A chromatin accessibility analysis method based on long-read sequencing

Abstract

The present invention relates to a chromatin accessibility analysis method based on long-read sequencing. The method is applicable to most animal and plant tissues and comprises the following steps: a) preparing a nonspecific m6A-MTase and measuring its enzymatic activity; b) extracting cell nuclei from animal or plant tissues; c) contacting the cell nuclei obtained in step b) with the nonspecific m6A-MTase and extracting nucleic acids; and d) constructing a HiFi library and sequencing the library.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology technology, and specifically relates to a chromatin accessibility analysis method based on long-read sequencing that is applicable to most animal and plant tissues. Background Art

[0002] ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a high-throughput technology widely used to study chromatin accessibility. It is widely used in fields such as gene regulation, developmental biology, disease mechanisms, and epigenetics. It can locate open chromatin regions (such as promoters and enhancers) genome-wide, revealing transcription factor binding sites and cell-type-specific regulatory elements. It can also be used to analyze dynamic chromatin changes during development, differentiation, or external stimuli. In disease research, ATAC-seq can be used to correlate GWAS risk loci and identify aberrant epigenetic regulation in diseases such as cancer. Integration with multi-omics data such as RNA-seq, ChIP-seq, or Hi-C allows for more systematic analysis of gene expression regulatory networks.

[0003] However, because the transposase Tn5 in the ATAC-seq experimental process prefers to insert into fragments approximately 200 bp (nucleosome-free regions) or periodic fragments (nucleosome-occupied regions), this can result in insufficient capture of long fragments (such as polynucleosome regions), thus affecting the interpretation of certain chromatin structures. Furthermore, nonspecific transposition or DNA damage can generate false-positive signals, requiring verification through biological replicates and control experiments.

[0004] The recently developed Fiber-seq technology (Andrew B. Stergachis et al., Science 2020) uses the nonspecific N6-adenine methyltransferase (m6A-MTase) to mark accessible chromatin regions. Combined with single-molecule long-read sequencing (e.g., the Pacific Biosciences platform), it enables nucleotide-level resolution of single chromatin fiber structure. However, this technology is only applicable to suspension cell lines or other living single cells, significantly limiting its applicability.

[0005] Therefore, the field urgently needs a more effective chromosome accessibility analysis technology to analyze chromatin accessibility and epigenetic information, and expand its application scenarios in multiple tissue types of animals and plants. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention aims to develop a chromatin accessibility analysis method based on long-read sequencing that is applicable to most animal and plant tissues. As mentioned above, the only sample types that Fiber-seq technology can be applied to are suspension cell lines or other living single cells. The present invention develops a chromatin accessibility analysis method applicable to most animal and plant tissues by extracting cell nuclei from animal or plant tissues. It is known that it is difficult to obtain cell nuclei from animal and plant tissues. The methods used in the prior art may cause cell nuclei to aggregate, making it difficult to achieve sufficient m6A labeling.

[0007] Therefore, the present invention provides a method for analyzing chromatin accessibility applicable to animal or plant tissues, comprising the following steps:

[0008] a) preparing a non-specific m6A-MTase and determining the enzymatic activity of the non-specific m6A-MTase;

[0009] b) extracting cell nuclei from animal or plant tissues;

[0010] c) contacting the cell nuclei obtained in step b) with the non-specific m6A-MTase and extracting nucleic acid; and

[0011] d) Construction of HiFi library and sequencing.

[0012] In some preferred embodiments, step b) comprises the following sub-steps:

[0013] If extracting nuclei from animal tissue, then b1) grind the animal tissue to a fine powder; b2) resuspend the fine powder in a buffer consisting of 8-12 mM Tris-HCl (pH=8.0), 2-4 mM MgCl2, 0.05-0.15% Tween-20, 8-10 mM NaCl, 0.5-1.5 mM DTT, and 0.01-0.03% NP-40; b3) incubate on ice for 5 min and immediately filter through a cell sieve; b4) centrifuge the filtrate at 500 g for 5 min and discard the supernatant; b5) resuspend the cells in PBS + 1% BSA solution and centrifuge at 500 g for 5 min and discard the supernatant; optionally, b6) repeat step b5);

[0014] If extracting nuclei from plant tissue, then b1) grind the plant tissue to a fine powder; b2) use a buffer consisting of 18-22 mM Tris-HCl (pH=6.8), 2-4 mM MgCl2, 4-6 mM KCl, 2-3 mM EDTA, 0.03-0.07% TritonX-100, and 0.5-1.5% PVP; b3) incubate on ice for 30 min and mix by inversion, then filter through a cell sieve; b4) centrifuge the filtrate at 3500g for 10 min and discard the supernatant; b5) resuspend the cells in the buffer from step b2) and centrifuge at 3500g for 10 min and discard the supernatant; optionally, b6) repeat step b5).

