Method for detecting chromatin accessibility or DNA-binding protein footprints in cells

Abstract

The present invention belongs to the field of biotechnology. Provided is a method for detecting chromatin accessibility or DNA-binding protein footprints in cells. Specifically, provided is a method for converting chromatin accessibility information into mutation information of a DNA sequence on the basis of the deamination effect of a DNA deaminase on a DNA in an chromatin accessibility region, thereby quantitatively determining the chromatin accessibility and the binding footprints of a DNA-binding protein such as a transcription factor on the single DNA molecule level. In addition, further provided is a method for detecting DNA-binding protein footprints in an accessibility region of a cell genome on the basis of a deaminase and a transposase, which method can enrich the chromatin accessibility region in a cell genome and detect DNA-binding protein footprints in the chromatin accessibility region.

Description

Methods for detecting chromatin accessibility or DNA-binding protein footprints in cells 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 chromatin accessibility information into mutation information of DNA sequences based on the deamination of DNA deaminase on DNA in chromatin open regions, thereby achieving 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 open regions of a cell genome based on deaminase and transposase, which can enrich the chromatin open regions of the cell genome and detect DNA binding protein footprints in the chromatin open regions.

[0002] Background of the Invention

[0003] Eukaryotic DNA forms a complex with histones, with approximately 147 base pairs of DNA coiled 1.65 times around a histone core particle to form the nucleosome structure. The nucleosome and its linker region together form a beaded structure, which undergoes further folding and supercoiling to form a dense chromatin structure, enclosed by the nuclear membrane and separated from the cytoplasm. Chromosome accessibility reflects the degree to which various regulatory factors can physically access genomic DNA. This is influenced by the arrangement of nucleosomes, the spatial structure of the surrounding chromatin, and the promotion or repression of chromatin structure by different chromatin-binding factors. Transcription factors dynamically compete with histones and other chromatin-binding proteins to regulate physiological processes such as gene transcription, thereby manifesting the characteristics of different cells. Therefore, studying chromatin accessibility, clarifying the chromatin states of different cells, and establishing a dynamic map of the mutual transformations between different cells in terms of transcription factor binding and nucleosome rearrangement are crucial for further understanding the processes of gene regulation in cells.

[0004] Most current methods for studying chromatin accessibility rely on the fact that open chromatin is more sensitive to enzymes or physical disruption. Common methods for measuring chromatin accessibility include MNase-seq, DNase-seq, and ATAC-seq, which rely on DNA fragment cleavage. These methods assess chromatin accessibility based on the enrichment of DNA sequences, thus providing only a relative estimate of accessibility. Furthermore, these enzymes exhibit sequence bias in DNA cleavage, potentially leading to biased experimental results. NOMe-seq and Fiber-seq, which measure accessibility based on enzyme modification activity, rely on methylases and m6A adenine methyltransferases, respectively. The methylases used in NOMe-seq can only methylate GpC sequences, resulting in poor resolution. Fiber-seq, on the other hand, cannot be directly amplified, requiring an enrichment step or requiring extremely large cell inputs to compensate for this amplification limitation.

[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 quantitativeness.

[0006] Summary 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 in accessible chromatin regions by DNA deaminase, chromatin accessibility information can be converted into DNA sequence mutation information, thereby enabling quantitative measurement of chromatin accessibility and the binding footprints of DNA-binding proteins, such as transcription factors, at the level of single DNA molecules (Figure 1). In addition, the qDeAC-ATAC-seq method, formed by combining qDeAC-seq and ATAC-seq, can enrich for accessible chromatin regions in the cellular genome and detect the footprints of DNA-binding proteins within accessible chromatin regions (Figure 21).

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

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

[0010] Figure 2. Separation of DddA / SsdA and their corresponding inhibitors by denaturation treatment with 8M urea.

[0011] Figure 3. Deaminase activity assay.

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

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

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

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

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

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

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

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

[0020] Figure 12. Effects of different library construction methods on the coverage at the center of the ATAC-seq library.

[0021] Figure 13. Effects of different DNA polymerases on PCR amplification of libraries.

[0022] Figure 14. Effect of cell permeabilization conditions on sample conversion efficiency.

[0023] Figure 15. Effect of deaminase reaction incubation time on deamination efficiency.

[0024] Figure 16. Effect of different deaminase concentrations on deamination efficiency.

[0025] Figure 17. Effects of different deaminase reaction systems on deamination efficiency.

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

[0027] Figure 19. Effect of adding UGI on sequencing depth at ATAC hubs.

[0028] Figure 20. Effect of adding UGI on conversion rate.

[0029] Figure 21. Schematic diagram of the qDeAC-ATAC-seq process. Strategy 1 is to first add Tn5 for treatment and then add SsdAtox for deamination. Strategy 2 is to first add SsdAtox for deamination and then add Tn5 to attack the open area.

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

[0031] Figure 23. Tn5 linker sequence diagram.

[0032] Figure 24. Effects of different Tn5 reaction buffers on the efficiency of open zone enrichment.

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

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

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

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

[0037] Figure 29. In K562 cells, different experimental strategies were tested to determine the effects of different transcription factors on TF depth (including separate qDeAC-seq data).

[0038] Detailed Description of the Invention

[0039] 1. 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. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0041] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed 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 word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed 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, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0043] Sequence "identity" has an art-recognized meaning, 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 nucleus of a cell, but also the organelle DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0045] 2. Methods for detecting chromatin accessibility and / or DNA-binding protein footprints in 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 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 maximize the preservation of chromatin accessibility and / or DNA-binding protein footprint information of the cell genome, the permeabilization buffer should be mild, which can destroy or partially destroy the cell wall / cell membrane, allowing DNA deaminases to enter the cell, but does not significantly disrupt the chromatin structure of the cell. The cell nucleus isolation should also be gentle and not significantly disrupt 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, particularly double-stranded) as a substrate and can catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. The DNA deaminase can deaminate C in open regions of genomic DNA to U, so that 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 described 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 described in the present invention can be derived 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 of 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. In some embodiments, the functional fragment of SsdA, such as SsdA tox, comprises the amino acid sequence of 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.

