Method for detecting chromatin accessibility or DNA binding protein footprint in cells

Abstract

The invention belongs to the technical field of biology. Specifically, the invention relates to a method for detecting chromatin accessibility or DNA binding protein footprint in cells. More specifically, the invention relates to a method for quantitatively determining chromatin accessibility of a single DNA molecule level and binding footprint of DNA binding proteins such as transcription factors by converting chromatin open information into mutation information of a DNA sequence based on deamination of DNA deaminase on chromatin open region DNA. In addition, the invention also relates to a method for detecting the DNA binding protein footprint in the open region of the cell genome based on deaminase and transposase, and the method can enrich the chromatin open region of the cell genome and detect the DNA binding protein footprint in the chromatin open region.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a method for detecting chromatin accessibility or DNA binding protein footprints in cells. More specifically, the present invention relates to a method for converting the chromatin accessibility information into the mutation information of the DNA sequence based on the deamination of DNA deaminase on the DNA in the chromatin open region, thereby realizing a method for quantitatively measuring the chromatin accessibility of a single DNA molecule and the binding footprints of DNA binding proteins such as transcription factors at the whole genome level. In addition, the present invention also relates to a method for detecting DNA binding protein footprints in the open region of the cell genome based on deaminase and transposase, which can enrich the chromatin open region of the cell genome and detect the DNA binding protein footprints in the chromatin open region. Background of the Invention

[0003] The DNA of eukaryotic organisms forms a complex with histones, and about 147bp of DNA is coiled around the histone core particle 1.65 times to form a nucleosome structure. The nucleosome and the connecting region together form a beaded structure, which is further folded and supercoiled to form a dense chromatin structure, which is wrapped by the nuclear membrane and separated from the cytoplasm. Chromosome accessibility reflects the degree to which various regulatory factors can physically contact genomic DNA, which is affected by the nucleosome arrangement, the spatial structure of the surrounding chromatin, and the promotion or inhibition of chromatin structure by different chromatin binding factors. Transcription factors dynamically compete with histones or other chromatin binding proteins to regulate physiological processes such as gene transcription, thereby showing the characteristics of different cells. Therefore, by studying chromatin accessibility, clarifying the state of chromatin in different cells, and establishing a dynamic change map of mutual transformation between different cells from the aspects of transcription factor binding and nucleosome rearrangement, it is very important to further clarify the gene regulation process of cells.

[0004] At present, most methods for studying chromatin accessibility rely on the property that open chromatin is more sensitive to enzymes or physical interruptions. Common methods for measuring chromatin accessibility include MNase-seq, DNase-seq, and ATAC-seq, which rely on cutting DNA fragments. These methods all judge the openness of chromatin based on the enrichment of DNA sequences, so they can only make relative judgments on openness. In addition, the above enzymes have sequence preferences for cutting DNA, which may also cause deviations in experimental results. NOMe-seq and Fiber-seq, which measure accessibility based on enzyme modification activity, rely on methylases and m6A adenine methyltransferases, respectively. The methylase used in NOMe-seq can only methylate GpC sequences, so the resolution is poor. However, Fiber-seq has the problem of not being able to amplify directly, which still requires an enrichment step, or uses a very large amount of cells to make up for the problem of not being able to amplify.

[0005] There is an urgent need for a method that can detect chromatin accessibility and / or transcription factor binding footprint information in cells with high resolution and quantitative analysis. Brief description of the invention

[0007] In the qDeAC-seq (Quantitative DNA Deaminase-Accessible Chromatin assay using sequencing) technology provided by the present invention, based on the deamination of DNA deaminase on the DNA in the chromatin accessibility region, the chromatin accessibility information can be converted into the mutation information of the DNA sequence, thereby achieving the quantitative determination of the chromatin accessibility at the level of a single DNA molecule and the binding footprint of DNA binding proteins such as transcription factors ( Figure 1 ). In addition, the qDeAC-ATAC-seq method formed by combining qDeAC-seq and ATAC-seq can enrich the chromatin open regions of the cell genome and detect the footprints of DNA binding proteins in the chromatin open regions ( Fig.21 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 .Schematic diagram of the qDeAC-seq workflow.

[0010] Figure 2 .8M urea denaturation treatment was used to separate DddA / SsdA and its corresponding inhibitor.

[0011] Figure 3 .Deaminase activity assay.

[0012] Figure 4 .qDeAC-seq cell processing workflow.

[0013] Figure 5 . Principle and process of single-stranded library construction using DNA Methylation Library Kit for Illumina V3.

[0014] Figure 6 .SsdA has no significant sequence preference for reaction sites.

[0015] Figure 7 .Comparison of Sanger sequencing results with ATAC-seq results.

[0016] Figure 8 . TSS region analysis and correlation analysis of MEF cells by qDeAC-seq second-generation sequencing.

[0017] Fig. 9.TSS and CTCF region data of R1 cells after qDeAC-seq treatment.

[0018] Fig.10 .There are obvious differences in conversion rates inside and outside the Klf4 and Thap11 binding motif sites.

[0019] Fig.11 .The conversion rates of SP1 and Nrf1 binding motif sites were significantly different between MEF and R1 cells.

[0020] Fig.12 .The impact of different library construction methods on the coverage of the center of the library ATAC-seq.

[0021] Fig.13 .The influence of different DNA polymerases on library PCR amplification.

[0022] Fig.14 .The effect of cell permeabilization conditions on sample conversion rate.

[0023] Fig.15 .The effect of deaminase reaction incubation time on the deamination effect.

[0024] Fig.16 .The effect of different concentrations of deaminase on the deamination effect.

[0025] Fig.17 .The influence of differences in deaminase reaction systems on the deamination effect.

[0026] Fig.18 .Optimization of deaminase reaction buffer.

[0027] Fig.19 .The effect of adding UGI on the sequencing depth at the ATAC center.

[0028] Fig. 20 The impact of adding UGI on conversion rate.

[0029] Fig.21 , qDeAC-ATAC-seq process diagram, strategy one is to add Tn5 treatment first, then add SsdAtox for deamination, strategy two is to add SsdAtox for deamination first, then add Tn5 to attack the open area.

[0030] Fig. 22 , Schematic diagram of the qDeAC-ATAC-seq library construction process.

[0031] Fig.23 , Tn5 linker sequence diagram.

[0032] Fig.24 , the effects of different Tn5 reaction buffers on the efficiency of enriching open areas.

[0033] Fig.25 , the effects of different Tn5 reaction buffers on the footprint depth of different transcription factors in the R1 cell line.

[0034] Fig.26 , qDeAC-ATAC-seq library fragment size distribution.

[0035] Fig. 27 Comparison of different TF depth values ​​under different deaminase treatment conditions in R1 cell line.

[0036] Fig.28 , Comparison of open zone enrichment effects between strategy 2 (the first group, and the fourth to eighth groups in the figure) and strategy 1 (the second and third groups in the figure).

[0037] Fig.29 , K562 cells, and tested the effects of different experimental strategies on the TF depth values ​​of different transcription factors (including separate qDeAC-seq data). DETAILED DESCRIPTION OF THE INVENTION

[0039] I. Definition

[0040] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0041] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0042] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered at this time, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0043] Sequence "identity" has a recognized meaning in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule.

[0044] "Genome" as used herein encompasses not only the chromosomal DNA present in the cell nucleus, but also the organellar DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0045] II. Methods for detecting chromatin accessibility and / or DNA binding protein footprints of cellular genomes

[0046] In one aspect, the present invention provides a method for detecting chromatin accessibility and / or DNA binding protein footprints of a cell genome, the method comprising:

[0047] a) treating at least one of the cells with a permeabilization buffer comprising a detergent, or isolating a cell nucleus from at least one of the cells;

[0048] b) treating the permeabilized cells or isolated cell nuclei obtained in step a) with a DNA deaminase, thereby allowing the DNA deaminase to fully react with the open regions of the genomic DNA in the cells;

[0049] c) isolating genomic DNA from the product of step b); and

[0050] d) sequencing the isolated genomic DNA or a portion thereof, and determining the chromatin accessibility and / or DNA binding protein footprint of the cell genome based on the sequencing results.

[0051] In order to preserve the chromatin accessibility and / or DNA binding protein footprint information of the cell genome to the greatest extent, the permeabilization buffer should be mild, which can destroy or partially destroy the cell wall / cell membrane, allowing DNA deaminase to enter the cell, but does not significantly destroy the chromatin structure of the cell. The cell nucleus isolation should also be mild and not significantly destroy the chromatin structure.

[0052] In some embodiments, the permeabilization buffer comprises NP40.

[0053] In some preferred embodiments, the permeabilization buffer comprises NP40, Tween-20, and Digitonin. In some preferred embodiments, the permeabilization buffer comprises about 0.1% NP40, about 0.1% Tween-20, and about 0.01% Digitonin.

[0054] In some embodiments, the permeabilization buffer further comprises Tris-HCl and / or PIC (protease inhibitor cocktail).

[0055] "Deaminase" refers to an enzyme that catalyzes a deamination reaction. As used herein, "DNA deaminase" refers to a deaminase that can accept DNA (single-stranded or double-stranded, especially double-stranded) as a substrate and can catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. The DNA deaminase can deaminate the C in the open region of genomic DNA to U, so in the subsequent amplification reaction for sequencing, the CG of the original genomic DNA is converted to TA. By detecting the conversion of CG to TA, the deamination site can be detected.

[0056] The DNA deaminase in the present invention can be single-stranded DNA deaminase A (SsdA) or double-stranded DNA deaminase A (DddA) or a functional fragment thereof. The DNA deaminase in the present invention can be from different species, for example, from Burkholderia cenocepacia or Phytophthora syringae.

[0057] As used herein, "functional fragment" refers to a fragment of a DNA deaminase that substantially retains its deamination activity, such as DddA tox or SsdA tox known in the art.

[0058] In some embodiments, the SsdA comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity with SEQ ID NO: 1. In some embodiments, the functional fragment of SsdA, such as SsdA tox, comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity with SEQ ID NO: 5.

[0059] In some embodiments, the DddA comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:2.

[0060] Due to its substantial lack of sequence preference, the DNA deaminase is preferably SsdA or a functional fragment thereof.

[0061] In some embodiments, the DNA deaminase is recombinantly produced. In some embodiments, the DNA deaminase also contains a fusion tag, such as a tag for separation and / or purification of the DNA deaminase. Methods for recombinant protein production are known in the art. And a variety of tags that can be used to separate and / or purify proteins are known in the art, including but not limited to His tags, smt3 tags, GST tags, etc. Generally speaking, these tags do not change the activity of the target protein. In some preferred embodiments, the DNA deaminase comprises a His tag and a smt3 tag.

[0062] In some specific embodiments, the SsdA or its functional fragment comprises an amino acid sequence shown in any one of SEQ ID NOs: 1, 3-5.

[0063] In some embodiments, step b) is performed in a reaction system of about 10 μl to about 100 μl, preferably 50 μl. The reaction system can be adjusted according to the number of cells or cell nuclei.

[0064] In some embodiments, the reaction system of step b) comprises a reaction buffer. For example, the reaction buffer can be a Tris-HCl buffer or a HEPES buffer. The pH of the reaction buffer is, for example, 7.4-8.0. Preferably, the pH of the reaction buffer is about 7.4.

[0065] In some embodiments, the reaction buffer comprises about 10 mM to about 20 mM, preferably about 10 mM Tris-HCl.

