Mapping of DNA binding sites

Abstract

This specification provides techniques for identifying the binding sites of DNA-binding proteins, and in particular, but not limited to, techniques for methods, systems, and kits for simultaneously mapping the binding sites of multiple proteins within the same cell.

Description

Cross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 540,174 filed on September 25, 2023, the entire disclosure of which is incorporated herein by reference in its entirety. [Sequence Listing]

[0002] This application contains a sequence listing submitted electronically in XML file format, which is incorporated by reference in its entirety. The XML copy was created on September 25, 2024, named "JHU_42334_601_SequenceListing.xml", and has a size of 148,729 bytes. Technical Field

[0003] The present specification provides techniques for identifying the binding positions of DNA-binding proteins, in particular, but not limited to, techniques relating to methods, systems, and kits for simultaneously mapping the binding sites of multiple proteins in the same cell. Background Art

[0004] Complex interactions between regulatory proteins and cis-regulatory elements control gene transcription. See, for example, Taverna (2007) "How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers," Nat Struct Mol Biol 14: 1025, and Ruthenburg (2007) "Multivalent engagement of chromatin modifications by linked binding modules," Nat Rev Mol Cell Biol 8: 983. These references are incorporated herein by reference. The regulation of gene transcription often involves the synchronous action of multiple proteins and diverse histone modifications. Examples include interactions between target genes, DNA binding sites, epigenetic modifications, and transcription factors.

[0005] New assays are being continuously developed and improved to address these challenges. The scientific community is actively working on developing and improving novel sequencing-based assays to identify and characterize binding sites on chromosomes.

[0006] Conventional ChIP-seq and similar techniques are widely used for identifying and mapping binding sites of transcription factors, cofactors, enzymes, and histone PTMs [References 1, 2, 3]. These methods involve fragmenting chromatin by physical or enzymatic means to generate fragmented chromatin. The fragmented chromatin is isolated using specific antibodies, a DNA library is constructed, and it is sequenced. Subsequently, bioinformatics analysis is performed to characterize the binding sites. Conventional ChIP-seq-based approaches require a large number of cells (over 1 million) and can introduce significant background noise and biological asynchronousness. Furthermore, the requirement for chromatin fragmentation presents a challenge when applying ChIP-seq at the single-cell level. As an alternative, CUT&RUN [Reference 20] and related assays [References 21, 22, 23] offer some solutions to the limitations of ChIP-seq. These alternative approaches selectively cleave only the target fragment using antibody-conjugated micrococcal nucleases (MNases), leaving the rest of the chromatin intact. This targeted fragmentation strategy substantially reduces background noise and improves the signal-to-noise ratio. Notably, because permeabilized cells can be preserved after digestion, the need for extensive chromatin fragmentation is minimized or eliminated, providing assays compatible with single-cell assays [References 22,23]. However, existing techniques require additional steps involving library preparation, sequencing, and adapter ligation for analysis.

[0007] This challenge is mitigated by CUT&Tag [Reference 4] and similar assays [References 12, 13]. These techniques use antibodies conjugated to a transposase (e.g., Tn5 or a similar enzyme) to simultaneously cleave target DNA and incorporate adapters into the ends of the cleaved DNA. This procedure is called "tagmentation" and simplifies library preparation. After tagmentation, an amplification step generates a library for sequencing. CUT&Tag uses a transposase-protein A fusion protein loaded with adapters, which interacts with antibodies specific to the target DNA binding. See, for example, Kaya-Okur (2019) "CUT&Tag for efficient epigenomic profiling of small samples and single cells" Nature Communications 10: 1930, International Patent Application Publication No. 2019060907 (disclosing the use of a specific binding agent containing a transposase and transposon in each transposome), and Gopalan (2021) "Simultaneous profiling of multiple chromatin proteins in the same cells" Molecular Cell 81: 4736. These are incorporated herein by reference. However, the dissociation of transposase-protein A fusion proteins and antibodies causes spurious tagging and increases background noise. Furthermore, in multiplex techniques that map multiple DNA binding targets using transposase-protein A fusion proteins loaded with multiple adapters and multiple antibodies, the swapping of adapter-loaded transposase-protein A fusion proteins and antibodies among their binding partners results in false (e.g., mixed) signals due to incorrect combinations of adapters and antibodies.

[0008] The regulation of gene transcription involves the synchronous action of multiple proteins and diverse histone modifications. Interactions between two proteins and / or histone post-translational modifications and their respective binding sites have been studied using multiple sequential chromatin immunoprecipitation (ChIP) assays [References 24, 25, 26]. However, these ChIP-seq-based techniques require multiple (e.g., at least two) immunoprecipitations with different antibodies, resulting in a heavy workload and large amounts of initial material. Furthermore, each ChIP round introduces significant background noise. A technique called SplitDamID offers an alternative for co-binding detection [Reference 27]. In this approach, the target protein is fused with a different subunit of DNA adenine methyltransferase (DAM). While SpDamID can detect co-binding between two proteins, it requires the construction of a fusion protein and therefore cannot analyze histone modifications. Consequently, SpDamID is limited to the identification of non-histone mark target pairs.

[0009] Multi-CUT&Tag [References 5,6], a derivative of CUT&Tag, allows for the identification of multiple targets within a single sample and a single experiment. This methodology combines an antibody with a protein A-Tn5 fusion protein, with the Tn5 component pre-loaded with a barcoded DNA adapter. Different antibody-Tn5 complexes are mixed and incubated simultaneously with cells. Analysis of captured chromosomal DNA using DNA barcoding and nucleotide sequencing data allows for the simultaneous decoding of multiple target proteins and histone marks by Multi-CUT&Tag. Similar to CUT&Tag, Multi-CUT&Tag can be used with a minimal number of cells, i.e., individual cells, and directly detects protein and / or histone modification interactions. A recently introduced multiplex technique known as MulTI-Tag [Reference 7] addresses the challenge of cross-contamination that can arise when simultaneously detecting different targets. To avoid this challenge, MulTI-Tag achieves multiplex functionality by performing multiple CUT&Tag operations sequentially. However, like ChIP-seq and CUT&Tag, MulTI-Tag cannot determine epitope colocalization. Furthermore, the time burden of continuous experiments limits its multiplexing capability, resulting in a highly laborious protocol.

[0010] A significant limitation of the cut-and-tag based approach is the increased background noise and potential for cross-contamination that occur when detecting multiple targets simultaneously. While not theoretically bound, background noise is thought to arise from the relatively weak interaction between protein A and antibodies. The protein A-Tn5 complex dissociates from the target, leading to ambiguous tagging. Furthermore, protein A does not universally bind to all antibody types, limiting the range of usable antibodies. In addition, since Tn5 binding and introduction to the antibody occurs several hours or days before use, the enzymatic activity of Tn5 is impaired.

[0011] In particular, new technologies are needed for multiplex DNA binding mapping. [Overview of the project] [Problems that the invention aims to solve]

[0012] This specification provides embodiments of a technique for mapping DNA binding sites to identify, for example, the binding sites of histone marks, histone modifying enzymes, transcription factors, and cofactors on chromosomes. In some embodiments, the technique enables the multiplex identification of multiple DNA binding sites (e.g., 1 to 500) on one or more targets (e.g., 1 to 500), allowing for the identification of multiple histone marks, histone variants, histone modifying enzymes, DNA modifying enzymes, chromatin-related proteins, transcription factors, RNA species, and cofactors within a genome (e.g., on one or more chromosomes). [Means for solving the problem]

[0013] In some embodiments, the subject matter of this disclosure provides a method for identifying a target nucleic acid binding site. The method comprises: (a) contacting the target bound to the nucleic acid binding site with the tagging composition to bind the tagging composition to the target, wherein the tagging composition comprises (i) an antibody or antibody fragment bound to the target, (ii) a heterocyclic compound bound to the antibody or antibody fragment, (iii) a protein complex, and (iv) two or more nucleic acids each containing a barcode nucleotide sequence, the two or more nucleic acids being bound to the heterocyclic compound; (b) contacting the two or more nucleic acids of the tagging composition with a transposase to form an antibody-barcode-transposase complex, wherein the antibody-barcode-transposase complex causes a double-strand break in the nucleic acid containing the nucleic acid binding site, thereby generating a nucleic acid fragment containing the nucleic acid binding site; (c) isolating the nucleic acid fragment; and (d) sequencing the nucleic acid fragment to identify the nucleic acid binding site of the target.

[0014] In some embodiments, the protein complex comprises avidin, streptavidin, or neutravidin. In some embodiments, the heterocyclic compound comprises biotin. In some embodiments, the transposase comprises a Tn5 transposase. In some embodiments, the two or more nucleic acids each further comprises a transposase mosaic sequence that binds to the transposase. In some embodiments, the transposase mosaic sequence binds to the Tn5 transposase. In some embodiments, the target comprises a DNA-binding protein. In some embodiments, the DNA-binding protein comprises a transcription factor, a regulatory element, a transcription repressor, a transcription activator, a polymerase, a nuclease, a nickase, a zinc finger protein, a transcription activator-like effector nuclease (TALEN), a glycosylase, a methylase, a ligase, a restriction enzyme, a replication protein, a helicase, or a kinase. In some embodiments, the antibody or antibody fragment is not directly bound to the two or more nucleic acids. In some embodiments, the protein complex is bound to the heterocyclic compound conjugated to the antibody or antibody fragment, and to the heterocyclic compound conjugated to the two or more nucleic acids. In some embodiments, the method further comprises adding magnesium to a sample containing the target and the tagging composition. In some embodiments, the two or more nucleic acids each further comprises an amplification handle. In some embodiments, the method further comprises amplifying the nucleic acid fragment to provide a sequencing library. In some embodiments, the amplification is polymerase chain reaction (PCR) amplification.

[0015] In some embodiments, the subject matter of the present disclosure provides a composition comprising (a) one or more antibodies or antibody fragments bound to a target, (b) a heterocyclic compound bound to the one or more antibodies or antibody fragments, (c) a protein complex comprising avidin, streptavidin, or neutravidin, and (d) two or more nucleic acids, each comprising (i) a barcode nucleotide sequence and (ii) a transposase mosaic sequence, wherein the two or more nucleic acids are bound to the heterocyclic compound, and the composition forms a complex in solution. In some embodiments, the protein complex comprises streptavidin. In some embodiments, the heterocyclic compound comprises biotin. In some embodiments, the transposase comprises Tn5 transposase. In some embodiments, the antibody or antibody fragment comprises a region bound to a DNA-binding protein. In some embodiments, the DNA-binding protein includes a transcription factor, regulatory element, transcription repressor, transcription activator, polymerase, nuclease, nickase, zinc finger protein, transcription activation-like effector nuclease (TALEN), glycosylase, methylase, ligase, restriction enzyme, replication protein, helicase, or kinase. In some embodiments, the protein complex binds to the heterocyclic compound.

[0016] In some embodiments, the subject matter of this disclosure provides a kit comprising a first container containing the composition described in claim 15, and a second container containing a transposase. In some embodiments, the kit further comprises a reagent for tagmentation. In some embodiments, the kit further comprises reagents and materials for DNA isolation and nucleic acid amplification. In some embodiments, the kit further comprises a cell capture scaffold. In some embodiments, the cell capture scaffold comprises magnetic beads, columns, concanavalin A beads, streptavidin beads, colloidal semiconductor nanocrystals, carbon nanotubes, or microfluidic devices.

[0017] In some embodiments, the subject matter of the present disclosure is a method for identifying two or more target binding sites on a nucleic acid, comprising: (a) providing two or more barcoding affinity reagents, each of which is an affinity reagent bound to a pair of adapters, wherein the first adapter comprises a first barcode nucleotide sequence and a first transposase-binding mosaic sequence, the second adapter comprises a second barcode nucleotide sequence and a second transposase-binding mosaic sequence, the first and second barcode nucleotide sequences are identical or different, none of the two or more barcoding affinity reagents contain a transposase, each of the two or more barcoding affinity reagents binds to a different target, and the first and second barcode nucleotide sequences of each barcoding affinity reagent are different from the first and second barcode nucleotide sequences of other barcoding affinity reagents that bind to different targets; and (b) providing the two or more barcoding affinity reagents, each (c) Adding affinity reagents to a sample containing targets, each target being bound to a nucleic acid at its respective target binding site, each barcode affinity reagent being bound to its respective target or to a primary affinity reagent bound to its respective target, and the binding of each affinity reagent occurring in the absence of transposase, (d) Adding unbound transposase and transposase activator to the sample, the unbound transposase being bound to the first transposase-bound mosaic sequence and the second transposase-bound mosaic sequence of each barcode affinity reagent, the bound transposase fragmenting the nucleic acid, and tagging the nucleic acid with the first barcode nucleotide sequence and the second barcode nucleotide sequence of each barcode affinity reagent to provide tagged nucleic acids, at least two tagged nucleic acids being provided, each corresponding to two or more barcode affinity reagents, each barcode affinity reagent corresponding to its respective target binding site, (d) Sequencing the tagged nucleic acids to provide nucleotide sequences.The present invention provides a method comprising (e) analyzing the nucleotide sequence to identify the target binding site on the nucleic acid. In some embodiments, the tagged nucleic acid includes the respective target binding site.

[0018] In some embodiments, the subject of this disclosure is a method for identifying one or more target binding sites on a nucleic acid, comprising: (a) providing one or more barcoding affinity reagents, each comprising an affinity reagent conjugated to a pair of adapters, wherein the first adapter comprises a first barcode nucleotide sequence and a first transposase-binding mosaic sequence, the second adapter comprises a second barcode nucleotide sequence and a second transposase-binding mosaic sequence, the first and second barcode nucleotide sequences are identical or different, none of the one or more barcoding affinity reagents contain a transposase, each barcoding affinity reagent binds to a different target, and the first and second barcode nucleotide sequences of each barcoding affinity reagent are different from the first and second barcode nucleotide sequences of other barcoding affinity reagents that bind to different targets; and (b) adding one or more barcoding affinity reagents to a sample containing the target of each affinity reagent, wherein each target is The method provides a method comprising: (c) adding a barcoding affinity reagent to a sample, wherein each barcoding affinity reagent is bound to a nucleic acid at its respective target binding site, and each barcoding affinity reagent is bound to its respective target or to a primary affinity reagent bound to its respective target, and the binding of each affinity reagent is performed in the absence of transposase; (d) adding an unbound transposase and a transposase activator to a sample, wherein the unbound transposase binds to the first transposase-bound mosaic sequence and the second transposase-bound mosaic sequence of each barcoding affinity reagent, the bound transposase fragments the nucleic acid, and tags the nucleic acid with the first barcode nucleotide sequence and the second barcode nucleotide sequence of each barcoding affinity reagent to provide a tagged nucleic acid, wherein at least one tagged nucleic acid is provided, which corresponds to the corresponding barcoding affinity reagent, and each barcoding affinity reagent corresponds to its respective target binding site; (e) sequencing the tagged nucleic acid to provide a nucleotide sequence; and analyzing the nucleotide sequence to identify the target binding site on the nucleic acid. In some embodiments, the tagged nucleic acid includes its respective target binding site.In some embodiments, two barcoding affinity reagents are provided, in which one tagged nucleic acid comprises two target binding sites corresponding to the two barcoding affinity reagents. In some embodiments, two barcoding affinity reagents are provided, in which two tagged nucleic acids each comprise the target binding sites of the corresponding barcoding affinity reagents.