[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 further comprises a fusion tag, such as a tag for isolating and / or purifying the DNA deaminase. Methods for recombinant protein production are known in the art. Various tags are known in the art that can be used for isolating and / or purifying proteins, including but not limited to His tags, SMT3 tags, GST tags, and the like. Generally speaking, these tags do not alter the activity of the target protein. In some preferred embodiments, the DNA deaminase comprises a His tag and an SMT3 tag.

[0062] In some specific embodiments, the SsdA or its functional fragment comprises the 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 besides 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 potential U-removal activity of enzymes present in the reaction system, thereby preventing the effects of U removal from the deamination reaction. Adding UGI can increase the enzyme dosage in the reaction while maintaining the signal-to-noise ratio, thereby shortening the reaction time. UGI useful in the methods of the present invention is commercially available.

[0069] In some embodiments, the at least one cell is 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 DNA deaminase is used for every about 50,000 cells or nuclei.

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

[0073] The enzyme activity unit (U) of the DNA deaminase used herein can be determined as described 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 oligomeric DNA substrate in a 10 μl total reaction system at 37°C within 1 hour.

[0074] In some embodiments, the cells or cell nuclei are treated with 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 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 chromatin accessibility and / or DNA binding protein footprints in 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 the group consisting of: 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 genome-wide 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 can be used to establish whole-genome sequencing libraries from isolated genomic DNA, including single-stranded DNA and double-stranded DNA libraries. Whole-genome DNA libraries can be established using commercially available kits, such as Noveocytogenes NE103 (Swift Biosciences Cat. No. 33096) for single-stranded libraries, Tn5 libraries (such as Noveocytogenes TD202) for double-stranded libraries, and Zymo Research D5455 & D5456 kits for WGBS libraries.

[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 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] Because DNA in open chromatin regions is more susceptible to DNA deaminase reactions, converting C to U, DNA bound by nucleosomes or DNA-binding proteins is protected from deamination. Therefore, by analyzing the distribution of deamination sites, we can determine the openness of chromatin, the arrangement of nucleosomes, and the positional information and binding dynamics of DNA-binding proteins.

[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 specific regions 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 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 a portion thereof can be a sequence of a genomic DNA or a portion thereof from a public database. Alternatively, the sequence of the control genomic DNA or a portion thereof can also be a sequence of a genomic DNA or a portion thereof from 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 from humans, mice, rats, cats, dogs, pigs, and cows; or, the cells are monocotyledonous or dicotyledonous plant cells, such as cells from rice, corn, wheat, sorghum, soybean, potato, and tomato; or, the cells are fungi, such as yeast cells.

[0097] The cells may be cell lines. Alternatively, the cells may be primary cells from different organs or tissues. For example, the cells may be from blood, cerebrospinal fluid, or biopsy tissue. The cells may be hepatocytes, cardiomyocytes, neurons, fibroblasts, epithelial cells, and the like. The cells may also be tumor cells, stem cells such as embryonic stem cells, or induced pluripotent stem cells.

[0098] In some embodiments, the cells are cells treated with a specific substance (compound) or condition or are at a specific developmental stage. Thus, the methods of the present invention can measure the effect of the specific substance (compound) or condition or specific developmental stage on genomic chromatin accessibility and / or DNA binding protein footprints in the cell. 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, 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 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 of 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 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 genomic DNA in the cells; or

[0111] ii) 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; treating the permeabilized cells or isolated cell nuclei treated with the DNA deaminase with a 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 regions are chromatin open regions. The term "chromatin open regions" as used herein has a well-known meaning in the art, and refers to regions where the compact chromatin structure 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 chromatin openness / accessibility.

[0115] In order to maximize the preservation of DNA-binding protein footprint information in the cell genome, the permeabilization buffer should be gentle, capable of disrupting or partially disrupting the cell wall / cell membrane, allowing DNA deaminases and / or Tn5 transposase to enter the cell, but not significantly disrupting the chromatin structure of the cell. Cell nuclear isolation should also be gentle, not significantly disrupting the chromatin structure.

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

[0117] In some embodiments, the cell nucleus isolation is performed using methods known in the art. In some embodiments, the cell nucleus is isolated 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, particularly double-stranded) as a substrate and can catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. The DNA deaminase can deaminate C in open regions of genomic DNA to U, so that 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 described 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 described in the present invention can be derived 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 of 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. In some embodiments, the functional fragment of SsdA, such as SsdA tox, comprises the amino acid sequence of 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.

[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 further comprises a fusion tag, such as a tag for isolating and / or purifying the DNA deaminase. Methods for recombinant protein production are known in the art. Various tags are known in the art that can be used for isolating and / or purifying proteins, including but not limited to His tags, SMT3 tags, GST tags, and the like. Generally speaking, these tags do not alter the activity of the target protein. In some preferred embodiments, the DNA deaminase comprises a His tag and an SMT3 tag.

[0125] In some specific embodiments, the SsdA or a functional fragment thereof comprises the 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 consists 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, thereby avoiding 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 is 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 per about 50,000 cells or cell nuclei. In some embodiments, about 1,000 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 Tn5ME 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%, or 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 Tn5ME sequence to form a transposome.

[0137] Suitable Tn5 transposases for nucleic acid sequencing library construction, methods for constructing transposomes with adapters, and corresponding commercial kits are already available in the art, and all of these can 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'-TGGTAGAGAGGGTGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 7) (the underlined portion is the double-stranded Tn5ME sequence, and the non-underlined portion is the single-stranded portion), which is formed by annealing a single-stranded Tn5adapter-F of the sequence shown in SEQ ID NO: 7 and a single-stranded Tn5adapter-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 commercially available Tn5 transposase tagmentation buffer. In some embodiments, the tagmentation buffer comprises DMF (dimethylformamide). In some embodiments, the tagmentation buffer does not contain a detergent. In some preferred embodiments, the tagmentation buffer comprises 10 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 10% DMF, and 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 cell population is small, step b)i) can be employed, which can result in a 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 employed 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 performed 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 genomic level.