[0066] In some embodiments, the reaction buffer comprises about 10 mM to about 20 mM, preferably about 10 mM Tris-HCl, and about 0.1% NP40, about 0.1% Tween-20, about 0.01% Digitonin and / or about 0.01% Triton-100. In some embodiments, the reaction buffer comprises about 10 mM to about 20 mM, preferably about 10 mM Tris-HCl, and about 0.01% Digitonin and about 0.1% Tween-20. In some embodiments, the reaction buffer does not comprise additional metal salts, such as NaCl or MgCl2.

[0067] In some embodiments, the reaction buffer does not contain additional reagents except Tris-HCl. In some embodiments, the reaction buffer consists of about 10 mM to about 20 mM, preferably about 10 mM Tris-HCl and water.

[0068] In some embodiments, step b) further comprises adding a uracil glycosylase inhibitor (UGI) to the reaction. Adding UGI can inhibit the activity of the enzyme present in the reaction system that may remove U, thereby avoiding the effect caused by the removal of U produced by the deamination reaction. Adding UGI can increase the amount of enzyme used in the reaction to shorten the reaction time while ensuring the signal-to-noise ratio. UGI that can be used in the method of the present invention can be commercially available.

[0069] In some embodiments, the at least one cell is from 1 cell to about 50,000 cells or more, such as about 100, about 1,000, about 10,000, about 50,000 cells or more.

[0070] In some embodiments, the cells are treated with about 0.5 U / μl to about 50 U / μl, preferably about 7.5 U / μl of DNA deaminase.

[0071] In some embodiments, about 25 U to about 2500 U, preferably about 375 U, of the DNA deaminase is treated for about every 50,000 cells or nuclei.

[0072] In some embodiments, about 0.5 U / μl to about 50 U / μl enzyme is used for about 1000 cells / μl.

[0073] The enzyme activity unit U of the DNA deaminase used herein can be determined as shown in the examples herein. For example, the enzyme activity unit U is defined as the amount of enzyme required to digest 0.1 μM double-stranded oligo DNA substrate in a total reaction system of 10 μl at 37°C within 1 hour.

[0074] In some embodiments, the cells or cell nuclei are treated with the DNA deaminase in step b) for about 5 minutes to about 60 minutes, preferably about 10 minutes to about 30 minutes, more preferably about 10 minutes.

[0075] In some embodiments, step b) is performed at a temperature of about 4°C to about 40°C, such as about 30°C to about 40°C, preferably about 37°C.

[0076] In some preferred embodiments, in step b), about 50,000 cells or nuclei from about 50,000 cells are treated with 7.5 U / μl of DNA deaminase in a 50 μl reaction system at about 37°C for about 30 minutes.

[0077] The isolation of genomic DNA in step c) can be performed by conventional methods known in the art, for example, by using a commercial genomic DNA extraction kit.

[0078] The method of the present invention can be used to detect the chromatin accessibility and / or DNA binding protein footprints of certain specific regions of the cell genome. In this case, only these specific regions need to be sequenced.

[0079] Thus, in some embodiments, step d) comprises amplifying and sequencing a specific portion of the genomic DNA to determine the chromatin accessibility and / or DNA binding protein footprint of the specific portion of the genome of the cell.

[0080] In some embodiments, the amplification is PCR amplification, such as PCR amplification performed using a U-tolerant DNA polymerase, for example, the U-tolerant DNA polymerase is selected from: HiFi V3 (component in the Novozymes single-stranded library construction kit, NE103), Phusion U (Thermo Scientific F555L), Phanta Uc (vazyme, P507-01), Q5U (NEB, M0515L) and Q6U (Biyuntian, D7239M).

[0081] The method of the present invention can also be used to detect the chromatin accessibility and / or DNA binding protein footprints of the cell genome at the whole genome level.

[0082] Therefore, in some embodiments, step d) comprises establishing a whole genome DNA library from the isolated genomic DNA, and performing sequencing based on the whole genome library, thereby determining the chromatin accessibility and / or DNA binding protein footprint of the cell genome at the whole genome level.

[0083] Various methods known in the art for establishing whole genome sequencing libraries from isolated genomic DNA can be used, including single-stranded DNA library construction and double-stranded DNA library construction. Whole genome DNA libraries can be established using commercial kits, such as Novagen NE103, Swift Biosciences Cat. No. 33096, etc. for single-stranded library construction; Tn5 library construction can be used for double-stranded library construction, such as Novagen TD202 kit; WGBS library construction can use Zymo Research D5455 & D5456 kit.

[0084] However, the present inventors surprisingly found that the method of constructing a library using single-stranded DNA can achieve a significantly higher library construction depth than the method of constructing a library using double-stranded DNA.

[0085] Therefore, in some preferred embodiments, a whole genomic DNA library is established from the isolated genomic DNA by single-stranded DNA library construction.

[0086] In some specific embodiments, by The single-stranded DNA library was constructed using the DNA Methylation Library Kit for Illumina V3NE103.

[0087] In some embodiments, a U-tolerant DNA polymerase is used for DNA amplification in the library establishment, for example, the U-tolerant DNA polymerase is selected from the group consisting of HiFi V3, Phusion U, Phanta Uc, Q5U and Q6U.

[0088] Sequencing described herein can be first generation sequencing such as Sanger sequencing, second generation sequencing (NGS) or other high throughput sequencing. For example, the second generation sequencing of Illumina can be used.

[0089] Since DNA in the open region of chromatin is more easily converted to U by DNA deaminase, DNA bound by nucleosomes or DNA binding proteins is protected and cannot be deaminated. Therefore, by analyzing the distribution of deamination sites, the openness of chromatin, the arrangement of nucleosomes, and the position information and binding dynamics of DNA binding proteins can be determined.

[0090] In some embodiments, the chromatin accessibility and / or DNA binding protein footprint of the genomic DNA or a portion thereof of the cell is determined by analyzing the presence of CG to TA transitions (i.e., C to T and G to A) in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

[0091] The presence of CG to TA transitions in a specific region of the genome indicates that chromatin in that region is accessible and / or not bound by DNA-binding proteins.

[0092] In some embodiments, the chromatin accessibility and / or DNA binding protein footprint of the genomic DNA or a portion thereof of the cell is determined by analyzing the position and / or density and / or conversion rate of CG to TA transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

[0093] The location and / or density of CG to TA transitions in the genome indicates the location and / or degree of open chromatin regions.

[0094] The sequence of the control genomic DNA or its part can be the sequence of the genomic DNA or its part from a public database. Alternatively, the sequence of the control genomic DNA or its part can also be the sequence of the genomic DNA or its part of the same type of cell that has not been treated with the DNA deaminase.

[0095] The DNA binding protein described herein can be a transcription factor, histone, etc., preferably a transcription factor, such as Klf4, Thap11, SP1, Nrf1, etc.

[0096] The cells described herein can be animal cells, plant cells, or microbial cells. For example, the cells are mammalian cells, including but not limited to cells of humans, mice, rats, cats, dogs, pigs, and cattle; or, the cells are monocotyledonous or dicotyledonous plant cells, such as cells of rice, corn, wheat, sorghum, soybean, potato, tomato, etc.; or, the cells are fungi such as yeast cells.

[0097] The cell may be a cell line cell. Alternatively, the cell may be a primary cell from a different organ or tissue. For example, the cell may be from blood, cerebrospinal fluid, or a biopsy. The cell may be a hepatocyte, a cardiomyocyte, a neuron, a fibroblast, an epithelial cell, or the like. The cell may also be a tumor cell, a stem cell such as an embryonic stem cell, or an induced pluripotent stem cell.

[0098] In some embodiments, the cell is a cell treated with a specific substance (compound) or condition or a cell at a specific developmental stage. Thus, the method of the present invention can measure the effect of the specific substance (compound) or condition or specific developmental stage on the chromatin accessibility of the cell genome and / or the footprint of DNA binding proteins. For example, the specific substance can be a drug.

[0099] In another aspect, the present invention provides a method for detecting binding between a DNA molecule and a DNA binding protein, comprising:

[0100] 1) forming a reaction mixture of DNA molecules and DNA binding proteins and allowing them to fully contact;

[0101] 2) adding DNA deaminase and optionally uracil glycosylase inhibitor (UGI) to the reaction mixture and allowing them to fully react with the DNA molecules;

[0102] 3) isolating DNA from the product of step b); and

[0103] 4) sequencing the isolated DNA or a portion thereof, and determining the binding of the DNA binding protein to the DNA molecule based on the sequencing results.

[0104] The DNA deaminase is as defined above. In some embodiments, the step 2) is performed in a reaction buffer as defined above.

[0105] In some embodiments, binding of the DNA binding protein to the DNA molecule is determined by analyzing the presence of a CG to TA transition in the DNA molecule or a portion thereof relative to a control DNA molecule (eg, a DNA molecule that has not been treated with a DNA deaminase).

[0106] In some embodiments, the position or sequence of the DNA molecule to which the DNA binding protein binds is determined by analyzing the position of the CG to TA transition in the DNA molecule or a portion thereof relative to a control DNA molecule (e.g., a DNA molecule that has not been treated with a DNA deaminase).

[0107] 3. Methods for detecting DNA binding protein footprints in open chromatin regions of cell genomes

[0108] In one aspect, the present invention provides a method for detecting DNA binding protein footprints in open regions in a cell genome, the method comprising:

[0109] a) treating at least one of the cells with a permeabilization buffer comprising a detergent, or isolating a cell nucleus from at least one of the cells;

[0110] b) i) treating the permeabilized cells or isolated cell nuclei obtained in step a) with Tn5 transposase, thereby allowing the Tn5 transposase to fully react with the open regions of the genomic DNA in the cells; treating the permeabilized cells or isolated cell nuclei treated with Tn5 transposase with DNA deaminase, thereby allowing the DNA deaminase to fully react with the open regions of the genomic DNA in the cells; or

[0111] ii) treating the permeabilized cells or isolated cell nuclei obtained in step a) with DNA deaminase, thereby allowing the DNA deaminase to fully react with the open regions of the genomic DNA in the cells; treating the permeabilized cells or isolated cell nuclei treated with DNA deaminase with Tn5 transposase, thereby allowing the Tn5 transposase to fully react with the open regions of the genomic DNA in the cells;

[0112] c) isolating genomic DNA from the product of step b); and

[0113] d) sequencing the isolated genomic DNA or a portion thereof, and determining the DNA binding protein footprints of the open regions of the cell genome based on the sequencing results.

[0114] In some embodiments, the open region is an open chromatin region. The open chromatin region herein has a meaning known in the art, and refers to a region where the compact structure of chromatin is opened. These regions allow trans-acting factors to bind to cis-regulatory elements such as promoters, enhancers, insulators, and silencers. This property of allowing trans-acting factors to bind is called the openness / accessibility of chromatin.

[0115] In order to retain the DNA binding protein footprint information of the cell genome to the maximum extent, the permeabilization buffer should be mild, which can destroy or partially destroy the cell wall / cell membrane, allowing DNA deaminase and / or Tn5 transposase to enter the cell, but does not significantly destroy the chromatin structure of the cell. Cell nuclear isolation should also be mild and not significantly destroy the chromatin structure.

[0116] In some embodiments, the permeation buffer comprises NP40. In some preferred embodiments, the permeation buffer comprises NP40, Tween-20 and Digitonin. In some preferred embodiments, the permeation buffer comprises about 0.1% NP40, about 0.1% Tween-20 and about 0.01% Digitonin. In some embodiments, the permeation buffer further comprises Tris-HCl and / or PIC (protease inhibitor cocktail).

