CN111154835A - Method for constructing ATAC-seq sequencing library - Google Patents
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Abstract
The invention belongs to the technical field of molecular biology, and particularly relates to a construction method of an ATAC-seq sequencing library. The construction process of the ATAC-seq sequencing library provided by the invention mainly comprises the processes of cell lysis and extraction of cell nucleus, transposition reaction and purification, PCR enrichment and purification, library quality inspection, computer installation and the like. The ATAC-seq sequencing library provided by the invention is suitable for cells, animals and plant tissues, is simple and convenient to operate, needs a small number of cells and has high sequencing flux.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a construction method of an ATAC-seq sequencing library.
Background
It is well known that most of the chromatin in genomes is tightly coiled around the nucleus, with only a small fraction of the area being relatively loose, and this fraction of the denuded DNA region of the anucleate body is called open chromatin (open chromatin), where DNA replication and gene transcription often occur. ATAC-seq (Assay for Transposase-Access chromosome with high-throughput sequencing of chromatin easy-open region) is a method for cutting a chromatin open region by using Tn5 Transposase, performing high-throughput sequencing by adding a sequencing primer, and identifying the positions of a transcription factor binding site and a nucleosome region through biological information analysis, thereby providing an effective method for researching gene regulation, DNA imprinting and the like. The acquisition of DNA sequences in an open state is a hotspot in epigenetic research, and ATAC-seq is the best interest for such research.
At present, most ATAC-seq sequencing experiments have certain requirements on the initial quantity of cells, the initial quantity generally required is 50000 cells, but the quantity of DNA contained in 50000 cells of different organisms is different, and the extracted cell nucleus needs to be stained and quantified by a fluorescence microscope. The commonly used nuclear stain DAPI is carcinogenic and fluorescence microscopes are expensive and not available in every laboratory. Counting with a fluorescence microscope requires a large amount of labor cost and time for a large sample size.
Chinese patent application CN109897885A discloses a library construction method suitable for ATAC-seq sequencing technology of cell lines and animal tissues, which is suitable for cell lines and animal tissues and can directly carry out quantitative operation through DNA content, however, the ATAC-seq library prepared by the method is only suitable for the condition that the DNA amount is 100ng-1000ng, and the method is not suitable for plant tissues.
In summary, when the sample size is large, the defects of large consumption of labor cost and time, narrow application range and high requirement on DNA quality generally exist in the prior art.
Disclosure of Invention
The invention aims to provide a method for constructing an ATAC-seq sequencing library aiming at the defects generally existing in the prior art. The ATAC-seq sequencing library provided by the invention is simple in operation method, wide in application range, applicable to cell samples, animal tissue samples and plant tissue samples and free of requirements on DNA quantity.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for constructing an ATAC-seq sequencing library comprises the following steps:
s1, cell lysis and cell nucleus extraction;
s2, transposition reaction and purification;
s3, PCR enrichment and purification;
s4, library quality inspection and computer installation.
Preferably, the extraction process of the cell lysis cell nucleus in step S1 is: for the cell sample, recovering and unfreezing the cells, and performing cell lysis to extract cell nuclei; for animal and plant tissue samples, cell suspensions are prepared and cell lysis is performed to extract cell nuclei.
Preferably, the specific process of cell nucleus lysis in step S1 is as follows: for cell samples, the samples are put in a water bath at 37 ℃ for resuscitation and thawing, are centrifuged for 5min at 500rpm and 4 ℃, are put on ice, are carefully sucked and discarded, and then are added with lysate for resuspension of cells, and are centrifuged for 5min at 500rpm and 4 ℃; for animal and plant tissue samples, taking about 30-50mg of tissue, grinding the tissue into powder under liquid nitrogen, placing the powder in a 2mL EP tube, quickly adding a resuspension liquid, fully resuspending the powder, and incubating the powder on ice for 10 min; filtering with 40uM cell sieve, sucking 1-1.5mL of filtrate into a new sterile 2mL EP tube, centrifuging at 500rpm and 4 ℃ for 5min, removing supernatant, and retaining precipitate; resuspending the precipitate, adding iodized solution of sandol, mixing, centrifuging at 4 deg.C and 1000rpm with a swing type centrifuge for 20min, discarding the supernatant, adding the resuspension solution, slightly blowing, sucking, and mixing.
