CN111763721A - ATAC-seq method suitable for plants - Google Patents
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Abstract
The invention discloses an ATAC-seq method suitable for plants, which comprises the steps of grinding plant tissues by liquid nitrogen, extracting complete cell nucleuses by cell lysis, transposition reaction and purification, PCR amplification, library quality inspection and on-machine sequencing, wherein the cell lysis process needs to be sequentially treated by a nuclear extraction buffer solution containing sucrose with different concentrations, and then a nuclear lysis solution is added for lysis. The method can effectively remove organelles such as chloroplast and the like, saccharides, phenols and the like in the plant tissue, reduces the influence of the organelles on the subsequent library construction and sequencing analysis result, reduces the sample loss, is convenient and quick to operate, has wide applicability, and is particularly suitable for the plant tissue with high polysaccharide and polyphenol content and small sample amount.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to an ATAC-seq method suitable for plants.
Background
The DNA of the eukaryote is not exposed outside but combined with the histone, namely the DNA is wound on the histone (namely forming nucleosome) to form a moniliform structure; then, further folding, compression, and with the assistance of other framework proteins, chromosomes are formed. The higher order structure of DNA is required to be solved during the process of DNA replication and gene transcription, and the partially opened chromatin is the open chromatin region which is an important binding site of transcription factors and transcription elements. Therefore, obtaining DNA sequences in an open state is a hotspot in epigenetics research, and can be used for researching the regulation mechanism of gene expression.
ATAC-seq (Assay for Transposase-Access chromosome with high-throughput sequencing) refers to a technique for detecting Transposase Accessible Chromatin using high-throughput sequencing. The regulatory regions are labeled by Tn5 transposase inserting the sequenced linker molecule into an "accessible" region on the genome, while the less sterically hindered accessible chromatin makes transposition unlikely, allowing the identification of transcription factor binding sites, the location of initiation of transcriptional activity, nucleosome and nucleosome modifications, enhancers and insulators in cell lines and tissue samples. Compared with the traditional experimental method, the ATAC-seq technology has stronger repeatability and simpler operation, and can obtain good signal to noise ratio only by a small amount of cells or tissues, thus being the first choice technical method for researching chromatin openness.
To date, there have been many studies relating to ATAC-seq technology, particularly with regard to the dynamic changes in chromatin opening regions at different stages of development. However, the ATAC-seq technology has wide application in cell lines and animal tissues, but not in plants, because ATAC-seq has not a good effect in some plant tissues with high polysaccharide polyphenol content. If this problem could be solved, future ATAC-seq technology could be applied to the study of multiple species or multiple biological processes.
Disclosure of Invention
In view of the above, the invention provides an ATAC-seq method suitable for plants, which has wide applicability, particularly has good detection effect on plant tissues with high polysaccharide and polyphenol content, and has the advantages of less sample consumption and short experiment time consumption.
The technical scheme of the invention is realized as follows:
the invention provides an ATAC-seq method suitable for plants, which comprises the following steps:
s1, grinding plant tissues by using liquid nitrogen;
s2, cell lysis and complete cell nucleus extraction, wherein the steps are sequentially carried out by treating with a cell nucleus extraction buffer solution containing sucrose with different concentrations, and then adding a cell nucleus lysis solution for lysis;
s3, transposition reaction and purification;
s4, PCR amplification:
s5, performing library quality inspection;
and S6, sequencing on the computer.
Based on the above technical solution, preferably, in step S2, the core Extraction buffers containing sucrose in different concentrations include a core Extraction Buffer a (NEB-a), a core Extraction Buffer B (NEB-B), and a core Extraction Buffer C (NEB-C), and the sucrose concentrations in the core Extraction Buffer A, B, C are 0.4M, 0.25M, and 1.7M, respectively.
Further, preferably, the nuclear extraction buffer A comprises 0.4M sucrose, 10mM Tris-HCl (pH8.0), 10mM MgCl25mM β -ME, 0.1mM PMSF and 1 × protease inhibitor, wherein the nuclear extraction buffer B comprises 0.25M sucrose, 10mM Tris-HCl (pH8.0), 10mM MgCl 21% (v/v) Triton X-100, 5mM β -ME, 0.1mM PMSF and 1 × protease inhibitor, wherein the nuclear extraction buffer C comprises 1.7M sucrose, 10mM Tris-HCl (pH8.0), 2mM MgCl20.15% (v/v) Triton X-100, 5mM β -ME, 0.1mM PMSF and 1 × protease inhibitor.