[0117] In some embodiments, the cell nucleus separation is performed using methods known in the art. In some embodiments, the cell nucleus is separated using a buffer comprising 10 mM Tris-HCl 7.4, 10 mM NaCl, 1% BSA, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin, 0.1 mM EDTA, 3 mM MgCl2, 1xPIC.

[0118] "Deaminase" refers to an enzyme that catalyzes a deamination reaction. As used herein, "DNA deaminase" refers to a deaminase that can accept DNA (single-stranded or double-stranded, especially double-stranded) as a substrate and can catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. The DNA deaminase can deaminate the C in the open region of genomic DNA to U, so in the subsequent amplification reaction for sequencing, the CG of the original genomic DNA is converted to TA. By detecting the conversion of CG to TA, the deamination site can be detected.

[0119] The DNA deaminase in the present invention can be single-stranded DNA deaminase A (SsdA) or double-stranded DNA deaminase A (DddA) or a functional fragment thereof. The DNA deaminase in the present invention can be from different species, for example, from Burkholderia cenocepacia or Phytophthora syringae.

[0120] As used herein, "functional fragment" refers to a fragment of a DNA deaminase that substantially retains its deamination activity, such as DddA tox or SsdA tox known in the art.

[0121] In some embodiments, the SsdA comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity with SEQ ID NO: 1. In some embodiments, the functional fragment of SsdA, such as SsdA tox, comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity with SEQ ID NO: 5.

[0122] In some embodiments, the DddA comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:2.

[0123] Due to its substantial lack of sequence preference, the DNA deaminase is preferably SsdA or a functional fragment thereof.

[0124] In some embodiments, the DNA deaminase is recombinantly produced. In some embodiments, the DNA deaminase also contains a fusion tag, such as a tag for separation and / or purification of the DNA deaminase. Methods for recombinant protein production are known in the art. And a variety of tags that can be used to separate and / or purify proteins are known in the art, including but not limited to His tags, smt3 tags, GST tags, etc. Generally speaking, these tags do not change the activity of the target protein. In some preferred embodiments, the DNA deaminase comprises a His tag and a smt3 tag.

[0125] In some specific embodiments, the SsdA or a functional fragment thereof comprises an amino acid sequence shown in any one of SEQ ID NOs: 1, 3-5.

[0126] In some embodiments, step b) is performed in a reaction system of about 10 μl to about 100 μl, preferably 50 μl. The reaction system can be adjusted according to the number of cells or cell nuclei.

[0127] In some embodiments, the DNA deaminase reacts sufficiently with the open regions of the genomic DNA in the cell to result in the deamination of C to U in the open regions of the genomic DNA in the cell.

[0128] In some embodiments, the DNA deaminase treatment is performed in a deamination reaction buffer.

[0129] For example, the deamination reaction buffer can be a Tris-HCl buffer or a HEPES buffer. The pH of the deamination reaction buffer is, for example, 7.4-8.0. Preferably, the pH of the deamination reaction buffer is about 7.4. In some embodiments, the deamination reaction buffer comprises about 10mM-about 20mM, preferably about 10mM Tris-HCl. In some embodiments, the deamination reaction buffer comprises about 10mM-about 20mM, preferably about 10mM Tris-HCl, and about 0.1% NP40, about 0.1% Tween-20, about 0.01% Digitonin and / or about 0.01% Triton-100. In some embodiments, the deamination reaction buffer comprises about 10mM-about 20mM, preferably about 10mM Tris-HCl, and about 0.01% Digitonin and about 0.1% Tween-20. In some embodiments, the deamination reaction buffer does not comprise additional metal salts, such as NaCl or MgCl2. In some embodiments, the deamination reaction buffer does not contain additional reagents except Tris-HCl. In some embodiments, the deamination reaction buffer is composed of about 10mM-about 20mM, preferably about 10mM Tris-HCl and water. In some embodiments, the deamination reaction buffer further comprises the addition of a uracil glycosylase inhibitor (UGI). Adding UGI can inhibit the activity of the enzyme present in the reaction system that may remove U, and avoid the effects caused by the removal of U produced by the deamination reaction. Adding UGI can increase the amount of enzyme used in the reaction to shorten the reaction time while ensuring the signal-to-noise ratio. UGI that can be used in the method of the present invention can be commercially available.

[0130] In some embodiments, the cells or cell nuclei are treated with about 0.5 U / μl to about 50 U / μl, preferably about 0.5-7.5 U / μl of DNA deaminase. In some embodiments, about 25 U to about 2500 U, preferably about 375 U of DNA deaminase is used for every 50,000 cells or cell nuclei. In some embodiments, about 1000 cells / μl are treated with about 0.5 U / μl to about 50 U / μl of DNA deaminase.

[0131] In some preferred embodiments, the cells or cell nuclei are treated with about 5 U / μl of DNA deaminase in step b)i). In some preferred embodiments, the cells or cell nuclei are treated with about 2 U / μl of DNA deaminase in step b)ii).

[0132] In some embodiments, the cells or cell nuclei are treated with the DNA deaminase for about 5 minutes to about 60 minutes, preferably about 10 minutes to about 30 minutes, and more preferably about 10 minutes.

[0133] In some embodiments, the cells or cell nuclei are treated with a DNA deaminase at a temperature of about 4°C to about 40°C, such as about 30°C to about 40°C, preferably about 37°C.

[0134] Herein, the Tn5 transposase fully reacts with the open region of the genomic DNA in the cell, resulting in the fragmentation of the genomic DNA in the open region and the addition of a linker containing a Tn5 ME sequence to the 5' end of the obtained DNA fragment (Tagmentation).

[0135] In some embodiments, the Tn5 transposase comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity to SEQ ID NO: 6. In some embodiments, the Tn5 transposase is capable of fragmenting substrate DNA and adding adapters to the fragmented DNA.

[0136] In some embodiments, the Tn5 transposase has been complexed with an adaptor containing a Tn5 ME sequence to form a transposome.

[0137] In the art, there are suitable Tn5 transposases for nucleic acid sequencing library construction, methods for constructing transpososomes with adapters, and corresponding commercial kits, which can all be applied to the present invention.

[0138] In some embodiments, the adapter added by the Tn5 transposase is compatible with subsequent library construction and sequencing steps. In some preferred embodiments, the adapter added by the Tn5 transposase does not contain C, and in particular, the sequence in the adapter other than the Tn5ME sequence does not contain C. In some embodiments, the adapter added by the Tn5 transposase comprises a nucleotide sequence as follows: 5'-TGGTAGAGAGGGTG AGATGTGTATAAGAGACAG-3' (SEQ ID NO:7) (the underlined part is the double-stranded Tn5 ME sequence, and the non-underlined part is the single-stranded part), which is formed by annealing the single-stranded Tn5 adapter-F of the sequence shown in SEQ ID NO:7 and the single-stranded Tn5 adapter-R of the sequence shown in SEQ ID NO:8 (5'-CTGTCTCTTATACACATCT-3').

[0139] In some embodiments, the Tn5 transposase treatment is performed in a Tagmentation buffer. The Tagmentation buffer can be a commercial Tn5 transposase Tagmentation buffer. In some embodiments, the Tagmentation buffer comprises DMF (dimethylformamide). In some embodiments, the Tagmentation buffer does not comprise a detergent. In some preferred embodiments, the Tagmentation buffer comprises 10 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 10% DMF, 33% PBS. The composition of PBS is: 155 mM NaCl, 3 mM Na2HPO4, and 1 mM KH2PO4.

[0140] In some embodiments, the Tagmentation buffer further comprises UGI, for example, the Tagmentation buffer used in step b) ii) comprises UGI.

[0141] In some embodiments, the concentration of the Tn5 transposase is about 50 nM to about 150 nM, such as 100 nM.

[0142] In some embodiments, the cells or cell nuclei are treated with Tn5 transposase for about 5 minutes to about 60 minutes, preferably about 10 minutes to about 30 minutes, and more preferably about 15 minutes.

[0143] In some embodiments, the cells or cell nuclei are treated with Tn5 transposase at a temperature of about 4°C to about 40°C, such as about 30°C to about 40°C, preferably about 37°C.

[0144] In some embodiments, if the amount of cells is small, step b)i) can be used, which can lead to higher enrichment of open regions of the cell genome. In some embodiments, for the detection of DNA binding proteins with weak DNA binding ability, step b)ii) can be used to avoid the effect of Tn5 transposase treatment causing DNA binding proteins to fall off the DNA.

[0145] In some embodiments, in step b) i), after the Tn5 transposase treatment, the cells or cell nuclei are separated by centrifugation and then subjected to the DNA deaminase treatment.

[0146] In some embodiments, in step b) ii), after the DNA deaminase treatment, the cells or cell nuclei are separated by centrifugation and then subjected to the Tn5 transposase treatment.

[0147] The isolation of genomic DNA in step c) of the method of the present invention can be carried out by conventional methods known in the art, for example, by using a commercial genomic DNA extraction kit.

[0148] In some embodiments, step d) comprises establishing a genomic DNA library from the isolated genomic DNA, and performing sequencing based on the genomic library, thereby determining the DNA binding protein footprints of the open regions of the cell genome at the genome level.

[0149] Various methods known in the art for establishing a genomic sequencing library from isolated genomic DNA can be used, including single-stranded DNA library construction and double-stranded DNA library construction. Genomic DNA libraries can be established using commercial kits, such as Novozyme NE103, Swift Biosciences Cat. No. 33096, etc. for single-stranded library construction; Tn5 library construction can be used for double-stranded library construction, such as Novozyme TD202 kit; WGBS library construction can use Zymo Research D5455 & D5456 kit.

[0150] The inventors surprisingly found that the method of building a library with single-stranded DNA can obtain a significantly higher library depth than the method of building a library with double-stranded DNA. Therefore, in some preferred embodiments, a genomic DNA library is established from the isolated genomic DNA by building a library with single-stranded DNA.

[0151] In some specific embodiments, by The single-stranded DNA library was constructed using the DNA Methylation Library Kit for Illumina V3NE103.

[0152] In some embodiments, a U-tolerant DNA polymerase is used for DNA amplification in the library establishment, for example, the U-tolerant DNA polymerase is selected from the group consisting of HiFi V3, Phusion U, Phanta Uc, Q5U and Q6U.

[0153] Sequencing described herein can be first generation sequencing such as Sanger sequencing, second generation sequencing (NGS) or other high throughput sequencing. For example, the second generation sequencing of Illumina can be used.

[0154] Since DNA in the open region of genomic chromatin is more susceptible to DNA deaminase reaction, resulting in C conversion to U, and DNA bound by DNA binding proteins is protected and cannot be deaminated, the positional information and binding dynamics of DNA binding proteins can be determined by analyzing the distribution of deamination sites.

[0155] In some embodiments, the DNA binding protein footprint of the genomic DNA or a portion thereof of the cell is determined by analyzing the presence of CG to TA transitions (i.e., C to T and G to A) in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

[0156] The presence of CG to TA transitions in a specific region of the genome indicates that the genomic DNA in that region is not bound by DNA binding proteins.

[0157] In some embodiments, the DNA binding protein footprint of the genomic DNA or a portion thereof is determined by analyzing the position and / or density and / or rate of CG to TA transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

[0158] The sequence of the control genomic DNA or its part can be the sequence of the genomic DNA or its part from a public database. Alternatively, the sequence of the control genomic DNA or its part can also be the sequence of the genomic DNA or its part of the same type of cell that has not been treated with the DNA deaminase.

