WO2025066245A1 - Method for detecting chromatin accessibility or dna-binding protein footprints in cells - Google Patents

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

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

Background of the Invention

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

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

There is an urgent need for a method that can detect chromatin accessibility and/or transcription factor binding footprint information in cells with high resolution and quantitative analysis.

Brief description of the invention

In the qDeAC-seq (Quantitative DNA Deaminase-Accessible Chromatin In the qDeAC-seq assay using sequencing technology, based on the deamination of DNA in the open region of chromatin by DNA deaminase, the openness information of chromatin can be converted into mutation information of DNA sequence, so that the chromatin accessibility and DNA binding protein binding footprints such as transcription factors at the level of single DNA molecules can be quantitatively measured (Figure 1). In addition, the qDeAC-ATAC-seq method formed by combining qDeAC-seq and ATAC-seq can enrich the open chromatin regions of the cell genome and detect the footprints of DNA binding proteins in the open chromatin regions (Figure 21).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic diagram of the qDeAC-seq workflow.

Figure 2. 8M urea denaturation treatment separates DddA/SsdA and its corresponding inhibitor.

Figure 3. Deaminase activity assay.

Figure 4. qDeAC-seq cell processing workflow.

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

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

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

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

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

Figure 10. There are obvious differences in conversion rates inside and outside the Klf4 and Thap11 binding motif sites.

Figure 11. There are obvious differences in the conversion rates of SP1 and Nrf1 binding motif sites between MEF and R1 cells.

Figure 12. The impact of different library construction methods on the coverage at the center of the ATAC-seq library.

Figure 13. Effects of different DNA polymerases on library PCR amplification.

Figure 14. Effect of cell permeabilization conditions on sample conversion rate.

Figure 15. Effect of deaminase reaction incubation time on deamination effect.

Figure 16. Effect of different concentrations of deaminase on deamination efficiency.

Figure 17. The impact of differences in deaminase reaction systems on deamination efficiency.

Figure 18. Optimization of deaminase reaction buffer.

Figure 19. Effect of adding UGI on sequencing depth at the ATAC center.

Figure 20 The impact of adding UGI on conversion rate.

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

Fig. 22. Schematic diagram of the qDeAC-ATAC-seq library construction process.

Figure 23. Tn5 linker sequence diagram.

FIG. 24 . Effects of different Tn5 reaction buffers on the efficiency of enriching open zones.

FIG. 25 . Effects of different Tn5 reaction buffers on the footprint depths of different transcription factors in the R1 cell line.

Fig. 26. qDeAC-ATAC-seq library fragment size distribution.

Figure 27. Comparison of different TF depth values under different deaminase treatment conditions in R1 cell line.

Figure 28. Opening area of strategy 2 (the first group, and the fourth to eighth groups in the figure) and strategy 1 (the second and third groups in the figure) Comparison of enrichment effects.

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

DETAILED DESCRIPTION OF THE INVENTION

I. Definition

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

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

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

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

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

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

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:

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;

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.

In order to preserve the chromatin accessibility and/or DNA binding protein footprint information of the cell genome to the greatest extent possible, the The permeabilization buffer should be gentle enough to disrupt or partially disrupt the cell wall/membrane, allowing DNA deaminases to enter the cells, but not significantly disrupt the chromatin structure of the cells. The nuclear isolation should also be gentle enough not to significantly disrupt the chromatin structure.

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).

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

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 from different species, for example, from Burkholderia cenocepacia or Phytophthora syringae.

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.

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

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 with SEQ ID NO:2.

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

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

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

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.

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.

In some embodiments, the reaction buffer comprises approximately 10 mM to approximately 20 mM, preferably approximately 10 mM Tris-HCl.

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 MgCl ₂ .

In some embodiments, the reaction buffer contains no additional reagents other than 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.

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

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

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.

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

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

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

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

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.

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

The separation of genomic DNA in step c) can be carried out by conventional methods known in the art, for example, The genomic DNA was extracted using a commercially available kit.

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

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.

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

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

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.

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

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.

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

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

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: HiFi V3, Phusion U, Phanta Uc, Q5U and Q6U.

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.

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

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.

The presence of C-G to T-A transitions in a specific region of the genome indicates that chromatin in that region is accessible and/or not bound by DNA-binding proteins.

In some embodiments, the chromatin accessibility and/or DNA binding protein footprint of the genomic DNA or a portion thereof of the cell is determined by analyzing the position and/or density and/or conversion rate of C-G to T-A transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

The location and/or density of C-G to T-A transitions in the genome indicates the location and/or degree of openness of chromatin regions.