[0019] In some embodiments, the transposase is Tn5, Tn3, Tn7, TnY, Sleeping Beauty, or PiggyBac, and the transposase activator is MgCl2. In some embodiments, the target is a DNA-binding protein such as a histone, a histone modifying enzyme, a transcription factor, a cofactor, or a chromatin-related protein. In some embodiments, the target is a post-translational modification on a histone or other chromatin-related protein, or a modified DNA base. In some embodiments, the modified DNA base is mC or 5hmC. In some embodiments, the nucleic acid is a part of chromatin, and the method further includes the simultaneous detection of histone marks, histone modifying enzymes, chromatin-related proteins, and transcription factors. In some embodiments, the chromatin-related protein is CTCF or cohesin.

[0020] In some embodiments, the affinity reagent comprises an antibody. In some embodiments, the affinity reagent is a target-specific affinity reagent. In some embodiments, the affinity reagent is a secondary affinity reagent specific to the primary target-specific affinity reagent. In some embodiments, the primary affinity reagent is barcode-free. In some embodiments, the method further comprises adding the primary affinity reagent to the sample.

[0021] In some embodiments, providing the barcoded affinity reagent comprising the affinity reagent bound to the pair of adapters comprises binding a first affinity moiety to the affinity reagent, providing a second affinity moiety to each of the first adapter and the second adapter, and allowing the first affinity moiety to specifically bind to the second affinity moiety. In some embodiments, the first affinity moiety and the second affinity moiety are a pair selected from the group consisting of: biotin and avidin, streptavidin, or neutravidin; a first reactive group and a second reactive group that react to form a covalent bond; a DNA-binding protein and a DNA sequence recognized by the DNA-binding protein; HaloTag and chloroalkane; SNAP-tag and O(6)-benzylguanine; as well as single-stranded DNA and its hybridizing DNA.

[0022] In some embodiments, the first adapter and the second adapter each further comprise an amplification handle. In some embodiments, analyzing the nucleotide sequence to identify the binding site of the target on the nucleic acid further comprises associating the barcode nucleotide sequence with the affinity reagent. In some embodiments, the method further comprises amplifying the tagmented nucleic acid to provide a sequencing library. In some embodiments, the amplification is polymerase chain reaction amplification. In some embodiments, each barcoded affinity reagent comprises a first handle and a second handle linked via a spacer, the first adapter is hybridized to the first handle, the second adapter is hybridized to the second handle, the first handle or the second handle comprises a first affinity moiety bound to a second affinity moiety of the affinity reagent, and the first adapter and the second adapter comprise different amplification handles.

[0023] In some embodiments, the sample is a cell, tissue, or cell-free DNA. In some embodiments, the method further comprises permeabilizing the cell or tissue.

[0024] In some embodiments, the presently disclosed subject matter is a multiplex method for identifying a plurality of binding sites of a plurality of targets on one or more nucleic acids, comprising: (a) providing a plurality of barcoded affinity reagents, wherein none of the plurality of barcoded affinity reagents comprises a transposase, and wherein each of the plurality of barcoded affinity reagents binds to a different target; (b) adding the plurality of barcoded affinity reagents to a sample; (c) adding the unloaded transposase and a transposase activating agent to the sample to provide a plurality of tagmented nucleic acids; (d) sequencing the plurality of tagmented nucleic acids to provide nucleotide sequences; and (e) analyzing the plurality of nucleotide sequences to identify the plurality of binding sites of the plurality of targets.

[0025] In some embodiments, the nucleic acid is part of chromatin, and the method further comprises determining a data fingerprint for a combination of two target binding sites, wherein the data fingerprint comprises (a) co-localization information of the two target binding sites, or the lack of interaction between the two target binding sites; (b) the distance between the two target binding sites or epitopes; and (c) the nucleotide sequence of the tagmented nucleic acid. In some embodiments, the data fingerprint further comprises (d) the polarity or order of modifications; (e) cis-regulatory elements; (f) proximity to or absence of a CpG island; (g) repetitive DNA sequences; and / or (h) the average DNA methylation level.

[0026] In some embodiments, the nucleic acid is part of chromatin. In some embodiments, the method further comprises simultaneously identifying a plurality of histone marks, histone variants, histone mark readers, histone modifying enzymes, DNA modifying enzymes, chromatin-associated proteins, transcription factors, RNA species, and / or cofactors in the genome.

[0027] In some embodiments of the methods disclosed herein, background IgG sequencing reads constitute less than 25%, less than 20%, less than 15%, or less than 10% of the total sequencing reads. In some embodiments, affinity reagent-specific signals are generated at less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, or less than 2% of the cross-contaminated signals between different antibodies. In some embodiments, the method further includes identifying the co-localization of two epitopes at a single intracellular locus. In some embodiments, the co-localization of H3K4me3 and H3K27me3 is identified.

[0028] In some embodiments of the methods disclosed herein, the methods further include identifying bivalent domain regions covered by two histone modifications in a sample. In some embodiments, the methods further include identifying the colocalization of two epitopes at the same location on the same chromosomal copy derived from the same chromosomal fragment within the same cell.

[0029] In some embodiments, barcoding multiple affinity reagents to provide multiple barcoded affinity reagents includes incubating each of the multiple affinity-labeled affinity reagents with a unique barcoded adapter in a separate reaction vessel to provide multiple individual barcoded affinity reagents. In some embodiments, the method further includes pooling the multiple individual barcoded affinity reagents to provide a mixture of barcoded affinity reagents. In some embodiments, analyzing the multiple nucleotide sequences to identify multiple binding sites of multiple targets further includes associating each barcoded nucleotide sequence of the multiple barcoded nucleotide sequences with each affinity reagent of the multiple affinity reagents. In some embodiments, the multiple targets include 2 to 500 targets.

[0030] In some embodiments of the methods disclosed herein, the methods further include isolating nuclei from cells, sorting single cells or single nuclei using flow cytometry or gel beads, lysing single cells or single nuclei, amplifying a single-cell / nucleus library including identification of signals from individual cells, pooling the single-cell / nucleus library, and sequencing the single-cell / nucleus library.

[0031] In some embodiments of the methods disclosed herein, the methods further include adding a drug to a sample, performing each of steps (a) to (e), and comparing how the drug perturbs the signature in vitro or in vivo.

[0032] In some embodiments, the subject matter of the present disclosure provides instructions to each include two or more barcoding affinity reagents; a pair of adapters, the first adapter comprising a first barcoding nucleotide sequence and a first transposase-binding mosaic sequence, and the second adapter comprising a second barcoding nucleotide sequence and a second transposase-binding mosaic sequence; affinity reagents; adapters, each comprising a barcoding nucleotide sequence and a transposase-binding mosaic sequence; an unbound transposase; and a transposase activator. In some embodiments, the affinity reagents, the adapters, the unbound transposase, and the transposase activator are contained in a container. In some embodiments, the kit further comprises one or more cell or nuclear permeabilization buffers and / or one or more wash buffers. In some embodiments, the buffers are contained in a container.

[0033] In some embodiments, the subject matter of the present disclosure provides a kit comprising two or more barcoding affinity reagents, an unbound transposase, and a transposase activator, wherein each of the two or more barcoding affinity reagents comprises a pair of adapters, the first adapter comprising a first barcoding nucleotide sequence and a first transposase-binding mosaic sequence, and the second adapter comprising a second barcoding nucleotide sequence and a second transposase-binding mosaic sequence. In some embodiments, the two or more barcoding affinity reagents, the unbound transposase, and the transposase activator are contained in a container. In some embodiments, the kit further comprises one or more cell or nuclear permeabilization buffers and / or one or more wash buffers. In some embodiments, the buffers are contained in a container.

[0034] In some embodiments, the kits disclosed herein further include controls. In some embodiments, the controls are recombinant nucleosomes bound to DNA and / or control affinity reagents. In some embodiments, the kits disclosed herein include a panel of affinity reagents. In some embodiments, the kits disclosed herein include a panel of cancer-specific affinity reagents. In some embodiments, the kits disclosed herein include a panel of epigenomic marking proteins and / or histone-specific affinity reagents. In some embodiments, the kits disclosed herein further include reagents and materials for DNA isolation and nucleic acid amplification. In some embodiments, the kits disclosed herein further include a cell capture scaffold. In some embodiments, the cell capture scaffold includes magnetic beads, columns, concanavalin A beads, streptavidin beads, colloidal semiconductor nanocrystals, carbon nanotubes, or microfluidic devices.

[0035] In embodiments relating to multiplex technology, the method includes pooling a plurality of individual and different barcoding affinity reagents (e.g., primary antibodies) and incubating a sample containing nucleic acids and a plurality of DNA binding targets (e.g., a sample containing permeabilized cells, nuclei, cell-free chromatin, cell-free DNA, or tissue) with the plurality of individual and different barcoding affinity reagents (e.g., primary antibodies).

[0036] In some embodiments, the method includes incubating the sample. In some embodiments, the method includes incubating the sample overnight (e.g., 8 to 16 hours (e.g., 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, or 16.0 hours)). In some embodiments, the method includes thoroughly washing the sample after incubation.

[0037] Embodiments of this technology can be used to map multiple DNA-binding targets using small amounts of starting material (e.g., small samples). In some embodiments, this technology can be used to map DNA-binding sites in single cells. In some embodiments, this technology can be used to map DNA-binding sites in cell-free DNA or chromatin preparations.

[0038] In some embodiments, the method involves biotinylating affinity reagents (for example, by adding approximately three biotin molecules (e.g., 1 to 5 molecules (e.g., 1, 2, 3, 4, or 5)) to each affinity reagent using N-hydroxysuccinimodobiotin at low stoichiometry to provide biotinylated affinity reagents). This approach is applicable to ligands of all subclasses and species. In some embodiments, each barcoding adapter oligonucleotide includes a biotin, a PCR handle, a barcode sequence (e.g., a barcode sequence of 10-15 nucleotides (e.g., 4-25 nucleotides (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides)), a nucleotide space (e.g., a space of 10-20 nucleotides (e.g., a space of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides)), and a double-stranded portion encoding a Tn5-binding mosaic sequence. In some embodiments, the features of these barcoding DNA adapters are arranged on the adapter from the 5' end to the 3' end in the following order: 5' terminal biotin, PCR handle, barcode sequence, nucleotide space, and double-stranded sequence encoding a Tn5-binding mosaic sequence. In some embodiments, barcoding affinity reagents for different targets are incubated in separate reaction vessels (e.g., tubes) to provide individual barcoding affinity reagents. In embodiments of the lowplex method, one or more unmodified primary ligands that bind to a specific target are provided, and a mixture of barcoding affinity reagents is used as a secondary ligand for the primary ligand. In embodiments of the highplex method, a mixture of barcoding affinity reagents is used as a primary affinity reagent that binds to a specific target. In some embodiments, amplifying tagged chromosomal DNA to produce one or more sequencing libraries involves using a polymerase chain reaction.

[0039] In some embodiments, the first affinity site is biotin, and the second affinity site is avidin, streptavidin, or neutravidin. In some embodiments, the first and second affinity sites react chemically to form a covalent bond (e.g., via click chemistry or maleimide or N-hydroxysuccinimide (NHS) crosslinking chemicals). Thus, in some embodiments, the first and second affinity sites include a click chemistry pair. In some embodiments, the first and second affinity sites include glutamine and amines, N-hydroxysuccinimide esters and primary amines, maleimide and sulfhydryls, Traut reagents and primary amines, or other known reactive groups that form a covalent bond. In some embodiments, the first and second affinity sites consist of a DNA-binding protein and a DNA sequence recognized by the DNA-binding protein. In some embodiments, the first and second affinity sites consist of a HaloTag and a chloroalkane. In some embodiments, the first and second affinity sites consist of a SNAP-tag and O(6)-benzylguanine.

[0040] In some embodiments, barcoding multiple affinity reagents to provide multiple barcoded affinity reagents includes incubating each of the multiple affinity-labeled affinity reagents with a unique barcoded adapter in a separate reaction vessel to provide multiple individual barcoded affinity reagents. In some embodiments, the method further includes pooling the multiple individual barcoded affinity reagents to provide a mixture of barcoded affinity reagents. In some embodiments, analyzing multiple nucleotide sequences to identify multiple binding sites of multiple targets further includes associating each barcoded nucleotide sequence of the multiple barcoded nucleotide sequences with each affinity reagent of the multiple affinity reagents. See, for example, Figures 7A and 7B. In some embodiments, the plurality of targets includes 2 to 50 targets (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 targets). In some embodiments, the multiple targets are 2 to 500 targets (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, Includes targets of 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, or 500.

[0041] The subject matter disclosed herein offers advantages over the prior art, as shown in each example and figure. For example, the subject matter disclosed herein offers the following advantages over existing techniques relating to the identification and characterization of binding sites on chromosomes: (1) Use of two or more barcoding affinity reagents, each containing an affinity reagent linked to a pair of adapters, wherein the first adapter contains a first barcode nucleotide sequence and a first transposase-binding mosaic sequence, the second adapter contains a second barcode nucleotide sequence and a second transposase-binding mosaic sequence, the first barcode nucleotide sequence and the second barcode nucleotide sequence are identical or different, and the two or more barcoding ligands do not contain transposases; (2) Affinity reagents linked to each adapter pair. For example, streptavidin-biotin binding between the affinity reagent and the adapter pair significantly reduces barcode dissociation or swapping. Strong binding affinity between streptavidin and biotin (dissociation constant K d is approximately 10 -14 (mol / liter) [Reference 8] provides a stable and specific affinity reagent-adapter conjugate. (3) Controlled tagmentation by free transposases (e.g., Tn5, Tn3, Tn7, TnY, Sleeping Beauty, piggyBac, etc.). Random tagmentation is minimized and / or eliminated by providing transposases that are not bound to adapters (adapter-free transposases). Affinity reagents are prepared individually without transposases, and transposases are added after each affinity reagent recognizes its target. This approach allows transposases to retain maximum enzymatic activity, maximizing efficient and precise tagmentation. (4) Extensive profiling of epigenetic regulators. Low noise and minimal cross-contamination provide a technique for detecting multiple targets in a single experiment using small amounts of starting material (e.g., single cells). This advantage is particularly useful for saving valuable samples. Furthermore, this technique provides a multiplex method for clearly identifying co-binding events, thus yielding comprehensive insights into complex regulatory interactions. This technique is used for the analysis of epigenetic landscapes and regulatory mechanisms.