[0159] The DNA binding protein described herein can be a transcription factor, a histone, etc., preferably a transcription factor. For example, the transcription factor is selected from NRF1, MYBL2, NFYA, RFX1, etc.

[0160] The cells described herein can be animal cells, plant cells, or microbial cells. For example, the cells are mammalian cells, including but not limited to cells of humans, mice, rats, cats, dogs, pigs, and cattle; or, the cells are monocotyledonous or dicotyledonous plant cells, such as cells of rice, corn, wheat, sorghum, soybean, potato, tomato, etc.; or, the cells are fungi such as yeast cells.

[0161] The cell may be a cell line cell. Alternatively, the cell may be a primary cell from a different organ or tissue. For example, the cell may be from blood, cerebrospinal fluid, or a biopsy. The cell may be a hepatocyte, a cardiomyocyte, a neuron, a fibroblast, an epithelial cell, or the like. The cell may also be a tumor cell, a stem cell such as an embryonic stem cell, or an induced pluripotent stem cell.

[0162] In some embodiments, the cell is a cell treated with a specific substance (compound) or condition or a cell at a specific developmental stage. Thus, the method of the present invention can measure the effect of the specific substance (compound) or condition or specific developmental stage on the chromatin accessibility of the cell genome and / or the footprint of DNA binding proteins. For example, the specific substance can be a drug.

[0163] IV. Test Kit

[0164] On the other hand, the present invention provides a kit for detecting chromatin accessibility and / or DNA binding protein footprints of a cell genome or for detecting the binding between a DNA molecule and a DNA binding protein, which comprises at least the DNA deaminase described herein above and an optional uracil glycosylase inhibitor (UGI).

[0165] In some embodiments, the kit is used to detect chromatin accessibility and / or DNA binding protein footprints of a cell genome by the method of the present invention, or is used to detect the binding between a DNA molecule and a DNA binding protein by the method of the present invention.

[0166] In some embodiments, the kit further comprises a permeabilization buffer comprising a detergent and / or a reagent for isolating cell nuclei. The permeabilization buffer is as defined above.

[0167] In some embodiments, the kit further comprises a reaction buffer that is conducive to the DNA deaminase reaction. The reaction buffer is as defined above.

[0168] In some embodiments, the kit further includes reagents for amplifying the genomic DNA portion of interest, such as specific primers.

[0169] In some embodiments, the kit further comprises reagents for establishing a genomic library.

[0170] In some embodiments, the kit further comprises reagents for sequencing, such as Sanger sequencing or next generation sequencing.

[0171] In another aspect, the present invention provides a kit for detecting DNA binding protein footprints in open regions of a cell genome, which comprises at least the DNA deaminase and Tn5 transposase described herein above.

[0172] In some embodiments, the kit is used to detect DNA binding protein footprints in open regions of a cell genome by the methods of the present invention.

[0173] In some embodiments, the kit further comprises a permeabilization buffer comprising a detergent and / or a reagent for isolating cell nuclei. The permeabilization buffer and the reagent for analyzing cell nuclei are as defined above.

[0174] In some embodiments, the kit further comprises a deamination reaction buffer that is conducive to the DNA deaminase reaction. The deamination reaction buffer is as defined above.

[0175] In some embodiments, the kit further comprises an adaptor comprising a Tn5 ME sequence, and a reagent for complexing the adaptor with the Tn5 transposase to form a transposome. The adaptor comprising a Tn5 ME sequence is as defined above.

[0176] In some embodiments, the kit further comprises a Tagmentation reaction buffer that facilitates the Tn5 transposase reaction. The Tagmentation reaction buffer is as defined above.

[0177] In some embodiments, the kit further comprises reagents for establishing a genomic library.

[0178] In some embodiments, the kit further comprises reagents for sequencing, such as Sanger sequencing or next generation sequencing.

[0179] The kit generally also includes a label indicating the intended use and / or method of use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit. Example

[0180] The techniques used in the following examples, including sequencing library establishment and high-throughput sequencing technology, as well as molecular biology techniques such as cell fixation and DNA purification, and cell culture and detection techniques, are conventional techniques known to those skilled in the art, unless otherwise specified. The instruments, equipment, reagents, and cell lines used, unless otherwise specified in this specification, are generally available to researchers and technicians in the field through public channels. In the examples listed in the specific implementation methods, the cell collection, processing, and single cell separation schemes and some buffer systems represent only a few of the many feasible treatment schemes.

[0181] Example 1: Detecting chromatin accessibility or DNA binding protein footprints in cells at the genome-wide level

[0182] General method description

[0183] 1). Construction of plasmid expressing deaminase protein

[0184] The pETDuet vector was used for protein expression and purification. The vector contains an ampicillin resistance gene and two multiple cloning sites, each of which contains a T7 promoter, a Lac operator and a ribosome binding site. The DddA / SsdA sequence or its tag sequence or its truncated sequence and its corresponding Inhibitor sequence were constructed into the two sites of the pETDuet vector respectively.

[0185] 2). Purification of deaminase protein

[0186] The vector was transformed into protein expression strains such as Rosetta (DE3), Rosetta Gami, and BL21 (DE3) using the heat shock method, and cultured under the conditions of ampicillin and chloramphenicol double resistance. Some bacteria were collected and resuspended in freezing solution (50% glycerol: double resistance 2YT = 1:1), and stored at -80°C after quick freezing in liquid nitrogen. Test the expression effect of different strains and select suitable strains for subsequent experiments. Use double resistance culture medium to culture the strain at a suitable speed, test the OD value of the bacterial solution at different times, and use IPTG to induce protein expression after the bacterial solution is cultured to a certain OD value. After a certain period of induction, the bacterial precipitate is collected by centrifugation and crushed by overpressure.

[0187] For affinity purification and denaturation, ①SsdA: centrifuge the broken bacterial solution and collect the inclusion bodies. Add a buffer containing 8M urea to dissolve the inclusion bodies, centrifuge to remove impurities, add Ni-NTA beads, and affinity purify the protein. ②DddA: centrifuge the broken bacterial solution, collect the supernatant, add Ni-NTA beads for incubation, and then add a buffer containing 8M urea to denature the protein. Treatment with 8M urea can separate DddA / SsdA from its corresponding inhibitor (see Figure 2 ), and then DddA / SsdA was renatured by reducing the urea concentration. Imidazole was used for elution, and Imidazole was removed by dialysis, and finally protein quantification was performed by Coomassie Brilliant Blue staining. Finally, the AKTA protein purification system was used to separate and purify the DddA / SsdA protein by ion exchange chromatography, molecular sieve, etc., and the corresponding components were concentrated to obtain the target protein sample.

[0188] 3). Verification of in vitro deaminase activity.

[0189] Design an oligonucleotide primer (5'-AATATAATATAATAACTCGCCATAATTTTAATTAAT-3', 5' with a 6-FAM fluorescent group) and its complementary sequence, and use annealing to generate a double-stranded oligonucleotide. According to the reaction system, different concentrations of deaminase were mixed with a double-stranded oligonucleotide substrate with a final concentration of 1nM, and incubated at 37°C for 1 hour to deaminize the cytosine on the substrate to form uracil. Add uracil DNA glycosylase, incubate at 37°C for 30 minutes, cut off uracil, and form a purine-free pyrimidine site. Then, the double-stranded oligonucleotide was unwound by high-temperature treatment with 100mM NaOH and formamide at 95°C, and the purine-free pyrimidine site was broken. Finally, the fragments were separated by 15% acrylamide gel electrophoresis containing 8M urea, and the deamination effect was judged by the fragment size. See details. Figure 3 .

[0190] The enzyme activity unit U is defined as the amount of enzyme required to digest 0.1 μM substrate in a total reaction system of 10 μl at 37°C for 1 hour.

[0191] The reaction system is:

[0192] 10x annealing buffer (100mM Tris pH 7.5, 500mM NaCl, 10mM EDTA)

[0193] Oligo1 and oligo2:stock 100μM

[0194]

[0195] Final concentration:10μM

[0196] Annealing program

[0197]

[0198] 4). Cell processing for qDeAC-seq ( Figure 4 )

[0199] (1) Cell collection and nuclear extraction:

[0200] Add appropriate amount of trypsin to digest cells, count after neutralization with medium, transfer to 1.5mL low-absorption EP tube, centrifuge at room temperature, 300×g for 4min, and discard supernatant. Add 1mL cold PBS to wash twice, centrifuge at 4℃, 300×g for 4min, and discard supernatant. Add 1mL cold Wash buffer (10mM Tris-HCl 7.4, 10mM NaCl, 1% BSA, 0.1mM EDTA, 3mM MgCl2, 1xPIC) to resuspend, and separate 50,000 cells × N groups. 4℃, 300×g, 4min, aspirate and discard supernatant as much as possible, and wash once in total.

[0201] Lysis: Resuspend the cells by flicking the bottom of the tube, then add 50ul Lysis Buffer (10mM Tris-HCl7.4, 10mM NaCl, 1% BSA, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin, 0.1mM EDTA, 3mM MgCl2, 1xPIC) to 50,000 cells, gently pipette 3 times, and place on ice for 3 minutes. Termination reaction: Add 1ml Final buffer (10mMTris-HCl 7.4, 10mM NaCl, 1% BSA, 0.1mM EDTA, 1xPIC), gently invert and mix 3 times, 4℃, 700×g, 5min, invert and centrifuge for 1min, remove the supernatant to about 100ul, invert and centrifuge again for 1min, and completely discard the supernatant.

[0202] (2) Deamination reaction:

[0203] According to the 50ul system (10mM Tris-HCl 7.4, 1mM DTT, 1xPIC), add enzyme, buffer and appropriate amount of lamda DNA to the punched cell sample, gently invert to mix and resuspend the cells. Incubate at 37℃ for 0.5h. Set the hot plate at 600rpm, 3min pause / 30s shaking.

[0204] (3) DNA extraction

[0205] Then, heat incubate the reaction system. Add 200ul gDNA Hot Elution Buffer to the 50ul reaction system and heat-shock for 1.5h at 65℃ and 1350rpm. Add gDNA and an equal volume (250ul) of phenol:chloroform:isoamyl alcohol DNA extraction solution and shake thoroughly to mix. Centrifuge at room temperature at 13000g for 5min. Obtain gDNA by isopropanol precipitation and measure the concentration by QUBIT.

[0206] 5). Library establishment

[0207] The total amount of starting DNA was selected based on the measured DNA concentration and experimental requirements. Taking 5 ng as an example, the genome was fragmented by ultrasound. DNA Methylation Library Kit for Illumina V3 NE103 kit for single-stranded DNA library construction ( Figure 5 ).

[0208] According to the kit steps, the 3'Adapter and 5'Adapter were connected respectively, and the primers were amplified to obtain the complete library. The DNA fragments were analyzed using the Agilent 2100 biochip analysis system. The constructed library was sent to the company for high-throughput sequencing. The sequencing machine used was Illumina X TEN, and the sequencing mode was double-end 150bp.

[0209] 6).qDeAC-seq base mutation identification.

[0210] By analyzing the sequencing data and calculating the base transition sites and numbers, we can determine the accessibility of the genome and the situation of protein binding at specific sites.

[0211] 1.1. qDeAC-seq to determine MEF genomic DNA accessibility

[0212] In order to clarify the selectivity of deaminases for genomic DNA, deaminases with different purification tags and auxiliary tags were purified in multiple strains, and mouse embryonic fibroblast stem cells were treated with different deaminases, and data analysis was performed: selected sites were amplified by PCR, and then all C sites were extracted, and the conversion rates of AC, TC, GC, and C in CC were calculated to determine the preference of SsdA for substrate conversion. The analysis results showed that DddA had a certain TC preference, while SsdA had basically no sequence preference ( Figure 6 ).