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

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

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

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

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

In another aspect, the present invention provides a method for detecting 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.

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

In some embodiments, the DNA binding is determined by analyzing the presence of a 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). Binding of the protein to the DNA molecule.

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 C-G to T-A transition in the DNA molecule or a portion thereof relative to a control DNA molecule (e.g., a DNA molecule that has not been treated with a DNA deaminase).

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

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

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.

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

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

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).

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

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

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 from different species, for example, from Burkholderia cenocepacia or Phytophthora syringae.

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.

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

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 with SEQ ID NO:2.

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

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

In some specific embodiments, the SsdA or its functional fragment comprises the amino acid sequence shown in any one of SEQ ID NO:1, 3-5.

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.

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.

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

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 10 mM to about 20 mM, preferably about 10 mM Tris-HCl. In some embodiments, the deamination 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 deamination reaction buffer contains 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 contain additional metal salts, such as NaCl or MgCl ₂ . 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 uracil glycosylase inhibitor (UGI). Adding UGI can inhibit the activity of enzymes present in the reaction system that may remove U, and avoid the effects caused by the removal of U produced by the deamination reaction. Adding UGI can increase the amount of enzyme in the reaction to shorten the reaction time while ensuring the signal-to-noise ratio. UGI that can be used in the method of the present invention can be purchased commercially.

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

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).

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.

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.

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).

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

In some embodiments, the Tn5 transposase has been complexed with an adaptor containing a Tn5ME sequence to form a transposome.

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

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 shown below: 5'-TGGTAGAGAGGGTG AGATGTGTATAAGAGACAG-3' (SEQ ID NO:7) (the underlined part is the double-stranded Tn5ME sequence, and the non-underlined part is the single-stranded part), which is formed by annealing the single-stranded Tn5adapter-F of the sequence shown in SEQ ID NO:7 and the single-stranded Tn5adapter-R of the sequence shown in SEQ ID NO:8 (5'-CTGTCTCTTATACACATCT-3').

In some embodiments, the Tn5 transposase treatment is performed in a Tagmentation buffer. The Tagmentation buffer may 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 comprise a detergent. In some preferred embodiments, the Tagmentation buffer comprises 10 mM Tris-HCl (pH 7.6), 5 mM MgCl ₂ , 10% DMF, 33% PBS. The composition of PBS is: 155 mM NaCl, 3 mM Na ₂ HPO ₄ , and 1 mM KH ₂ PO ₄ .

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

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

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.

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.

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

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.

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.

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

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

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

The inventors surprisingly found that the method of building a library with single-stranded DNA can obtain more Therefore, in some preferred embodiments, a genomic DNA library is established from the isolated genomic DNA by single-stranded DNA library construction.

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

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: HiFi V3, Phusion U, Phanta Uc, Q5U and Q6U.

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.

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

In some embodiments, the DNA binding protein footprint of the cell's genomic DNA or a portion thereof is determined by analyzing the presence of C-G to T-A transitions (i.e., C to T and G to A) in the cell's genomic DNA or a portion thereof relative to a control genomic DNA or a portion thereof.

The presence of C-G to T-A transitions in a specific region of the genome indicates that the genomic DNA in that region is not bound by DNA binding proteins.

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 conversion rate of C-G to T-A transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof.

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

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.

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

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

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

IV. Test Kit

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).

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

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.

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.

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

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

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

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

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

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.

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.

In some embodiments, the kit further comprises an adaptor comprising a Tn5ME sequence, and a reagent for complexing the adaptor with Tn5 transposase to form a transposome. The adaptor comprising a Tn5ME sequence is as defined above.

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.

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

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

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

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

Example 1: Detecting chromatin accessibility or DNA binding protein footprints in cells at the whole genome level

General method description

1). Construction of plasmid expressing deaminase protein

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

2). Purification of deaminase protein

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

For affinity purification and denaturation, ①SsdA: centrifuge the broken bacterial solution and collect the inclusion bodies. Add a buffer containing 8M urea to dissolve the inclusion bodies, centrifuge to remove impurities, add Ni-NTA beads, and affinity purify the protein. ②DddA: centrifuge the broken bacterial solution, collect the supernatant, add Ni-NTA beads for incubation, and then add a buffer containing 8M urea to denature the protein. Treatment with 8M urea can separate DddA/SsdA and its corresponding inhibitor (see Figure 2), and then DddA/SsdA is renatured by reducing the urea concentration. Elute with Imidazole, dialyze to remove Imidazole, and finally quantify the protein by Coomassie Brilliant Blue staining. Finally, use the AKTA protein purification system to separate and purify the DddA/SsdA protein by ion exchange chromatography, molecular sieves, etc., and concentrate the corresponding components to obtain the target protein sample.