[0042] During the development of embodiments of this technology, data demonstrated that the technology generates very low background signals, minimizing or eliminating signal mixing and ambiguity between adapter-affinity reagent pairs. Furthermore, benchmarking using the ENCODE database showed that embodiments of this technology recover a large portion of the ENCODE peaks. Embodiments of this technology enable the simultaneous detection of histone marks, histone modifying enzymes, and transcription factors. This technology provides a technique for identifying multiple bivalent binding events within the same cell, verifying the formation of histone codes, and relating histone coding information to the distribution of histone modifying enzymes and transcription factors.

[0043] In parts of this specification, embodiments of the technology are described in terms of algorithms and symbolic representations of operations on information. These algorithmic descriptions and representations are commonly used by those skilled in the field of data processing to effectively communicate the essence of their work to others skilled in the field. These operations are described functionally, computationally, or logically, but are understood to be implemented by computer programs or equivalent electronic circuits, microcode, etc. Furthermore, for convenience, sequences of these operations may be referred to as modules, but this does not diminish generality. The described operations and associated modules may be embodied in software, firmware, hardware, or any combination thereof.

[0044] The specific steps, operations, or processes described herein may be performed or implemented by one or more hardware or software modules, either alone or in combination with other devices. In some embodiments, the software module is implemented as a computer program product comprising a computer-readable medium containing computer program code, which is executable by a computer processor to perform any or all of the steps, operations, or processes described herein.

[0045] In some embodiments, the system includes a computer and / or virtually provided data storage (e.g., as a cloud computing resource). In certain embodiments, the technology includes utilizing cloud computing to provide a virtual computer system that includes or performs the functions of the computer components described herein. Thus, in some embodiments, cloud computing provides the infrastructure, applications, and software described herein over a network and / or the internet. In some embodiments, computing resources (e.g., data analysis, computation, data storage, application programs, file storage, etc.) are provided remotely over a network (e.g., the internet and / or a cellular network).

[0046] Embodiments of this technology may also relate to devices that perform the operations described herein. Such devices may be specifically constructed for a desired purpose (e.g., application-specific integrated circuits or field-programmable gate arrays), or they may include general-purpose computing devices (e.g., microcontrollers, microprocessors) that are selectively operated or reconfigured by computer programs stored in a computer. Such devices may be configured to perform one or more steps, operations, and / or functions described herein, which may be provided, for example, as instructions in a computer program. Such computer programs may be stored in non-temporary, tangible computer-readable storage media or any type of media suitable for storing electronic instructions and connected to a computer system bus. Furthermore, any computing system referred to herein may include a single processor, or it may be an architecture employing a multi-processor design to increase computing power.

[0047] Additional embodiments will be apparent to those skilled in the art based on the teachings contained herein. [Brief explanation of the drawing]

[0048] Each patent or application file must include at least one color drawing. A copy of the published patent or patent application, including the color drawing, is available upon request from the Office, with payment of the prescribed fee.

[0049] These and other features, aspects, and advantages of the present invention will be better understood with reference to the following drawings.

[0050] [Figure 1A]This figure shows the simultaneous and effective characterization of multiple chromatin proteins using Hi-PlexCUT&Tag. Hi-PlexCUT&Tag Workflow: 1) Barcoded primary antibodies are constructed by incubating a barcoded adapter containing biotinylated antibodies, streptavidin, and Tn5-conjugated mosaic (orange). 2) Multiple barcoded antibodies are mixed and incubated with immobilized cells. Different targets are simultaneously bound by their respective antibodies. Unbound antibodies are removed by washing. 3) Tn5 and MgCl2 activate tagmentation, inserting barcodes for different targets into nearby genomic DNA. 4) Fragment libraries are enriched by PCR and sequenced with Illumina next-generation sequencing. [Figure 1B] This figure shows the simultaneous and effective characterization of multiple chromatin proteins using Hi-PlexCUT&Tag. It displays genome browser signal tracks (RPM: reads per million reads) of IgG negative control in ChIP-seq, CUT&RUN, CUT&Tag, and Hi-PlexCUT&Tag. Hi-PlexCUT&Tag exhibits the lowest IgG background signal. [Figure 1C] This figure shows the simultaneous and effective characterization of multiple chromatin proteins using Hi-PlexCUT&Tag. It also shows a scatter plot illustrating the high correlation between replicated reads of H3K4me3, RNAPII, and H3K27me3. [Figure 1D] This figure shows the simultaneous and effective characterization of multiple chromatin proteins using Hi-PlexCUT&Tag. It displays genome browser signal tracks (within the same genomic region) of H3K27me3, RNAPII, and H3K4me3 singleton reads from Hi-PlexCUT&Tag, MulTI-Tag, ChIP-seq, and general ATAC-seq. Hi-PlexCUT&Tag profiles are similar to most methods but differ from general ATAC-seq, which measures accessibility. [Figure 1E]This figure shows the simultaneous and effective characterization of multiple chromatin proteins using Hi-PlexCUT&Tag. A heatmap shows the enrichment of two mutually exclusive target pairs (H3K9me3 vs. H3K9ac, H3K27me3 vs. H3K27ac). Note that there is no overlap in the genomic localization of these exclusive epitopes. [Figure 1F] This figure shows the simultaneous and effective characterization of multiple chromatin proteins using Hi-PlexCUT&Tag. It displays genome browser signal tracks for H3K4me3 and RNAPII obtained by ChIP-seq, and H3K4me3 / RNAPII heterotone (tagged sequence reads having different barcode sequences at both ends) obtained by Hi-PlexCUT&Tag. Peaks highlighted in green indicate that the Hi-PlexCUT&Tag heterotone signal can represent the overlap of two related individual ChIP-seq signals. Peaks highlighted in orange indicate that the Hi-PlexCUT&Tag heterotone read can only identify cases where two target epitopes overlap. [Figure 1G] This figure shows the simultaneous and effective characterization of multiple chromatin proteins using Hi-PlexCUT&Tag. A heatmap shows the enrichment of RNAPII versus H3K4me3. There is some overlap in the genomic localization of these targets. [Figure 1H] This figure shows the simultaneous and effective characterization of multiple chromatin proteins using Hi-PlexCUT&Tag. The stacked bar graph summarizes the peak overlap between H3K4me3 / H3K27me3 heterotones obtained by Hi-PlexCUT&Tag and individual H3K4me3 and H3K27me3 reads obtained by ChIP-seq. Hi-PlexCUT&Tag identifies more bivalent events than ChIP-seq. [Figure 1I]This figure shows the simultaneous and effective characterization of multiple chromatin proteins using Hi-PlexCUT&Tag. It displays genome browser signal tracks of H3K4me3 (blue) and H3K27me3 (red) by ChIP-seq, and H3K4me3 / H3K27me3 heterotone (purple) by Hi-PlexCUT&Tag. The enlarged view at the bottom highlights epitope duplication clearly detected at the promoter by Hi-PlexCUT&Tag. [Figure 1J] This figure shows the simultaneous and effective characterization of multiple chromatin proteins using Hi-PlexCUT&Tag. The genome browser signal tracks (top) and SEACR peaks (bottom rectangles) for H3K4me3 and H3K27me3 detected by ChIP-seq, and for the H3K27me3 / H3K4me3 heterotone detected by Hi-PlexCUT&Tag. Hi-PlexCUT&Tag identifies bivalent events not detected by ChIP-seq and is highlighted in blue. [Figure 2A] This figure illustrates the complexity of the Hi-PlexCUT&Tag dataset. It's a cartoon illustration of the information packed into the Hi-Plex technology measurements. Hi-Plex technology can measure the co-localization of two targets on the genome and further map them to various regulatory elements. The lengths of sequence fragments are also clustered and can be explained by the measured number of nucleosomes, relating to the function of the targets involved in the fragment. [Figure 2B] This figure shows the complexity of the Hi-PlexCUT&Tag dataset. The heatmap shows the number of peaks called using SEACR for each target pair obtained from 36 different epigenetic marks, including histone modifications, RNA polymerase II, epigenetic writers, and transcription factors. [Figure 2C] This figure shows the complexity of the Hi-PlexCUT&Tag dataset. Box plots show the distribution of cis-regulatory and repeating elements between euchromatin mark pairs and heterochromatin mark pairs. Significance tests of differences between euchromatin and heterochromatin marks are labeled at the top. [Figure 2D] This figure illustrates the complexity of the Hi-PlexCUT&Tag dataset. It shows stacked bar graphs of peak annotations for cis-regulatory elements, repeating elements, and mean DNA methylation for selected target pairs including euchromatin marks, bivalent marks, and heterochromatin marks. Hierarchical clustering identifies four major groups. [Figure 2E] This figure shows the complexity of the Hi-PlexCUT&Tag dataset. It is a stacked bar graph of the fragment length distribution. It shows 20 target pairs, organized by the highest levels of subnucleosomes, mononucleosomes, and dinucleosomes. Targets with the highest subnucleosome levels are typically associated with transcription factor-related marks, while targets with the highest dinucleosome levels are generally associated with histone modification-related marks. [Figure 2F] This figure shows the complexity of the Hi-PlexCUT&Tag dataset. [Figure 3A] This is a diagram of single-cell Hi-PlexCUT&Tag profiling. It is a schematic diagram of the single-cell Hi-PlexCUT&Tag (scHi-PlexCUT&Tag) method. [Figure 3B] This figure shows single-cell Hi-PlexCUT&Tag profiling. It compares the chromatin landscape of the bulk Hi-PlexCUT&Tag map and the scHi-Plex CUT&Tag map (aggregation of all single cells and individual cells) in the enriched region of H3K27me3 homotone (tagged sequence reads with identical barcode sequences at both ends). Cells are ordered by read coverage within the depicted region. [Figure 4] This figure outlines the 37 barcoded antibodies used in Hi-PlexCUT&Tag. We barcoded a panel of 37 antibodies targeting 12 common histone marks (orange), 14 histone modifying enzymes (light blue), 8 human transcription factors (gray), CTCF (dark blue), PolII (pSer2) (yellow), and rabbit IgG negative control (green). [Figure 5] This figure shows the Low-Plex CUT&Tag workflow. 1. Barcoded secondary antibodies (2° antibodies) are constructed by incubating biotinylated antibodies, streptavidin, and a barcoded adapter. Tn5-conjugated mosaic (orange) is on the adapter. Different antibodies are prepared individually before mixing. 2. Cells immobilized with concanavalin A-coated beads are first incubated with the primary antibody, then with the barcoded secondary antibody. 3. Tn5 and MgCl2 are introduced to activate tagmentation. The barcode is inserted into nearby genomic DNA. 4. The fragment library is enriched by PCR and sequenced by Illumina sequencing. [Figure 6A] This figure shows the size distribution of Hi-PlexCUT&Tag fragments. The gel image shows a ladder-like pattern in the Hi-PlexCUT&Tag library. Fragments of different sizes are labeled as sub-, mono-, di-, and tri+- (related to the number of nucleosomes occupying the endogenous fragment). [Figure 6B] This figure shows the size distribution of Hi-PlexCUT&Tag fragments. It represents a size distribution analysis of all fragments obtained from the Hi-PlexCUT&Tag library. Different size names are labeled at the top of each peak. [Figure 7A]This figure shows embodiments of modified and barcoded antibodies described herein. As shown in Figure 7A, the embodiments provide antibodies modified with one or more first affinity sites ("A"). As shown in Figure 7A, the affinity sites may be bound to the antibody via one or more linkers. Furthermore, the antibody may be barcoded with one or more barcode adapters containing a second affinity site ("B") and different barcode sequences. The binding pairs (e.g., affinity sites) A ​​and B are as described herein (e.g., covalent bonds (click chemistry (by click chemistry pairs), glutamine and amine, N-hydroxysuccinimide ester and primary amine, maleimide and sulfhydryl, Trout's reagent and primary amine, and other covalent chemistry known to those skilled in the art); avidin and biotin, neutravidin and biotin, streptavidin and biotin, DNA-binding proteins and their recognition DNA sequences, HaloTag and chloroalkane, or SNAP-tag and O(6)-benzylguanine). [Figure 7B] This figure shows an embodiment of a modified antibody conjugate including multiple (e.g., two) adapters. Two adapters, including lead 1 and lead 2, are conjugated to the same antibody using a single-stranded DNA handle (brown in the figure) with a spacer between the two hybridize regions. The binding pairs (e.g., affinity sites) A ​​and B are as described herein (e.g., covalent bonding (click chemistry (by click chemistry pairs), glutamine and amine, N-hydroxysuccinimide ester and primary amine, maleimide and sulfhydryl, Trout reagent and primary amine, and other covalent bonding chemistry known to those skilled in the art); avidin and biotin, neutravidin and biotin, streptavidin and biotin, DNA-binding protein and its recognition DNA sequence, HaloTag and chloroalkane, or SNAP-tag and O(6)-benzylguanine). In some embodiments, the melting temperature (Tm) of each hybridized region within the handle is 42°C to 49°C (e.g., 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, or 49°C).

[0051] Please understand that the figures are not necessarily drawn to scale, and the scales of the objects in the figures are not necessarily consistent. Each figure is intended to provide clarity and understanding of the embodiments of the various devices, systems, and methods disclosed herein. Wherever possible, the same reference numerals are used throughout the drawings to refer to identical or similar parts. Furthermore, please understand that the drawings are not intended to limit the scope of the invention in any way. [Modes for carrying out the invention]

[0052] Current CUT&Tag and multi-CUT&Tag Tn5 transposase-based genome-wide sequencing technologies localize chromatin-related factors such as histone marks, transcription factors, and cofactors, relying on guiding pre-loaded Tn5-protein A fusions to specific chromatin regions where tagmentation is possible. These methods use protein A as a connector to bind Tn5 to factor-specific antibodies. However, pre-loaded transposases can desorb and perform random tagmentation, resulting in high background noise. This problem is more pronounced when multiple pre-assembled antibody-Tn5-pA complexes are used simultaneously, as signals from different targets are unintentionally mixed. This limitation significantly restricts the ability to multiplex. Given the strong desire to detect binding sites for many important histone marks, transcription factors, and cofactors, we developed a novel technology called Hi-PlexCUT&Tag, enabling higher-order plex detection of dozens of histone marks, histone modifying enzymes, and transcription factors. In this method, a DNA barcode adapter containing transposase-binding mosaic is directly bound to a predetermined antibody via biotin-streptavidin interaction, without pre-loading Tn5. This process includes incubation with a pooled barcoded primary antibody. After removing unbound antibody by washing, unloaded transposases are introduced and activated. These transposases bind to the binding mosaic on the adapter, initiating the tagmentation process. The Hi-PlexCUT&Tag method allows for profiling of numerous targets with small amounts of starting material and can be extended to the single-cell level. Data analysis of Hi-PlexCUT&Tag confirmed that this novel technique produces very low background signal, and more importantly, extremely low cross-contamination between dozens of different antibodies used in the same assay. Using the ENCODE database, we also benchmarked that our novel method can actually reproduce most of the ENCODE peaks.The ability to simultaneously detect numerous histone marks, histone modifying enzymes, and transcription factors allows for the identification of multiple bivalent events within the same cell, verification of histone coding formation, and correlation of this information with the distribution of histone modifying enzymes and transcription factors.

