Chromatin accessibility analysis method based on long read length sequencing

Abstract

The invention relates to a chromatin accessibility analysis method based on long read length sequencing. The method is suitable for most animal and plant tissues and comprises the following steps: a) preparing non-specific m6A-MTase and determining the enzyme activity of the non-specific m6A-MTase, b) extracting cell nucleuses of animal or plant tissues, c) contacting the cell nucleuses obtained in the step b) with the non-specific m6A-MTase and extracting nucleic acids, and d) constructing a HiFi library and sequencing.

Description

Technical Field

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

[0002] ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a high-throughput technology widely used for studying chromatin accessibility, and is widely applied in fields such as gene regulation, developmental biology, disease mechanisms, and epigenetics. It can locate open chromatin regions (such as promoters, enhancers, etc.) across the genome, reveal transcription factor binding sites and cell type-specific regulatory elements, and be used to analyze dynamic chromatin changes during development, differentiation, or under external stimuli. In disease research, ATAC-seq can associate GWAS risk loci and discover abnormal epigenetic regulation in diseases such as cancer. By integrating with multi-omics data such as RNA-seq, ChIP-seq, or Hi-C, the gene expression regulatory network can be more systematically analyzed.

[0003] However, since the transposase Tn5 in the ATAC-seq experimental process prefers to insert into approximately 200 bp (nucleosome-free regions) or periodic fragments (nucleosome-occupied regions), it may lead to insufficient capture of long fragments (such as multi-nucleosome regions), thereby affecting the analysis of certain chromatin structures. In addition, non-specific transposition or DNA damage may generate false positive signals, which need to be verified through biological replicates and control experiments.

[0004] The recently developed Fiber-seq technology (Andrew B. Stergachis et al., Science 2020) marks chromatin accessible regions through non-specific N6-adenine methyltransferase (m6A-MTase), and combines single-molecule long-read sequencing (such as the Pacific Biosciences platform) to achieve the analysis of single chromatin fiber structures at nucleotide-level resolution. However, the sample types applicable to this technology are only suspension cell lines or other living single cells, which greatly limits the applicable sample types of this technology.

[0005] Therefore, there is an urgent need in the field for a more effective chromosome accessibility analysis technology for analyzing chromatin accessibility and epigenetic information, and expanding its application scenarios in multiple tissue types of animals and plants. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention aims to develop a chromatin accessibility analysis method based on long-read sequencing and applicable to most animal and plant tissues. As mentioned above, the Fiber-seq technology can only be applied to sample types such as suspension cell lines or other living single cells. The present invention has developed a chromatin accessibility analysis method applicable to most animal and plant tissues through the step of extracting cell nuclei from animal or plant tissues. It is known that it is difficult to obtain cell nuclei from animal and plant tissues, and 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 chromatin accessibility analysis method applicable to animal or plant tissues, which comprises the following steps: a) Preparing a non-specific m6A-MTase and measuring the enzyme activity of the non-specific m6A-MTase; b) Extracting cell nuclei from animal or plant tissues; c) Contacting the cell nuclei obtained in step b) with the non-specific m6A-MTase and extracting nucleic acids; and d) Constructing a HiFi library and sequencing.

[0008] In some preferred embodiments, step b) comprises the following sub-steps: If extracting cell nuclei from animal tissues, then b1) grinding the animal tissues into fine powder; b2) resuspending the fine powder with a buffer solution having a composition 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, 0.01 - 0.03% NP-40; b3) immediately performing cell sieve filtration after incubating on ice for 5 min; b4) centrifuging the filtrate at 500 g for 5 min and discarding the supernatant; b5) resuspending the cells with PBS + 1% BSA solution and then centrifuging at 500 g for 5 min and discarding the supernatant; optionally b6) repeating step b5). If extracting cell nuclei from plant tissues, then b1) grinding the plant tissues into fine powder; b2) using a buffer solution having a composition 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, 0.5 - 1.5% PVP; b3) incubating on ice for 30 min, inverting and mixing well, and then performing cell sieve filtration; b4) centrifuging the filtrate at 3500 g for 10 min and discarding the supernatant; b5) resuspending the cells with the buffer solution in step b2) and then centrifuging at 3500 g for 10 min and discarding the supernatant; optionally b6) repeating step b5).