3). Verification of in vitro deaminase activity.

Design oligonucleotide primers (5'-AATATAATATAATAACTCGCCATAATTTTAATTAAT-3', 5' with 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 double-stranded oligonucleotide substrates with a final concentration of 1 nM and incubated at 37°C for 1 hour. When cytosine on the substrate is deaminated to form uracil. Uracil DNA glycosylase was added and incubated at 37°C for 30 minutes to cut off uracil and form a purine-free pyrimidine site. After that, the double-stranded oligonucleotide was unwound and broken at the purine-free pyrimidine site by high temperature treatment with 100mM NaOH and formamide at 95°C. Finally, the fragments were separated by 15% acrylamide gel electrophoresis containing 8M urea, and the deamination effect was judged by the fragment size. See Figure 3 for details.

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

The reaction system is:

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

4). Cell processing for qDeAC-seq (Figure 4)

(1) Cell collection and nuclear extraction:

Add appropriate amount of trypsin to digest cells, count after neutralization with medium, transfer to 1.5mL low-absorption EP tube, centrifuge at room temperature, 300×g for 4min, and discard supernatant. Add 1mL cold PBS to wash twice, centrifuge at 4℃, 300×g for 4min, and discard supernatant. Add 1mL cold Wash buffer (10mM Tris-HCl 7.4, 10mM NaCl, 1% BSA, 0.1mM EDTA, 3mM MgCl ₂ , 1xPIC) to resuspend, and separate 50,000 cells × N groups. 4℃, 300×g, 4min, aspirate and discard supernatant as much as possible, and wash once in total.

Lysis: Resuspend the cells by flicking the bottom of the tube, then add 50ul Lysis Buffer (10mM Tris-HCl 7.4, 10mM NaCl, 1% BSA, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin, 0.1mM EDTA, 3mM MgCl ₂ , 1xPIC) to 50,000 cells, gently pipette 3 times, and place on ice for 3 minutes. Terminate the reaction: Add 1ml Final buffer (10mM Tris-HCl 7.4, 10mM NaCl, 1% BSA, 0.1mM EDTA, 1xPIC), gently invert to mix 3 times, 4℃, 700×g, 5min, centrifuge for 1min, remove the supernatant to about 100ul, invert and centrifuge again for 1min, and completely discard the supernatant.

(2) Deamination reaction:

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

(3) DNA extraction

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

5). Library establishment

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

According to the kit steps, the 3'Adapter and 5'Adapter were connected respectively, and finally 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. The sequencing machine used was Illumina X TEN, and the sequencing mode was double-end 150bp.

6).qDeAC-seq base mutation identification.

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

1.1. qDeAC-seq to determine MEF genomic DNA accessibility

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

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

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

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

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

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

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

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

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

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

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

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

The whole genome DNA of the cells after deamination treatment by deaminase SsdA was collected and ultrasonically treated to obtain fragmented double-stranded DNA. A certain amount of DNA was taken as the starting library, and the double-stranded DNA was denatured at 95°C and immediately placed on ice to obtain fragmented single-stranded DNA. After connecting the 3' end adapter to the 3' end of the single-stranded DNA using 3' adapter ligase, an extension system was added for reaction, and then 1.2x DNA purification magnetic beads (Novozymes, N411, VAHTS DNA Clean Beads) were used to obtain a double-stranded DNA product with a 3' end adapter connected. The 5' adapter ligase was used to connect the adapter to the 5' end of the double-stranded DNA, and then purified using 1x DNA magnetic beads to obtain a double-stranded DNA product with a double-end adapter connected. Finally, Novozymes amplification primers (including but not limited to N321/N322, VAHTS Multiplex Oligos Set The library was amplified by PCR for an appropriate number of rounds and purified using 0.85x DNA purification magnetic beads to obtain the final library.

Double-chain library construction process

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

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

Finally, use Novazon amplification primers (including but not limited to TD204, TruePrep Index Kit V4for Illumina) and a uracil (U)-tolerant DNA polymerase (including but not limited to Phusion U) to perform appropriate rounds of PCR amplification, and magnetic bead purification or gel recovery of fragments between 300-500bp to obtain the final purified library.