[0149] Various methods known in the art can be used to establish genomic sequencing libraries from isolated genomic DNA, including single-stranded DNA and double-stranded DNA library construction. Genomic DNA libraries can be established using commercially available kits, such as Noveocytogenes NE103 (Swift Biosciences Cat. No. 33096) for single-stranded libraries; Tn5 libraries can be established using Noveocytogenes TD202 kits for double-stranded libraries; and WGBS libraries can be established using Zymo Research D5455 & D5456 kits.

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

[0151] In some 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] Because DNA in open chromatin regions is more susceptible to DNA deaminase reactions, converting C to U, DNA bound by DNA-binding proteins is protected from deamination. Therefore, analyzing the distribution of deamination sites can reveal the location and binding dynamics of DNA-binding proteins.

[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 specific regions 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 a portion thereof can be a sequence of a genomic DNA or a portion thereof from a public database. Alternatively, the sequence of the control genomic DNA or a portion thereof can also be a sequence of a genomic DNA or a portion thereof from 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 from humans, mice, rats, cats, dogs, pigs, and cows; or, the cells are monocotyledonous or dicotyledonous plant cells, such as cells from rice, corn, wheat, sorghum, soybean, potato, and tomato; or, the cells are fungi, such as yeast cells.

[0161] The cells may be cell lines. Alternatively, the cells may be primary cells from different organs or tissues. For example, the cells may be from blood, cerebrospinal fluid, or biopsy tissue. The cells may be hepatocytes, cardiomyocytes, neurons, fibroblasts, epithelial cells, and the like. The cells may also be tumor cells, stem cells such as embryonic stem cells, or induced pluripotent stem cells.

[0162] In some embodiments, the cells are cells treated with a specific substance (compound) or condition or are at a specific developmental stage. Thus, the methods of the present invention can measure the effect of the specific substance (compound) or condition or specific developmental stage on genomic chromatin accessibility and / or DNA binding protein footprints in the cell. For example, the specific substance can be a drug.

[0163] 4. 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 to detect 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 comprises 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, comprising 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 using 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 a linker comprising a Tn5ME sequence and a reagent for complexing the linker with Tn5 transposase to form a transposome. The linker comprising a Tn5ME 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 technologies, as well as molecular biology techniques such as cell fixation and DNA purification, and cell culture and detection techniques, are all 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 all 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 isolation schemes and some buffer systems represent only a few of the many possible processing 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 a plasmid expressing deaminase protein

[0184] Protein expression and purification were performed using the pETDuet vector, which contains an ampicillin resistance gene and two multiple cloning sites. Each multiple cloning site includes a T7 promoter, a Lac operator, and a ribosome binding site. The DddA / SsdA sequence, its tag sequence, or its truncated sequence, along with its corresponding inhibitor sequence, was constructed into two sites of the pETDuet vector.

[0185] 2). Purification of deaminase protein

[0186] Use the heat shock method to transform the vector into protein-expressing strains such as Rosetta (DE3), Rosetta Gami, and BL21 (DE3). Cultivate under the dual-antibody conditions of ampicillin and chloramphenicol. Collect some bacteria, resuspend them in freezing solution (50% glycerol: dual-antibody 2YT = 1:1), snap-freeze in liquid nitrogen, and store at -80°C. Test the expression effects of different strains and select suitable strains for subsequent experiments. Cultivate the strains using dual-antibody medium at an appropriate rotation speed. Measure the OD value of the culture solution at different times. Once the culture solution reaches a certain OD value, use IPTG to induce protein expression. After a certain period of induction, collect the bacterial pellet by centrifugation and crush it by superpressure.

[0187] For affinity purification and denaturation, ①SsdA: The disrupted bacterial suspension was centrifuged and the inclusion bodies were collected. The inclusion bodies were solubilized with a buffer containing 8 M urea, and impurities were removed by centrifugation. Ni-NTA beads were then added for affinity purification. ②DddA: The disrupted bacterial suspension was centrifuged, the supernatant was collected, and incubated with Ni-NTA beads. The protein was then denatured by adding a buffer containing 8 M urea. Treatment with 8 M urea allowed the separation of DddA / SsdA from its corresponding inhibitor (see Figure 2). Subsequently, the DddA / SsdA was renatured by decreasing the urea concentration. Imidazole was eluted and dialyzed to remove the imidazole. Finally, protein quantification was performed by Coomassie Brilliant Blue staining. Finally, the DddA / SsdA protein was isolated and purified using an AKTA protein purification system through ion exchange chromatography and molecular sieve separation. The corresponding fractions were concentrated to obtain the target protein sample.

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

[0189] Design oligonucleotide primers (5'-AATATAATATAATAACTCGCCATAATTTTAATTAAT-3', 5' with a 6-FAM fluorescent group) and their complementary sequences, and use annealing to generate double-stranded oligonucleotides. 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 and incubate at 37°C for 30 minutes to 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 fragment size. See Figure 3 for details.

[0190] The enzyme activity unit (U) was defined as the amount of enzyme required to digest 0.1 μM substrate in a 10 μl total reaction 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] 4) Cell processing for qDeAC-seq (Figure 4)

[0194] (1) Cell collection and nuclear extraction:

[0195] Add an appropriate amount of trypsin to digest the cells, neutralize with culture medium, count the cells, transfer to a 1.5 mL low-binding EP tube, centrifuge at 300 × g for 4 min at room temperature, and discard the supernatant. Wash twice with 1 mL of cold PBS, centrifuge at 300 × g for 4 min at 4°C, and discard the supernatant. Resuspend in 1 mL of cold wash buffer (10 mM Tris-HCl 7.4, 10 mM NaCl, 1% BSA, 0.1 mM EDTA, 3 mM MgCl2, 1x PIC), and divide into 50,000 cells × N groups. Centrifuge at 300 × g for 4 min at 4°C, and discard the supernatant as much as possible. Wash once in total.