[0213] By analyzing the ATAC-seq data in the public database, we selected a region with high openness, designed primers for PCR in this region, connected the product to the T vector, and then performed Sanger sequencing. The trend of the sequencing results is consistent with the ATAC-seq results, and the sequence shows a mark similar to the binding of transcription factor SP1 ( Figure 7 ).

[0214] After processing the mouse embryonic fibroblast genome with qDeAC-seq, the next generation sequencing library was constructed. The sequencing results clearly reflected the high openness of the TSS (transcription start site) region, and up to the third nucleosome position after the TSS could be seen. Correlation analysis with ATAC-seq revealed a correlation between qDeAC-seq and ATAC ( Figure 8 ).

[0215] 1.2. qDeAC-seq determination of R1 cell genomic DNA accessibility and specific TF site binding

[0216] The genome was processed with qDeAC-seq and then subjected to next-generation sequencing to construct a library. The results clearly reflected the high openness of the TSS region, and the third nucleosome position after the TSS could be seen. At the same time, clear openness fluctuations on both sides of CTCF (CCCTC binding factor) could be observed ( Fig. 9 ).

[0217] In addition, the binding regions of known specific TFs (Klf4, Thap11) were aligned according to the binding motif and sorted according to the degree of openness. It can be clearly observed that protein binding affects the deaminase reaction and the conversion rate of the central site decreases. This shows that qDeAC-seq can intuitively and high-resolutionly see the imprint of transcription factor binding. And for specific transcription factors, qDeAC-seq can see the difference between positive and negative chains. ( Fig.10 )

[0218] 1.3. Differences in TF binding between R1 and MEF genomes by qDeAC-seq

[0219] After processing the genomes of the two cells using qDeAC-seq, the next generation sequencing library was constructed. Through the analysis of the sequencing results, the known specific TF binding regions were aligned according to the binding motif as the center, and sorted according to the openness of the center of the R1 cell line. At the same time, the corresponding data of MEF were sorted in this order, and it can be seen that there are significant differences between the two cells. Fig.11 Comparison of the two transcription factors, SP1 and Nrf1, shows obvious differences.

[0220] Therefore, it can be seen from the experimental results of Examples 1-3 above that the experimental data obtained by using the qDeAC-seq of the present invention to perform cell genome openness and transcription factor binding imprint detection have high repeatability, and the conversion rate can be intuitively seen to be affected by nucleosome occupancy, and the occupancy of specific transcription factor binding sites can be observed. It shows that the qDeAC-seq method of the present invention can intuitively see the changes in the degree of chromatin openness and the occupancy of specific transcription factor binding sites, which greatly improves the resolution compared with other methods, and makes the data intuitively observable, more real and reliable, and can see the difference between the positive and negative chains of the specific transcription factor binding site, and infer the directionality of its binding. This is currently impossible to accomplish with other methods.

[0221] 1.4. Comparison of the effects of different library construction methods on qDeAC-seq results

[0222] In this example, the effects of single-stranded and double-stranded library construction on qDeAC-seq results were compared. The library construction process is as follows: Single-chain library construction process:

[0223] The qDeAC-seq single-strand library construction method complies with the instructions of the EpiArt DNA Methylation Library Kit for Illumina V3 (NE103).

[0224] The whole genome DNA of the cells treated with deamination by deaminase SsdA was collected and ultrasonically treated to obtain fragmented double-stranded DNA. A certain amount of DNA was taken as the starting library, and the double-stranded DNA was immediately placed on ice after being denatured at 95°C to obtain fragmented single-stranded DNA. After the 3' end adapter was connected to the 3' end adapter at the 3' end of the single-stranded DNA using 3' adapter ligase, an extension system was added for reaction, and then 1.2x DNA purification magnetic beads (N411, VAHTS DNA Clean Beads) were used to obtain a double-stranded DNA product connected to the 3' end adapter. The 5' adapter ligase was used to connect the adapter at the 5' end of the double-stranded DNA, and then purified using 1x DNA magnetic beads to obtain a double-stranded DNA product connected to the double-end adapter. Finally, the library was amplified by PCR for an appropriate number of rounds using N321 / N322, VAHTS Multiplex Oligos Set4 / 5for Illumina, and the final library was purified using 0.85x DNA purification magnetic beads.

[0225] Double-chain library construction process

[0226] The qDeAC-seq double-strand library construction method complies with the instructions of the TruePrep DNA Library PrepKit V2 for Illumina kit TD502.

[0227] The whole genome DNA of cells treated with deamination by deaminase SsdA was collected, 5 ng of which was added with transposase Tn5 for treatment, and then purified using 2x AMP XP magnetic beads to obtain the DNA fragments connected to the street by transposase Tn5.

[0228] Finally, use Novagen amplification primers (including but not limited to TD204, TruePrep Index Kit V4 for Illumina) and a uracil (U)-tolerant DNA polymerase (including but not limited to Phusion U) to perform appropriate rounds of PCR amplification, and magnetic bead purification or gel recovery of fragments between 300-500 bp to obtain the final purified library.

[0229] The two database construction methods have the following differences:

[0230] 1) The DNA state of the starting library is different. The starting library of single-stranded library construction is single-stranded DNA, and the starting library of double-stranded DNA is double-stranded DNA.

[0231] 2) The library is fragmented in different ways. Single-stranded library construction uses the physical effect of ultrasound to fragment intact DNA, while double-stranded library construction uses the DNA cutting activity of the transposase Tn5 itself to fragment DNA.

[0232] 3) The methods of connecting adapters are different. The single-stranded library construction uses a ligase-based method to connect the 3' and 5' adapters in two rounds of reactions. The double-stranded library construction uses the adapter-connecting activity of the transposase Tn5 itself to connect the adapters during the fragmentation of DNA.

[0233] For the original sequence that has been converted by deaminase, there will be more UG mismatch regions on the double-stranded DNA sequence, which will affect the attack efficiency of Tn5 on the DNA sequence, resulting in more edited regions being less likely to be attacked by Tn5 and a lower library depth. Single-stranded library construction can significantly improve this phenomenon.

[0234] DNA samples of MEF cell lines were treated with 2.8U SsdA for 30 minutes, and double-stranded DNA libraries and single-stranded DNA libraries were constructed using the above method, and then sequenced. Analysis showed that the coverage of the DNA library obtained by single-stranded library construction was significantly improved in the center of the peak obtained by the published ATAC data analysis, which was significantly higher than that of the double-stranded DNA library sample. ( Fig.12 )

[0235] 1.5 Optimization of qDeAC-seq

[0236] Choice of DNA polymerase

[0237] Due to the deamination effect of the deaminase, the obtained DNA before amplification contains a large amount of uracil bases (Uracil, U). Therefore, when performing the final PCR amplification of the library, a DNA polymerase that tolerates U is required. The inventors tested different U-tolerant DNA polymerases and analyzed the differences in amplification effects, and found that the effects of different enzymes were similar ( Fig.13 ). The enzymes tested included: HiFi V3, Phusion U, Phanta Uc, Q5U, Q6U.

[0238] Optimization of cell punching conditions

[0239] The commonly used perforation conditions in various other omics methods were analyzed, and it was found that 0.5% NP-40 and 0.1% NP-40, 0.1% Tween-20 and 0.01% Digitonin (referred to as Mix in the figure) were the two most commonly used detergent conditions in the perforation reaction. The cells were treated with these two perforation buffers, and it was found that both perforation methods had the effect of making the cell membrane permeable. However, under the same enzyme dosage conditions, the conversion rate of the Mix group was significantly higher than that of the 0.5% NP40 group, and there was a higher base conversion rate at the open area. ( Fig.14 )

[0240] Deaminase incubation time

[0241] The deaminase reaction incubation time of 10 minutes and 30 minutes were tested respectively, and both time lengths produced samples with significant conversion rates. The 30-minute reaction condition produced a higher genome conversion rate. Fig.15 )

[0242] Deaminase concentration

[0243] Using different concentrations of deaminase to treat genomic DNA, it can be seen that as the enzyme concentration increases, the overall genomic base conversion rate also increases. Through analysis, as the enzyme concentration increases, the signal-to-noise ratio of the open area and the adjacent area shows a trend of first increasing and then decreasing. For this batch of enzymes, the conversion rate and signal-to-noise ratio are higher at an enzyme concentration of 7.5U / μl ( Fig.16 ). The data of the 15U group and the 30U group are not shown, but the signal-to-noise ratio of the 30U group is lower than that of the 15U group.

[0244] Deaminase reaction system

[0245] The total volume of the basic enzyme reaction system is 50μl and 50,000 cells are used for the reaction. Through experimental testing, the components in the reaction were reduced in equal proportions to a total volume of 10μl, and 10,000 cells were treated for the reaction. Through analysis, it was found that the overall conversion rate of the 50μl basic reaction system was significantly higher than that of the 10μl system group. This shows that the reduced system can also react, but the basic reaction system reaction is more stable. ( Fig.17 )

[0246] Optimization of reaction buffer

[0247] First, the effect of additional components in the test reaction buffer on SsdA activity was tested (see "In vitro deaminase activity verification" above). The grouped experimental conditions are as follows:

[0248]

[0249] The results are as follows Fig.18 As shown in A. Group 4 has the best deamination effect.

[0250] In addition, the effects of NaCl and MgCl2 on SsdA activity were also tested. The grouping experimental conditions are as follows:

[0251]

[0252] The results are as follows Fig.18 As shown in B.

[0253] Add UGI optimization

[0254] The inventors surprisingly found that the prepared deaminase has the activity of cleaving off U produced by deamination, thereby affecting the reliability of subsequent reactions and detection. Therefore, the inventors tested the effect of adding uracil glycosylase inhibitor (UGI) to the reaction system.

[0255] The effects of 30U enzyme reaction for 30 minutes, 15U enzyme reaction for 30 minutes, and 30U enzyme reaction for 10 minutes were compared with or without UGI. Fig.19 and 20 .in Fig.19 It shows that after adding UGI, the depth of the ATAC peak center is significantly increased. Fig. 20 The results show that the addition of UGI significantly increased the conversion rate, which indicates that the addition of UGI allows the use of higher enzyme amounts, longer reaction times, and better results.

[0256] Deaminase truncated variants

[0257] The inventors also tested the activities of a truncated variant of SsdA deaminase, SsdA-tox, and the full-length SsdA deaminase in a variety of different buffer systems and reaction temperatures.

[0258] The results showed that SsdA deaminase and its truncated variant SsdA-tox had good deamination activity in a variety of commonly used buffer systems and at temperatures as low as 4°C, greatly expanding its range of applications.

[0259] In summary, the present invention provides a method and system for determining the chromatin openness state and the position information and binding dynamics of DNA binding proteins such as transcription factors at the whole genome level. This technology creates a new qDEAC-seq (Quantitative DNA Deaminase-Accessible Chromatin assay using sequencing) method, which treats permeabilized cells or cell nuclei with single-stranded DNA deaminase A (SsdA) or double-stranded DNA deaminase A (DddA). After the deaminase comes into contact, the DNA deaminase causes the exposed cytosine base on the DNA sequence to be deaminated into uracil; and in the subsequent PCR amplification, uracil complements adenine and replaces the original cytosine with thymine. Then, sanger sequencing, next-generation sequencing (NGS) or single-molecule long-read sequencing is used to detect the location of base mutations in genomic DNA. Given that DNA in open chromatin regions is more susceptible to the reaction of deaminases, while DNA bound to nucleosomes or DNA-binding proteins is protected, by analyzing the distribution of deamination sites, it is possible to determine the openness of chromatin, the arrangement of nucleosomes, and the positional information and binding dynamics of DNA-binding proteins at the genome-wide level.