Preferably, the concrete operation of the transposition reaction in step S2 is: the nucleus extracted in step S1 was added to a cell containing 5 XTBL 10 μ L, TTE Mix V505 μ L, ddH2O35. mu.L of the transposition reaction system is placed in a PCR instrument and reacted for 30min at the temperature of 37 ℃.
Preferably, the purification process in step S2 is: beads purification was performed by adding 50. mu.L of DNA followed by 2 washes with freshly prepared 80% volume ethanol solution and finally ddH2And (4) eluting with O.
Preferably, the specific operation of PCR enrichment in step S3 is: using the DNA product purified in step S2 as a template, a 50. mu.L reaction system was prepared: 5 XTAB 10. mu.L, PPM 5. mu.L, N5XX 5. mu.L, N7XX 5. mu.L, TAE 1. mu.L, 24. mu.L of purified DNA, mixed well and amplified according to the following procedure: first, extension at 72 ℃ for 3min, denaturation at 98 ℃ for 30s, followed by amplification for 15 cycles according to the following parameters: 15s at 98 ℃, 30s at 60 ℃ and 3min at 72 ℃; finally, extension was carried out at 72 ℃ for 5min, 4 ℃ Hold.
Preferably, in the purification in step S3, the product obtained by PCR amplification is added to magnetic beads and sorted twice at 0.55X and 1X to obtain the target size DNA.
The ATAC-seq sequencing library can be applied to cell lines, animal tissues and plant tissues.
The invention provides a library construction method suitable for ATAC-seq sequencing of cells, animals and plant tissues, and relates to the technical field of molecular biology.
Compared with the prior art, the ATAC-seq sequencing library provided by the invention has the following advantages:
(1) the method is simple and convenient to operate, accurate weighing of cells or animal and plant tissues is not required in early-stage treatment, transposition reaction is adopted, the number of required cells is small, and the flux of treated samples is high;
(2) when animal and plant tissue samples are treated by the method, animal and plant cell nucleuses are obtained by separating and purifying the tissue samples by using iodixanol solution, so that the separation efficiency and the cell nucleus purity are improved, and the method is harmless to human bodies;
(3) the method adopts twice magnetic bead sorting and purification after PCR amplification, so that the purification efficiency is higher, the automation degree is better, time and labor are saved, the length range of the DNA fragment required by sorting is more accurate, and a foundation is laid for successful library construction;
(4) the method has wide application range and is suitable for cell samples, animal tissue samples and plant tissue samples.
Drawings
FIG. 1 is a library quality control diagram of DNA of a cell sample obtained by the method of the present invention;
FIG. 2 is a library quality control chart of DNA of an animal or plant tissue sample obtained by the method of the present invention.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1 library construction method for ATAC-seq sequencing of cell samples
The library construction method for ATAC-seq sequencing of the cell sample comprises the following steps:
s1, cell lysis and cell nucleus extraction:
(1) cell thawing and reviving: placing the sample into a water bath kettle at 37 ℃ and slightly shaking to rapidly melt the cells;
(2) centrifuging at 4 deg.C for 5min at 500rpm, placing on ice, and carefully sucking off the supernatant;
(3) adding lysis solution to resuspend cells, centrifuging at 500rpm and 4 deg.C for 5min to obtain cell nucleus precipitate; the lysis solution is composed of 10mM Tris-Cl, pH7.4; 10mM NaCl; 3mM MgCl2(ii) a 0.1% (v/v) Igepal CA-630.