Further, preferably, the process of extracting the whole cell nucleus by cell lysis in step S2 specifically includes the following steps: transferring the ground tissue into a pre-cooled nuclear extraction buffer solution A, and cracking for 15 min; filtering with double-layer Miracloth, collecting liquid in a new centrifuge tube, centrifuging and removing supernatant; resuspending the precipitate with 1mL of a nuclear extraction buffer B, centrifuging, removing the supernatant, and repeatedly washing the precipitate for 3-5 times; resuspending the precipitate with 300. mu.L of the nuclear extraction buffer C, adding it to 300. mu.L of the precooled nuclear extraction buffer C in a new centrifuge tube, centrifuging 3 times with density gradient, and removing the supernatant; resuspend the pellet with 1mL of pre-cooled NP-40 free buffer; cell nuclei were counted using trypan blue staining; putting the NP-40-free buffer solution suspension with the volume corresponding to 50000 cell nuclei into a new centrifugal tube, centrifuging and removing supernatant; resuspending the pellet with 50. mu.L of nuclear lysate, and reacting on ice for 15 min; add 1mL of pre-cooled NP-40 free buffer and mix well, centrifuge and remove supernatant to get intact nuclei.
Further, preferably, the density gradient centrifugation is performed at 2200g and 4 ℃ for 15 min.
On the basis of the technical proposal, the device comprises a shell,preferably, in step S2, the nuclear lysate includes 10mM Tris-HCl (pH7.4), 10mM NaCl, 3mM MgCl20.1% (v/v) NP-40 and 1 × protease inhibitors.
On the basis of the above technical solution, preferably, in step S3, the transposition reaction is specifically performed by: immediately, 2.5. mu.L of Tn5 transposase, 25. mu.L of TD transposase buffer and 22.5. mu.L of enzyme-free water were added to the intact nuclei extracted in step S2, and incubated at 37 ℃ for 30 min.
Based on the above technical solution, preferably, in step S4, the reaction system of the PCR amplification is: 24 μ L of the DNA purified in step S3, 10 μ L of 5 XTAB, 5 μ L of PPM, 5 μ L N5, 5 μ L N7, 1 μ L of TAE.
Based on the above technical solution, preferably, in step S4, the reaction procedure of PCR amplification is: heating the cover at 105 deg.C, and extending at 72 deg.C for 5 min; denaturation at 98 ℃ for 30 s; then amplifying for 11-15 cycles according to the following parameters: 10s at 98 ℃, 30s at 63 ℃ and 1min at 72 ℃; finally, extension is carried out for 5min at 72 ℃.
Compared with the prior art, the ATAC-seq method suitable for plants has the following beneficial effects:
(1) the invention can effectively reduce the ratio of plant cell fragments to secondary metabolites by the nuclear extraction buffer solution A and the double-layer membrane filtration, and simultaneously, Mg in the buffer solution2+Chromatin that stabilizes the nucleus; secondly, Triton X-100 in the buffer solution B for nuclear extraction is used as a nonionic detergent, so that the release of cell nuclei can be promoted, a chloroplast membrane can be damaged, and the proportion of pigments in chromatin can be effectively reduced through multiple washing of the buffer solution B; in addition, in the nuclear extraction buffer solution C, the sedimentation coefficients of organelle DNA and chromatin in high-concentration sucrose are different, and the proper centrifugal rotating speed is adopted, so that the residual rate of the organelle DNA can be greatly reduced;
(2) the method adopts Tn5 transposase to segment the nuclear DNA, carries out quantitative operation according to the DNA content, and has wide applicability;
(3) the library construction method adopted by the invention eliminates the complicated steps of DNA fragmentation, terminal repair, adaptor connection and the like in the traditional method, simplifies the steps into one-step enzymatic reaction, shortens the experimental time and improves the library construction efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a quality control chart of ATAC library obtained by the method of the present invention.
FIG. 2 is a graph showing the results of the length distribution of the insert fragments in the ATAC library obtained by the method of the present invention.
FIG. 3 is a schematic representation of the enrichment of peaks by sequencing according to the method of the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
This example proposes an ATAC-seq method suitable for plants, specifically taking cotton ovules as an example, aiming at the specificity of plant tissues, and the method comprises the following steps:
s1 grinding plant tissues by liquid nitrogen
A sample of 0.1g of clean cotton ovules was taken and ground with liquid nitrogen.