[0053] Eukaryotic DNA wraps around histone proteins to form mononucleosome subunits of chromatin, which can act as physical barriers to transcription. Approximately 3 × 10⁷ nucleosomes are distributed throughout the chromatin of each human cell. Genome-wide sequencing studies over the past 20 years have suggested that dozens of different post-translational histone modification (PTM) combinations can coexist on a single nucleosome, and that nucleosomes with different PTM combinations are located at different loci on chromatin. Histone PTMs are added by histone modifying enzymes that read, write, and erase them, functioning as docking sites for chromatin-associated complexes, regulating gene transcription and influencing the functional state of chromatin. These chromatin-associated complexes often consist of histone modifying enzymes and nucleosome remodelers, coordinating the proper access and function of proteins on chromosomal DNA. A fundamental problem in elucidating these combination events is that without a holistic understanding of the co-localization of epigenetic modifications and regulators, robust prediction of gene expression or the resulting phenotype is impossible. Since most sequencing analyses can only analyze one PTM or epigenetic regulator at a time, how specific chromatin-related complexes interact with histone PTM combinations to promote appropriate chromatin structure and gene expression remains largely unknown.

[0054] ChIP-seq (chromatin immunoprecipitation-followed sequencing) has historically been the most common method for globally profiling DNA-binding proteins (e.g., transcription factors (TFs) and cofactors) and histone PTMs, but it has drawbacks such as being able to profile only one target at a time, low sensitivity, high cost, low efficiency, and inability to map epitopes at the single-cell level [References 1,2,3]. Recently developed CUT&Tag (Cleavage Under Targets and Tagmentation) uses a protein A-fusion Tn5 transposase to induce Tn5 loaded with an adapter onto an antibody already bound to the target protein (e.g., TF or PTM) within the cell. 2+ When activated, the transposase cleaves chromosomal DNA nearby, releasing small DNA fragments that are then sequenced on a next-generation sequencing platform. Compared to ChIP-seq, CUT&Tag requires fewer cells, offers higher resolution, and is applicable to single-cell analysis [Reference 4]. Furthermore, the recently developed Multi-CUT&Tag allows for simultaneous profiling of up to three targets in a single experiment by pre-forming antibody-protein A::Tn5 fusion complexes loaded with sequencing adapters containing specific DNA barcode sequences, and then pooling different antibodies and incubating them with permeabilized cells. Multiplexing allows for simultaneous verification of three epitopes and their combinations within the same cell [References 5,6].

[0055] However, CUT&Tag technology and its derivatives have several significant drawbacks. 1) Tn5: Protein A complex has a relatively weak interaction with the antibody (K D =10 -81) It dissociates due to M) and acts as an ATAC reagent, so high background signal is common. 2) Cross-contamination can be serious in multiplex assays because "swapping" occurs between DNA adapters of different antibodies. These problems greatly limit the ability to perform higher-order plexing [Reference 7]. Furthermore, the design principle is such that epitope co-localization can only be detected in a very small portion of Multi-CUT&Tag and MulTI-Tag data [References 5,6,7].

[0056] To minimize background signaling and cross-contamination, enhance multiplexing capabilities tenfold, and increase the possibility of detecting epitope co-localization within the same cell, we invented a novel technique called Hi-PlexCUT&Tag, enabling simultaneous pairwise genome-wide positioning of up to 40 targets using next-generation sequencing.

[0057] To reduce background signaling and cross-contamination, we employed a different strategy for antibody (Ab) barcoding and improved the tagmentation procedure. Tetramer-type streptavidin was used as a connector to conjugate biotinylated and barcoded DNA adapter sequences to biotinylated antibodies. A mixture of these individually barcoded antibodies was incubated with permeabilized cells or nuclei at room temperature (RT) for 1 hour. After removing unbound antibodies by rigorous washing, Tn5 and MgCl2 were simultaneously added to the sample, and incubated at 37°C for 1 hour. Finally, genomic DNA was extracted, tagged DNA was amplified by PCR, and library preparation and next-generation sequencing were performed (Figure 1A). The biotin-streptavidin interaction is nearly irreversible (K D <10 ―14Considering that it is M) [Reference 8], the likelihood of the biotinylated DNA adapter sequence dissociating from the antibody to produce a nonspecific ATAC-like background signal, or swapping with a different adapter sequence to produce a cross-contamination signal, is extremely low. Our improved tagmentation procedure further reduces the background signal and improves Tn5 activity by simultaneously adding Tn5 and MgCl2 after removing the unbound antibody, thereby avoiding long-term (overnight) incubation.

[0058] Our data demonstrate that Hi-PlexCUT&Tag represents a step forward in multiplex chromatin profiling, showing improved specificity and sensitivity in detecting multiple targets. Its streamlined workflow and precise control of the tagging process make it a valuable tool for studying chromatin biology and protein interactions in various biological contexts.

[0059] While embodiments of this technology describe an example of linking an affinity reagent to a DNA adapter via a streptavidin-biotin linkage, this technology is not limited to this linkage pair or linkage mode. This technology includes linkage and linkage modes such as ionic (e.g., electrostatic) interactions, affinity linkage (e.g., protein-protein (e.g., antibody-antigen, etc.), protein-nucleic acid (e.g., nucleic acids and nucleic acid-binding proteins), glycans-lectins, metal-chelators), direct (e.g., covalent) conjugation (e.g., click chemistry (e.g., triazole formation by azido-alkynes, transcyclooctene and tetrazine, Staudinger ligation, azido-cyclooctin cyclization, reverse electron-required Diels-Alder reaction, etc.)), and nucleic acid hybridization (e.g., hydrogen bonding) (as described herein). For example, see Dugal-Tessier (2021) "Antibody-Oligonucleotide Conjugates: A Twist to Antibody-Drug Conjugates" J. Clin. Med. 10: 838 (incorporated herein by reference), Dovgan (2019) "Antibody-Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents" Bioconjugate Chemistry 30: 2483 (incorporated herein by reference), etc.

[0060] Binding pairs and binding modes include, but are not limited to, pairs that interact via covalent and non-covalent bonds (ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, etc.). Binding pairs include, but are not limited to, receptor / affinity reagent pairs, affinity reagent and receptor affinity reagent binding sites, antibody / antigen pairs, antigen-antibody antigen-binding fragments, antibody or antibody fragment and hapten, lectin / glycan pairs, enzyme / substrate pairs, biotin / avidin, biotin / streptavidin, digoxin / anti-digoxin, DNA or RNA aptamer binding pairs, peptide aptamer binding pairs, etc.

[0061] In some embodiments, the DNA adapter is bound to the affinity reagent using covalent bonds. In some embodiments, covalent bonds are formed using click chemistry, glutamine and amine, N-hydroxysuccinimide ester and primary amine, maleimide and sulfhydryl, Trout reagent and primary amine, and other covalent linking chemistry known in the art. In some embodiments, the DNA adapter is bound to the affinity reagent using binding pairs. In some embodiments, binding pairs such as avidin and biotin, neutravidin and biotin, streptavidin and biotin, DNA-binding proteins and their recognition DNA sequences, HaloTag and chloroalkane, SNAP-tag and O(6)-benzylguanine are used.

[0062] In some embodiments, one DNA adapter binds to a single site on the antibody. In some embodiments, multiple DNA adapters (e.g., 2, 3, 4, 5 or more) bind to a single site on the antibody. In some embodiments, one or more DNA adapters (e.g., 1, 2, 3, 4, 5 or more) bind to each of multiple sites (e.g., 2, 3, 4, 5 or more) on the antibody.

[0063] In some embodiments, antibodies are modified at specific sites. In some embodiments, antibodies are modified nonspecifically.

[0064] In some embodiments, a structure having a spacer between two or more hybridization regions on a single-stranded DNA handle is used to conjugate (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) adapters to the same antibody. In some embodiments, a structure having a spacer between two hybridization regions on a single-stranded DNA handle is used to conjugate (e.g., 2 adapters to the same antibody) (Figure 7B). The conjugation pairs A and B are as described herein (e.g., click chemistry, glutamine and amine, N-hydroxysuccinimide ester and primary amine, maleimide and sulfhydryl, Trout reagent and primary amine, and other known covalent linking chemistry; avidin and biotin, neutravidin and biotin, streptavidin and biotin, DNA-binding proteins and their recognition DNA sequences, HaloTag and chloroalkane, SNAP-tag and O(6)-benzylguanine).

[0065] During the development of the technology described herein, data were obtained demonstrating that this technology brings improvements to multiplex chromatin profiling. In particular, experiments showed improved specificity and sensitivity in the detection of multiple targets compared to existing technologies. Furthermore, this technology provides a streamlined workflow and precise control of the tagmentation process. Therefore, embodiments of this technology are useful for studying chromatin biology and protein interactions in various biological contexts.

[0066] The detailed descriptions of each embodiment in this specification include numerous specific details for illustrative purposes and to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will understand that these embodiments are implementable with or without these specific details. In other examples, structures and apparatus are shown in block diagram form. Furthermore, those skilled in the art will readily understand that the particular order in which the methods are presented and implemented is illustrative, that the order is modifiable, and that it remains within the spirit and scope of each embodiment disclosed herein.

[0067] All documents and similar materials cited in this application (including, but not limited to, patents, patent applications, articles, books, professional texts, and internet web pages) are expressly incorporated in their entirety by reference. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. In the event of any discrepancy between the definitions of terms in the cited documents and those in this specification, the definitions in this specification shall prevail. Section headings used herein are for organizational purposes only and do not limit the subject matter described in any way.

[0068] definition To facilitate understanding of this technology, numerous terms and phrases are defined below. Additional definitions are provided throughout the detailed explanation.

[0069] In this specification and in the claims, the following terms shall have the meanings expressly associated herein unless the context requires a clearly different meaning. The phrase “in one embodiment” does not necessarily refer to the same embodiment, but it may be so. Similarly, the phrase “in another embodiment” does not necessarily refer to a different embodiment, but it may be so. Thus, various embodiments of the present invention can be readily combined without departing from the scope or spirit of the invention, as described below.

[0070] Furthermore, as used herein, "or" is an inclusive "or" operator and is equivalent to "and / or" unless the context requires a distinctly different meaning. The term "based on" is not exclusive and may be based on additional factors not listed unless the context requires a distinctly different meaning. Also, throughout this specification, the meanings of "a," "an," and "the" include plural references. The meaning of "in" includes both "in" and "on."

[0071] In this specification, the terms “about,” “approximately,” “substantially,” and “significantly” are understood by those skilled in the art and have meanings that vary to some extent depending on the context in which they are used. Where the use of these terms is not clear to those skilled in the art from the context, “about” and “approximately” mean within ±10% of the value of the particular term, while “substantially” and “significantly” mean more than ±10% of the value of the particular term.

[0072] In this specification, a range disclosure includes the disclosure of all values ​​across the entire range and further subdivided ranges (including endpoints and sub-ranges given to the range). In this specification, a numerical range disclosure includes endpoints and each numerical value of the same precision between them. For example, the range 6 to 9 includes 7 and 8 in addition to 6 and 9, and the range 6.0 to 7.0 explicitly includes 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0.

[0073] In this specification, the suffix "-free" refers to an embodiment of the technology in which the core characteristics of the word to which "-free" is attached are omitted. That is, the term "X-free" in this specification means "without X," where X is a characteristic of the technology that is omitted in the "X-free" technology. For example, a "calcium-free" composition does not contain calcium, and a "mixing-free" method does not include a mixing step, etc.

[0074] Terms such as "first," "second," and "third" may be used herein to describe various processes, elements, compositions, components, areas, layers, and / or sections, but these processes, elements, compositions, components, areas, layers, and / or sections should not be limited by these terms unless otherwise explicitly stated. These terms are used to distinguish one process, element, composition, component, area, layer, and / or section from other processes, elements, compositions, components, areas, layers, and / or sections. Where numerals such as "first" and "second" are used herein, they do not imply order or sequence unless explicitly indicated by the context. Therefore, a first process, element, composition, component, area, layer, or section described herein may be referred to as a second process, element, composition, component, area, layer, or section without departing from the art.

[0075] In this specification, the terms “presence” or “absence” (or alternatively, “present” or “absent”) are used in a relative sense to describe the quantity or level of a particular entity (e.g., component, action, element). For example, when an entity is “present,” it means that the level or quantity of that entity is above a predetermined threshold, and conversely, when it is “absent,” it means that the level or quantity of that entity is below a predetermined threshold. The predetermined threshold may be a detectability threshold related to a particular test used to detect the entity, or any other threshold. When an entity is “detected,” it is “present,” and when it is “not detected,” it is “absent.”

[0076] In this specification, “increase” or “decrease” means a detectable (e.g., measured) positive or negative change in the value of a variable compared to a previously measured value, a preset value, and / or a standard control value. An increase is a positive change of preferably at least 10%, more preferably 50%, even more preferably 2 times, even more preferably at least 5 times, and most preferably at least 10 times, compared to a previously measured value, a preset value, and / or a standard control value. Similarly, a decrease is a negative change of preferably at least 10%, more preferably 50%, even more preferably at least 80%, and most preferably at least 90%, compared to a previously measured value, a preset value, and / or a standard control value. Other terms indicating quantitative changes or differences, such as “more” or “less,” are used in the same manner as above.

[0077] In this specification, the term “binding site” refers to a portion of a nucleic acid to which a nucleic acid binding (e.g., chromatin binding) target binds or can bind (e.g., where sufficient conditions for binding exist). The binding site may be single-stranded or double-stranded. The binding site may include two or more portions of the nucleic acid to which the target binds (e.g., in the case of a nucleic acid binding target that forms a dimer or a higher-order complex). The binding site may include both the portion of the nucleic acid to which the target directly binds and the portions of the nucleic acid adjacent to the upstream and / or downstream sides of the target. In some embodiments, the binding site may include up to about 1000 base pairs (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 base pairs) adjacent to the upstream and / or downstream sides of the nucleic acid portion that directly interacts with the target.