[0009] In some specific embodiments, step b) includes the following sub-steps: If extracting the cell nuclei of animal tissues, then b1) grind the animal tissues into fine powder; b2) resuspend the fine powder with a buffer solution with the composition of 10 mM Tris-HCl (pH = 8.0), 3 mM MgCl2, 0.1% Tween-20, 10 mM NaCl, 1 mM DTT, 0.02% NP-40; b3) immediately perform cell sieve filtration after incubating on ice for 5 min; b4) centrifuge the filtrate at 500 g for 5 min and discard the supernatant; b5) resuspend the cells with PBS + 1% BSA solution and then centrifuge at 500 g for 5 min and discard the supernatant; optionally, b6) repeat step b5).

[0010] If extracting the cell nuclei of plant tissues, then b1) grind the plant tissues into fine powder; b2) use a buffer solution with the composition of 20 mM Tris-HCl (pH = 6.8), 3 mM MgCl2, 5 mM KCl, 2.5 mM EDTA, 0.05% TritonX-100, 1% PVP; b3) incubate on ice for 30 min, invert and mix well, and then perform cell sieve filtration; b4) centrifuge the filtrate at 3500 g for 10 min and discard the supernatant; b5) resuspend the cells with the buffer solution in step b2) and then centrifuge at 3500 g for 10 min and discard the supernatant; optionally, b6) repeat step b5).

[0011] Through the above steps of extracting cell nuclei from animal or plant tissues, intact and independent cell nuclei can be obtained, which are suitable for subsequent m6A labeling.

[0012] In some specific embodiments, the GenBank accession number of the gene sequence information of the non-specific m6A-MTase is JF268249.

[0013] In some embodiments, the enzyme activity assay of the non-specific m6A-MTase uses a DNA substrate with the sequence shown in SEQ ID NO:1. The DNA substrate is a fragment sequence with a length of 831 bp from the pET-28α plasmid, and this fragment contains 4 specific cleavage sites "GATC" of DpnI endonuclease. The enzyme activity assay uses non-specific m6A-MTase with different gradient dilutions, specifically diluted to 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, and make up to 1 μl with enzyme-free water. The criterion for determining the enzyme activity of the non-specific m6A-MTase is that the highest enzyme dilution that can completely methylate 1 μg of substrate DNA is defined as 1 U.

[0014] In some embodiments, the amount of the non-specific m6A-MTase enzyme in step c) is 200 U.

[0015] In some embodiments, the animal or plant tissue is cryopreserved and step b) is carried out on ice or at 4 °C.

[0016] In some embodiments, the animal tissue is derived from a mouse liver.

[0017] In some embodiments, the plant tissue is derived from a maize leaf.

[0018] In some embodiments, nucleic acids are extracted using the CTAB method in step c).

[0019] In some embodiments, the sequencing is performed using the PacBio sequencing platform of Pacific Biosciences or the Nanopore sequencing platform of ONT. Description of the Drawings

[0020] Figure 1 It is a detection graph of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0021] Figure 2 It is a detection graph of the determination of non-specific m6A-MTase enzyme activity.

[0022] Figure 3 It is an image of the nucleus after DAPI staining of animal tissue extraction, where the upper figure is the result obtained after extraction using a nuclear extraction buffer containing 0.1% NP-40, and the lower figure is the result obtained after extraction using a nuclear extraction buffer containing 0.02% NP-40.

[0023] Figure 4 It is an image of the nucleus after DAPI staining of plant tissue extraction, where the upper figure is the result obtained after extraction using a nuclear extraction buffer containing 0.05% TritonX-100, and the lower figure is the result obtained after extraction using a nuclear extraction buffer containing 0.1% TritonX-100. Detailed Description of the Invention

[0024] The exemplary embodiments of the present invention are described in detail, which should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention.

[0025] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, the preferred materials and methods are described below.