[0196] Lysis: Resuspend cells by gently flicking the tube bottom. Then, add 50,000 cells to 50 μL of Lysis Buffer (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, 1x PIC). Gently pipette up and down three times. Incubate on ice for 3 minutes. Terminate the reaction by adding 1 ml of Final Buffer (10 mM Tris-HCl 7.4%, 10 mM NaCl, 1% BSA, 0.1 mM EDTA, 1x PIC). Gently invert the tube three times to mix thoroughly. Centrifuge at 700 × g for 5 minutes at 4°C. End-up centrifuge for 1 minute. Remove the supernatant to approximately 100 μL. End-up centrifuge again for 1 minute. Discard the supernatant completely.

[0197] (2) Deamination reaction:

[0198] Add enzyme, buffer, and an appropriate amount of lambda DNA to the punched cells in a 50 µL volume (10 mM Tris-HCl 7.4, 1 mM DTT, 1x PIC). Gently invert to mix and resuspend the cells. Incubate at 37°C for 0.5 h. Set the hot plate to 600 rpm, with a 3-min pause and 30-s shake.

[0199] (3): DNA extraction

[0200] After heat incubation, add 200 μl of gDNA Hot Elution Buffer to a 50 μl reaction volume and incubate at 65°C, 1350 rpm, and shake for 1.5 hours. Add gDNA and an equal volume (250 μl) of phenol:chloroform:isoamyl alcohol DNA extraction solution and shake thoroughly to mix. Centrifuge at 13,000 g for 5 minutes at room temperature. Obtain gDNA by isopropanol precipitation, and measure its concentration using QUBIT.

[0201] 5). Library establishment

[0202] The total amount of starting DNA was selected based on the measured DNA concentration and experimental requirements. Taking 5 ng starting amount as an example, the genome was fragmented by ultrasound. The DNA Methylation Library Kit for Illumina V3NE103 was used for single-stranded DNA library construction (Figure 5).

[0203] The 3' and 5' adapters were ligated according to the kit instructions, and primer amplification was performed to obtain a complete library. DNA fragments were analyzed using the Agilent 2100 Biochip Analysis System. The constructed library was sent to the company for high-throughput sequencing using an Illumina X TEN machine, using a paired-end 150bp sequencing mode.

[0204] 6).qDeAC-seq base mutation identification.

[0205] By analyzing the sequencing data and calculating the base transition sites and numbers, the accessibility of the genome and the situation of protein binding at specific sites can be determined.

[0206] 1.1. qDeAC-seq determination of MEF genomic DNA accessibility

[0207] To clarify the deaminase's selectivity for genomic DNA, deaminases with different purification tags and auxiliary tags were purified from multiple strains. Mouse embryonic fibroblasts were then treated with each deaminase. Data analysis was performed: selected sites were amplified by PCR, and all C sites were extracted. The conversion rates of C in AC, TC, GC, and CC were calculated to determine SsdA's substrate conversion preference. The results showed that DddA exhibited a moderate TC preference, while SsdA exhibited virtually no sequence preference (Figure 6).

[0208] By analyzing ATAC-seq data from public databases, we selected a region with high accessibility. We designed primers for PCR in this region, ligated the product to a T-vector, and subsequently performed Sanger sequencing. The sequencing results showed a consistent trend with the ATAC-seq results, and a signature similar to that of transcription factor SP1 binding was observed in the sequence (Figure 7).

[0209] After processing the mouse embryonic fibroblast genome using qDeAC-seq, a next-generation sequencing library was constructed. The sequencing results clearly demonstrated high accessibility of the TSS (transcription start site) region, with up to the third nucleosome position after the TSS visible. Correlation analysis with ATAC-seq revealed a correlation between qDeAC-seq and ATAC (Figure 8).

[0210] 1.2. qDeAC-seq determination of genomic DNA accessibility and specific TF site binding in R1 cells

[0211] The genome was processed using qDeAC-seq and then subjected to next-generation sequencing library construction. The results clearly demonstrated high accessibility in the TSS region, with coverage extending up to the third nucleosome position after the TSS. Clear fluctuations in accessibility were also observed on both sides of the CTCF (CCCTC binding factor) region (Figure 9).

[0212] Furthermore, by aligning the binding regions of known specific TFs (Klf4 and Thap11) based on their binding motifs and sorting them by degree of openness, we can clearly see how protein binding affects the deaminase reaction, reducing the conversion rate at the central site. This demonstrates that qDeAC-seq can visually and high-resolutionly visualize the imprint of transcription factor binding. Furthermore, for specific transcription factors, qDeAC-seq can reveal differences between the positive and negative strands (Figure 10).

[0213] 1.3. Differences in TF binding between R1 and MEF genomes by qDeAC-seq

[0214] After processing the genomes of both cell lines using qDeAC-seq, libraries were constructed using next-generation sequencing. Analysis of the sequencing results revealed significant differences between the two cell lines by aligning known TF binding regions around their binding motifs and ranking them by the degree of openness of their centers in the R1 cell line. The corresponding MEF data were also sorted in this order. Figure 11 shows a comparison of the two transcription factors, SP1 and Nrf1, demonstrating significant differences.

[0215] Therefore, from the experimental results of Examples 1-3 above, it can be seen 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 are highly reproducible, and the influence of nucleosome occupancy on conversion rate can be intuitively seen, as well as the occupancy of specific transcription factor binding sites can be observed. This shows that the qDeAC-seq method of the present invention can intuitively visualize changes in chromatin openness and the occupancy of specific transcription factor binding sites, greatly improving the resolution compared to other methods, and making the data intuitively observable, more realistic and reliable. It can also show the difference between the positive and negative strands of specific transcription factor binding sites, and infer the directionality of their binding. This is something that other methods currently cannot achieve.