The two database construction methods have the following differences:

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

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

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

For the original sequence that has been converted by deaminase, there will be more U-G mismatch regions on the double-stranded DNA sequence, which will affect the attack efficiency of Tn5 on the DNA sequence, making the edited regions more difficult to be attacked by Tn5 and the library construction depth lower. Single-stranded library construction can significantly improve this phenomenon.

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

1.5 Optimization of qDeAC-seq

Choice of DNA polymerase

Due to the deamination effect of the deaminase, the obtained pre-amplification DNA contains a large amount of uracil bases (Uracil, U), so when the library is amplified by PCR at the end, a DNA polymerase that tolerates U is required. The inventors tested different U-tolerant DNA polymerases, analyzed the differences in amplification effects, and found that the effects of different enzymes were similar (Figure 13). The enzymes tested include: HiFi V3, Phusion U, Phanta Uc, Q5U, and Q6U.

Optimization of cell punching conditions

We analyzed the punching conditions commonly used in various other omics methods and found that 0.5% NP-40 and 0.1% NP-40, 0.1% Tween-20 and 0.01% Digitonin (referred to as Mix in the figure) are the two most commonly used detergent conditions in the punching reaction. We used these two punching buffers to treat cells and found that both punching methods had the effect of permeabilizing the cell membrane. However, under the same enzyme dosage conditions, the conversion rate of the Mix group was significantly higher than that of the 0.5% NP40 group, and there was a higher base conversion rate at the open area. (Figure 14)

Deaminase incubation time

The deaminase reaction incubation time of 10 minutes and 30 minutes was tested respectively, and samples with significant conversion rate were obtained under both incubation times. The genome conversion rate obtained under the 30-minute reaction condition was higher. (Figure 15)

Deaminase concentration

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

Deaminase reaction system

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

Optimization of reaction buffer

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

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

In addition, the effects of NaCl and MgCl ₂ on SsdA activity were also tested. The grouping experimental conditions are as follows:

The results are shown in Figure 18B.

Add UGI optimization

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

The effects of adding or not adding UGI were compared under the conditions of 30U enzyme reaction for 30 minutes, 15U enzyme reaction for 30 minutes, and 30U enzyme reaction for 10 minutes. The results are shown in Figures 19 and 20. Figure 19 shows that after adding UGI, the depth at the center of the ATAC peak is significantly improved. Figure 20 shows that the conversion rate is significantly improved after adding UGI. It can be seen that adding UGI allows the use of higher enzyme amounts, longer reaction times, and better results.

Deaminase truncated variants

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

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

In summary, the present invention provides a method and system for determining the chromatin accessibility state and the position information and binding dynamics of DNA binding proteins such as transcription factors at the whole genome level. This technology creates a new qDEAC-seq (Quantitative DNA Deaminase-Accessible Chromatin assay using sequencing) method, which treats permeabilized cells or cell nuclei with single-stranded DNA deaminase A (SsdA) or double-stranded DNA deaminase A (DddA). After deaminase contact, the DNA deaminase causes the exposed cytosine bases on the DNA sequence to be deaminated into uracil; and in the subsequent PCR amplification, uracil complements adenine and replaces the original cytosine with thymine. Subsequently, Sanger sequencing, next-generation sequencing (NGS) or single-molecule long-read sequencing is used to detect the location of base mutations in genomic DNA. Given that the DNA in the open chromatin region is more easily reacted by deaminase, DNA bound by nucleosomes or DNA-binding proteins will be protected. Therefore, 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.

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

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

As shown in Example 1, the inventors have realized the detection of transcription factor footprints at the whole genome level using single-stranded DNA cytosine deaminase (SsdA), which is called qDeAC-seq. However, since qDeAC-seq is a whole genome level detection, it has a high requirement for sequencing amount. 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 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.

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

General method description

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

Strategy 1:

(A) Permeabilization or nuclear extraction

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

(B) Tn5 attack open zone reaction

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

(C) Deaminase reaction

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

(D) DNA extraction and single-stranded DNA library construction

After the reaction is terminated, genomic DNA is extracted. No ultrasonic disruption is required. Use Novozyme The DNA Methylation Library Kit for Illumina V3NE103 kit is used to construct a single-stranded DNA library. No 5' ligation is required, and the sample can be directly amplified after extension. First, the first step of amplification is performed using primers containing a Tn5 adapter homologous sequence and an i7 primer. After adding a fragment with a homologous sequence to the i5 primer at the 5' end of the library by overlap PCR, PCR amplification uses i5 and i7 primers for the second step of amplification. The schematic diagram of the library construction process is shown in Figure 22.