[0078] In this specification, “system” means a set of tangible and / or abstract components that work together for a common purpose. In some embodiments, “system” is an integrated collection of hardware and / or software components. In some embodiments, each component of the system interacts with or is associated with one or more other components. In some embodiments, the system refers to a combination of components and software for controlling and directing a method. For example, “system” or “subsystem” may include one or any combination of the following: a machine, hardware, hardware components, circuits, networks, logic designs, logic components, software, software modules, components of software or software modules, software procedures, software instructions, software routines, software objects, software functions, software classes, software programs, files containing software, etc., for performing the functions of the system or subsystem. Accordingly, the methods and apparatus of the embodiments, or specific aspects or parts thereof, may take the form of program code (e.g., instructions) embodied on a tangible medium such as a floppy disk, CD-ROM, hard drive, flash memory, or other machine-readable storage medium, and when such program code is loaded and executed on a machine such as a computer, the machine becomes an apparatus for carrying out the embodiments. When the program code is executed on a programmable computer, the computer generally includes a processor, a processor-readable storage medium (e.g., volatile and non-volatile memory and / or memory elements), at least one input device, and at least one output device. One or more programs may implement or utilize the processes described in relation to the embodiments, for example, using an application programming interface (API), reusable controls, etc. Such programs are preferably implemented in a high-level procedural or object-oriented programming language for communication with the computer system.However, the program may be implemented in assembly language or machine code, if necessary. In either case, the language may be compiled or interpreted and may be combined with a hardware implementation.

[0079] explanation The technologies provided herein relate to the identification of binding sites of DNA-binding proteins, and more particularly, but not limited to, methods, systems, and kits for simultaneously mapping the binding sites of multiple proteins within the same cell using affinity reagent-specific barcodes.

[0080] As used herein, “affinity reagent” refers to any molecule that specifically binds to another molecule (also referred to herein as “target”). For example, an affinity reagent may be an antibody, antibody fragment, nanobody, aptamer, small molecule, synthetic antigen-binding reagent, oligonucleotide, DARPin, peptamer, tetramer, protein scaffold, or other similar ligand or molecule that binds to the target. In some embodiments, the affinity reagent may include an antibody or a fragment thereof (e.g., a monoclonal antibody). The antibody or its fragment may include Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody, triabody, tetrabody, polyspecific antibody formed from antibody fragments, single-domain antibody (sdAb), single chain containing complementary scFv (tandem scFv) or bispecific tandem scFv, Fv construct, disulfide-bonded Fv, bivariable-domain immunoglobulin (DVD-Ig) binding protein or nanobody, aptamer, afibody, affin, afitin, affimer, alphabody, antikalin, avimer, DARPin, finomer, Knitz domain peptide, monobody, or any combination thereof. In this specification, "antibody" refers to monoclonal antibodies, synthetic antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR-transplanted antibodies, polyspecific conjugates that bind to two or more targets, bispecific antibodies, bispecific antibodies or polyspecific antibodies, affinity-mature antibodies, single antibody chains or scFv fragments, diabodies, single chains containing complementary scFv (tandem scFv) or bispecific tandem scFv, Fv constructs, disulfide-bonded Fv, Fab constructs, and Fab' constructs. This refers to F(ab')2 constructs, Fc constructs, monovalent or bivalent constructs (with domains not essential for monoclonal antibody function removed), single-chain molecules (containing one VL, one VH antigen-binding domain, and one or two constant "effector" domains, optionally linked by a linker domain), univalent antibodies lacking a hinge region, single-domain antibodies, bivariable-domain immunoglobulin (DVD-Ig) binding proteins, or nanobodies."Label" also refers to antibody mimetic bodies such as affibodies, which are a class of engineered affinity proteins consisting of small, single-domain proteins, generally about 6.5 kDa, that can be isolated to have high affinity and specificity for any target protein. In some embodiments, the affinity reagent is a single-domain antibody. In some embodiments, the affinity reagent is an antibody against protein A used in CUT&Tag. See Kaya-Okur (2020) Nat Protoc. 15:3264 (incorporated herein by reference).

[0081] In some embodiments, the affinity reagent binds to a target (e.g., a biomolecule). In some embodiments, the target includes, but is not limited to, peptides, proteins, antibodies or antibody fragments, aphibodies, ribonucleic acid sequences or deoxyribonucleic acid sequences, aptamers, lipids, polysaccharides, lectins, or chimeric molecules consisting of multiple homogeneous or heterogeneous parts. In some embodiments, the target is a protein. In some embodiments, the affinity reagent is not an antibody against protein A.

[0082] As used herein, "target" refers to a DNA-related protein or a chromatin-related protein. In some embodiments, the target is a protein on or related to chromatin in a sample. Chromatin is composed of cellular DNA and related proteins. Histone proteins and DNA are present in nearly equal amounts in eukaryotic chromatin, and non-histone proteins are also present. The basic structural unit of chromatin is the nucleosome, a structure composed of DNA and histone proteins that is repeated throughout the genetic material. Histones are highly conserved basic proteins, and their positive charge facilitates the binding of DNA to the negatively charged phosphate backbone.

[0083] In some embodiments, the targets are ALC1, androgen receptor, Bmi-1, BRD4, Brg1, coREST, c-Jun, c-Myc, CTCF, EED, EZH2, Fos, histone H1, histone H3, histone H4, heterochromatin protein-1γ, heterochromatin protein-1, HMGN2 / HMG-17, HP1α, HP1γ, hTERT, Jun, KLF4, K-Ras, Max, MeCP2, MLL / HRX, NPAT, p300, Nanog, NFAT-1, Oct4, P53, PolII (8WG16), RNAPolII Ser2P, RNAPolII Ser5P, RNA Pol II Ser2+5P, RNA Pol IISer7P, Rb, RNA polymerase II, SMCI, Sox2, STAT1, STAT2, STAT3, Suz12, Tip60, UTF1, H1S27ph, H1K25mel, H1K2 5me2, H1K25me3, H1K26me, H2(A)K4ac, H2(A)K5ac, H2(A)K7ac, H2(A)S1ph, H2(A)T119ph, H2(A)S122ph, H 2(A)S129ph, H2(A)S139ph, H2(A)K119ub, H2(A)K126su, H2(A)K9bi, H2(A)K13bi, H2(B)K5ac, H2(B)K11a c, H2(B)K12ac, H2(B)K15ac, H2(B)K16ac, H2(B)K20ac, H2(B)S10ph, H2(B)S14ph, H2(B)33ph, H2(B)K120u b, H2(B)K123ub, H3K4ac, H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K56ac, H3K4mel, H3K4me2, H3K 4me3, H3R8me, H3K9mel, H3K9me2, H3K9me3, H3R17me, H3K27mel, H3K27me2, H3K27me3, H3K36me, H3K79mel This includes H3K79me2, H3K79me3, H3K122ac, H3T3ph, H3S10ph, H3T1ph, H3S28ph, H3K4bi, H3K9bi, H3K18bi, H4K5ac, H4K8ac, H4K12ac, H4K16ac, H4K91ac, H4R3me, H4K20me, H4K59me, H4S1ph, H4K12bi, H4n-terminal tail ubiquitination, etc. In some embodiments, the affinity reagent binds to epitopes containing monomethylated (me1), dimethylated (me2), trimethylated (me3), phosphorylated (ph), ubiquitinated (ub), sumolated (su), biotinylated (bi), acetylated (ac), ADP-ribosylated, O-glycosylated, citrullinated, butyrylated, succinylated, or crotonylated histone residues.

[0084] In some embodiments, the target includes transcription factors, regulatory elements, transcription repressors, transcription activators, polymerases, nucleases, nickases, zinc finger proteins, transcription activator-like effector nucleases (TALENs), glycosylases, methylases, ligases, restriction enzymes, replication proteins, helicases, or kinases. In some embodiments, the target is a DNA-binding protein such as a histone, a histone modifying enzyme, a transcription factor, a cofactor, or a chromatin-related protein. In some embodiments, the target is a post-translational modification on a histone or other chromatin-related protein, or a modified DNA base. In some embodiments, the modified DNA base is mC or 5hmC.

[0085] In some embodiments, the target includes histones, e.g., H1, H2A, H2B, H3, H4, and H5. See Annunziato (2008) DNA Packaging: Nucleosomes and Chromatin. Nature Education 1(1):26 (incorporated herein by reference). Post-translational modified histones may also be targets. For example, histones containing phosphorylated serine or threonine, histones containing methylated lysine or arginine, histones containing acetylated and / or deacetylated lysine, histones containing ubiquitinated lysine, histones containing smolated lysine, etc. In some embodiments, the target is RNA polymerase. In some embodiments, the target is H2AK5ac, H2AK9ac, H2BK120ac, H2BK12ac, H2BK15ac, H2BK20ac, H2BK5ac, H2Bub, H3, H3ac, H3K14ac, H3K18ac, H3K23ac, H3K23me2, H3K27mel, H3K27me2, H3K36ac, H3K36mel, H3K36me2, H3K4ac, H3K56ac, H3K79mel, H3K79m e3, H3K9acS10ph, H3K9me2, H3S10ph, H3T1lph, H4, H4ac, H4K12ac, H4K16ac, H4K5ac, H4K8ac, H4K91ac, H3F3A, H3K27me3, H3K36me3, H3K4mel, H3K79me2, H3K9mel, H3K9me2, H3K9me3, H4K20mel, H2AFZ, H3K27ac, H3K4me2, H3K4me3, or H3K9ac.

[0086] In some embodiments, the target is a transcription factor (TF), TF cofactor, or putative transcription factor. A list of known and putative human transcription factors is provided in Lambert (2018) The Human Transcription Factors. Cell. 172: 650 (incorporated herein by reference). A list of human TFs is provided in Table 1 of International Patent Application Publication No. 2023081863. A list of representative human targets is provided in Table 2 of the same document, a list of representative mouse targets is provided in Table 3, and a list of Drosophila targets is provided in Table 4. During the development of embodiments of the technology described herein, experiments were performed to assay the targets listed in Table 1 below.

[0087] In some embodiments, the target is specifically bound by a first affinity reagent (e.g., a primary antibody), and a second affinity reagent (e.g., a secondary antibody) specifically binds to the first affinity reagent. Thus, in some embodiments, the second affinity reagent binds indirectly to the target. Thus, in some embodiments, the affinity reagent is a secondary antibody specific to the primary antibody species and isotype. For example, in some embodiments, the affinity reagent is anti-IgA, anti-IgD, anti-IgE, anti-IgG, or anti-IgM. Also, in embodiments using a secondary antibody, the secondary antibody may be prepared against a primary antibody of any species such as human, mouse, rat, or rabbit. The affinity reagent can be independently selected from any type of antibody and / or affinity reagent described herein and known to those skilled in the art.

[0088] The embodiments include the use of transposases. In some embodiments, transposases are used for tagmentation. A "transposase" is an enzyme that binds to the end of a transposon and catalyzes its movement to other parts of the genome via a cut-and-paste mechanism or a replication-type transposition mechanism. Representative transposases include Tn5 transposase, Tn3 transposase, Tn7 transposase, TnY transposase, Sleeping Beauty, piggyBac, Hyperactive Tn5 transposase, Mu transposase, IS5 transposase, IS91 transposase, Tn552 transposase, Ty1 transposase, Tn / O transposase, IS10 transposase, Mariner transposase, Tel transposase, P-element transposase, Tn3 transposase, bacterial insertion sequence transposase, retrovirus transposase, yeast retrotransposon transposase, ISS transposase, Tn1O transposase, Tn903 transposase, or combinations thereof.

[0089] In this specification, “transposon” refers to a nucleic acid molecule that can be incorporated into nucleic acids by a transposase. A transposon comprises two transposon ends (also called “arms,” “mosaic ends,” or “ME”). In some embodiments, the two transposon ends are flanked by a sequence long enough to form a loop in the presence of a transposase. Depending on the transposase, the transposon may contain double-stranded, single-stranded, or both single-stranded and double-stranded regions. In the case of the Tn5 transposase, the transposon ends are double-stranded, and the binding sequence is single-stranded or double-stranded. “Mosaic” or “binding mosaic” refers to a sequence region that interacts with a transposase.

[0090] In some embodiments, the transposase is an enzyme that is a member of the RNase superfamily of proteins, which includes retroviral integrases. Examples of transposases include Tn3, Tn5, and their hyperactive mutants. Tn5 is found in bacteria of the genera Shewanella and Escherichia. Examples of hyperactive Tn5 mutants include those containing the E54K and / or L372P mutations. In some embodiments, the transposase is Tn5. In some embodiments, the transposase is TnY, which is a hyperactive transposase mutant from Vibrio parahemolyticus, containing the P50K and M53Q mutations. The inner and outer ends of the transposon contain the same sequences as the inner and outer ends of the Tn5 transposon (see International Patent Application Publication No. 2021011433, incorporated herein by reference). Other transposases used in embodiments of this technology include P. luminescens, L. pneumophila, L. longbeachae, C. glomeribacter, V. prahemolyticus transposases, and Tn5 HA and sarSeaEAK transposases known to those skilled in the art.

[0091] The nucleotide sequence encoding the Tn5 transposase is provided by (SEQ ID NO: 1): [ka] [ka]

[0092] The amino acid sequence of Tn5 transposase is provided by (SEQ ID NO: 2): [ka]

[0093] The nucleotide sequence encoding the TnY transposase is provided by (SEQ ID NO: 3): [ka] [ka]

[0094] The amino acid sequence of TnY transposase is provided by (SEQ ID NO: 4): [ka]

[0095] In some embodiments, the technology involves the use of an adapter containing a transposase-binding sequence known to those skilled in the art as a “mosaic” or “binding mosaic.” The mosaic sequence is known to those skilled in the art to be used, for example, with the Tn5 transposase. A typical upper chain of a mosaic sequence used with the Tn5 transposase is AGATGTGTATAAGAGACAG (SEQ ID NO: 5). In some embodiments, the mosaic sequence is located at the 5' end of the adapter, the 3' end of the adapter, or both the 5' and 3' ends of the adapter. See, for example, Picelli (2014) Genome Research 24: 2033 (incorporated herein by reference).

[0096] In some embodiments, the adapter includes an amplification handle or a primer binding site. In some embodiments, the adapter includes a sequencing priming region, e.g., a P5 or P7 sequence for Illumina sequencing. In some embodiments, the adapter includes specific priming sequences, e.g., mRNA-specific priming sequences (e.g., poly-T sequences for reverse transcription priming of RNA), targeted priming sequences, and / or random priming sequences. In some embodiments, the adapter includes a promoter for T7 RNA polymerase, e.g., enabling in vitro transcription during sample processing.