[0216] 1.4. Comparison of the effects of different library construction methods on qDeAC-seq results

[0217] This example compares the effects of single-stranded and double-stranded library construction on qDeAC-seq results. The library construction process is as follows: Single-stranded library construction process:

[0218] The qDeAC-seq single-strand library construction method complies with the instructions of the Novozymes kit NE103 (EpiArt DNA Methylation Library Kit for Illumina V3).

[0219] Whole-cell genomic DNA, deaminized by the deaminase SsdA, was collected and sonicated to obtain fragmented double-stranded DNA. A certain amount of DNA was used as the starting library. The double-stranded DNA was denatured at 95°C and immediately placed on ice to obtain fragmented single-stranded DNA. 3' adapters were ligated to the 3' end of the single-stranded DNA using 3' adapter ligase. An extension system was added for reaction. 1.2x DNA purification magnetic beads (N411, VAHTS DNA Clean Beads) were then used to obtain the double-stranded DNA product with 3' adapters attached. 5' adapter ligase was used to ligate adapters to the 5' end of the double-stranded DNA. The double-stranded DNA product was then purified using 1x DNA magnetic beads to obtain the double-stranded DNA product with double-end adapters attached. Finally, the library was amplified for an appropriate number of rounds of PCR using N321 / N322 primers from VAHTS Multiplex Oligos Set 4 / 5 for Illumina. The final library was purified using 0.85x DNA purification magnetic beads.

[0220] Double-chain library construction process

[0221] The qDeAC-seq double-strand library construction method complies with the instructions of the TruePrep DNA Library Prep Kit V2 for Illumina TD502.

[0222] The whole genomic DNA of the cells was collected after deamination by the deaminase SsdA, and 5 ng was added to treat the transposase Tn5. The DNA fragments connected to the street by the transposase Tn5 were purified using 2x AMP XP magnetic beads.

[0223] 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) for appropriate rounds of PCR amplification, and magnetic bead purification or gel recovery of fragments between 300-500 bp to obtain the final purified library.

[0224] The two database construction methods have the following differences:

[0225] 1) The DNA state of the starting library is different. The starting library for single-stranded library construction is single-stranded DNA, and the starting library for double-stranded DNA is double-stranded DNA.

[0226] 2) The library fragmentation methods are different. Single-stranded library construction uses the physical effect of ultrasound to fragment the intact DNA, while double-stranded library construction uses the DNA cutting activity of the transposase Tn5 itself to fragment the DNA.

[0227] 3) The methods of connecting adapters are different. Single-stranded library construction uses a ligase-based method to connect 3' and 5' adapters in two rounds of reactions. Double-stranded library construction uses the adapter ligation activity of transposase Tn5 itself to connect adapters during the DNA fragmentation process.

[0228] For the original sequence that has been converted by deaminase, there will be many UG mismatch regions on the double-stranded DNA sequence. These regions will affect the efficiency of Tn5 attack on the DNA sequence, making the regions with more edits less likely to be attacked by Tn5 and resulting in a lower library depth. Single-stranded library construction can significantly improve this phenomenon.

[0229] DNA samples from MEF cells were treated with 2.8U of SsdA for 30 minutes. Double-stranded and single-stranded DNA libraries were constructed using the aforementioned method and sequenced. Analysis revealed that the single-stranded DNA library had significantly higher coverage at the center of the peak identified in published ATAC data analysis, significantly higher than that of the double-stranded DNA library (Figure 12).

[0230] 1.5 Optimization of qDeAC-seq

[0231] Choice of DNA polymerase

[0232] Due to the deamination effect of the deaminase, the resulting pre-amplified DNA contains a large amount of uracil (U). Therefore, a U-tolerant DNA polymerase is required during the final PCR amplification of the library. The inventors tested different U-tolerant DNA polymerases and analyzed the differences in amplification performance, finding that the different enzymes had similar effects (Figure 13). The enzymes tested included: HiFi V3, Phusion U, Phanta Uc, Q5U, and Q6U.

[0233] Optimization of cell punching conditions

[0234] Analysis of commonly used perforation conditions in various omics methods revealed that 0.5% NP-40 and 0.1% NP-40, and 0.1% Tween-20 and 0.01% Digitonin (referred to as Mix in the figure) are the two most commonly used detergent conditions in perforation reactions. Treating cells with these two perforation buffers revealed that both perforation methods permeabilized the cell membrane. However, at the same enzyme dosage, the Mix group exhibited significantly higher conversion rates than the 0.5% NP40 group, with higher base conversion rates in the open region (Figure 14).

[0235] Deaminase incubation time

[0236] We tested deaminase reaction incubation times of 10 minutes and 30 minutes, and both yielded samples with significant conversion rates. The 30-minute reaction yielded a higher genome conversion rate (Figure 15).

[0237] Deaminase concentration

[0238] 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 enzyme, the conversion rate and signal-to-noise ratio are higher at an enzyme concentration of 7.5U / μl (Figure 16). The data for the 15U and 30U groups are not shown, but the signal-to-noise ratio of the 30U group is less than that of the 15U group.

[0239] Deaminase reaction system

[0240] The basic enzyme reaction system consisted of 50 μl of reaction volume and 50,000 cells. Experimental testing reduced the reaction volume to 10 μl for each component, treating 10,000 cells. Analysis revealed that the overall conversion rate in the 50 μl basic reaction system was significantly higher than that in the 10 μl system. This suggests that the reaction can proceed in a reduced volume system, but the basic reaction system is more stable. (Figure 17)

[0241] Optimization of reaction buffer

[0242] First, we tested the effect of additional components in the reaction buffer on SsdA activity (see "In vitro deaminase activity verification" above). The experimental conditions were grouped as follows:

[0243] The results are shown in Figure 18 A. Group 4 had the best deamination effect.