Strategy 2:

(A) Permeabilization or nuclear extraction

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

(B) SsdAtox deamination reaction

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.

(C) Tn5 attack open zone reaction

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

(D) DNA extraction and single-stranded DNA library construction

After the reaction is terminated, genomic DNA is extracted. No ultrasonic disruption is required. Use Novozyme The DNA Methylation Library Kit for Illumina V3NE103 kit is used to construct a single-stranded DNA library. No 5' ligation is required, and the sample can be directly amplified after extension. First, the first step of amplification is performed using primers containing a Tn5 adapter homologous sequence and an i7 primer. After adding a fragment with a homologous sequence to the i5 primer at the 5' end of the library by overlap PCR, PCR amplification uses i5 and i7 primers for the second step of amplification. The schematic diagram of the library construction process is shown in Figure 22.

Specific experimental methods

1). Design Tn5 embedding linker sequence

In order to prevent the linker from being deaminated by the 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.

2).qDeAC-ATAC-seq process

Strategy 1: Tn5 reaction first and then SsdAtox deamination reaction

(1) Cell collection and nuclear extraction:

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

(2) Tn5 interruption reaction:

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

(3) SsdAtox deamination reaction:

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

(4) Genomic DNA extraction

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

Strategy 2: SsdAtox deamination reaction first and then Tn5 reaction

(1) Cell collection and nuclear extraction:

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

(2) SsdAtox deamination reaction:

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

(3) Tn5 interruption reaction:

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

(4) Genomic DNA extraction

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

3). Library establishment

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

According to the steps of the kit, the 3'Adapter was connected and Extension was performed. After purification, the first step of amplification could be performed. The program was set to amplify 4 rounds, followed by a 1x beads purification step, and the second step of amplification. Taking 50ng as an example, the second step of amplification was performed for 8 rounds, for a total of 12 rounds. After obtaining the complete library, nucleic acid agarose gel electrophoresis was performed, and then fragments of 300-500bp were selected for gel cutting and recovery to obtain the final library with appropriate concentration (see Figure 22 for details).

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

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

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

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

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

Table 1. Tn5 reaction buffer grouping conditions

The results are shown in Figures 24 and 25. It was found that Tn5 had a higher efficiency of enriching the open area under the condition of containing DMF. Finally, it was concluded that the condition of 10mM Tris-HCl 7.6, 5mM MgCl ₂ , 10% DMF, 33% PBS had the least effect on TF depth. Adding detergent to the reaction buffer would reduce the enrichment efficiency.

From the library sequencing results obtained by qDeAC-ATAC-seq, the results can show the distribution pattern of nucleosomes more clearly (Figure 26), and the increase in the depth of the library at the ATAC peak center can be seen, indicating an enrichment effect on the open area (Figure 24).

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

2.2. qDeAC-ATAC-seq reaction sequence test

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

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 cells (Figure 28, Groups 2-3), but due to the previous treatment of Tn5, transcription factors with weak DNA binding ability fall off the DNA, resulting in low TF depth values of these transcription factors (such as NFYA, RFX1, etc. in Figure 29). Strategy 2 is more accurate and realistic in capturing transcription factor footprints, but due to the changes in DNA bases caused by deamination, the subsequent ability of Tn5 to attack open areas is reduced, resulting in poor enrichment of open areas in cells. It is obvious that the enrichment efficiency of Tn5 for open areas will decrease with the increase in the total amount of deaminase added (Figure 28, Groups 1 and 4-7).

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 cutting activity of Tn5, the amount of enzyme used in Strategy 2 should be lower than that in Strategy 1 (Figures 28 and 29).

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

Sequence information involved in this article:

>SEQ ID NO:1 SsdA

>SEQ ID NO:2 DddAtox

>SEQ ID NO:3 His-SsdA

>SEQ ID NO:4 His-smt3-SsdA

>SEQ ID NO:5 SsdA tox

>SEQ ID NO:6 Tn5 transposase

>SEQ ID NO:7 Tn5 adapter-F

>SEQ ID NO:8 Tn5 adapter-R

Claims (57)