[0260] By combining with ATAC-seq, ChIP-seq or CUT&TAG and other technologies, it is possible to further enrich chromatin open areas or specific protein binding areas, and then analyze the protein binding imprints at these sites. By comprehensively analyzing protein imprint information with transcription factor-related databases and transcriptome information, the transcriptional regulatory network of cells can be constructed more accurately. By combining with CRISPR technology, it is expected to analyze the impact of any protein, including transcription factors, on the transcriptional regulatory network at the whole genome level.

[0261] Example 2: Detection of DNA-binding protein footprints in open regions of the cell genome

[0262] As shown in Example 1, the inventors have realized the detection of transcription factor footprints at the whole genome level using single-stranded DNA cytosine deaminase (SsdA), which is called qDeAC-seq. However, since qDeAC-seq is a whole genome level detection, it has a high requirement for sequencing quantity. Transcription factors appear more frequently in open regions of chromatin, so by enriching open regions, more transcription factor footprint information can be obtained under lower sequencing quantity conditions. The present invention combines a method for detecting transcription factor footprints at the whole genome level based on deaminase (qDeAC-seq) and a sequencing analysis method for transposase-accessible chromatin (Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) to create a new method for specifically analyzing transcription factor footprints in open regions of chromatin, called qDeAC-ATAC-seq.

[0263] This method uses Tn5 and SsdA to treat permeabilized cells or cell nuclei in sequence, and uses the transposase activity of Tn5 to interrupt the chromatin DNA in the more open area, and at the same time, adds a special Tn5 adapter to the 5' end of the chromatin DNA (considering the deamination effect of SsdA on cytosine bases, a Tn5 adapter sequence without cytosine is used here, so that the DNA fragment located in the open area of ​​chromatin can be specifically amplified by using the Tn5 adapter sequence primer in the subsequent process to achieve the enrichment of chromatin in the open area). At the same time, the activity of single-stranded DNA deaminase A (SsdA) to deaminize cytosine in double-stranded DNA is used: after SsdA contacts genomic DNA, the exposed cytosine base on the DNA sequence is deaminated and converted into uracil base. In the subsequent PCR amplification process, uracil is recognized as thymine, complementary to adenine, and the cytosine base deaminated by the deaminase is eventually replaced by thymine. This mutation can be detected by first-generation Sanger sequencing, second-generation sequencing (Next Generation Sequencing, NGS) or single-molecule long-read sequencing. Given that DNA in the open region of chromatin is regulated by more transcription factors, the DNA fragments in the open region are enriched by Tn5, and combined with the transcription factor footprints produced by the deamination reaction, the binding site analysis of transcription factors can be achieved under low sequencing cost conditions.

[0264] General method description

[0265] The qDeAC-ATAC-seq technology of the present invention can include two experimental strategies ( Fig.21 ), the basic steps are as follows:

[0266] Strategy 1:

[0267] (A) Permeabilization or nuclear extraction

[0268] Treat cells with a permeabilization buffer containing a certain concentration of detergent to make holes in the cell membrane to increase membrane permeability, or remove the cytoplasm to extract and permeabilize the cell nucleus.

[0269] (B) Tn5 attack open zone reaction

[0270] The cells or nuclear samples collected in step (A) are incubated with Tn5 in which the adapter sequence is embedded in advance, so that the transposase can fully react with the open chromatin DNA.

[0271] (C) Deaminase reaction

[0272] The Tn5 reaction system is removed by centrifugation, and the precipitated cells / nuclei are collected. The deaminase reaction system is then added to allow the deaminase to fully react with the chromatin DNA in the cells, and then the genomic DNA is extracted using phenol chloroform.

[0273] (D) DNA extraction and single-stranded DNA library construction

[0274] After the reaction is terminated, genomic DNA is extracted. No ultrasonic disruption is required. Use Novozyme DNAMethylation Library Kit for Illumina V3 NE103 is used to construct single-stranded DNA libraries. No 5' ligation is required. Samples can be directly amplified after extension. First, primers containing Tn5 linker homologous sequences and i7 primers are used for the first step of amplification. After a fragment with homologous sequences to the i5 primer is added to the 5' end of the library by overlap PCR, PCR amplification is performed using i5 and i7 primers for the second step of amplification. The schematic diagram of the library construction process is shown in Fig. 22 .

[0275] Strategy 2:

[0276] (A) Permeabilization or nuclear extraction

[0277] Treat cells with a permeabilization buffer containing a certain concentration of detergent to make holes in the cell membrane to increase membrane permeability, or remove the cytoplasm to extract and permeabilize the cell nucleus.

[0278] (B) SsdAtox deamination reaction

[0279] The cells or nuclear samples collected in step (A) are incubated with SsdAtox to allow the deaminase to fully react with the open chromatin DNA.

[0280] (C) Tn5 attack open zone reaction

[0281] The SsdAtox reaction system was removed by centrifugation, and the precipitated cells / nuclei were collected. Then, the Tn5 reaction system was added to allow the Tn5 transposase to fully react with the DNA in the open chromatin region of the cells, and then the genomic DNA was extracted using phenol chloroform.

[0282] (D) DNA extraction and single-stranded DNA library construction

[0283] After the reaction is terminated, genomic DNA is extracted. No ultrasonic disruption is required. Use Novozyme DNAMethylation Library Kit for Illumina V3 NE103 is used to construct single-stranded DNA libraries. No 5' ligation is required. Samples can be directly amplified after extension. First, primers containing Tn5 linker homologous sequences and i7 primers are used for the first step of amplification. After a fragment with homologous sequences to the i5 primer is added to the 5' end of the library by overlap PCR, PCR amplification is performed using i5 and i7 primers for the second step of amplification. The schematic diagram of the library construction process is shown in Fig. 22 .

[0284] Specific experimental methods

[0285] 1). Design Tn5 embedding linker sequence

[0286] In order to avoid the adapter being deaminated by the subsequent SsdAtox, the adapter sequence that does not contain cytosine used by Tn5 in sci-MET was used as a reference ( Fig.23 ), which was annealed with the Tn5 ME sequence and then incubated with Tn5 to complete the embedding of the Tn5 linker.

[0287] 2).qDeAC-ATAC-seq process

[0288] Strategy 1: Tn5 reaction first and then SsdAtox deamination reaction

[0289] (1) Cell collection and nuclear extraction:

[0290] Add appropriate amount of trypsin to digest cells, count after neutralization with culture medium, transfer from cell room to sequencing room, transfer to 1.5mL low adsorption EP tube, centrifuge at room temperature, 300×g for 4min, discard supernatant. Add 1mL cold PBS + 0.04% BSA to wash once, centrifuge at 4°C, 300×g for 4min, discard supernatant, and try to discard completely. Add corresponding volume of NucleiExtraction Buffer (10mM Tris-HCl 7.4, 10mM NaCl, 1% BSA, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin, 0.1mM EDTA, 3mM MgCl2, 1xPIC) according to 50μl / 50000 cells, resuspend, gently pipette 3 times, and place on ice for 3min. Stop the reaction: add 1 ml Final buffer (10 mM Tris-HCl 7.4, 10 mM NaCl, 1% BSA, 0.1 mM EDTA, 3 mM MgCl2, 1xPIC), gently invert and mix 3 times, 4°C, 700 × g, 5 min, centrifuge for 1 min, remove the supernatant to about 100 ul, invert and centrifuge again for 1 min, and completely discard the supernatant.

[0291] (2) Tn5 interruption reaction:

[0292] Prepare the required mix for the reaction according to 50 μl system (10 mM Tris-HCl 7.6, 5 mM MgCl2, 10% Dimethyl Formamide, 33% DPBS, 100 mM Tn5), resuspend the cell nuclei using the reaction system, react at 37°C for 15 min, and set the hot plate to shake at 1000 rpm.

[0293] (3) SsdAtox deamination reaction:

[0294] Add 500ul 1x SsdA Reaction Buffer (10mM Tris-HCl 7.4, 1mM DTT, 1x PCI) to dilute the Tn5 reaction system, then centrifuge at 4°C, 700×g, 5min, invert for 1min, remove the supernatant to about 100μl, invert again for 1min, and completely discard the supernatant. Add 50μl deaminase reaction system (10mM Tris-HCl 7.4, 1mM DTT, 1x PCI, 0.1U / μl UGI, 5U / μl SsdAtox), react at 37°C for 10min, and set the hot plate to shake at 1000rpm.

[0295] (4) Genomic DNA extraction

[0296] Then, heat incubate the reaction system. Add 200ul gDNA Hot Elution Buffer to the 50ul reaction system and heat-shock for 1.5h at 65℃ and 1350rpm. Add gDNA and an equal volume (250μl) of phenol:chloroform:isoamyl alcohol DNA extraction solution and shake thoroughly to mix. Centrifuge at room temperature at 13000g for 5min. Obtain gDNA by isopropanol precipitation and measure the concentration by QUBIT.

[0297] Strategy 2: SsdAtox deamination reaction first and then Tn5 reaction

[0298] (1) Cell collection and nuclear extraction:

[0299] Add appropriate amount of trypsin to digest cells, count after neutralization with culture medium, transfer from cell room to sequencing room, transfer to 1.5mL low adsorption EP tube, centrifuge at room temperature, 300×g for 4min, discard supernatant. Add 1mL cold PBS + 0.04% BSA to wash once, centrifuge at 4°C, 300×g for 4min, discard supernatant, and try to discard completely. Add corresponding volume of NucleiExtraction Buffer (10mM Tris-HCl 7.4, 10mM NaCl, 1% BSA, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin, 0.1mM EDTA, 3mM MgCl2, 1xPIC) according to 50μl / 50000 cells, resuspend, gently pipette 3 times, and place on ice for 3min. Stop the reaction: add 1 ml Final buffer (10 mM Tris-HCl 7.4, 10 mM NaCl, 1% BSA, 0.1 mM EDTA, 1xPIC), gently invert and mix 3 times, 4 ° C, 700 × g, 5 min, centrifuge for 1 min, remove the supernatant to about 100 ul, invert and centrifuge again for 1 min, and completely discard the supernatant.

[0300] (2) SsdAtox deamination reaction:

[0301] Prepare the required mix for the reaction according to the 50ul system (10mM Tris-HCl 7.4, 1mM DTT, 1xPIC, 0.1U / μl UGI, 2U / μl SsdAtox), resuspend the cell nuclei using the reaction system, react at 37°C for 10min, and set the hot stage to shake at 1000rpm.

[0302] (3) Tn5 interruption reaction:

[0303] Add 500ul 1x SsdA Reaction Stop Buffer (10mM Tris-HCl 7.4, 1mM DTT, 1xPCI, 5mM MgCl2) to dilute the deamination reaction system, then centrifuge at 4°C, 700×g, 5min, invert for 1min, remove the supernatant to about 100ul, invert again for 1min, and completely discard the supernatant. Add 50μl Tn5 reaction system (10mM Tris-HCl7.6, 5mMMgCl2, 10% Dimethyl Formamide, 33% DPBS, 100mM Tn5, 0.1U / μl UGI), react at 37°C for 15min, set the hot plate to shake at 1000rpm.