[0244] In addition, the effects of NaCl and MgCl2 on SsdA activity were also tested. The grouped experimental conditions are as follows:

[0245] The results are shown in FIG18B .

[0246] Add UGI optimization

[0247] The inventors unexpectedly discovered that the prepared deaminase has the activity of cleaving off the U produced by deamination, thereby affecting the reliability of subsequent reactions and detection. Therefore, the inventors tested the effect of adding a uracil glycosylase inhibitor (UGI) to the reaction system.

[0248] The effects of adding UGI were compared with and without the addition of UGI for the reaction conditions of 30 U enzyme for 30 minutes, 15 U enzyme for 30 minutes, and 30 U enzyme for 10 minutes. The results are shown in Figures 19 and 20. Figure 19 shows that the depth of the ATAC peak center significantly increased after the addition of UGI. Figure 20 shows that the addition of UGI significantly improved the conversion rate. This indicates that the addition of UGI allows the use of higher enzyme dosages and longer reaction times, resulting in better results.

[0249] Deaminase truncation variants

[0250] The present 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.

[0251] The results showed that SsdA deaminase and its truncated variant SsdA-tox have good deamination activity in a variety of commonly used buffer systems and at temperatures as low as 4°C, greatly expanding its application range.

[0252] In summary, the present invention provides a method and system for determining the chromatin accessibility state and the location and binding dynamics of DNA-binding proteins, such as transcription factors, at the genome-wide 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 deaminase contact, the DNA deaminase causes the exposed cytosine bases in the DNA sequence to be deaminated and converted to uracil; and in the subsequent PCR amplification, uracil complements adenine, replacing the original cytosine with thymine. Subsequently, Sanger sequencing, next-generation sequencing (NGS), or single-molecule long-read sequencing is used to detect the location of base mutations in the genomic DNA. Given that DNA in open chromatin regions is more susceptible to the reaction of deaminases, while DNA bound by nucleosomes or DNA-binding proteins is protected, by analyzing the distribution of deamination sites, the openness of chromatin, the arrangement of nucleosomes, and the positional information and binding dynamics of DNA-binding proteins can be determined at the whole genome level.

[0253] By combining this with techniques such as ATAC-seq, ChIP-seq, or CUT&TAG, it is possible to further specifically enrich for open chromatin regions or specific protein-binding regions, and then analyze the protein binding imprints at these sites. By integrating protein imprint information with transcription factor-related databases and transcriptome information, the cell's transcriptional regulatory network can be more accurately constructed. By combining this with CRISPR technology, it is possible to analyze the impact of any protein, including transcription factors, on the transcriptional regulatory network at the genome-wide level.

[0254] Example 2: Detection of DNA-binding protein footprints in open regions of the cell genome

[0255] As shown in Example 1, the present inventors have achieved 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 chromatin regions, 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 chromatin regions, called qDeAC-ATAC-seq.

[0256] This method utilizes Tn5 and SsdA to sequentially treat permeabilized cells or nuclei. The transposase activity of Tn5 disrupts chromatin DNA in more open regions. A specific Tn5 linker is then added to the 5' end of the chromatin DNA. Given SsdA's deamination of cytosine bases, a Tn5 linker sequence containing no cytosine is used. This allows for the subsequent amplification of DNA fragments located in open chromatin regions using primers containing the Tn5 linker sequence, thereby enriching for chromatin in these regions. Furthermore, the single-stranded DNA deaminase A (SsdA) deaminates cytosine in double-stranded DNA. Upon contact with genomic DNA, SsdA deaminates exposed cytosine bases in the DNA sequence to uracil. During the subsequent PCR amplification process, uracil is recognized as thymine, where it pairs complementary to adenine. Ultimately, the deaminated cytosine base is replaced by thymine. This mutation can be detected using first-generation Sanger sequencing, second-generation sequencing (NGS), or single-molecule long-read sequencing. Given that DNA within open chromatin regions is regulated by more transcription factors, Tn5 enrichment of DNA fragments within open regions, combined with the transcription factor footprints generated by deamination reactions, enables analysis of transcription factor binding sites at low sequencing costs.

[0257] General method description

[0258] The qDeAC-ATAC-seq technology of the present invention can include two experimental strategies ( FIG. 21 ), the basic steps of which are as follows:

[0259] Strategy 1:

[0260] (A) Permeabilization or nuclear extraction

[0261] Treat cells with a permeabilization buffer containing a certain concentration of detergent to perforate the cell membrane to increase membrane permeability, or remove the cytoplasm to extract and permeabilize the cell nucleus.

[0262] (B) Tn5 attack open area reaction

[0263] The cells or nuclear samples collected in step (A) are incubated with Tn5 pre-embedded with the adapter sequence to allow the transposase to fully react with the open chromatin DNA.

[0264] (C) Deaminase reaction

[0265] 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. The genomic DNA is then extracted using phenol-chloroform.

[0266] (D) DNA extraction and single-stranded DNA library construction

[0267] After the reaction is terminated, genomic DNA is extracted. No ultrasonic shearing is required. The DNA Methylation Library Kit for Illumina V3NE103 is used for single-stranded DNA library construction, eliminating the need for 5' ligation. Samples can be directly amplified after extension. The first amplification step involves using primers containing Tn5 adaptor homology and the i7 primer. A fragment with homology to the i5 primer is added to the 5' end of the library via overlap PCR. A second amplification step, using PCR, is performed using the i5 and i7 primers. A schematic diagram of the library construction process is shown in Figure 22.

[0268] Strategy 2:

[0269] (A) Permeabilization or nuclear extraction

[0270] Treat cells with a permeabilization buffer containing a certain concentration of detergent to perforate the cell membrane to increase membrane permeability, or remove the cytoplasm to extract and permeabilize the cell nucleus.

[0271] (B) SsdAtox deamination reaction

[0272] 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.