[0015] In some specific embodiments, step b) comprises the following sub-steps:

[0016] If the nuclei are to be extracted from animal tissue, then b1) grind the animal tissue to a fine powder; b2) resuspend the fine powder in a buffer consisting of 10 mM Tris-HCl (pH 8.0), 3 mM MgCl2, 0.1% Tween-20, 10 mM NaCl, 1 mM DTT, and 0.02% NP-40; b3) incubate on ice for 5 min and immediately filter the cells through a cell sieve; b4) centrifuge the filtrate at 500 g for 5 min and discard the supernatant; b5) resuspend the cells in PBS + 1% BSA solution and centrifuge at 500 g for 5 min and discard the supernatant; optionally, b6) repeat step b5).

[0017] If extracting nuclei from plant tissue, then b1) grind the plant tissue to a fine powder; b2) use a buffer consisting of 20 mM Tris-HCl (pH = 6.8), 3 mM MgCl2, 5 mM KCl, 2.5 mM EDTA, 0.05% TritonX-100, and 1% PVP; b3) incubate on ice for 30 min and mix by inversion before filtering through a cell sieve; b4) centrifuge the filtrate at 3500g for 10 min and discard the supernatant; b5) resuspend the cells in the buffer of step b2) and centrifuge at 3500g for 10 min and discard the supernatant; optionally, b6) repeat step b5).

[0018] The above-mentioned nuclear extraction steps from animal or plant tissues can obtain complete and independent cell nuclei, which are suitable for subsequent m6A labeling.

[0019] In some specific embodiments, the GenBank accession number of the gene sequence information of the non-specific m6A-MTase is JF268249.

[0020] In some embodiments, the enzymatic activity of the non-specific m6A-MTase is determined using a DNA substrate having a sequence as shown in SEQ ID NO: 1. The DNA substrate is derived from an 831 bp fragment sequence of the pET-28α plasmid, which contains four specific restriction sites "GATC" for the DpnI endonuclease. The enzymatic activity is determined using different gradient dilutions of non-specific m6A-MTase, specifically 0.1, 0.05, 0.02, 0.0175, 0.015, 0.0125, 0.01, 0.005, 0.001, and 0 μl of non-specific m6A-MTase, supplemented with enzyme-free water to 1 μl. The enzymatic activity of the non-specific m6A-MTase is determined by defining the highest enzyme dilution that can completely methylate 1 μg of substrate DNA as 1U.

[0021] In some embodiments, the amount of the non-specific m6A-MTase in step c) is 200 U.

[0022] In some embodiments, the animal or plant tissue is frozen and step b) is performed on ice or at 4°C.

[0023] In some embodiments, the animal tissue is derived from mouse liver.

[0024] In some embodiments, the plant tissue is derived from corn leaves.

[0025] In some embodiments, the nucleic acid is extracted using the CTAB method in step c).

[0026] In some embodiments, the sequencing uses the PacBio sequencing platform of Pacific Biosciences or the Nanopore sequencing platform of ONT. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) detection diagram.

[0028] Figure 2 This is a detection diagram of nonspecific m6A-MTase activity assay.

[0029] Figure 3 These are images of cell nuclei extracted from animal tissues after DAPI staining. The upper image shows the result after extraction using a cell nuclear extraction buffer containing 0.1% NP-40, and the lower image shows the result after extraction using a cell nuclear extraction buffer containing 0.02% NP-40.

[0030] Figure 4These are images of cell nuclei extracted from plant tissues after DAPI staining. The upper image shows the result after extraction using a cell nucleus extraction buffer containing 0.05% TritonX-100, and the lower image shows the result after extraction using a cell nucleus extraction buffer containing 0.1% TritonX-100. DETAILED DESCRIPTION

[0031] While exemplary embodiments of the present invention are described in detail, they should not be considered as limitations of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the scope are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded within the scope. Although any methods and materials similar or equivalent to those described herein can be used in the practice or test of the present disclosure, preferred materials and methods are described below.