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. 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. The method of claim 2, wherein the SsdA comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:1; or wherein the SsdA tox comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:5. The method of any one of claims 1-3, wherein the permeabilization buffer comprises NP40. The method of claim 4, wherein the permeabilization buffer comprises NP40, Tween-20 and Digitonin. The method of claim 5, wherein the permeabilization buffer comprises about 0.1% NP40, about 0.1% Tween-20, and about 0.01% Digitonin. 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. 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. The method according to any one of claims 1 to 8, wherein in step b), the cells or cell nuclei are treated with a DNA deaminase for about 5 minutes to about 60 minutes, preferably for about 10 minutes to about 30 minutes, more preferably for about 10 minutes. 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. The method according to any one of claims 1 to 9, wherein step d) comprises establishing a whole genome DNA library from the isolated genomic DNA, and sequencing based on the whole genome library, thereby determining the Describes the chromatin accessibility and/or DNA binding protein footprints of the cellular genome. The method of claim 11, wherein a whole genomic DNA library is established from the isolated genomic DNA by single-stranded DNA library construction. The method of claim 11, wherein by The single-stranded DNA library was constructed using the DNA Methylation Library Kit for Illumina V3 NE103 kit. The method of claim 11 or 12, wherein a U-tolerant DNA polymerase is used for DNA amplification in the establishment of the library, for example, the U-tolerant DNA polymerase is selected from: HiFi V3, Phusion U, Phanta Uc, Q5U and Q6U. 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 C-G to T-A transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof. 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 C-G to T-A transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof. The method of any one of claims 1 to 16, wherein the cell is an animal cell, a plant cell or a microbial cell. The method of claim 17, wherein the cell is a mammalian cell, including but not limited to a cell of a human, mouse, rat, cat, dog, pig, or cow; or, the cell is a monocot or dicot cell, such as a cell of rice, corn, wheat, sorghum, soybean, potato, or tomato; or, the cell is a fungus such as a yeast cell. 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 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. 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. 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. 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. 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%, at least 99.9% sequence identity to SEQ ID NO:1. 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). 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. The method of claim 25, wherein the Tn5 transposase fully reacts with the open region of the genomic DNA in the cell to cause the genomic DNA in the open region to be fragmented and a linker containing the Tn5 ME sequence is added to the 5' end of the obtained DNA fragment. 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 with SEQ ID NO:6. 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 linker does not contain C in sequences other than the Tn5 ME sequence. 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. The method of any one of claims 25 to 29, wherein the Tn5 transposase treatment is performed in a Tagmentation buffer comprising DMF (dimethylformamide). The method of claim 30, wherein the tagmentation buffer comprises 10 mM Tris-HCl (pH 7.6), 5 mM MgCl ₂ , 10% DMF, and 33% PBS. The method of claim 30 or 31, wherein the Tagmentation buffer further comprises a uracil glycosylase inhibitor (UGI). 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. 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. The method of any one of claims 25 to 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. The method of any one of claims 25-35, 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. The method of claim 36, wherein the SsdA comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:1; or wherein the SsdA tox comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to SEQ ID NO:5. 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. 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 . The method of claim 39, wherein the deamination reaction buffer further comprises a uracil glycosylase inhibitor (UGI). 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. The method of any one of claims 25 to 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. The method of any one of claims 25-42, wherein the permeabilization buffer comprises NP40. The method of claim 43, wherein the permeabilization buffer comprises NP40, Tween-20 and Digitonin. The method of claim 44, wherein the permeabilization buffer comprises about 0.1% NP40, about 0.1% Tween-20, and about 0.01% Digitonin. The method of any one of claims 25 to 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. The method of any one of claims 25 to 46, wherein step d) comprises establishing a genomic DNA library from the isolated genomic DNA, and sequencing based on the genomic library, thereby determining the Footprints of DNA-binding proteins in open regions of the cellular genome. The method of claim 47, wherein a genomic DNA library is established from said isolated genomic DNA by single-stranded DNA library construction. The method of claim 48, wherein The single-stranded DNA library was constructed using the DNA Methylation Library Kit for Illumina V3 NE103 kit. The method of any one of claims 47-49, wherein a U-tolerant DNA polymerase is used for DNA amplification in the establishment of the library, for example, the U-tolerant DNA polymerase is selected from: HiFi V3, Phusion U, Phanta Uc, Q5U and Q6U. 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 C-G to T-A transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof. The method of claim 51, wherein the 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 C-G to T-A transitions in the genomic DNA or a portion thereof of the cell relative to a control genomic DNA or a portion thereof. The method of any one of claims 25-52, wherein the cell is an animal cell, a plant cell or a microbial cell. 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. 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. 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. A kit for detecting DNA binding protein footprints in a cell genome by the method according to any one of claims 25 to 56, comprising at least the DNA deaminase and the Tn5 transposase.