appear as mixed species cluster, which represents doublets or potentially cross-contaminated droplets in the sequencing process.

[0113] FIG. 9 shows the size profile of ATAC library derived from the current single-cell multiomic methodology. The fragment size profile is constructed from the mappedsequencing reads of the ATAC library. It reflects the accessibility of chromatin across a genome and provides insight into nucleosome positioning and chromatin organization. The short fragments of size -<100 bp correspond to nucleosome-free regions, where chromatin is open and accessible to transposase enzymes. The second peak corresponds to the mononucleosome fragments of -150-200 bp. These fragments represent DNA wrapped around a single nucleosome. The spikes of the peak are typical tagmentation signature of Tn5 transposase.Table 1. Single-cell ATACseq performance metricsTranscription Fraction of Reads Median Unique Start Site (TSS) in Peaks (FRiP) Nuclear Fragments Enrichment Score per CellMultiomicATACseqdataset 4.1 0.39 12096

[0114] Table 1 shows several key single-cell ATACseq performance metrics of multiomic ATACseq dataset generated by the workflow of FIG. 6. Transcription Start Site (TSS) Enrichment Score reflects how well the assay captures open chromatin regions near transcription start sites (TSS), which are typically sites of active transcription regulation. The higher TSS score means the higher data quality. The typical TSS score in single-cell ATACseq is 4 or higher. Fraction of Reads in Peaks (FRiP) measures the proportion of sequencing reads that fall within identified peaks, which represent regions of open chromatin. In single-cell ATAC-seq, acceptable FRiP scores can vary but are generally >0.1 (10%) for high-quality libraries. Median Unique Nuclear Fragments per Cell evaluates the overall quality and depth of chromatin accessibility data collected for individual cells. High-quality single-cell ATAC-seq data normally can over 10,000 median unique nuclear fragments per cell. Mapped Mitochondrial Read Pairs refers to the proportion or count of sequencing reads that align to the mitochondrial genome. Less than 5-20% mitochondrial reads is considered acceptable for high-quality single-cell ATACseq data. All above metrics indicates the FIG. 6 workflow can yield mid to high quality single-cell ATACseq data.

[0115] FIG. 10 shows the barnyard plot of cDNA library derived from the current singlecell multiomic methodology. Single-cell library-prep of the FIG. 6 workflow was performedon a mixed species experiment to validate the purity and percentage crosstalk of the current experimental workflow. The designated clusters provide a graphical representation of the sequencing fragment origin, distinguishing between species of sources in the mixed-species experiments. Cells expressing only human RNA transcript fragments cluster along the X-axis. Cells expressing only mouse RNA transcript fragments cluster along the Y-axis. Cells carry RNA transcript fragments that map to both species appear in the mixed species cluster, which represents doublets or potentially cross-contaminated droplets in the sequencing process.

[0116] FIG. 11 shows the gene transcript body coverage plot the cDNA library derived from the current single-cell multiomic methodology of FIG. 6, assessing the uniformity of sequencing read distribution across the length of transcript bodies. The X-axis represents the normalized position along the gene body, ranging from the 5' to the 3' end of the gene divided into percentages. The Y-axis indicates the relative coverage or read density at each position of the reads derived by the current method.Table 2. Single-cell RNAseq performance metrics.Percentage of Percentage of Percentage of Percentage of reads falling reads falling reads falling reads falling into the genic into the into rRNA into region intergenic region mitochondrial region region MultiomicRNAseqdataset 50.42 13.23 2.85 2.24

[0117] Table 2 shows several key single-cell RNAseq performance metrics of multiomic RNAseq dataset generated by the workflow in the current invention. The percentage of reads falling into the genic region is a uality control metric in single-cell RNA-seq that measures the proportion of sequencing reads mapping to annotated gene regions (both exonic and intronic) in the genome. It helps assess the quality of RNA-seq data and the proportion of biologically meaningful reads. Higher than 50% of reads mapping to genic regions is typically acceptable in single-cell RNAseq. Percentages of reads falling into the intergenic region, rRNA region, mitochondrial region measures the noise in the single-cell RNAseq data. This table indicates the FIG. 6 workflow can yield acceptable single-cell RNAseq data with low percentages of noise.

[0118] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein, including patents, patent applications, non-patent literature, and Genbank accession numbers, is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.SEQUENCES SEQ ID NO: 1: - WT Tn5 sequence MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSEAMQEGAYR FIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGS IQDKSRGW WVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGSMMSNVIAV CDREADIHAYLQDKLAHNERFWRSKHPRKDVESGLYLYDHLKNQPELGGYQIS I PQKGWD KRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLA QALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRESFTL PQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGG FMDSKRTGIASWGALWEGWEALQSKLDGFLAAKDLMAQGIKISEQ ID NO:2 - commercially available tagmentase MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAM QEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGK LGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLA AAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGL YLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITL NAVLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGA GAERQRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSA ETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGA LWEGWEALQSKLDGFLAAKDLMAQGIKI SEQ ID NO:3 - tagmentase mutant of the disclosure MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAAQ EGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKL GSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAA AATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSRHRRKDVESGLY LYDHLKNQPELGGYQISIPQKGVVDKRRKRKNRPARKASLSLRSGRITLKQGNITLN AVLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAG VERQRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGAL WEGWEALQSKLDGFLAAKDLMAQGIKI SEQ ID NO: 4 - transferred transposon end sequence5' AGATGTGTATAAGAGACAG 3'SEQ ID NO:5- mosaic end’’ or “ME” sequence5' CTGTCTCTTATACACATCT 3'