[0273] (C) Tn5 attack open zone reaction

[0274] The SsdAtox reaction system was removed by centrifugation, and the precipitated cells / nuclei were collected. The Tn5 reaction system was then added to allow the Tn5 transposase to fully react with the DNA in the open chromatin region of the cells. The genomic DNA was then extracted using phenol-chloroform.

[0275] (D) DNA extraction and single-stranded DNA library construction

[0276] After the reaction is terminated, genomic DNA is extracted. No ultrasonic shearing is required. The DNA Methylation Library Kit for Illumina V3NE103 is used for single-stranded DNA library construction, eliminating the need for 5' ligation. Samples can be directly amplified after extension. The first amplification step involves using primers containing Tn5 adaptor homology and the i7 primer. A fragment with homology to the i5 primer is added to the 5' end of the library via overlap PCR. A second amplification step, using PCR, is performed using the i5 and i7 primers. A schematic diagram of the library construction process is shown in Figure 22.

[0277] Specific experimental methods

[0278] 1). Design Tn5 embedding linker sequence

[0279] In order to prevent the linker from being deaminated by subsequent SsdAtox, the adapter sequence that does not contain cytosine used by Tn5 in sci-MET was referenced (Figure 23). After annealing with the Tn5ME sequence, it was incubated with Tn5 to complete the linker embedding of Tn5.

[0280] 2).qDeAC-ATAC-seq process

[0281] Strategy 1: Tn5 reaction first followed by SsdAtox deamination reaction

[0282] (1) Cell collection and nuclear extraction:

[0283] Add an appropriate amount of trypsin to digest the cells, neutralize with culture medium, count, transfer from the cell culture chamber to the sequencing chamber, transfer to a 1.5 mL low-binding EP tube, centrifuge at 300 × g for 4 min at room temperature, and discard the supernatant. Wash once with 1 mL of cold PBS + 0.04% BSA, centrifuge at 300 × g for 4 min at 4°C, and discard the supernatant as completely as possible. Resuspend in 50 μl of Nuclei Extraction Buffer (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, 1x PIC) at a ratio of 50 μl per 50,000 cells. Gently pipette three times and place on ice for 3 min. To terminate the reaction, add 1 ml of Final buffer (10 mM Tris-HCl 7.4, 10 mM NaCl, 1% BSA, 0.1 mM EDTA, 3 mM MgCl2, 1x PIC), gently invert three times to mix, centrifuge at 4°C, 700 × g, for 5 min, remove the supernatant to approximately 100 μl, centrifuge again for 1 min, and discard the supernatant completely.

[0284] (2) Tn5 interruption reaction:

[0285] Prepare the required reaction mix 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 in the reaction system, and then react at 37°C for 15 minutes, setting the hot plate to shake at 1000 rpm.

[0286] (3) SsdAtox deamination reaction:

[0287] Add 500 μl of 1x SsdA Reaction Buffer (10 mM Tris-HCl 7.4, 1 mM DTT, 1x PCI) to dilute the Tn5 reaction system. Centrifuge at 700 × g for 5 min at 4°C, end over end for 1 min. Remove the supernatant to approximately 100 μl. Centrifuge again end over end for 1 min, and discard the supernatant completely. Add 50 μl of deaminase reaction system (10 mM Tris-HCl 7.4, 1 mM DTT, 1x PCI, 0.1 U / μl UGI, 5 U / μl SsdAtox). Incubate at 37°C for 10 min, shaking at 1000 rpm on a hot plate.

[0288] (4) Genomic DNA extraction

[0289] After heat incubation, add 200 μl of gDNA Hot Elution Buffer to a 50 μl reaction volume and incubate at 65°C, 1350 rpm, and shake for 1.5 hours. Add gDNA and an equal volume (250 μl) of phenol:chloroform:isoamyl alcohol DNA extraction solution and shake thoroughly to mix. Centrifuge at 13,000 g for 5 minutes at room temperature. Obtain gDNA by isopropanol precipitation, and measure its concentration using QUBIT.

[0290] Strategy 2: SsdAtox deamination reaction first and then Tn5 reaction

[0291] (1) Cell collection and nuclear extraction:

[0292] Add an appropriate amount of trypsin to digest the cells, neutralize with culture medium, count, transfer from the cell culture chamber to the sequencing chamber, transfer to a 1.5 mL low-binding EP tube, centrifuge at 300 × g for 4 min at room temperature, and discard the supernatant. Wash once with 1 mL of cold PBS + 0.04% BSA, centrifuge at 300 × g for 4 min at 4°C, and discard the supernatant as completely as possible. Resuspend in 50 μl of Nuclei Extraction Buffer (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, 1x PIC) at a ratio of 50 μl per 50,000 cells. Gently pipette three times and place on ice for 3 min. To terminate the reaction, add 1 ml of Final buffer (10 mM Tris-HCl 7.4, 10 mM NaCl, 1% BSA, 0.1 mM EDTA, 1x PIC), gently invert three times to mix, centrifuge at 4°C, 700 × g, for 5 min, centrifuge for 1 min, remove the supernatant to approximately 100 μl, centrifuge again for 1 min, and completely discard the supernatant.

[0293] (2) SsdAtox deamination reaction:

[0294] Prepare the required reaction mix 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, and then react at 37°C for 10min, setting the hot plate to shake at 1000rpm.

[0295] (3) Tn5 interruption reaction:

[0296] Add 500 μl of 1x SsdA Reaction Stop Buffer (10 mM Tris-HCl 7.4, 1 mM DTT, 1x PCI, 5 mM MgCl2) to dilute the deamination reaction system. Centrifuge at 700 × g for 5 min at 4°C, end-over-end for 1 min. Remove the supernatant to approximately 100 μl. Centrifuge again end-over-end for 1 min, and discard the supernatant completely. Add 50 μl of Tn5 reaction system (10 mM Tris-HCl 7.6, 5 mM MgCl2, 10% Dimethyl Formamide, 33% DPBS, 100 mM Tn5, 0.1 U / μl UGI) and incubate at 37°C for 15 min, shaking at 1000 rpm on a hot plate.