[0033] The sequences used in the present invention are as follows:

[0034]

[0035] Example 1: Non-specific DNA adenine N 6 Preparation of m6A-methyltransferase (m6A-MTase)

[0036] The gene sequence of the nonspecific m6A-MTase used in this invention has been published in the literature (Marrek Drozdzet et al., Nucleic Acids Research 2012) and its GenBank accession number is JF268249. Double-stranded DNA molecules containing this gene sequence were synthesized, and primers were designed to enrich the DNA molecules and introduce dual restriction sites for NdeI and XhoI endonucleases.

[0037] The above-mentioned nonspecific m6A-MTase gene was inserted into the pET-30a(+) plasmid vector by double enzyme digestion. Specifically, the pET-30a(+) plasmid vector was first cut into linear molecules using NdeI endonuclease and XhoI endonuclease and recovered. The nonspecific m6A-MTase gene was then subjected to the same double enzyme digestion to obtain a gene sequence with specific sticky ends. Finally, it was connected to the linear pET-30a(+) plasmid vector that had completed double enzyme digestion using ligase to obtain a circular gene expression vector.

[0038] The gene expression vector was transferred into E. coli Positive clones from the ER2566 strain were selected for protein expression and purification. The overnight culture was diluted 1:100 into 500 mL of fresh LB medium (containing kanamycin) and cultured at 37°C with shaking until the OD600 reached 1.0. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the culture was continued at 20°C for 4 hours. The cells were harvested by centrifugation and resuspended in buffer A (50 mM HEPES, pH 8.0; 300 mM NaCl; containing 1 mM PMSF, 10 mM β-mercaptoethanol, 10% glycerol, and 0.5% Triton X-100). After sonication, the cells were disrupted and centrifuged at 40,000 × g for 1 hour to remove debris. The supernatant was then applied to a 6 mL nickel column (Ni-NTA agarose) pre-equilibrated in buffer A containing 20 mM imidazole. The recombinant protein was washed sequentially with buffer A containing 50 mM and 70 mM imidazole, and finally eluted with buffer A containing 150 mM imidazole. The purity was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1 ).

[0039] Depend on Figure 1 A single band at approximately 34 kDa was observed, demonstrating that the nonspecific m6A-MTase was well prepared.

[0040] Example 2: Determination of nonspecific m6A-MTase activity

[0041] The enzyme activity of nonspecific m6A-MTase was determined as follows: the highest enzyme dilution that could completely methylate 1 μg of substrate DNA was defined as 1 U. Under this condition, no complete DNA bands should be present after DpnI digestion.

[0042] The substrate DNA used in this invention is derived from an 831-bp fragment of the pET-28α plasmid (SEQ ID NO: 1). This fragment contains four specific restriction sites for the DpnI endonuclease, "GATC." The specific experimental steps are as follows: Prepare 60 μl of a nonspecific m6A-MTase reaction system containing 1 μg of substrate DNA. Dispense 1 μl of various dilutions of the nonspecific m6A-MTase solution. The enzyme dilution series is as follows: 0.1, 0.05, 0.02, 0.0175, 0.015, 0.0125, 0.01, 0.005, 0.001, and 0 μl of nonspecific m6A-MTase, and top up to 1 μl with enzyme-free water. The reaction buffer was Buffer A (15 mM Tris-HCl (pH 8.0), 15 mM NaCl, 60 mM KCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), and 0.5 mM spermidine), supplemented with 0.8 mM S-adenosylmethionine (NEB B9003S). Mix by gently pipetting 10 times and incubate at 37°C for 1 hour (with the heated lid set to 40°C). After completion of the reaction, DNA was purified using 1x magnetic beads (SPRIselect 60ml Reagent, B23318) and eluted with 20 μl of enzyme-free water. Ten restriction enzyme digestion systems were prepared: 15 μl of purified DNA sample was added with 1 μl of DpnI (NEB R0176S) and 4 μl of 10X CutSmart Buffer (NEB), and the total volume was made up to 40 μl with enzyme-free water. Mix by gently flicking and incubate at 37°C for 1 hour. After the reaction, 10 μl of the product was taken, added with 6.67 μL of 6× Loading Buffer (Takara, 9156), and mixed thoroughly. The mixture was then electrophoresed on a 1.2% agarose gel at 160 V for approximately 30 minutes. The images were taken using the Universal hood II gel imaging system ( Figure 2 ).