S2 Capture of intact nuclei
(1) Solution preparation
Preparing NEB-A buffer solution, wherein the NEB-A buffer solution comprises: 0.4M sucrose, 10mM Tris-HCl (pH8.0), 10mM MgCl25mM β -ME, 0.1mM PMSF, 1 × protease inhibitor;
preparing NEB-B buffer solution, which comprises the following steps: 0.25M sucrose, 10mM Tris-HCl (pH8.0), 10mM MgCl21% (v/v) Triton X-100, 5mM β -ME, 0.1mM PMSF, 1 × protease inhibitor;
preparing NEB-C buffer solution, which comprises the following steps: 1.7M sucrose, 10mM Tris-HCl (pH8.0), 2mM MgCl20.15% (v/v) Triton X-100, 5mM β -ME, 0.1mM PMSF, 1 × protease inhibitor;
preparing a buffer without NP-40, wherein the buffer comprises: 10mM Tris-HCl (pH7.4), 10mM NaCl, 3mM MgCl 21 × protease inhibitors;
preparing a nuclear lysis solution, wherein the method comprises the following steps: 10mM Tris-HCl (pH7.4), 10mM NaCl, 3mM MgCl20.1% (v/v) NP-40, 1 × protease inhibitor;
(2) transferring the ground sample into 1.5mL of precooled NEB-A buffer solution, and carrying out lysis for 15 min;
(3) filtering with double-layer Miracloth, collecting liquid in a new centrifuge tube, centrifuging at 6000g and 4 deg.C for 10min, and removing supernatant;
(4) resuspending the precipitate with 1mL of NEB-B buffer, centrifuging at 12000g and 4 deg.C for 10min, removing supernatant, and washing the precipitate for 3-5 times until pigment is washed off;
(5) resuspending the pellet with 300. mu.L of NEB-C buffer, adding 300. mu.L of precooled NEB-C buffer to a new 1.5mL centrifuge tube, centrifuging at 2200g and 4 ℃ for 15min, centrifuging the step with density gradient for 3 times, and removing the supernatant;
(6) resuspend the pellet with 1mL of pre-cooled NP-40 free buffer;
(7) cell nuclei were counted under microscope using trypan blue staining at a final concentration of 0.04%;
(8) placing the suspension without NP-40 buffer solution with the volume corresponding to 50000 cell nucleus into a new EP tube, centrifuging at 12000g and 4 ℃ for 10min, and removing the supernatant;
(9) resuspending the pellet with 50. mu.L of nuclear lysate, and reacting on ice for 15 min; adding 1mL of precooled NP-40-free buffer solution, mixing uniformly, centrifuging at 12000g and 4 ℃ for 10min, and removing supernatant to obtain treated cell nuclei.
S3 transposition reaction and purification
Adding 2.5 μ L of Tn5 transposase, 25 μ L of TD transposable buffer solution and 22.5 μ L of enzyme-free water immediately into cell nucleus, and incubating at 37 deg.C for 30 min;
the transposition reaction was immediately followed by Purification with Qiagen MinElute PCR Purification Kit and 25. mu.L of Elution Buffer was added to elute the DNA.
S4 PCR amplification
Preparing a PCR reaction system: mu.L of the above eluted DNA, 10. mu.L of 5 XTAB, 5. mu.L of PPM, 5. mu. L N5, 5. mu. L N7, 1. mu.L of TAE; wherein, the nucleotide sequence of the primer N5 is 5 '-AATGATACGGCGACCACCGAGATCTACAC [ TAGATCGC ] TCGTCGGCAGCGTC-3', and the nucleotide sequence of the primer N7 is 5 '-CAAGCAGAAGACGGCATACGAGAT [ ATCACGTT ] GTCTCGTGGGCTCGG-3'.
PCR reaction procedure: heating the cover at 105 deg.C, and extending at 72 deg.C for 5 min; denaturation at 98 ℃ for 30 s; amplification was performed for 11-15 cycles with the following parameters: 10s at 98 ℃, 30s at 63 ℃ and 1min at 72 ℃; finally, extension is carried out for 5min at 72 ℃.
The library was purified using the Qiagen MinElute PCR Purification Kit and was finally eluted with 20. mu.L of ElutionBuffer.
S5 library quality control
And (3) carrying out concentration and quality detection on the purified library, wherein the concentration and quality detection mainly comprises the following steps:
and (3) concentration detection: with Life Invitrogen
3.0 detecting the concentration of the library;
and (3) quality detection: the quality of the library was checked with Q-sep1 analysts.
S6, sequencing on computer
The constructed library was sequenced using the Illumina HiSeq 4000 platform for PE 150.
Example 2
1. Library quality detection
Nucleosome-winding DNA consists of two parts: the segment wrapped around the nucleosome, 146bp in length, and the segment with naked edges, of variable length.