Claims

WHAT IS CLAIMED IS:

1. A transposase comprising an amino acid sequence at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1 and comprising lysine (K) at position 54, arginine (R) at position 212, arginine (R) at position 214, arginine (R) at position 251, valine (V) at position 338, and proline (P) at position 372 as numbered with reference to SEQ ID NO:1.

2. The transposase of claim 1, wherein position 56 is A.

3. The transposase of claim 1, wherein the transposase comprises SEQ ID NO:3.

4. A kit comprising the transposase of any one of claims 1-3.

5. The kit of claim 4, further comprising oligonucleotide adaptors comprising a mosaic end (ME) sequence.

6. A transposome composed of the transposase of any one of claims 1-3 and oligonucleotide adaptors comprising a mosaic end (ME) sequence.

7. A solution comprising polyethylene glycol (PEG) and the transposome of claim 6..

8. The solution of claim 7, wherein the transposase comprises SEQ ID NO:3.

9. The solution of claim 7 or 8, wherein the PEG has an average molecular weight between 200-10,000 g / mol, optionally 400-8000 g / mol.

10. The solution of claim 7 or 8 or 9, wherein the PEG is at a concentration of 0.5-10% w / v, optionally 4-8%.

11. The solution of any one of claims 7-10, wherein the solution comprises fixed and permeabilized cell.

12. A nucleic acid encoding the tagmentase of any one of claims 1-3.

13. An expression cassette comprising a promoter operably linked to the nucleic acid of claim 12.

14. A cell comprising the nucleic acid of claim 13 or the expression cassette of claim 14.

15. A method of producing the tagmentase of claim 14, the method comprising incubating the cell under conditions resulting in expression of the tagmentase in the cell, and optionally purifying the tagmentase from the cell.

16. A method of tagmenting DNA / RNA duplex nucleic acids in the absence of dimethylformamide (DMF), the method comprisingcontacting DNA / RNA duplex nucleic acids in a solution with a transposome composed of a transposase and oligonucleotide adaptors under conditions such that the transposase fragments the nucleic acids and introduces the oligonucleotide adapters into breakpoints in the fragmented nucleic acids, wherein the contacting is performed in the presence of polyethylene glycol (PEG) and the absence of DMF.

17. The method of claim 16, wherein the transposase comprises an amino acid sequence at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1 and comprising K at position 54. R at position 212, R at position 214, R at position 251, V at position 338, and P at position 372 as numbered with reference to SEQ ID NO: 1.

18. The method of claim 16, wherein the transposase comprises SEQ ID NO:

319. The method of claim 16 or 17 or 18, wherein the PEG has an average molecular weight between 200-10,000 g / mol, optionally 400-8000 g / mol.

20. The method of claim 16 or 17 or 18 or 19, wherein the PEG is at a concentration of 0.5-10% w / v, optionally 4-8%.

21. The method of any one of claims 16-20, wherein the solution comprises fixed and permeabilized cells.

22. A method of preparing tagmented cDNA and gDNA from a cell, the method comprising,providing fixed and permeabilized cells comprising RNA and gDNA; diffusing into the cells a first transposome, composed of a first transposase and a first and second partially double-stranded oligonucleotide adaptor, that selectivelyfragments the gDNA while leaving the RNA intact, wherein the first transposase introduces the oligonucleotide adapters onto 5' ends at breakpoints in the fragmented gDNA, wherein the first and second partially double-stranded oligonucleotide adaptors have different first and second single-stranded 5’ sequences, respectively;performing reverse transcription of the RNA in the cells by contacting the RNA with oligonucleotide primers and a reverse transcriptase, annealing the oligonucleotide primers to the RNA and extending the oligonucleotide primers with the reverse transcriptase to form first strand cDNA / RNA duplexes;optionally removing the oligonucleotide primers; andcontacting the first strand cDNA / RNA duplexes with a solution comprising a second transposome, composed of a second transposase and two copies of the first partially double-stranded oligonucleotide adaptor, that fragments cDNA / RNA duplexes, wherein the second transposase introduces the first oligonucleotide adapters onto 5’ ends of breakpoints in the fragmented cDNA,thereby generating cells comprising tagmented cDNA and gDNA.

23. The method of claim 22, wherein the cells are in a bulk solution during the contacting with the first and second transposomes and the performing of reverse transcription.

24. The method of claim 23, following the contacting the cDNA / RNA duplexes with a second transposome, partitioning the cells to form partitions containing single cells.