[0304] (4) Genomic DNA extraction

[0305] Then, heat incubate the reaction system. Add 200ul gDNA Hot Elution Buffer to the 50ul reaction system and heat-shock for 1.5h at 65℃ and 1350rpm. Add gDNA and an equal volume (250μl) of phenol:chloroform:isoamyl alcohol DNA extraction solution and shake thoroughly to mix. Centrifuge at room temperature at 13000g for 5min. Obtain gDNA by isopropanol precipitation and measure the concentration by QUBIT.

[0306] 3). Library establishment

[0307] The total amount of starting DNA was selected based on the measured DNA concentration and experimental requirements, and Novozymes DNA Methylation Library Kit for Illumina V3 NE103 was used for single-stranded DNA library construction.

[0308] According to the kit steps, connect the 3'Adapter and perform Extension. After purification, the first step of amplification can be performed. Set the program to amplify 4 rounds, then perform a 1x bead purification, and perform the second step of amplification. Taking 50ng as an example, the second step of amplification is 8 rounds, a total of 12 rounds. After obtaining the complete library, perform nucleic acid agarose gel electrophoresis, then select 300-500bp fragments for gel excision and recovery, and obtain the final library with appropriate concentration (see Fig. 22 ).

[0309] 4) Analyze qDeAC-ATAC-seq data to analyze transcription factor binding in open regions

[0310] Through bioinformatics analysis, the enrichment of open chromatin regions in cells is analyzed, and the base transition sites and numbers are calculated to determine the situation of protein binding at specific sites.

[0311] 2.1. Using strategy 1 for qDeAC-ATAC-seq can enrich open regions and detect transcription factor binding in open regions

[0312] In this example, strategy 1 was used to process the R1 cell line and construct a library for sequencing.

[0313] First, in order to study the effects of different components in the Tn5 reaction buffer on the binding of different transcription factors in the open region, the inventors tried to react Tn5 in different Tn5 reaction buffers and then add deaminase to treat the cells. The experimental groups are shown in Table 1 below.

[0314] Table 1. Tn5 reaction buffer grouping conditions

[0315]

[0316] The results are as follows Fig.24 and Fig.25 As shown in the figure, it was found that Tn5 had a higher efficiency of enriching the open area under the condition of DMF. Finally, it was concluded that the condition of 10mM Tris-HCl 7.6, 5mM MgCl2, 10% DMF, and 33% PBS had the least effect on TFdepth. Adding detergent to the reaction buffer would reduce the enrichment efficiency.

[0317] The library sequencing results obtained by qDeAC-ATAC-seq can clearly show the distribution pattern of nucleosomes ( Fig.26 ), and the depth of the library at the ATAC peak center can be seen to increase, indicating that there is an enrichment effect on the open area ( Fig.24 ).

[0318] In addition, by testing the addition of different amounts of deaminase (5U / μl, 7.5U / μl, 15U / μl, 30U / μl), comparing the overall deamination efficiency and the capture of transcription factor footprints between groups, it was found that for the mouse embryonic stem cell R1 cell line, a deaminase concentration of 5U / μl could achieve the best capture of transcription factor binding footprints ( Fig. 27 ). For different cell lines, the optimal enzyme amount needs further testing.

[0319] 2.2. qDeAC-ATAC-seq reaction sequence test

[0320] In this example, the Tn5 attack was first performed on K562 cells, followed by the addition of deaminase treatment (strategy 1), and the deamination reaction was first performed and then the Tn5 treatment of chromatin was added (strategy 2). Since deaminase, like Tn5, preferentially attacks the chromatin DNA in the open region, the deamination rate of the DNA in the open region is the highest, and the base mutation caused by deamination will destroy the complete double-stranded structure of DNA, so it is necessary to balance the two and select the experimental strategy according to the experimental purpose.

[0321] Strategy 1 and 2 have their own advantages and disadvantages. Strategy 1 has a higher enrichment degree for open areas and can better show the open areas in cells ( Fig.28 , Group 2-3), but due to the previous treatment with Tn5, the transcription factors with weaker DNA binding ability fell off the DNA, resulting in low TF depth values ​​of these transcription factors (e.g. Fig.29 Strategy 2 is more accurate and realistic in capturing transcription factor footprints, but due to the change in DNA bases caused by deamination, the subsequent ability of Tn5 to attack the open area is reduced, resulting in poor enrichment of the open area in the cell. It is obvious that the enrichment efficiency of Tn5 for the open area will decrease with the increase of the total amount of deaminase added ( Fig.28 , Groups 1 and 4-7).

[0322] In addition, the inventors also tried to test different amounts of deaminase under the experimental conditions of strategy 2 ( Figure 8 , Groups 1 and 4-7), by comparing strategy 1 ( Fig.28 The amount of deaminase used in strategy 2 should be lower than that in strategy 1 in order to enrich the open region as much as possible and improve the cleavage activity of Tn5. Fig.28 , Fig.29 ).

[0323] As can be seen from the above experimental results, the qDeAC-ATAC-seq experimental method of the present invention can simultaneously capture the open chromatin area of ​​the cell genome and detect the binding footprint of transcription factors in the open chromatin area, and the experimental data obtained have high repeatability. Compared with the method based on deaminase to detect chromatin openness and transcription factor footprints at the whole genome level, the sequencing cost is reduced, and the binding information of transcription factors can still be captured under the condition of lower sequencing depth, thereby reducing the sequencing cost. Moreover, by combining qDeAC-seq with ATAC-seq technology, it is expected that the unique advantage of qDeAC-seq in capturing transcription factor footprints can be brought into play at the single cell level, thereby achieving the capture of multi-omics information such as chromatin accessibility, transcription factor footprints, and transcriptomes at a higher throughput level, which is of great significance for constructing gene regulatory networks in dynamic changes such as cell fate transformation, development, and carcinogenesis.

[0324] Sequence information involved in this article:

[0325] >SEQ ID NO:1SsdA

[0326] MSAAARVNDPIEHTGSLTGLLAGLAIGAIGAALVVGTGGLAAVAIVGASAATGAGVGQLIGSLSCCNH

[0327] QTGQIVSGSSNVYINGEPAARAHADQAKCDEHSSRPQVIAQGSSNVYINGHPAARVGDRTACDAKIVV

[0328] GSSNVFIGGGTETTDPINPEVPELLERGILLVGLASAFVLASPVIVIAGLVGGIAGGTVGSMGGAQLF

[0329] GEGTDGQKLMAFGGALLGGGLGAKGGKWFDTRYDIKVQGVGSNLGNLKITPKGAAKVSNIAESEAALG

[0330] RASQARADLPQSKELKVKTVSSNDKKTLSGWGNKKPEGYERISAEQVKAKSEEIGHEVKSHPYDRDYK

[0331] GQYFSSHAEKQMSIASPNHPLGVSKPMCTDCQGYFSQLAKYSKVEQTVADPKAIRIFKTDGSVETIMRSE.

[0332] >SEQ ID NO:2DddAtox

[0333] EASIGLNGGANVYHYAPNPVGWVDPWGLAGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKV

[0334] FSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC*

[0335] >SEQ ID NO:3His-SsdA

[0336] MGSSHHHHHHSQDPLEVLFQGPEAYMSAAARVNDPIEHTGSLTGLLAGLAIGAIGAALVVGTGGLAAV

[0337] AIVGASAATGAGVGQLIGSLSCCNHQTGQIVSGSSNVYINGEPAARAHADQAKCDEHSSRPQVIAQGS

[0338] SNVYINGHPAARVGDRTACDAKIVVGSSNVFIGGGTETTDPINPEVPELLERGILLVGLASAFVLASP

[0339] VIVIAGLVGGIAGGTVGSMGGAQLFGEGTDGQKLMAFGGALLGGGLGAKGGKWFDTRYDIKVQGVGSN

[0340] LGNLKITPKGAAKVSNIAESEAALGRASQARADLPQSKELKVKTVSSNDKKTLSGWGNKKPEGYERIS

[0341] AEQVKAKSEEIGHEVKSHPYDRDYKGQYFSSHAEKQMSIASPNHPLGVSKPMCTDCQGYFSQLAKYSK

[0342] VEQTVADPKAIRIFKTDGSVETIMRSE

[0343] >SEQ ID NO:4His-smt3-SsdA

[0344] MGSSHHHHHHSQDPMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAK

[0345] RQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGALEVLFQGPEAYMSAAAARVNDPIE

[0346] HTGSLTGLLAGLAIGAIGAALVVGTGGLAAVAIVGASAATGAGVGQLIGSLSCCNHQTGQIVSGSSNV

[0347] YINGEPAARAHADQAKCDEHSSRPQVIAQGSSNVYINGHPAARVGDRTACDAKIVVGSSNVFIGGGTE

[0348] TTDPINPEVPELLERGILLVGLASAFVLASPVIVIAGLVGGIAGGTVGSMGGAQLFGEGTDGQKLMAF

[0349] GGALLGGGLGAKGGKWFDTRYDIKVQGVGSNLGNLKITPKGAAKVSNIAESEAALGRASQARADLPQS

[0350] KELKVKTVSSNDKKTLSGWGNKKPEGYERISAEQVKAKSEEIGHEVKSHPYDRDYKGQYFSSHAEKQM

[0351] SIASPNHPLGVSKPMCTDCQGYFSQLAKYSKVEQTVADPKAIRIFKTDGSVETIMRSE

[0352] >SEQ ID NO:5SsdA tox

[0353] KVSNIAESEAAALGRASQARADLPQSKELKVKTVSSNDKKTLSGWGNKKPEGYERISAEQVKAKSEEIG

[0354] HEVKSHPYDRDYKGQYFSSHAEKQMSIASPNHPLGVSKPMCTDCQGYFSQLAKYSKVEQTVADPKAIR

[0355] IFKTDGSVETIMRSE

[0356] >SEQ ID NO:6 Tn5 transposase

[0357] MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPN

[0358] VSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATT

[0359] FRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHN

[0360] ERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITL

[0361] KQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAER

[0362] QRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYL

[0363] DKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLDGFLAAKDLMAQGIKI>SEQ ID NO:7 Tn5 adapter-F

[0364] TGGTAGAGAGGGTGAGATGTGTATAAGAGACAG

[0365] >SEQ ID NO:8 Tn5 adapter-R

[0366] CTGTCTCTTATACACATCT

Claims

1. A method for detecting chromatin accessibility and / or DNA binding protein footprints of a cell genome, the method comprising: a) treating at least one of the cells with a permeabilization buffer comprising a detergent, or isolating a cell nucleus from at least one of the cells; b) treating the permeabilized cells or isolated cell nuclei obtained in step a) with a DNA deaminase, thereby allowing the DNA deaminase to fully react with the open regions of the genomic DNA in the cells, preferably, a uracil glycosylase inhibitor (UGI) is also added in the reaction; c) isolating genomic DNA from the product of step b); and d) sequencing the isolated genomic DNA or a portion thereof, and determining the chromatin accessibility and / or DNA binding protein footprint of the cell genome based on the sequencing results.

2. The method of claim 1, wherein the DNA deaminase is single-stranded DNA deaminase A (SsdA) or double-stranded DNA deaminase A (DddA) or a functional fragment thereof, preferably SsdA or a functional fragment thereof, such as SsdAtox.

3. The method of claim 2, wherein the SsdA comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:1; or wherein the SsdAtox comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:

5.