S2, transposition reaction and purification:
transposition reaction: prepare 50 μ L of reaction system: 5 XTTBL 10. mu.L, TTE Mix V505. mu.L, ddH2O35 mu L, then placing the mixture into a PCR instrument, and reacting for 30min at 37 ℃ to obtain nuclease-free water;
and (3) purification: adding 100 mu L of DNA Beads, uniformly mixing, standing at room temperature for 5min, putting on a magnetic frame, standing for 5min, carefully absorbing and discarding the supernatant, washing the magnetic Beads for 2 times by using an ethanol solution with the volume fraction of 80%, adding 26 mu L of nuclease-free water after the magnetic Beads are dried, uniformly mixing, standing at room temperature for 5min, putting on the frame for 5min, and absorbing 24 mu L for later use.
S3, PCR enrichment and purification:
PCR enrichment: a50. mu.L PCR system was prepared as shown in Table 1 below.
TABLE 1PCR enrichment System formulation
| Components | Volume of |
| Nuclease-free water obtained in S2 | 24μL |
| 5×TAB | 10μL |
| PPM | 5μL |
| N5XX | 5μL |
| N7XX | 5μL |
| TAE | 1μL |
| Total | 50μL |
Placing the mixture in a PCR instrument for reaction, wherein the reaction conditions are as follows: 3min at 72 ℃, 30s at 98 ℃ (15 s at 98 ℃, 30s at 60 ℃, 3min at 72 ℃) for 15 cycles, 5min at 72 ℃ and 4 ℃ for storage.
And (3) purification process: adding 27.5 mu L of DNA Beads, uniformly mixing, standing at room temperature for 5min, putting on a magnetic frame, standing for 5min, carefully sucking 72uL of supernatant, adding into a new sterile EP tube, adding 50 mu L of DNA Beads, uniformly mixing, standing at room temperature for 5min, putting on the magnetic frame, standing for 5min, carefully sucking and discarding the supernatant, washing the magnetic Beads with 80% ethanol for 2 times, drying the magnetic Beads in the air, adding 17 mu L of nuclease-free water, uniformly mixing, standing at room temperature for 5min, putting on the frame for 5min, sucking 15 mu L of nuclease-free water for later use.
S4, library quality inspection and computer installation: the library quality test was performed using Agilent 2100 bioanalyzer, and the results are shown in FIG. 1, wherein the size range of the library fragment is 200 and 700bp, and the main peaks are about 200bp, 500bp and 700bp, and are in the shape of sawtooth peaks.
Example 2 library construction method for ATAC-seq sequencing of plant or animal tissues
The method for constructing the ATAC-seq sequenced library of the plant or animal tissue comprises the following steps:
s1, cell lysis and cell nucleus extraction:
(1) grinding 30-50mg of tissue into powder under liquid nitrogen, and placing the powder in a 2mL EP tube;
(2) quickly adding the resuspension liquid, fully resuspending the powder, and incubating on ice for 10 min;
(3) filtering the sample of step 2 through a 40 μ M cell sieve, and aspirating about 1mL of filtrate into a fresh sterile 2mL EP tube;
(4) centrifuging at 1000rpm and 4 deg.C for 5min, discarding supernatant, and keeping precipitate;
(5) adding PBS as a resuspension, carefully resuspending the pellet;
(6) adding iodixanol solution, blowing, sucking and mixing;
(7) centrifuging at 10000rpm and 4 deg.C for 20 min;
(8) discarding the supernatant to leave a layer liquid;
(9) adding the heavy suspension, and gently blowing, sucking and mixing uniformly to obtain the cell nucleus precipitate.
S2, transposition reaction and purification:
transposition reaction: prepare 50 μ L of reaction system: 5 × TTBL10μL，TTE Mix V50 5μL，ddH2O35 mu L, then placing the mixture into a PCR instrument, and reacting for 30min at 37 ℃ to obtain nuclease-free water;
and (3) purification process: adding 100 mu L of DNA Beads, uniformly mixing, standing at room temperature for 5min, putting on a magnetic frame, standing for 5min, carefully absorbing and discarding the supernatant, washing the magnetic Beads for 2 times by using an ethanol solution with the volume fraction of 80%, adding 26 mu L of nuclease-free water after the magnetic Beads are dried, uniformly mixing, standing at room temperature for 5min, putting on the frame for 5min, and absorbing 24 mu L for later use.