[0026] The sequences used in the present invention are as follows:

[0027] Example 1: Preparation of N 6 -methyltransferase (m6A-MTase) of non-specific DNA adenine The gene sequence information of the non-specific m6A-MTase used in the present invention has been published in the literature (Marrek Drozdz et al., Nucleic Acids Research 2012), and its GenBank accession number is JF268249. A double-stranded DNA molecule of this gene sequence is obtained by artificial synthesis, and DNA molecule enrichment and the introduction of double digestion sites of NdeI endonuclease and XhoI endonuclease are carried out through primer design.

[0028] The above non-specific m6A-MTase gene is inserted into the pET-30a(+) plasmid vector by double digestion. Specifically, the pET-30a(+) plasmid vector is first cut into a linear molecule with NdeI endonuclease and XhoI endonuclease and recovered, then the non-specific m6A-MTase gene is subjected to the same double digestion to obtain a gene sequence with specific sticky ends, and finally it is ligated with the linear pET-30a(+) plasmid vector that has completed double digestion through a ligase to obtain a circular gene expression vector.

[0029] Transfer the above gene expression vector to E. coliIn the ER2566 strain, after selecting positive clone strains, protein expression and purification were carried out. Specifically: The overnight culture broth was diluted 1:100 to 500 mL of fresh LB medium (containing kanamycin), and cultured with shaking at 37 °C until the OD600 reached 1.0. Isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1 mM was added, and the culture was continued at 20 °C for 4 hours. After centrifuging to collect the bacterial cells, the cell pellet was resuspended with 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 ultrasonic cell disruption, debris was removed by centrifugation at 40,000×g for 1 hour. The supernatant was added to a 6 mL nickel column (Ni-NTA agarose) pre-equilibrated with buffer A containing 20 mM imidazole. It was washed successively with buffer A containing 50 mM and 70 mM imidazole, and finally the recombinant protein was eluted with buffer A containing 150 mM imidazole, and the purity was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( Figure 1 ).

[0030] It can be seen that there is a single band at about 34 kDa, proving that the preparation of non-specific m6A-MTase is in good condition. Figure 1

[0031] Example 2: Enzyme activity assay of non-specific m6A-MTase The criteria for determining the enzyme activity of non-specific m6A-MTase are as follows: The highest enzyme dilution factor that can completely methylate 1 μg of substrate DNA is defined as 1 U, and there should be no intact DNA band after DpnI digestion under this condition.

[0032] ​The substrate DNA used in the present invention is a fragment sequence (SEQ ID NO: 1) with a length of 831 bp from the pET-28α plasmid, and this fragment contains 4 specific cleavage sites "GATC" of DpnI endonuclease. The specific experimental steps are as follows: Prepare a 60 μl non-specific m6A-MTase reaction system, with each system containing 1 μg of substrate DNA. Take 1 μl of different gradient dilutions of non-specific m6A-MTase. The enzyme gradient dilution method 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 non-specific m6A-MTase, and supplement nuclease-free water to 1 μl. The reaction buffer is 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 0.8 mM S-adenosylmethionine (NEB B9003S) is added. Gently pipette 10 times to mix evenly, and react at 37 °C for 1 h on a PCR instrument (the hot lid is set at 40 °C). After the reaction is completed, DNA purification is performed using magnetic beads with a 1-fold multiplier (SPRIselect 60 ml Reagent, B23318), and elute with 20 μl of nuclease-free water. Prepare 10 groups of restriction enzyme digestion systems: 15 μl of the purified DNA sample, add 1 μl of DpnI (NEB R0176S), 4 μl of 10X CutSmart Buffer (NEB), and supplement nuclease-free water to a total volume of 40 μl. Flick to mix evenly and react at 37 °C for 1 hour. After the reaction is completed, take 10 μl of the product, add 6.67 μL of 6× Loading Buffer (Takara, 9156) to mix evenly, and use it for electrophoresis at 160 V in a 1.2% agarose gel for about 30 minutes. Use the Universal hoodII gel imaging system for photography ( Figure 2 )

[0033] It can be seen that Figure 2 under the condition of 0.01 μl of non-specific m6A-MTase enzyme amount, the DNA substrate can still be completely cleaved by DpnI. However, under the condition of the next gradient of 0.005 μl of non-specific m6A-MTase enzyme amount, the DNA substrate can no longer be completely cleaved by DpnI, and a main band of 831 bp appears. Based on this, the enzyme activity of this non-specific m6A-MTase is calibrated as 0.01 μl = 1 U, that is, 100 U / μl.