[0043] Depend on Figure 2 It can be seen that under the condition of 0.01μl nonspecific m6A-MTase enzyme amount, the DNA substrate can still be completely cut by DpnI, while under the condition of 0.005μl nonspecific m6A-MTase enzyme amount in the next gradient, the DNA substrate can no longer be completely cut by DpnI, and a main band of 831bp appears. The enzyme activity of this nonspecific m6A-MTase is demarcated as 0.01μl=1U, that is, 100U / μl.

[0044] Example 3: Extraction of cell nuclei from animal tissues

[0045] This example involves extracting nuclei from frozen mouse liver tissue. Specifically, the frozen tissue is first ground into a fine powder under liquid nitrogen conditions. The liquid nitrogen-preserved animal tissue powder is then dissolved and resuspended in 5 mL of ice-cold nuclear extraction buffer containing 10 mM Tris-HCl (pH 8.0), 3 mM MgCl2, 0.1% Tween-20, 10 mM NaCl, 1 mM DTT, and 0.02% NP-40 or 0.1% NP-40. The mixture is incubated on ice for 5 minutes and immediately filtered through a 30 μm pore size cell sieve. The filtrate is centrifuged at 500 g for 5 minutes at 4°C, the supernatant discarded, and the cell pellet resuspended and washed with 300 μl of PBS + 1% BSA solution. The pellet is then centrifuged at 500 g for 5 minutes at 4°C, the supernatant discarded, and the washing cycle repeated once to obtain a nuclear pellet. After extraction, the nuclear pellet is resuspended, and the nuclei are counted and observed under a microscope. Figure 3 ).

[0046] Depend on Figure 3 It can be seen that the cell nuclei appear blue, and the cell nuclei obtained after extraction with a cell nuclear extraction buffer containing 0.1% NP-40 are overly lysed and contain a lot of impurities and fragments; extraction with a cell nuclear extraction buffer containing 0.02% NP-40 can obtain relatively pure and regularly shaped cell nuclei.

[0047] Example 4: Extraction of cell nuclei from plant tissues

[0048] This example involves extracting nuclei from frozen corn leaf tissue. Specifically, the frozen tissue is ground into a fine powder under liquid nitrogen. The liquid nitrogen-preserved plant tissue powder is then dissolved and resuspended in ice-cold nuclear extraction buffer consisting of 20 mM Tris-HCl (pH 6.8), 3 mM MgCl2, 5 mM KCl, 2.5 mM EDTA, 0.05% Triton X-100 or 0.1% Triton X-100, and 1% PVP. The mixture is incubated on ice for 30 minutes, mixing by inversion every 10 minutes. The entire solution is then filtered through a 30 μm pore size cell sieve. The filtrate is collected and centrifuged at 3500 g for 10 minutes at 4°C. The supernatant is discarded, and the pellet is resuspended and washed in 10 mL of the ice-cold nuclear extraction buffer. The pellet is then centrifuged at 3500 g for 10 minutes at 4°C. The supernatant is discarded, and the same washing procedure is repeated once more to obtain a nuclear pellet. After extraction, the cell nucleus pellet was resuspended and the cell nuclei were counted and observed under a microscope ( Figure 4 ).

[0049] Depend on Figure 4It can be seen that the cell nuclei appear blue, and extraction with a nuclear extraction buffer containing 0.05% TritonX-100 can obtain relatively pure cell nuclei with regular shapes. After extraction with a nuclear extraction buffer containing 0.1% TritonX-100, the cell nuclei are overlyzed, and more irregularly shaped and smaller fluorescent signals appear.

[0050] Example 5: Nonspecific m6A-MTase reaction

[0051] Up to 6 million cell nuclei prepared in Example 3 or 4 were used for the subsequent nonspecific m6A-MTase treatment reaction. The specific steps were as follows: Resuspend up to 6 million cell nuclei in 57.5 μl of Buffer A (15 mM Tris-HCl (pH 8.0), 15 mM NaCl, 60 mM KCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), 0.5 mM spermidine) and transfer to a 37°C PCR instrument. Add 200 U of the nonspecific m6A-MTase determined in Example 2 and 1.5 μl of SAM (final concentration 0.8 mM) to each tube, then mix by pipetting up and down 10 times using a wide-bore pipette tip. Incubate the reaction at 37°C for 10 minutes, then terminate the reaction by adding 6 μl of 10% SDS (final concentration 1%). Transfer all reaction products to a 15 ml tube pre-filled with 5 ml of 2X CTAB lysis buffer for subsequent CTAB method nucleic acid extraction.