S3, PCR enrichment and purification
And (3) PCR enrichment process: a50. mu.L PCR system was prepared as shown in Table 1 below.
TABLE 1PCR enrichment System formulation
Placing the mixture in a PCR instrument for reaction, wherein the reaction conditions are as follows: 3min at 72 ℃, 30s at 98 ℃ (15 s at 98 ℃, 30s at 60 ℃, 3min at 72 ℃) for 15 cycles, 5min at 72 ℃ and 4 ℃ for storage.
And (3) purification process: adding 27.5 mu L of DNA Beads, uniformly mixing, standing at room temperature for 5min, putting on a magnetic frame, standing for 5min, carefully sucking 72uL of supernatant, adding into a new sterile EP tube, adding 50 mu L of DNA Beads, uniformly mixing, standing at room temperature for 5min, putting on the magnetic frame, standing for 5min, carefully sucking and discarding the supernatant, washing the magnetic Beads with 80% ethanol for 2 times, drying the magnetic Beads in the air, adding 17 mu L of nuclease-free water, uniformly mixing, standing at room temperature for 5min, putting on the frame for 5min, sucking 15 mu L of nuclease-free water for later use.
S4, library quality inspection and computer installation: the library quality test was performed using Agilent 2100 bioanalyzer, and the results are shown in FIG. 2, wherein the size range of the library fragment is 200 and 700bp, and the main peaks are about 200bp, 500bp and 700bp, and have a sawtooth peak shape.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. A method for constructing an ATAC-seq sequencing library is characterized by comprising the following steps:
s1, cell lysis and cell nucleus extraction;
s2, transposition reaction and purification;
s3, PCR enrichment and purification;
s4, library quality inspection and computer installation.
2. The method for constructing the ATAC-seq sequencing library of claim 1, wherein the extraction process of the cell lysis nuclei in the step S1 is as follows: for the cell sample, recovering and unfreezing the cells, and performing cell lysis to extract cell nuclei; for animal and plant tissue samples, cell suspensions are prepared and cell lysis is performed to extract cell nuclei.
3. The method for constructing the ATAC-seq sequencing library of claim 2, wherein the specific process of cell nucleus lysis in step S1 is as follows: for cell samples, the samples are put in a water bath at 37 ℃ for resuscitation and thawing, are centrifuged for 5min at 500rpm and 4 ℃, are put on ice, are carefully sucked and discarded, and then are added with lysate for resuspension of cells, and are centrifuged for 5min at 500rpm and 4 ℃; for animal and plant tissue samples, taking about 30-50mg of tissue, grinding the tissue into powder under liquid nitrogen, placing the powder in a 2mL EP tube, quickly adding a resuspension liquid, fully resuspending the powder, and incubating the powder on ice for 10 min; filtering with 40uM cell sieve, sucking 1-1.5mL of filtrate into a new sterile 2mLEP tube, centrifuging at 500rpm and 4 ℃ for 5min, removing supernatant, and retaining precipitate; resuspending the precipitate, adding iodized solution of sandol, mixing, centrifuging at 4 deg.C and 1000rpm with a swing type centrifuge for 20min, discarding the supernatant, adding the resuspension solution, slightly blowing, sucking, and mixing.
4. As in claimThe method for constructing the ATAC-seq sequencing library as claimed in claim 1, wherein the transposition reaction in step S2 is specifically performed by: the nucleus extracted in step S1 was added to a mixture containing 5 XTBL 10. mu.L, TTE Mix V505. mu.L, ddH2O35 μ L in a transposition reaction system, and reacting for 30min at 37 ℃ in a PCR instrument.