[0097] In certain embodiments, the adapter further includes a barcode sequence ("target barcode") that identifies the target of the affinity reagent. The target barcode sequence is used to identify the affinity reagent and / or the target. The target barcode sequence is a unique sequence that makes it possible to identify a particular affinity reagent to be tested or used. Embodiments provide target barcodes of arbitrary length obtained using polynucleotide synthesis techniques, where the barcode length limits the number of formulations that can be tested simultaneously. For example, a 10-base pair barcode provides a total of 1,048,576 different unique barcode sequences. Thus, in some embodiments, the length of the barcode sequence is from 4 nt to 100 nt, e.g., from 10 nt to 20 nt, e.g., 10 nt. In some embodiments, the length of the barcode sequence is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nt. In some embodiments, affinity reagents (e.g., antibodies) are modified (e.g., conjugated) with an adapter or multiple adapters. See, for example, Figures 7A and 7B.

[0098] For example, as shown in Figure 7A, the embodiment provides an antibody modified with one or more first affinity sites ("A"). As shown in Figure 7A, the affinity sites may be bound to the antibody with one or more linkers. Furthermore, the antibody may be barcoded with a second affinity site ("B") and one or more barcode adapters containing different barcode sequences. The binding pairs (e.g., affinity sites) A ​​and B are as described herein (e.g., covalent bonds (e.g., provided by click chemistry, glutamine and amine, N-hydroxysuccinimide ester and primary amine, maleimide and sulfhydryl, Trout reagent and primary amine, and other covalent chemistry known to those skilled in the art); avidin and biotin, neutravidin and biotin, streptavidin and biotin, DNA-binding protein and the DNA sequence recognized by that DNA-binding protein, HaloTag and chloroalkane, or SNAP-tag and O(6)-benzylguanine).

[0099] For example, as shown in Figure 7B, each embodiment provides two adapters comprising lead 1 and lead 2. Leads 1 and 2 are conjugated to the same antibody using a single-stranded DNA handle (brown in the figure) having a spacer between the two hybridization regions. The binding pairs A and B are as described herein (e.g., covalent bonds (e.g., provided by click chemistry, glutamine and amine, N-hydroxysuccinimide ester and primary amine, maleimide and sulfhydryl, Trout reagent and primary amine, and other covalent chemistry known to those skilled in the art); avidin and biotin, neutravidin and biotin, streptavidin and biotin, DNA-binding protein and the DNA sequence recognized by that DNA-binding protein, HaloTag and chloroalkane, or SNAP-tag and O(6)-benzylguanine). In some embodiments, the handle has a length greater than 22 base pairs.

[0100] This technology will be used in research, medicine, and other fields. For example, next-generation CUT&Tag technology enables multiplex characterization of epiproteomic epitopes at the single-cell level. Thus, embodiments of this technology enable the verification of dozens of chromatin-related biological events, mechanisms, or markers occurring at the single-cell level. These events may occur at one or more locations within the genome of a single cell and may differ from similar loci in the genomes of other cells in the same culture, tissue, or preparation. Regarding chromatin-related events associated with DNA damage, DNA damage is specifically programmed on a single-cell basis through many biological pathways, including VDJ rearrangement, selection of origins of replication during DNA replication, hotspots and productive or unproductive recombination events during meiosis, and DNA breaks observed in differentiating neurons. Currently, it is difficult to verify such DNA damage beyond a small number of epiproteomic epitopes (e.g., 1, 2, 3) at single-cell sites. Due to a lack of technologies providing epiproteome resolution in this field, the molecular mechanisms and related biology associated with programmed DNA damage—including its initiation, consequence, and resolution—are not fully understood. Furthermore, next-generation CUT&Tag technology provides insights into the differences in the level and location of DNA damage events between normal and cancer cells, as well as DNA damage that occurs during disease treatment. In some embodiments, this technology uses non-invasive methods to analyze epiproteomes and circulating extracellular chromatin fragments obtained from blood and liquid biopsies to gain insights into the origin, developmental stage, and metastatic potential of cancer.

[0101] While the disclosures described herein refer to certain illustrated embodiments, it should be understood that these embodiments are illustrative and not limiting. [Examples]

[0102] Examples This specification provides an improved DNA binding mapping technique compared to existing techniques (e.g., ChIP-Seq, CUT&RUN, SplitDamID, CUT&Tag, Multi-CUT&Tag, CoBATCH, scChIC-Seq, ACT-Seq, Co-ChIP). In particular, this technique does not employ protein-A fusion or nanobody methods for conjugating preloaded transposases (e.g., Tn5) to affinity reagents (e.g., antibodies) (e.g., this technique is protein-A fusion-free, and in some embodiments, preloaded transposase-free). Therefore, this technique minimizes or eliminates ambiguity of background and cross-signals.

[0103] Materials and methods Biological materials and reagents. K562 cells were cultured in RPMI medium (Gibco, 11875119) and supplemented with 10% FBS (Gemini Bio, 100-602-500) and 1% penicillin-streptomycin (ThermoFisher, 15140122). For sodium butyrate treatment, newly growing K562 cells were seeded in 6-well plates at a cell density of 0.1 million / mL. For cell treatment, 1 mM sodium butyrate (Millipore Sigma, 19-137) was added to the cell culture and incubated for 72 hours. Distilled water (ThermoFisher, 10977023) was added to the control cells. All antibodies used in this study are shown in Table 1. All reagents and materials used in this study are shown in Table 2. All oligonucleotides used for barcoding in this study were ordered from Integrated DNA Technologies and are shown in Tables 3 and 4.

[0104] [Table 1]

[0105] [Table 2]

[0106] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

[0107] [Table 4]

[0108] Antibody barcoding. The antibody should preferably be in PBS buffer before the reaction. Incubate the antibody and NHS-PEG12-biotin (ThermoFisher, A35389) at a molar ratio of 1:0.1 to 1:100 overnight at 4°C. The next day, replace the biotinylated antibody buffer with PBS three times using a 40K Zeba® desalting column or plate (ThermoFisher, 87767, 87775). For adapter annealing: Prepare a stock of Tn5MErev oligo at 500 μM in water. Prepare P5 and P7 adapter oligos at 100 μM in water. Mix 10 μL of Tn5MErev oligo, 50 μL of either adapter oligo, and 40 μL of distilled water (ThermoFisher, 10977023), incubate at 95°C for 2 minutes, and gradually cool to room temperature. To prepare a barcode adapter pair, mix equal volumes of either P5 or P7 adapters. In this study, P5 and P7 adapters with the same number were paired to contain the same barcode. For antibody barcoding: Prepare each antibody individually in separate tubes or wells. Mix 10 μg of biotinylated antibody, 0.39 μL of streptavidin (ThermoFisher, 21122), and 2.34 μL of adapter pair, and adjust the total volume to 100 μL with PBS. Incubate the mixture at room temperature for 1 hour. Add 2.25 μM of D-biotin (ThermoFisher, B20656) to the mixture and incubate at room temperature for 30 minutes. Pool all antibody mixtures. Concentrate through a 30K Amicon centrifuge filter (Millipore Sigma, UFC503096, UFC803096) and store at 4°C.

[0109] High-plex cut and tag method. The primary antibody is prepared using the method described above. Different antibodies are loaded with different barcode adapter pairs. Start with 100,000 cells and use 10 μL of concanavalin A-coated magnetic beads (Polysciences, 86057-3). Activate the concanavalin A beads by washing twice with binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). Wash 100,000 fresh K562 cells once with PBS and once with washing buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitor cocktail). Resuspend the cells in 0.5 mL of washing buffer and transfer to the activated concanavalin A beads. Incubate the cells and beads in a rotator at room temperature for 15 minutes. Remove the buffer by placing the tube on a magnetic stand. Resuspend the cells in 100 μL of washing buffer with the antibody mixture (pooled at 1 μg per antibody), 0.05% digitonin, and 2 mM EDTA. Incubate in a rotator at room temperature for 1 hour. Wash the beads four times with Dig-med buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, 1× protease inhibitor cocktail). Resuspend the beads in 100 μL of Dig-med buffer with 10 mM MgCl2 and 5 μg of Tn5 (Diagenode, C01070010-20). Incubate in a rotator at 37°C for 1 hour. To stop tagmentation, add 3.33 μL of 0.5 M EDTA, 1 μL of 10% SDS, and 0.33 μL of 20 mg / mL proteinase K (ThermoFisher, EO0491). Vortex and incubate at 50°C for 1 hour. Perform DNA purification as follows: Add 100 μL of phenol-chloroform-isoamyl alcohol (pH 8) (ThermoFisher, 17908) and mix well. Transfer the sample to a phase-lock tube (ThermoFisher, NC1093153) and centrifuge at 16000 g for 3 minutes at room temperature. Add 100 μL of chloroform to the aqueous layer and centrifuge at 16000 g for 5 minutes.Transfer the aqueous layer to a new tube and add 250 μL of 100% ethanol and 8.75 μL of 20 mg / mL glycogen. Incubate overnight at -80°C. The next day, centrifuge at 16000 g for 15 minutes at 4°C. Wash the pellet with 1 mL of 100% ethanol. Centrifuge at 16000 g for 5 minutes at 4°C. After air-drying the pellet, dissolve it in 23 μL of 10 mM Tris-HCl pH 8 (containing 1 / 100 RNAse A (ThermoFisher, EN0531)). Incubate at 37°C for 10 minutes. For library amplification, mix 21 μL of purified DNA with 2 μL each of barcoded i5 primer (10 μM) and i7 primer (10 μM) (use a different combination for each sample). The sequences of the i5 and i7 primers are shown below. The barcode sequences follow the previous paper [Reference 19]. Add 25 μL of NEBNext Ultra II Q5 Master Mix (NEB, M0544S) and mix gently. Incubate in a thermocycler using the following program: 72°C for 5 minutes, 98°C for 30 seconds (1 cycle); 98°C for 10 seconds, 63°C for 10 seconds (17 cycles); 72°C for 1 minute, hold at 4°C (1 cycle). Clean up the library using AMPure XP beads (Beckman, A63881) in a 1:1.1 ratio, following the manual. The library is ready for sequencing.

[0110] i5 primer (SEQ ID NO: 85): 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNNTCGTCGGCAGCGTC-3'(N:11nt barcode)

[0111] i7 primer (SEQ ID NO: 86): 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNNNNNNGTCTCGTGGGCTCGG-3'(N:11nt barcode)

[0112] Each of the i5 and i7 primers contains an 11nt barcode indicated by NNNNNNNNNNN (SEQ ID NO: 87) in the above sequence. The various barcode sequences for the i5 and i7 primers are described in Mezger (2018) "Hi-plex chromatin accessibility profiling at single-cell resolution" Nat Commun 9: 3647, which are incorporated herein by reference.

[0113] Single-cell Hi-PlexCutTag method. To obtain at least approximately 5,000 cells, 100,000 K562 cells were collected and the sample was prepared in bulk as follows: Wash the cells once with PBS and once with washing buffer. Activate 10 μL of concanavalin A beads by washing twice with binding buffer. Resuspend the cells in 1 mL of NP-washing buffer (washing buffer, 0.01% digitonin, 0.01% NP-40), add 20 mM sodium butyrate, and incubate with the activated beads in a rotator at room temperature for 15 minutes. Remove the buffer and resuspend the beads in 100 μL of NP-washing buffer containing 2 mM EDTA. Add the barcode-loaded antibody mixture to the beads and incubate in a rotator at room temperature for 1 hour. Wash the beads four times with NP-Dig-med buffer (Dig-med buffer, 0.01% NP-40). Resuspend the beads in 100 μL of NP-Dig-med buffer with 10 mM MgCl2 and 5 μg of Tn5. Incubate in a rotator at 37°C for 1 hour. Replace the buffer with 1 mL of 10 mM Tris-Cl (containing 10 μg / mL DAPI (ThermoFisher, D1306)). Transfer the beads through a cell strainer to a round-bottom tube (Falcon, 352235). Sort the samples into a 384-well plate using a MoFlo XDP instrument, ensuring one cell per well. Centrifuge the plate at 3000 g for 3 minutes at 4°C. Store the cells at -80°C until the next step. Add the reagents to the 384-well plate using an Echo 650 Acoustic Liquid Handler. Add 1 μL of 0.095% SDS to each well. Centrifuge the plate at 3000 g for 3 minutes. Incubate at 58°C for 1 hour. Add 0.5 μL of 2.5% Triton X-100 and 0.5 μL of i5 / i7 primer mix (10 μM) to each well. Assign a unique index pair to each well. Add 2 μL of NEBNext Ultra II Q5 Master Mix (NEB, M0544S) to each well. Centrifuge the plate at 3000 g at 4°C for 3 minutes.Incubate in a thermocycler using the following program: 58°C for 5 minutes, 72°C for 5 minutes, 98°C for 30 seconds (1 cycle); 98°C for 10 seconds, 63°C for 10 seconds (17 cycles); 72°C for 1 minute, hold at 4°C (1 cycle). Pool the library using Single-well Deep Well Plates (Miltenyi Biotec, 130-114-966). Invert the 384-well plate onto the deep well plate and centrifuge at 1000g at 4°C for 1 minute. Repeat this process until the library is recovered from all 384-well plates. Transfer the pooled library to a new tube. Clean up the library with AMPure XP beads (Beckman, A63881) in a 1:1.1 ratio, following the instructions. The library can be used for sequencing.

[0114] [Example 1 - Hi-PlexCUT&Tag enables simultaneous and effective profiling of multiple nucleosomes and their binding regulatory factors.] To evaluate the performance and multiplexing capabilities of this new technology, we assigned barcodes to a panel of 36 monoclonal antibodies (mAbs), each targeting 12 common histone marks, 14 histone modifying enzymes, 8 human transcription factors (TFs), CTCF, and PolII (pSer2) (Figure 4). Rabbit IgG was also included as a negative control. Next, all 37 barcoded antibodies were pooled and applied to K562 cells permeabilized without the addition of Tn5 (10 5 Cells were incubated with unbound Tn5 and MgCl2 at room temperature for 1 hour. To detect the global binding sites of these antibodies, the cells were thoroughly washed and incubated with unbound Tn5 and MgCl2 at 37°C for 1 hour. Finally, genomic DNA was extracted, tagged DNA was amplified by PCR, and library preparation and next-generation sequencing were performed (Figure 1A). This assay was performed in duplicate, yielding approximately 200 million reads.

[0115] Next, an antibody ID was assigned to each read, the sequencing data was demultiplexed, and the inserts were mapped to the genome. 92.63% of the reads were successfully demultiplexed. To investigate background signaling, all reads containing at least one rabbit IgG barcode (referred to as single tones) were extracted, and these accounted for only 0.07% of all reads, indicating very low background. Furthermore, a comparison of our IgG tracks with IgG tracks obtained from ChIP-seq, CUT&Run, and CUT&Tag in K562 cells showed that our IgG reads were significantly more dilute and at lower levels than these [References 9,10,4]. As an example, a 2Mbp region on chromosome 3 is shown in Figure 1B. In addition, the reproducibility of the dataset was evaluated by scatter plot analysis of the duplicate assays. The calculated correlation coefficients ranged from 0.934 to 0.994, indicating that this assay has high reproducibility (e.g., Figure 1C).