25. The method of claim 24, wherein partition-specific barcode oligonucleotides are attached to common sequences at the end of the tagmented cDNA / RNA duplexes and gDNA in the partitions.

26. The method of claim 25, wherein the partition-specific barcode oligonucleotides are linked to a solid support and are combined with the cells comprising tagmented cDNA / RNA duplexes and gDNA and wherein the partitioning comprises partitioning the solid supports with the cells.

27. The method of claim 25 or 26, comprising in the partitions, optionally raising the temperature of the tagmented gDNA and cDNA / RNA duplexes to displace the second tagmentases;gap-filling with a first polymerase the first and second single-stranded 5’ sequences of the tagmented gDNA and the first single-stranded 5’ sequences of the tagmented cDNA / RNA duplexes to form 3’ ends;annealing the partition-specific barcode oligonucleotides to the 3’ ends formed from gap-filling the first single-stranded 5 ’ sequences on the gDNA and the cDNA / RNA duplexes and extending the partition-specific barcode oligonucleotides with the first polymerase or a second polymerase to form barcoded tagmented cDNA / RNA duplexes and barcoded tagmented gDNA; andgenerating copies of barcoded tagmented cDNA / RNA duplexes by extending the partition-specific barcode oligonucleotide and a cDNA-specific reverse primer with a second polymerase and generating copies of barcoded tagmented gDNA by extending the partition-specific barcode oligonucleotide and a gDNA-specific reverse primer with the second polymerase,combining contents of the partitions, thereby forming a solution comprising copies of barcoded tagmented first strand cDNA and copies of barcoded tagmented gDNA.

28. The method of claim 27, further comprising,aliquoting a first aliquot of the solution comprising copies of barcoded tagmented first strand cDNA and copies of barcoded tagmented gDNA into a first container and specifically amplifying the barcoded tagmented first strand cDNA using an oligonucleotide comprising the cDNA-specific reverse primer; andaliquoting a second aliquot of the solution comprising copies of barcoded tagmented first strand cDNA and copies of barcoded tagmented gDNA into a second container and specifically amplifying the barcoded tagmented first strand gDNA using an oligonucleotide comprising the gDNA-specific reverse primer,thereby forming a first solution comprising amplified barcoded tagmented first strand cDNA and a second solution comprising amplified barcoded tagmented first strand gDNA.

29. The method of claim 27, wherein one of the cDNA-specific reverse primer or the gDNA-specific reverse primer is biotinylated such that one of the copies of barcoded tagmented cDNA / RNA duplexes or the copies of barcoded tagmented gDNA are biotinylated; and the method further comprises separating biotinylated copies fromunbiotinylated copies, thereby forming a first solution comprising barcoded tagmented first strand cDNA and a second solution comprising barcoded tagmented first strand gDNA.

30. The method of claim 29, wherein the gDNA-specific reverse primer is biotinylated such that the copies of barcoded tagmented gDNA are biotinylated and wherein the copies of barcoded tagmented cDNA / RNA duplexes are not biotinylated.

31. The method of claim 29, wherein the cDNA-specific reverse primer is biotinylated such that the copies of barcoded tagmented cDNA / RNA duplexes are biotinylated and wherein the copies of barcoded tagmented gDNA are not biotinylated.

32. The method of any one of claim 29-31, wherein the separating comprises contacting the biotinylated and unbiotinylated copies with a solid support linked to streptavidin under conditions in which the biotinylated copies bind to the solid support and the unbiotinylated copies do not bind to the solid support, and removing unbiotinylated copies in a solution from the solid support and subsequently eluting the biotinylated copies from the solid support thereby forming separate solutions of biotinylated and unbiotinylated copies.

33. The method of claim 33, wherein the solid support is a magnetic or paramagnetic bead.

34. The method of any one of claims 22-33, wherein the oligonucleotide primers in the reverse transcription comprise a selection of hexamer primers.

35. The method of any one of claims 22-34, wherein the diffusing of the first transposome occurs in the absence of exogenous Mg++and the method further comprises subsequently diffusing Mg++into the cell, thereby activating the tagmentase.

36. The method of any one of claims 22-27, wherein the removing of the oligonucleotides primers comprises diffusing a single-stranded exonuclease into the cells that digests non-annealed oligonucleotide primers following reverse transcription.

37. The method of any one of claims 22-36, wherein the solution comprising the second transposome further comprises polyethylene glycol.

38. The method of claim 37, wherein the PEG has an average molecular weight between 200-10,000 g / mol. optionally 400-8000 g / mol.

39. The method of claim 37 or 38, wherein the PEG is at a concentration of 2-10% w / v, optionally 4-8%.

40. The method of any one of claims 22-39, wherein the second transposase comprises an amino acid sequence at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1 and comprising K at position 54, R at position 212, R at position 214, R at position 251, V at position 338, and P at position 372 as numbered with reference to SEQ ID NO:1.

41. The method of claim 40, wherein the second transposase comprises SEQ ID NO:3.

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