[0052] Example 6: Sequencing and analysis

[0053] The nucleic acid obtained in Example 5 was used to construct a HiFi library and sequenced. The data were analyzed using fibertools and compared with published data (see Mitchell R. Vollger et al., Synchronized long-read genome, methylome, epigenome and transcriptome profiling resolve a Mendelian condition. Nat Genet 57, 469–479 (2025). https: / / doi.org / 10.1038 / s41588-024-02067-0). The results showed that the marker ratios were basically consistent with those of the control group, indicating that the data obtained in this invention can be used for chromosome accessibility analysis.

[0054] Table 1:

[0055]

[0056] Note: HG001 is a human sample standard cell line, and its data are derived from the aforementioned literature (Mitchell R. Vollger et al., Nat Genet, 2025). The inventors established the following reference standard based on experience: if the m6A Percentage is close to the literature data and does not exceed 15%, it is considered qualified.

[0057] In summary, the present invention provides a chromatin accessibility analysis method based on long-read sequencing that is applicable to most animal and plant tissues. Its process and parameters are optimized, and ultimately achieve similar results and data output as existing methods that are only applicable to cell line samples.

[0058] It should be noted that while the above examples illustrate certain features of the present invention, they are not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. The reagents and reaction conditions involved in long-range PCR reactions and single-molecule sequencing library construction can be adjusted and modified accordingly based on specific needs. Therefore, those skilled in the art will appreciate that several simple substitutions can be made without departing from the concepts and principles of the present invention, all of which are intended to be within the scope of protection of the present invention.

Claims

1. A method for analyzing chromatin accessibility in animal or plant tissues, comprising the following steps: a) preparing nonspecific m6A-MTase and determining the enzymatic activity of the nonspecific m6A-MTase; b) extracting cell nuclei from animal or plant tissues, wherein the animal tissue is derived from mouse liver and the plant tissue is derived from corn leaves; c) contacting the cell nuclei obtained in step b) with the non-specific m6A-MTase and extracting nucleic acid; and d) constructing HiFi libraries and sequencing; Wherein step b) comprises the following sub-steps: If the nuclei of animal tissue are to be extracted, then b1) the animal tissue is ground into a fine powder; b2) the fine powder is resuspended in a buffer comprising 8-12 mM Tris-HCl, 2-4 mM MgCl2, 0.05-0.15% Tween-20, 8-10 mM NaCl, 0.5-1.5 mM DTT, and 0.02% NP-40 at pH 8.0; b3) the cells are incubated on ice for 5 minutes and then immediately filtered through a cell sieve; b4) the filtrate is centrifuged at 500 g for 5 minutes and the supernatant is discarded; b5) the cells are resuspended in PBS + 1% BSA solution and then centrifuged at 500 g for 5 minutes and the supernatant is discarded; If the nuclei of plant tissue are to be extracted, b1) grinding the plant tissue to a fine powder; b2) using a buffer solution comprising 18-22 mM Tris-HCl, 2-4 mM MgCl2, 4-6 mM KCl, 2-3 mM EDTA, 0.05% Triton X-100, and 0.5-1.5% PVP at pH 6.8; b3) incubating on ice for 30 min and mixing by inversion, then filtering through a cell sieve; b4) centrifuging the filtrate at 3500 g for 10 min and discarding the supernatant; b5) resuspending the cells in the buffer used in step b2), centrifuging at 3500 g for 10 min and discarding the supernatant.

2. The method according to claim 1, wherein step b) further comprises the sub-step: b6) repeating step b5).

3. The method according to claim 1, wherein the enzymatic activity of the non-specific m6A-MTase is determined using a DNA substrate whose sequence is shown in SEQ ID NO:

1.

4. The method according to claim 1 or 2, wherein the animal or plant tissue is frozen and the step b) is performed on ice or at 4°C.

5. The method according to claim 1, wherein the amount of non-specific m6A-MTase in step c) is 200 U. The method according to claim 1 , wherein the nucleic acid is extracted using the CTAB method in step c).

7. The method according to claim 1, wherein the sequencing uses the PacBio sequencing platform of Pacific Biosciences or the Nanopore sequencing platform of ONT.

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