5. The method for constructing the ATAC-seq sequencing library of claim 1, wherein the purification process in step S2 is: beads purification was performed by adding 50. mu.L of DNA followed by 2 washes with freshly prepared 80% volume ethanol solution and finally ddH2And (4) eluting with O.
6. The method for constructing the ATAC-seq sequencing library of claim 1, wherein the PCR enrichment of step S3 is performed by the following steps: using the DNA product purified in step S2 as a template, a 50. mu.L reaction system was prepared: 5 XTAB 10. mu.L, PPM 5. mu.L, N5XX 5. mu.L, N7XX 5. mu.L, TAE 1. mu.L, 24. mu.L of purified DNA, mixed well and amplified according to the following procedure: first, extension at 72 ℃ for 3min, denaturation at 98 ℃ for 30s, followed by amplification for 15 cycles according to the following parameters: 15s at 98 ℃, 30s at 60 ℃ and 3min at 72 ℃; finally, extension was carried out at 72 ℃ for 5min, 4 ℃ Hold.
7. The method of claim 6, wherein the purification in step S3 is performed by adding the PCR amplified product to magnetic beads and performing 0.55X and 1X sorting to obtain DNA of desired size.
8. Use of the ATAC-seq sequencing library of any of claims 1-7 in cell line, animal tissue and plant tissue studies.
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| CN111763721A (en) * | 2020-07-14 | 2020-10-13 | 武汉爱基百客生物科技有限公司 | ATAC-seq method suitable for plants |
| CN112410403A (en) * | 2020-11-04 | 2021-02-26 | 苏州京脉生物科技有限公司 | Preparation method and kit of ATAC-seq library with accurate quantification |
| CN112662737A (en) * | 2020-12-31 | 2021-04-16 | 广州基迪奥生物科技有限公司 | Preparation method and application of animal cell nucleus suspension |
| WO2022056704A1 (en) * | 2020-09-16 | 2022-03-24 | 深圳华大生命科学研究院 | Method for analyzing cell epigenomics from multiple dimensions |
| CN114395638A (en) * | 2021-12-25 | 2022-04-26 | 武汉爱基百客生物科技有限公司 | ATAC-seq method suitable for high-starch fruits |
| CN114686564A (en) * | 2020-12-31 | 2022-07-01 | 深圳华大生命科学研究院 | DNA sample preparation method suitable for micro sample |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111763721A (en) * | 2020-07-14 | 2020-10-13 | 武汉爱基百客生物科技有限公司 | ATAC-seq method suitable for plants |
| WO2022056704A1 (en) * | 2020-09-16 | 2022-03-24 | 深圳华大生命科学研究院 | Method for analyzing cell epigenomics from multiple dimensions |
| CN112410403A (en) * | 2020-11-04 | 2021-02-26 | 苏州京脉生物科技有限公司 | Preparation method and kit of ATAC-seq library with accurate quantification |
| CN112662737A (en) * | 2020-12-31 | 2021-04-16 | 广州基迪奥生物科技有限公司 | Preparation method and application of animal cell nucleus suspension |
| CN112662737B (en) * | 2020-12-31 | 2021-08-24 | 广州基迪奥生物科技有限公司 | Preparation method and application of animal cell nucleus suspension |
| CN114686564A (en) * | 2020-12-31 | 2022-07-01 | 深圳华大生命科学研究院 | DNA sample preparation method suitable for micro sample |
| CN114686564B (en) * | 2020-12-31 | 2023-10-24 | 深圳华大生命科学研究院 | DNA sample preparation method suitable for micro sample |
| CN114395638A (en) * | 2021-12-25 | 2022-04-26 | 武汉爱基百客生物科技有限公司 | ATAC-seq method suitable for high-starch fruits |
| CN114395638B (en) * | 2021-12-25 | 2024-02-09 | 武汉爱基百客生物科技有限公司 | ATAC-seq method suitable for high starch fruits |
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