[0034] Example 3: Extraction of cell nuclei from animal tissues In this example, the nuclei were extracted from a cryopreserved mouse liver tissue. Specifically: First, the cryopreserved tissue was ground into fine powder under liquid nitrogen conditions. Subsequently, 5 mL of ice-cold nuclear extraction buffer with the composition of 10 mM Tris-HCl (pH = 8.0), 3 mM MgCl2, 0.1% Tween-20, 10 mM NaCl, 1 mM DTT, 0.02% NP-40 or 0.1% NP-40 was used to dissolve and resuspend the fine powder of the animal tissue in the liquid nitrogen state. After incubating on ice for 5 min, it was immediately filtered through a cell sieve with a pore size of 30 μm. The filtrate was centrifuged at 4°C and 500 g for 5 minutes, and the supernatant was discarded. Then, it was resuspended and washed with 300 μL of PBS + 1% BSA solution, centrifuged at 4°C and 500 g for 5 minutes, and the supernatant was discarded. The washing was repeated once, and finally, a nuclear precipitate was obtained. After the extraction was completed, the nuclear precipitate was resuspended, and nuclear counting and microscopic observation were carried out ( Figure 3 )

[0035] As Figure 3 can be seen, the nuclei were blue, and the nuclei obtained after extraction with the nuclear extraction buffer containing 0.1% NP-40 were over-lysed with more impurity fragments; the nuclei extracted with the nuclear extraction buffer containing 0.02% NP-40 were relatively pure and regular in shape.

[0036] Example 4: Extraction of nuclei from plant tissues In this example, the nuclei were extracted from a cryopreserved maize leaf tissue. Specifically: First, the cryopreserved tissue was ground into fine powder under liquid nitrogen conditions. Subsequently, the ice-cold nuclear extraction buffer with the composition of 20 mM Tris-HCl (pH = 6.8), 3 mM MgCl2, 5 mM KCl, 2.5 mM EDTA, 0.05% TritonX-100 or 0.1% TritonX-100, 1% PVP was used to dissolve and resuspend the fine powder of the plant tissue in the liquid nitrogen state. After incubating on ice for 30 min, it was inverted and mixed evenly every 10 min during this period. Subsequently, the entire solution was filtered through a cell sieve with a pore size of 30 μm. After collecting the filtrate, it was centrifuged at 4°C and 3500 g for 10 minutes, and the supernatant was discarded. The precipitate was resuspended and washed with 10 mL of the above-mentioned ice-cold nuclear extraction buffer, centrifuged at 4°C and 3500 g for 10 minutes, and the supernatant was discarded. The same washing operation was repeated once again, and finally, a nuclear precipitate was obtained. After the extraction was completed, the nuclear precipitate was resuspended, and nuclear counting and microscopic observation were carried out ( Figure 4 )

[0037] As Figure 4It can be seen that the cell nuclei appear blue, and the use of nuclear extraction buffer containing 0.05% TritonX-100 can obtain relatively pure cell nuclei with regular shapes; after extraction with nuclear extraction buffer containing 0.1% TritonX-100, the cell nuclei are overly lysed, and more irregularly shaped and smaller fluorescent signals appear.

[0038] Example 5: Nonspecific m6A-MTase reaction Take up to 6 million cell nuclei prepared in Example 3 or 4 for subsequent non-specific m6A-MTase treatment reaction. The specific steps are as follows: resuspend up to 6 million cell nuclei in 57.5 μl Buffer A (15 mM Tris-HCl (pH8.0), 15 mM NaCl, 60 mM KCl, 1 mM EDTA (pH8.0), 0.5 mM EGTA (pH8.0), 0.5 mM spermidine), and transfer to a 37°C PCR instrument. Add 200U non-specific m6A-MTase and 1.5 μl SAM (final concentration 0.8 mM) determined in Example 2 to each tube, and then use a wide-mouth pipette tip to pipette up and down 10 times to mix. After the reaction system is incubated at 37°C for 10 minutes, 6 μl 10% SDS (final concentration 1%) is added to terminate the reaction. Transfer all reaction products to a 15 ml tube pre-filled with 5 ml 2X CTAB lysis buffer for subsequent CTAB nucleic acid extraction.