[0297] (4) Genomic DNA extraction

[0298] After heat incubation, add 200 μl of gDNA Hot Elution Buffer to a 50 μl reaction volume and incubate at 65°C, 1350 rpm, and shake for 1.5 hours. Add gDNA and an equal volume (250 μl) of phenol:chloroform:isoamyl alcohol DNA extraction solution and shake thoroughly to mix. Centrifuge at 13,000 g for 5 minutes at room temperature. Obtain gDNA by isopropanol precipitation, and measure its concentration using QUBIT.

[0299] 3). Library establishment

[0300] The total amount of starting DNA was selected based on the measured DNA concentration and experimental requirements. DNA Methylation Library Kit for Illumina V3NE103 was used for single-stranded DNA library construction.

[0301] Following the kit instructions, 3' Adapters were attached and extension was performed. After purification, the first amplification step was performed. The program was set for four rounds of amplification, followed by a one-step 1x bead purification, and then the second amplification step. For a 50 ng input, eight rounds of second amplification were performed, for a total of 12 rounds. After obtaining the complete library, nucleic acid was subjected to agarose gel electrophoresis, and fragments of 300-500 bp were selected for gel excision and recovery to obtain the final library of appropriate concentration (see Figure 22 for details).

[0302] 4) Analyze qDeAC-ATAC-seq data to analyze transcription factor binding in open regions

[0303] Through bioinformatics analysis, the enrichment level 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.

[0304] 2.1. Using Strategy 1 for qDeAC-ATAC-seq can enrich open regions and detect transcription factor binding within open regions

[0305] In this example, Strategy 1 was used to process the R1 cell line and construct a library for sequencing.

[0306] First, to investigate the effects of different components in the Tn5 reaction buffer on the binding of different transcription factors within the open region, the inventors attempted to treat cells with deaminase after reacting Tn5 in different Tn5 reaction buffers. The experimental groups are shown in Table 1 below.

[0307] Table 1. Tn5 reaction buffer grouping conditions

[0308] The results, shown in Figures 24 and 25, show that Tn5 enrichment efficiency in open regions is higher in the presence of DMF. Ultimately, the results suggest that the conditions of 10 mM Tris-HCl 7.6, 5 mM MgCl2, 10% DMF, and 33% PBS have the least effect on TF depth. Adding detergent to the reaction buffer reduces enrichment efficiency.

[0309] The library sequencing results obtained by qDeAC-ATAC-seq clearly demonstrate the distribution of nucleosomes (Figure 26), and the increased depth of the library at the ATAC peak center can be seen, indicating an enrichment effect for open regions (Figure 24).

[0310] Furthermore, by testing different amounts of deaminase (5 U / μl, 7.5 U / μl, 15 U / μl, and 30 U / μl) and comparing the overall deamination efficiency and transcription factor footprint capture between groups, we found that for the mouse embryonic stem cell R1 cell line, a deaminase concentration of 5 U / μl achieved the best capture of transcription factor binding footprints (Figure 27). The optimal enzyme dosage for different cell lines requires further testing.

[0311] 2.2. qDeAC-ATAC-seq reaction sequence test

[0312] This example tested K562 cells by first performing a Tn5 attack followed by a deaminase treatment (Strategy 1). Also tested was a deamination reaction followed by a Tn5 treatment of the chromatin (Strategy 2). Because deaminases, like Tn5, preferentially attack chromatin DNA in open regions, the deamination rate of DNA in open regions is highest. However, base mutations caused by deamination can disrupt the intact double-stranded structure of DNA. Therefore, it is important to balance these two factors and select the experimental strategy based on the experimental objectives.

[0313] Compared with strategies 1 and 2, each has its own advantages and disadvantages. Strategy 1 has a higher degree of enrichment for open areas and can better show the open areas in the cell (Figure 28, groups 2-3). However, due to the early treatment with Tn5, transcription factors with weak DNA binding ability fall off the DNA, resulting in low TF depth values ​​for these transcription factors (such as NFYA, RFX1, etc. in Figure 29). Strategy 2 is more accurate and realistic in capturing the footprints of transcription factors, but due to the changes in DNA bases caused by deamination, the ability of subsequent Tn5 to attack open areas is reduced, resulting in poor enrichment of open areas in cells. It can be clearly seen that the enrichment efficiency of Tn5 for open areas decreases with the increase of the total amount of deaminase added (Figure 28, groups 1 and 4-7).

[0314] 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 the amount of deaminase used in Strategy 1 (Figure 28, Groups 2-3), it can be seen that in order to enrich the open area to the greatest extent possible and improve the cleavage activity of Tn5, the amount of enzyme used in Strategy 2 should be lower than that in Strategy 1 (Figures 28 and 29).

[0315] 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 areas of the cell genome and detect the binding footprints of transcription factors in the open chromatin areas, and the experimental data obtained are highly reproducible. 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 at a 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 to the single-cell level, thereby achieving the capture of multi-omics information such as chromatin accessibility, transcription factor footprints, and transcriptomes at higher throughput levels, which is of great significance for constructing gene regulatory networks in dynamic changes such as cell fate transition, development, and carcinogenesis.

[0316] Sequence information involved in this article:

[0317] >SEQ ID NO:1 SsdA

[0318] >SEQ ID NO:2 DddAtox

[0319] >SEQ ID NO:3 His-SsdA

[0320] >SEQ ID NO:4 His-smt3-SsdA

[0321] >SEQ ID NO:5 SsdA tox

[0322] >SEQ ID NO:6 Tn5 transposase

[0323] >SEQ ID NO:7 Tn5 adapter-F

[0324] >SEQ ID NO:8 Tn5 adapter-R

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 SsdA tox.

3. The method of claim 2, 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.

4. The method of any one of claims 1-3, 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 Tn5 ME 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 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.

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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