[0116] To evaluate the usefulness of the Hi-PlexCUT&Tag map, silent regions and actively transcribed regions were identified. Single-tone tracks of H3K27me3, RNAPII, and H3K4me3 were extracted from Hi-PlexCUT&Tag reads and compared with the multi-CUT&Tag dataset (H3K27me3&RNAPII), the ENCODE database (H3K4me3), and ATAC-seq data from the same cells. As shown in Figure 1D, the H3K27me3 and RNAPII tracks showed excellent agreement between Hi-PlexCUT&Tag and multi-CUT&Tag. Similarly, the H3K4me3 track obtained from Hi-PlexCUT&Tag was almost identical to that obtained by the conventional ChIP-seq method (blue track, Figure 1D). Importantly, while both tracks largely overlap with the ATAC-seq track, an additional ATAC track was found in the region covered by the H3K27me3 track (black track; Figure 1D). These analyses demonstrated that the Hi-PlexCUT&Tag technology can generate antibody-specific signals with minimal background noise from Tn5 transposase action alone. It was also confirmed that the H3K27me3 and RNAPII tracks are mutually exclusive. In fact, global analyses of mutually exclusive marks such as H3K9me3 vs. H3K9ac and H3K27me3 vs. H3K27ac showed minimal overlapping signals, suggesting significant exclusion of cross-contamination between different antibodies (Figure 1E).

[0117] To elucidate the synergistic effects of multiple epitopes at a single locus, we investigated whether Hi-PlexCUT&Tag could accurately identify the co-localization of two epitopes. As an example, for the transcription-related epitopes H3K4me3 and RNAPII, we stratified reads using only reads containing barcodes representing both epitopes at both ends (referred to as heterotones) and compared them with existing H3K4me3 and RNAPII ChIP-seq tracks in ENCODE (Figure 1F). The H3K4me3 / RNAPII heterotone track was mainly located in the region where individual H3K4me3 and RNAPII ChIP-seq tracks overlapped (green shaded region; Figure 1F). On the other hand, when the H3K4me3 ChIP-seq track was absent in the NEAT1 gene itself or other regions, the H3K4me3 / RNAPII heterotone track also disappeared (orange shaded region; Figure 1F). Furthermore, global analysis also revealed some overlapping signals between the two targets, supporting the accuracy of detecting co-localization events between H3K4me3 and RNA PII (Figure 1G).

[0118] Recent studies have shown that histone marks with opposing biological functions, such as H3K4me3 (euchromatin) and H3K27me3 (facultative heterochromatin), exist adjacent to each other, and these are called "bivalent" domains [Reference 11]. To verify whether Hi-Plex CUT&Tag can easily detect this type of colocalization, heterotone reads with H3K4me3 and H3K27me3 barcodes at both ends were extracted, yielding more than 5,000 peaks (Figure 1H). To identify bivalent domains in K562 cells, existing single H3K4me3 and H3K27me3 ChIP-seq data were aligned, and overlapping regions were identified (multi-CUT&Tag or sequential ChIP-seq data were not obtained). Approximately 950 H3K4me3 and H3K27me3 bivalent domains were identified from overlapping ChIP-seq data, and 36% of them were covered by our H3K4me3 / H3K27me3 heterotone peak (Figure 1H, left bar plot). For example, a strong H3K4me3 / H3K27me3 heterotone peak extending approximately 1,100 bp was observed in the NBPF1 promoter, and individual H3K4me3 and H3K27me3 ChIP-seq peaks were also observed at the same location, although the H3K27me3 peak was considerably weaker. Surprisingly, 94.9% of the H3K4me3 / H3K27me3 heterotone peaks could not be identified in individual ChIP-seq data (Figure 1H). Further investigation revealed that this technique is highly sensitive to the detection of such combined events. For example, seven H3K4me3 / H3K27me3 heterotone peaks were identified in the PLEKHG5 gene itself, and two and nine H3K4me3 and H3K27me3 ChIP-seq peaks were observed individually (blue shaded region; Figure 1J). No bivalent domains were identified in the overlap analysis with the above ChIP-seq data (Figure 1J). Note that the reads of heterotone events with mixed barcodes originate from the same chromosomal fragment within the same cell, and therefore reflect the true colocalization of both epitopes at the same position on the same chromosomal copy.These results demonstrate that Hi-PlexCUT&Tag is highly sensitive to epitope colocalization mapping.

[0119] To verify whether this technology improves the possibility of detecting epitope co-localization within the same cell, we aggregated the number of reads from all 630 (=36 × 35 / 2) heterotone and 36 homotone events (reads with the same barcode at both ends). Approximately 80% were heterotone (data not shown). The number of reads for each combination varied considerably, partially reflecting the endogenous abundance of epitopes on the chromatin. This result supports the finding that Hi-PlexCUT&Tag significantly improves the detection of co-localization of two epitopes.

[0120] Overall, the Hi-PlexCUT&Tag method provides compelling data demonstrating its ability to detect hundreds of different co-localized epitope pairs within the same cell with high sensitivity by significantly reducing background signaling and cross-contamination.

[0121] In addition to large-scale profiling, this method can also be applied to limited targets of up to three targets depending on the available secondary antibodies. To distinguish it from Hi-PlexCUT&Tag, this approach is called Low-PlexCUT&Tag (Figure 5). As shown in Figure 5, the process involves incubation with an unlabeled primary antibody followed by signal enhancement by binding of a barcoded secondary antibody. Subsequent steps (introduction and activation of unbound transposase, DNA purification, and library preparation) are the same as those for Hi-PlexCUT&Tag. Using the same data processing as Hi-PlexCUT&Tag, it was confirmed that Low-PlexCUT&Tag can also effectively minimize background noise and cross-contamination (data not shown).

[0122] [Example 2 - Anatomy of the complexity of the Hi-Plex CUT&Tag dataset] Unlike existing methods such as ChIP-seq, ATAC-seq, and CUT&Tag, each sequence read generated by Hi-PlexCUT&Tag requires two tagging events to occur simultaneously within the same cell, and the length of the tagged chromosomal DNA provides an approximation of the distance between the two epitopes. This applies to all heterotone reads (i.e., reads tagged with two different barcodes). In other words, each Hi-PlexCUT&Tag sequence read contains information about the epitope combination that generated the fragment, the approximate chromosomal distance between the two epitopes, and genetic information conserved in the tagged sequence. The structure of the new dataset can be represented by three information axes: genomic DNA sequence, epitope combination, and the distance of each combination (Figure 2A). Furthermore, the polarity / order of modifications can also be determined from the heterotone data.

[0123] Theoretically, using 36 different antibodies allows us to investigate 36 homotone and 630 (=36 × 35 / 2) heterotone events. The number of reads generated varies significantly among antibodies, reflecting differences in epitope abundance, chromatin stability, and antibody affinity. This trend became clearer when a heatmap was created using SEACR to display the number of peaks for each epitope combination derived from the 36 mAbs (Figure 2B). As shown, H3K4me1, H3K4me3, H3K9me3, and H3K27me3 were involved in the most homotone and heterotone reads, followed by RNAPII, CBP, and SUV39H1. On the other hand, many transcription factors produced few reads, indicating they are very dilute or unstable on chromatin. Note that chromatin cross-linking was not performed to maintain epitope accessibility and availability. Therefore, we filtered out epitope combinations with peaks below the 20th percentile of the read number distribution, and focused on 501 epitope combinations in future analyses. Furthermore, 68% of the eligible peaks were heterotone events, indicating that this new technology significantly improves the potential of epitope colocalization mapping. It should also be noted that homotone events with internucleosome distances of two or more (e.g., greater than 300 bp) are also likely to be generated by two antibodies, as their barcode sequence lengths are 66 or 72 bp.

[0124] Next, we investigated whether each epitope combination was associated with specific DNA sequence types, such as cis-regulatory elements and repetitive DNA sequences, and whether there were differences in CpG methylation levels. Hierarchical clustering analysis using peak annotation stacked bar graphs was performed along with annotated cis-regulatory elements, repetitive elements, and mean DNA methylation from the same cell line [Reference 14]. All 501 epitope combinations were annotated with ENCODE cis-regulatory elements. Four major clusters were identified, and the top eight combinations in each cluster are shown in Figure 2D.

[0125] In the first cluster, epitope combinations were primarily associated with enhancer-like and promoter-like elements. This cluster mainly consisted of euchromatin marks and included heterotone pairs such as H3K4me3 / RNAPII, H3K4m3 / H3K27ac, and H3K4m3 / H3K36me3. Regarding repeat elements, this group is characterized by a high proportion of simple repeats and a low proportion of transposon elements (Figure 2D).

[0126] The second cluster has a high proportion of gene bodies. It is mainly characterized by epitope combinations related to RNA polymerase II and histone acetylation marks, including H3K14ac / RNAPII and RNAPII / RNAPII. The dominance of these features suggests that RNA polymerase II actively transcribes genes, and that acetylation plays a crucial role in transcriptional elongation, maintaining open chromatin structure and promoting efficient transcription. On the other hand, this group has a high proportion of SINEs and LINEs. In fact, previous studies have shown that K562 cells express full-length L1 mRNA and L1 coding proteins [References 15;16]. The activity of L1 elements in K562 cells is higher than in many other cell types, which is consistent with the increased retrotransposon activity commonly observed in cancer cell lines. The Alu element, the most common SINE in humans, is also actively transcribed in K562 cells. Li et al. showed that Alu repeats in K562 cells are abnormally hypomethylated and transcribed much more actively than in other human cell lines and somatic tissues [Reference 17].

[0127] The third cluster mainly consists of epitope combinations containing H3K27me3PTM (post-translational histone modification). This cluster has a high proportion of gene components and low-DNase regions, and exhibits repressive function. Repeat elements are predominantly SINE, LINE, and LTR.

[0128] In the fourth cluster, most combinations contain either an H3K9me3 or H3K9me2 mark, and the underlying genome sequence is predominantly composed of repetitive elements such as sine, line, and long-term tracer (LTR) and a high proportion of satellite DNA.

[0129] Significant differences were observed among the four clusters regarding mean DNA methylation levels. The first and second clusters (epitope combinations containing many open histone marks) had low mean DNA methylation levels, while the third and fourth clusters showed a broader range of DNA methylation levels. This is in good agreement with the function of CpG methylation in gene silencing [Reference 18].

[0130] Based on these observations, we investigated whether epitope combinations containing annotated euchromatin and heterochromatin marks showed significant differences in their association with cis-regulatory elements and repeat elements, respectively. Box plot analysis revealed that euchromatin marks containing H3K4me3 and H3K27ac were highly enriched in dELS (distal enhancer-like signature), pELS (proximal enhancer-like signature), and PLS (promoter-like signature), while heterochromatin marks containing H3K9me3 and H3K27me3 were significantly associated with gene sequences and low DNase-accessible regions. Regarding repeat elements, combinations containing heterochromatin marks were more enriched in LINE, SINE, satellite DNA, LTR, and DNA repeats than in euchromatin marks (excluding simple repeats) (Figure 2C). These results are in good agreement with literature reports, but further analysis is needed to determine whether this correlation applies to each individual epitope combination (see Figure 2D).

[0131] Interestingly, after PCR amplification of tagged species, a distinct ladder-like pattern appeared instead of a smear (Figure 6A). Each tagged species not only anchors two tagging events on chromatin but also provides an estimate of the distance between the two events, allowing us to investigate whether there are length-specific features for tagged species. Histogram analysis of all eligible reads revealed peaks at approximately 60, 200, and 380 bp, with deep dips in between, and the signal rapidly attenuating above 600 bp (Figure 6B). The distance difference between adjacent peaks was approximately 150 bp, which corresponds to the DNA length wrapped around a single nucleosome. Therefore, we refer to these peaks as sub (0–120 bp), mono (120–300 bp), die (300–460 bp), and tri+ (>460 bp) nucleosome fragments.

[0132] Next, epitope combinations were ranked based on the proportion of sub, mono, and di+nucleosome species. The top examples for each category are shown in stacked bar graphs (Figure 2E). Transcription factors (YY1, NRF1, cFos, USF2, etc.) had the highest proportion of tagging events of less than 80 bp, which reflects that TFs have a short footprint on chromatin due to their sequence-specific binding activity. Since two adjacent tagging events are required for read generation, these short reads may represent homodimer binding events. In fact, YY1, NRF1, cFos, USF2, and Jun are known to form homodimers. On the other hand, the top combinations with many reads exceeding 300 bp were pairs between euchromatin histone marks (e.g., H3K27me3 / H3K4me3) and / or their writers (e.g., EP300 / H3K27ac, EP300 / H3K9ac). This phenomenon represents the spreading of histone modifications across multiple nucleosomes, suggesting the generation of longer fragments.

[0133] [Example 3 - Establishment of a protocol for Hi-PlexCUT & Tag profiling in single cells] Previous studies have shown that CUT&Tag and multi-CUT&Tag are useful for profiling chromatin regulatory factors at the single-cell level [References 4, 5, 6, 12, 13]. Accordingly, we developed a protocol for applying Hi-PlexCUT&Tag to single-cell profiling (Figure 3A). First, nuclei were isolated from cells, and Hi-PlexCUT&Tag was performed in bulk according to the procedure described above, including tagging. The nuclei were then stained with DAPI, and single nuclei were sorted into 384-well plates by flow cytometry. Single-cell library preparation began with lysis of single nuclei with SDS, followed by inactivation of SDS with Triton X-100. Amplification of the sequencing library was performed using different index primer pairs to enable identification of signals derived from individual cells. After adding the PCR reaction mixture, library amplification was performed in each well. The libraries from each cell were pooled, purified with Ampure XP beads, and prepared for sequencing.

[0134] As a proof-of-concept, 16 out of 36 targets were evaluated in K562 cells. These included six histone modifications (H3K4me3, H3K9me3, H3K9ac, H3K14ac, H3K27me3, H3K27ac), ten transcription factors (CTCF, RNAPII S2P, c-Jun, c-Fos, Max, Myc, USF1, USF2, NRF1, YY1), and a negative control (rabbit IgG). A total of two replications were performed, with each replication consisting of 1,536 cells. Using the same method as for data analysis in the bulk experiment, reads with identical H3K27me3 barcodes at both ends (referred to as homotones) were processed from each cell (Figure 3B). Next, when the accumulation signals from all single cells were evaluated, the enrichment of the pseudo-bulk reed matched that of the bulk reads at numerous sites, demonstrating the high specificity of single-cell Hi-PlexCUT&Tag (Figure 3B).