[0039] Example 6: Sequencing and analysis The nucleic acid obtained in Example 5 was subjected to HiFi library construction and sequencing. The data was analyzed by fibertools and compared with the published data (see Mitchell R. Vollger et al., Synchronized long-read genome, methylome, epigenome and transcriptomeprofiling 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 ratio was basically consistent with that of the control group, indicating that the data obtained by the present invention can be used for chromosome accessibility analysis.

[0040] Table 1:

[0041] Note: HG001 is a human sample standard cell, and its data is sourced from the aforementioned literature Mitchell R. Vollger et al., Nat Genet, 2025. The inventors established the following reference standards based on experience: if the m6A Percent ratio is close to the literature data and does not exceed 15%, it is considered qualified.

[0042] In summary, the present invention provides a chromatin accessibility analysis method based on long-read sequencing applicable to most animal and plant tissues, optimized its process and parameters, and finally achieved similar effects and data outputs as the existing methods only applicable to cell line samples.

[0043] It should be noted that although some features of the present invention have been clarified through the above embodiments, they cannot be used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. The reaction reagents, reaction conditions, etc. involved in the long-fragment PCR reaction and single-molecule sequencing library construction can be adjusted and changed according to specific needs. Therefore, for those skilled in the art, within the scope of the concept and principle of the present invention, several simple substitutions can be made, and these should all be included in the protection scope of the present invention.

Claims

1. A method for chromatin accessibility analysis applicable to animal or plant tissues, comprising the following steps: a) Prepare a non-specific m6A-MTase and determine the enzyme activity of the non-specific m6A-MTase; b) Extract the cell nuclei of animal or plant tissues; c) Contact the cell nuclei obtained in step b) with the non-specific m6A-MTase and extract nucleic acids; and d) Construct a HiFi library and perform sequencing.

2. The method according to claim 1, wherein step b) comprises the following sub-steps: If extracting the cell nuclei of animal tissues, then b1) Grind the animal tissues into fine powder; b2) Resuspend the fine powder with a buffer solution composed of 8-12 mM Tris-HCl with pH 8.0, 2-4 mM MgCl2, 0.05-0.15% Tween-20, 8-10 mM NaCl, 0.5-1.5 mM DTT, 0.01-0.03% NP-40; b3) Immediately perform cell sieve filtration after incubating on ice for 5 min; b4) Centrifuge the filtrate at 500 g for 5 min and discard the supernatant; b5) Resuspend the cells with PBS + 1% BSA solution and then centrifuge at 500 g for 5 min and discard the supernatant; If extracting the cell nuclei of plant tissues, then b1) Grind the plant tissues into fine powder; b2) Use a buffer solution composed of 18-22 mM Tris-HCl with pH = 6.8, 2-4 mM MgCl2, 4-6 mM KCl, 2-3 mM EDTA, 0.03-0.07% TritonX-100, 0.5-1.5% PVP; b3) Incubate on ice for 30 min, invert and mix well, and then perform cell sieve filtration; b4) Centrifuge the filtrate at 3500 g for 10 min and discard the supernatant; b5) Resuspend the cells with the buffer solution in step b2) and then centrifuge at 3500 g for 10 min and discard the supernatant.

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

4. The method according to claim 1, wherein the enzyme activity determination of the non-specific m6A-MTase uses a DNA substrate with a sequence as shown in SEQ ID NO:

1.

5. The method according to claim 2 or 3, wherein the animal or plant tissues are cryopreserved and step b) is performed on ice or at 4°C.

6. The method according to claim 1, wherein the animal tissues are derived from mouse liver.

7. The method according to claim 1, wherein the plant tissues are derived from corn leaves.

8. The method according to claim 1, wherein the enzyme amount of the non-specific m6A-MTase in step c) is 200 U.

9. The method according to claim 1, wherein the CTAB method is used to extract nucleic acids in step c).

10. 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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