4. The method of any one of claims 1 to 3, wherein the permeabilization buffer comprises NP40.

5. The method of claim 4, wherein the permeabilization buffer comprises NP40, Tween-20 and Digitonin.

6. The method of claim 5, wherein the permeabilization buffer comprises about 0.1% NP40, about 0.1% Tween-20, and about 0.01% Digitonin.

7. The method according to any one of claims 1 to 6, wherein step b) is carried out in a reaction system of about 10 μl to about 100 μl, preferably 50 μl.

8. The method of any one of claims 1 to 7, wherein the cells or cell nuclei are treated with about 0.5 U / μl to about 50 U / μl, preferably about 7.5 U / μl of DNA deaminase; or, wherein about 25 U to about 2500 U, preferably about 375 U of DNA deaminase is treated per about 50,000 cells or cell nuclei.

9. The method of any one of claims 1 to 8, wherein the cells or cell nuclei are treated with a DNA deaminase in step b) for about 5 minutes to about 60 minutes, preferably for about 10 minutes to about 30 minutes, more preferably for about 10 minutes.

10. The method of any one of claims 1 to 9, wherein step d) comprises amplifying a specific portion of the genomic DNA and sequencing it to thereby determine the chromatin accessibility and / or DNA binding protein footprint of the specific portion of the genome of the cell.

11. The method of any one of claims 1 to 9, wherein step d) comprises establishing a whole-genome DNA library from the isolated genomic DNA, and performing sequencing based on the whole-genome library, thereby determining the chromatin accessibility and / or DNA-binding protein footprints of the cell genome at the whole-genome level.

12. The method of claim 11, wherein a whole genomic DNA library is established from the isolated genomic DNA by single-stranded DNA library construction.

13. The method of claim 11, wherein The single-stranded DNA library was constructed using the DNA Methylation Library Kit for Illumina V3 NE103 kit.

14. The method of claim 11 or 12, wherein a U-tolerant DNA polymerase is used for DNA amplification in the library establishment, for example, the U-tolerant DNA polymerase is selected from the group consisting of HiFi V3, Phusion U, Phanta Uc, Q5U and Q6U.

15. The method of any one of claims 1 to 14, wherein the chromatin accessibility and / or DNA binding protein footprint of the genomic DNA or a portion thereof of the cell is determined by analyzing the presence of CG to TA transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

16. The method of claim 15, wherein the chromatin accessibility and / or DNA binding protein footprint of the genomic DNA or a portion thereof of the cell is determined by analyzing the position and / or density of CG to TA transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

17. The method of any one of claims 1 to 16, wherein the cell is an animal cell, a plant cell or a microbial cell.

18. The method of claim 17, wherein the cell is a mammalian cell, including but not limited to cells of humans, mice, rats, cats, dogs, pigs, and cows; or, the cell is a monocotyledonous or dicotyledonous plant cell, such as cells of rice, corn, wheat, sorghum, soybean, potato, tomato, etc.; or, the cell is a fungus such as a yeast cell.

19. The method of claim 17, wherein the cell is a cell line cell; or, the cell is a primary cell from a different organ or tissue, for example, the cell is from blood, cerebrospinal fluid, biopsy tissue. The cell can be a hepatocyte, a cardiomyocyte, a neuron cell, a fibroblast, an epithelial cell; or, the cell is a tumor cell, a stem cell such as an embryonic stem cell or an induced pluripotent stem cell.

20. The method of claim 17, wherein the cell is a cell treated with a specific substance or condition or a cell at a specific developmental stage, for example, the specific substance is a drug.

21. A method for detecting the binding between a DNA molecule and a DNA binding protein, comprising: 1) forming a reaction mixture of DNA molecules and DNA binding proteins and allowing them to fully contact; 2) adding DNA deaminase and optionally uracil glycosylase inhibitor (UGI) to the reaction mixture and allowing them to fully react with the DNA molecules; 3) isolating DNA from the product of step b); and 4) sequencing the isolated DNA or a portion thereof, and determining the binding of the DNA binding protein to the DNA molecule based on the sequencing results.

22. The method of claim 21, wherein the DNA deaminase is single-stranded DNA deaminase A (SsdA) or double-stranded DNA deaminase A (DddA), preferably SsdA.

23. The method of claim 22, wherein the SsdA comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:

1.

24. A kit for detecting chromatin accessibility and / or DNA binding protein footprints of a cell genome or for detecting the binding between a DNA molecule and a DNA binding protein, comprising at least a DNA deaminase and optionally a uracil glycosylase inhibitor (UGI).

25. A method for detecting DNA binding protein footprints in an open region of a cell genome, the method comprising: a) treating at least one of the cells with a permeabilization buffer comprising a detergent, or isolating a cell nucleus from at least one of the cells; b) i) treating the permeabilized cells or isolated cell nuclei obtained in step a) with Tn5 transposase, thereby allowing the Tn5 transposase to fully react with the open regions of the genomic DNA in the cells; treating the permeabilized cells or isolated cell nuclei treated with Tn5 transposase with DNA deaminase, thereby allowing the DNA deaminase to fully react with the open regions of the genomic DNA in the cells; or ii) treating the permeabilized cells or isolated cell nuclei obtained in step a) with DNA deaminase, thereby allowing the DNA deaminase to fully react with the open regions of the genomic DNA in the cells; treating the permeabilized cells or isolated cell nuclei treated with DNA deaminase with Tn5 transposase, thereby allowing the Tn5 transposase to fully react with the open regions of the genomic DNA in the cells; c) isolating genomic DNA from the product of step b); and d) sequencing the isolated genomic DNA or a portion thereof, and determining the DNA binding protein footprints of the open regions of the cell genome based on the sequencing results.

26. The method of claim 25, wherein the Tn5 transposase reacts sufficiently with the open region of the genomic DNA in the cell to cause the genomic DNA in the open region to be fragmented and an adapter containing a Tn5 ME sequence is added to the 5' end of the obtained DNA fragments.

27. The method of claim 25 or 26, wherein the Tn5 transposase comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:

6.

28. The method of any one of claims 25-27, wherein the Tn5 transposase has been complexed with a linker containing a Tn5 ME sequence to form a transposome, preferably, the linker does not contain C, more preferably, the sequence of the linker other than the Tn5ME sequence does not contain C.

29. The method of any one of claims 25-28, wherein the linker added by the Tn5 transposase comprises the nucleotide sequence shown in SEQ ID NO:

7.

30. The method of any one of claims 25-29, wherein the Tn5 transposase treatment is performed in a Tagmentation buffer comprising DMF (dimethylformamide).

31. The method of claim 30, wherein the Tagmentation buffer comprises 10 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 10% DMF, 33% PBS.

32. The method of claim 30 or 31, wherein the Tagmentation buffer further comprises a uracil glycosylase inhibitor (UGI).

33. The method of any one of claims 25 to 32, wherein the concentration of the Tn5 transposase is about 50 mM to about 150 mM, such as 100 mM.

34. The method of any one of claims 25 to 33, wherein the cells or cell nuclei are treated with Tn5 transposase for about 5 minutes to about 60 minutes, preferably about 10 minutes to about 30 minutes, more preferably about 15 minutes.

35. The method of any one of claims 25-34, wherein the cells or cell nuclei are treated with Tn5 transposase at a temperature of about 4°C to about 40°C, such as about 30°C to about 40°C, preferably about 37°C.

36. The method of any one of claims 25 to 35, wherein the DNA deaminase is single-stranded DNA deaminase A (SsdA) or double-stranded DNA deaminase A (DddA) or a functional fragment thereof, preferably SsdA or a functional fragment thereof, such as SsdA tox.

37. The method of claim 36, wherein the SsdA comprises the amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:1; or wherein the SsdA tox comprises the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:

5.

38. The method of any one of claims 25 to 37, wherein the cells or cell nuclei are treated with about 0.5 U / μl to about 50 U / μl, preferably about 0.5 to 7.5 U / μl of DNA deaminase.

39. The method of any one of claims 25 to 38, wherein the DNA deaminase treatment is performed in a deamination reaction buffer that does not contain additional metal salts, such as NaCl or MgCl2.

40. The method of claim 39, wherein the deamination reaction buffer further comprises a uracil glycosylase inhibitor (UGI).

41. The method of any one of claims 25 to 40, wherein the cells or cell nuclei are treated with the DNA deaminase for about 5 minutes to about 60 minutes, preferably about 10 minutes to about 30 minutes, more preferably about 10 minutes.

42. The method of any one of claims 25-41, wherein the cells or cell nuclei are treated with a DNA deaminase at a temperature of about 4°C to about 40°C, such as about 30°C to about 40°C, preferably about 37°C.

43. The method of any one of claims 25-42, wherein the permeabilization buffer comprises NP40.

44. The method of claim 43, wherein the permeabilization buffer comprises NP40, Tween-20, and Digitonin.

45. The method of claim 44, wherein the permeabilization buffer comprises about 0.1% NP40, about 0.1% Tween-20, and about 0.01% Digitonin.

46. ​​The method of any one of claims 25-45, wherein Step b) i) after the Tn5 transposase treatment, separating the cells or cell nuclei by centrifugation, and then subjecting them to the DNA deaminase treatment; or Step b) ii) After the DNA deaminase treatment, the cells or cell nuclei are separated by centrifugation and then subjected to the Tn5 transposase treatment.

47. The method of any one of claims 25-46, wherein step d) comprises establishing a genomic DNA library from the isolated genomic DNA, and performing sequencing based on the genomic library, thereby determining the DNA binding protein footprints of the open regions of the cell genome at the genomic level.

48. The method of claim 47, wherein a genomic DNA library is established from the isolated genomic DNA by single-stranded DNA library construction.

49. The method of claim 48, wherein The single-stranded DNA library was constructed using the DNA Methylation Library Kit for Illumina V3 NE103 kit.

50. The method of any one of claims 47-49, wherein a U-tolerant DNA polymerase is used for DNA amplification in the establishment of the library, for example, the U-tolerant DNA polymerase is selected from the group consisting of: HiFi V3, Phusion U, PhantaUc, Q5U and Q6U.

51. The method of any one of claims 25-50, wherein the DNA binding protein footprint of the genomic DNA or a portion thereof of the cell is determined by analyzing the presence of a CG to TA transition in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

52. The method of claim 51, wherein the DNA binding protein footprint of the cellular genomic DNA or a portion thereof is determined by analyzing the position and / or density of CG to TA transitions in the cellular genomic DNA or a portion thereof relative to a control genomic DNA or a portion thereof.

53. The method of any one of claims 25-52, wherein the cell is an animal cell, a plant cell, or a microbial cell.

54. The method of claim 53, wherein the cell is a mammalian cell, including but not limited to cells of humans, mice, rats, cats, dogs, pigs, and cows; or, the cell is a monocotyledonous or dicotyledonous plant cell, such as cells of rice, corn, wheat, sorghum, soybean, potato, tomato, etc.; or, the cell is a fungus such as a yeast cell.

55. The method of claim 53, wherein the cell is a cell line cell; or, the cell is a primary cell from a different organ or tissue, for example, the cell is from blood, cerebrospinal fluid, biopsy tissue. The cell can be a hepatocyte, a cardiomyocyte, a neuronal cell, a fibroblast, an epithelial cell; or, the cell is a tumor cell, a stem cell such as an embryonic stem cell or an induced pluripotent stem cell.

56. The method of claim 53, wherein the cell is a cell treated with a specific substance or condition or a cell at a specific developmental stage, for example, the specific substance is a drug.

57. A kit for detecting DNA binding protein footprints in a cell genome by the method of any one of claims 25 to 56, comprising at least the DNA deaminase and the Tn5 transposase.

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