[0135] This method can also be used in combination with 10x Genomics' Chromium Single Cell ATAC gel beads and Chromium Next GEM Single Cell Multiome ATAC + Gene Expression gel beads to further increase cell count and perform simultaneous RNA profiling (data not shown).

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[0137] All documents and patents cited in the above specification are incorporated herein by reference in their entirety. Various modifications and variations of the compositions, methods and techniques described are possible without departing from the scope and spirit of the Art, and should be understood that the claimed invention should not be unduly limited to such specific embodiments, although the Art has been described in relation to specific embodiments. Indeed, various modifications of embodiments of the invention that would be obvious to those skilled in the Art are intended to be included in the following claims.

Claims

1. A method for identifying a target nucleic acid binding site, (a) The tagging composition is attached to the target by bringing the target, which is bound to the nucleic acid binding site, into contact with the tagging composition, wherein the tagging composition is (i) an antibody or antibody fragment that binds to the target, (ii) A heterocyclic compound bound to the antibody or antibody fragment, (iii) Protein complex, and (iv) Two or more nucleic acids, each containing a barcode nucleotide sequence, which are bonded to the heterocyclic compound, to be bonded. (b) Contacting the two or more nucleic acids of the tagging composition with a transposase to form an antibody-barcode-transposase complex, wherein the antibody-barcode-transposase complex causes a double-strand break in the nucleic acid containing the nucleic acid binding site, thereby generating a nucleic acid fragment containing the nucleic acid binding site. (c) Isolating the nucleic acid fragment, and (d) Sequencing the nucleic acid fragment to identify the nucleic acid binding site of the target, Methods that include...

2. The method according to claim 1, wherein the protein complex comprises avidin, streptavidin, or neutravidin.

3. The method according to claim 1, wherein the heterocyclic compound comprises biotin.

4. The method according to claim 1, wherein the transposase comprises Tn5 transposase.

5. The method according to claim 1, wherein each of the two or more nucleic acids further comprises a transposase mosaic sequence that binds to the transposase.

6. The method according to claim 5, wherein the transposase mosaic sequence binds to Tn5 transposase.

7. The method according to claim 1, wherein the target comprises a DNA-binding protein.

8. The method according to claim 7, wherein the DNA-binding protein comprises a transcription factor, a regulatory element, a transcription repressor, a transcription activator, a polymerase, a nuclease, a nickase, a zinc finger protein, a transcription activator-like effector nuclease (TALEN), a glycosylase, a methylase, a ligase, a restriction enzyme, a replication protein, a helicase, or a kinase.

9. The method according to claim 1, wherein the antibody or antibody fragment is not directly bound to the two or more nucleic acids.

10. The method according to claim 1, wherein the protein complex is bound to the heterocyclic compound bound to the antibody or antibody fragment, and is also bound to the heterocyclic compound bound to the two or more nucleic acids.

11. The method according to claim 1, further comprising adding magnesium to a sample containing the target and the tagging composition.

12. The method according to claim 1, wherein each of the two or more nucleic acids further comprises an amplification handle.

13. The method according to claim 1, further comprising amplifying the nucleic acid fragment to provide a sequencing library.

14. The method according to claim 13, wherein the amplification is polymerase chain reaction (PCR) amplification.

15. (a) One or more antibodies or antibody fragments that bind to the target, (b) A heterocyclic compound conjugated to one or more antibodies or antibody fragments, (c) Protein complexes comprising avidin, streptavidin, or neutravidin, and (d) Two or more nucleic acids, each of which (i) barcode nucleotide sequence; and (ii) Two or more nucleic acids containing a transposase mosaic sequence, In a composition containing, The two or more nucleic acids are bonded to the heterocyclic compound, and the composition forms a complex in solution. composition.

16. The composition according to claim 15, wherein the protein complex comprises streptavidin.

17. The composition according to claim 15, wherein the heterocyclic compound comprises biotin.

18. The composition according to claim 15, wherein the transposase comprises Tn5 transposase.

19. The composition according to claim 15, wherein the antibody or antibody fragment includes a region that binds to a DNA-binding protein.

20. The composition according to claim 19, wherein the DNA-binding protein comprises a transcription factor, a regulatory element, a transcription repressor, a transcription activator, a polymerase, a nuclease, a nickase, a zinc finger protein, a transcription activator-like effector nuclease (TALEN), a glycosylase, a methylase, a ligase, a restriction enzyme, a replication protein, a helicase, or a kinase.

21. The composition according to claim 20, wherein the protein complex is bound to the heterocyclic compound.

22. A first container containing the composition described in claim 15, and Second container containing transposase, A kit that includes this.

23. The kit according to claim 22, further comprising a reagent for tagging.

24. The kit according to claim 22, further comprising reagents and materials for the isolation of DNA and amplification of nucleic acids.

25. The kit according to claim 22, further comprising a cell capture scaffold.

26. The kit according to claim 25, wherein the cell capture scaffold comprises magnetic beads, a column, concanavalin A beads, streptavidin beads, colloidal semiconductor nanocrystals, carbon nanotubes, or a microfluidic device.

27. A method for identifying two or more target binding sites on a nucleic acid, (a) To provide two or more barcoding affinity reagents, The two or more barcoding affinity reagents are each affinity reagents bound to a pair of adapters. The first adapter includes a first barcode nucleotide sequence and a first transposase-binding mosaic sequence. The second adapter includes a second barcode nucleotide sequence and a second transposase-binding mosaic sequence. The first barcode nucleotide sequence and the second barcode nucleotide sequence may be the same or different. None of the two or more barcoding affinity reagents mentioned above contain transposase. The two or more barcoding affinity reagents each bind to a different target. The first and second barcode nucleotide sequences of each barcoding affinity reagent are different from the first and second barcode nucleotide sequences of other barcoding affinity reagents that bind to different targets. (b) Adding the two or more barcoding affinity reagents to a sample containing the target of each affinity reagent, Each target is bound to nucleic acid at its respective target-binding site. Each barcoded affinity reagent binds to its respective target or to the primary affinity reagent bound to its respective target, and the binding of each affinity reagent occurs in the absence of transposase. (c) Adding an unbound transposase and a transposase activator to the sample, The unbound transposase binds to the first transposase-bound mosaic sequence and the second transposase-bound mosaic sequence of each barcoding affinity reagent. The bound transposase fragments the nucleic acid, and the nucleic acid is tagged with the first barcode nucleotide sequence and the second barcode nucleotide sequence of the respective barcoding affinity reagent to provide tagged nucleic acid. At least two tagged nucleic acids are provided, each corresponding to two or more barcoding affinity reagents, and each barcoding affinity reagent corresponds to its respective target binding site, to be added. (d) Sequencing the tagged nucleic acid to provide a nucleotide sequence, and (e) Analyzing the nucleotide sequence to identify the target binding site on the nucleic acid, A method that includes this.

28. The transposase is Tn5, Tn3, Tn7, TnY, Sleeping Beauty, or piggyBac, and the transposase activator is MgCl ２ The method according to claim 27.

29. The method according to claim 27, wherein the target is a DNA-binding protein such as a histone, a histone modifying enzyme, a transcription factor, a cofactor, or a chromatin-related protein.

30. The method according to claim 27, wherein the target is a post-translational modification on a histone or other chromatin-related protein, or a modified DNA base.

31. The method according to claim 30, wherein the modified DNA base is mC or 5hmC.

32. The nucleic acid is part of chromatin, The method according to claim 27, further comprising simultaneously detecting histone marks, histone modifying enzymes, chromatin-related proteins, and transcription factors.

33. The method according to claim 32, wherein the chromatin-related protein is CTCF or cohesin.

34. The method according to claim 27, wherein the affinity reagent comprises an antibody.

35. The method according to claim 27, wherein the affinity reagent is a target-specific affinity reagent.

36. The method according to claim 27, wherein the affinity reagent is a secondary affinity reagent specific to the primary target-specific affinity reagent.

37. The method according to claim 36, wherein the primary affinity reagent is barcode-free.

38. The method according to claim 36, further comprising adding the primary affinity reagent to the sample.

39. To provide the barcoded affinity reagent, which includes the affinity reagent coupled to the pair of adapters, The first affinity site is bound to the affinity reagent, The method includes providing a second affinity site to each of the first and second adapters, and specifically binding the first affinity site to the second affinity site. The method according to claim 27.

40. The first affinity site and the second affinity site are Biotin and avidin, streptavidin, or neutravidin; A first reactant and a second reactant that react to form a covalent bond; DNA-binding protein, and DNA sequence recognized by the DNA-binding protein; Halotags and chloroalkanes; SNAP-tag and O(6)-benzylguanine; and Single-stranded DNA and its hybridized DNA, The method according to claim 39, wherein the pair is selected from the group consisting of the following.

41. The method according to claim 27, wherein the first adapter and the second adapter each further include an amplification handle.

42. The method according to claim 27, further comprising analyzing the nucleotide sequence to identify the binding site of the target on the nucleic acid, and associating the barcode nucleotide sequence with an affinity reagent.

43. The method according to claim 27, further comprising amplifying the tagged nucleic acid to provide a sequencing library.

44. The method according to claim 43, wherein the amplification is polymerase chain reaction amplification.

45. Each barcoding affinity reagent includes a first handle and a second handle connected via a spacer. The first adapter is hybridized to the first handle, The second adapter is hybridized to the second handle, The method according to claim 39, wherein the first handle or the second handle includes a first affinity site bound to a second affinity site of the affinity reagent, and the first adapter and the second adapter include different amplification handles.

46. The method according to claim 27, wherein the sample is a cell, tissue, or cell-free DNA.

47. The method according to claim 46, further comprising permeabilizing cells or tissues.

48. The method described above is a multiplex method for identifying multiple binding sites of multiple targets on one or more nucleic acids. (a) To provide a plurality of barcoding affinity reagents, None of the aforementioned barcoding affinity reagents contain transposases. The aforementioned multiple barcoding affinity reagents each bind to different targets, and provide them. (b) Adding the plurality of barcoding affinity reagents to the sample, (c) To provide a plurality of tagged nucleic acids by adding the unbound (unloaded) transposase and the transposase activator to the sample. (d) Sequencing the plurality of tagged nucleic acids to provide a nucleotide sequence, and (e) Analyzing the multiple nucleotide sequences to identify multiple binding sites of multiple targets, The method according to claim 27, including the method described in claim 27.

49. The nucleic acid is a part of chromatin, and the method further includes determining a data fingerprint for a combination of two target binding sites, the data fingerprint is (a) Colocalization information of two target binding sites, or lack of interaction between two target binding sites; (b) the distance between two target binding sites or epitopes; (c) The method according to claim 27, comprising the nucleotide sequence of the tagged nucleic acid.

50. The aforementioned data fingerprint is (d) Polarity or order of modifications; (e) Sys control element; (f) Proximity to or absence of a CpG island; (g) Repeat DNA sequences; and / or The method according to claim 49, further comprising (h) average DNA methylation level.

51. The method according to claim 46, wherein the nucleic acid is a part of chromatin.

52. The method according to claim 46, further comprising simultaneously identifying multiple histone marks, histone variants, histone mark readers, histone modifying enzymes, DNA modifying enzymes, chromatin-related proteins, transcription factors, RNA species, and / or cofactors within a genome.

53. The method according to any one of claims 27 to 52, wherein the background IgG sequencing reads are less than 25%, less than 20%, less than 15%, or less than 10% of the total sequencing reads.

54. The method according to any one of claims 27 to 52, wherein the affinity reagent-specific signal is generated in less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, and less than 2% of the cross-contaminated signal between different antibodies.

55. A method according to claim 27 or claim 48, further comprising identifying the colocalization of two epitopes at a single locus within a cell.

56. The method according to claim 55, for identifying the colocalization of H3K4me3 and H3K27me3.

57. The method according to claim 27 or claim 48, further comprising identifying a bivalent domain region covered by two histone modifications in the sample.

58. The method according to claim 27 or claim 48, further comprising identifying the colocalization of two epitopes at the same location on the same chromosome copy originating from the same chromosome fragment within the same cell.

59. The method according to claim 48, wherein providing multiple barcoded affinity reagents by barcoding multiple affinity reagents includes providing multiple individual barcoded affinity reagents by incubating each of the multiple affinity-labeled affinity reagents with a unique barcoding adapter in a separate reaction vessel.

60. The method according to claim 59, further comprising pooling the plurality of individual barcoding affinity reagents to provide a mixture of barcoding affinity reagents.

61. The method according to claim 48, wherein the analysis of the plurality of nucleotide sequences to identify multiple binding sites of multiple targets further comprises associating each barcode nucleotide sequence of the plurality of barcode nucleotide sequences with each affinity reagent of the plurality of affinity reagents.

62. The method according to claim 48, wherein the plurality of targets includes 2 to 500 targets.

63. Isolating the nucleus from a cell, Sorting single cells or single nuclei using flow cytometry or gel beads. Lysis of a single cell or single nucleus, Amplifying single-cell / nuclear libraries, including identifying signals from individual cells. Pooling single-cell / nuclear libraries, and Sequencing the aforementioned single-cell / nuclear library, The method according to claims 27 and 48, further comprising:

64. Adding drugs to the sample, (a) to (e) must be carried out. The method according to claims 27 and 48, further comprising comparing how the drug perturbs the signature in vitro or in vivo.

65. Instructions providing two or more barcoding affinity reagents, affinity reagents, An adapter comprising a barcode nucleotide sequence and a transposase-binding mosaic sequence, Unbound transposase and Transposase activators and A kit that includes, The two or more barcoding affinity reagents are, A pair of adapters, The first adapter includes a first barcode nucleotide sequence and a first transposase-binding mosaic sequence, The second adapter includes a pair of adapters, each containing a second barcode nucleotide sequence and a second transposase-binding mosaic sequence. kit.

66. Cell or nuclear permeabilization buffer, and / or The kit according to claim 65, further comprising a washing buffer.

67. Two or more barcoding affinity reagents, Unbound transposase and Transposase activators and A kit that includes, The two or more barcoding affinity reagents are, A pair of adapters, The first adapter comprises a first barcode nucleotide sequence and a first transposase-binding mosaic sequence. The second adapter includes a pair of adapters, each containing a second barcode nucleotide sequence and a second transposase-binding mosaic sequence. kit.

68. Cell or nuclear permeabilization buffer, and / or The kit according to claim 67, further comprising a washing buffer.

69. The kit according to claim 65 or claim 67, further comprising a control.

70. The kit according to claim 65, wherein the control is a recombinant nucleosome bound to DNA and / or a control affinity reagent.

71. The kit according to claim 65 or claim 67, comprising a panel of affinity reagents.

72. The kit according to claim 65 or 67, comprising a panel of cancer-specific affinity reagents.

73. The kit according to claim 65 or claim 67, comprising a panel of affinity reagents specific to epigenome marking proteins and / or histones.

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