According to the ATAC-Seq experiment of example 1, the obtained DNA fragment (about 60bp) between nucleosomes and about 200bp fragment of 136bp linker are the first peak; the fragment 146bp wound with a nucleosome is added with a joint fragment of 136bp and a fragment with naked edges, and a second peak appears at about 350-400 bp; the fragment 146bp x 2, which is the intertwined two nucleosomes, plus the 136bp linker, the 60bp fragment between the two nucleosomes and the denuded-margin fragment, gave a third peak around 550-600 bp. Because of the varying size of the segment where the edge is exposed, a range of fluctuations in the position of each peak can occur. As a result, as shown in FIG. 1, peaks 1, 2 and 3 appeared at about 190bp, 350bp and 650bp, respectively, and the results were all normal. Meanwhile, the distribution length of the sequencing was counted by bioinformatics analysis, and the fragment size was consistent with the theoretical size (fig. 2).
Enrichment of peaks in the vicinity of TSS
Most of the open regions obtained by ATAC-seq are transcription active regions, so peaks can be enriched near the transcription starting point, and FIG. 3 shows the enrichment condition of peaks, and the darker the region closer to TSS, the better the enrichment effect. This result further demonstrates the effectiveness of the method of the invention.
Example 3
In this example, ATAC-seq data of cotton were obtained based on the method of example 1, as shown in Table 1:
TABLE 1 ATAC-seq data for Cotton
As can be seen from the data in Table 1, the ATAC-Seq data of cotton obtained by the ATAC-Seq method of the invention has high comparison rate.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. An ATAC-seq method suitable for plants, comprising the steps of:
s1, grinding plant tissues by using liquid nitrogen;
s2, cell lysis and complete cell nucleus extraction, wherein the steps are sequentially carried out by treating with a cell nucleus extraction buffer solution containing sucrose with different concentrations, and then adding a cell nucleus lysis solution for lysis;
s3, transposition reaction and purification;
s4, PCR amplification:
s5, performing library quality inspection;
and S6, sequencing on the computer.
2. The ATAC-seq method suitable for plants according to claim 1, wherein: in step S2, the core extraction buffers containing different concentrations of sucrose include a core extraction buffer a, a core extraction buffer B, and a core extraction buffer C, and the concentrations of sucrose contained in the core extraction buffer A, B, C are 0.4M, 0.25M, and 1.7M, respectively.
3. The ATAC-seq method suitable for plants according to claim 2, wherein: the nuclear extraction buffer solution A comprises 0.4M sucrose, 10mM Tris-HCl (pH8.0), 10mM MgCl25mM β -ME, 0.1mM PMSF and 1 × protease inhibitor, wherein the nuclear extraction buffer B comprises 0.25M sucrose, 10mM Tris-HCl (pH8.0), 10mM MgCl21% (v/v) Triton X-100, 5mM β -ME, 0.1mM PMSF and 1 × protease inhibitor, wherein the nuclear extraction buffer C comprises 1.7M sucrose, 10mM Tris-HCl (pH8.0), 2mM MgCl20.15% (v/v) Triton X-100, 5mM β -ME, 0.1mM PMSF and 1 × protease inhibitor.
4. The ATAC-seq method suitable for plants as claimed in claim 3, wherein the process of extracting the complete cell nucleus by cell lysis in step S2 comprises the following steps: transferring the ground tissue into a pre-cooled nuclear extraction buffer solution A, and cracking for 15 min; filtering with double-layer Miracloth, collecting liquid in a new centrifuge tube, centrifuging and removing supernatant; resuspending the precipitate with 1mL of a nuclear extraction buffer B, centrifuging, removing the supernatant, and repeatedly washing the precipitate for 3-5 times; resuspending the precipitate with 300. mu.L of the nuclear extraction buffer C, adding it to 300. mu.L of the precooled nuclear extraction buffer C in a new centrifuge tube, centrifuging 3 times with density gradient, and removing the supernatant; resuspend the pellet with 1mL of pre-cooled NP-40 free buffer; cell nuclei were counted using trypan blue staining; putting the NP-40-free buffer solution suspension with the volume corresponding to 50000 cell nuclei into a new centrifugal tube, centrifuging and removing supernatant; resuspending the pellet with 50. mu.L of nuclear lysate, and reacting on ice for 15 min; add 1mL of pre-cooled NP-40 free buffer and mix well, centrifuge and remove supernatant to get intact nuclei.
5. The ATAC-seq method suitable for plants according to claim 4, wherein: the density gradient centrifugation condition is 2200g, 4 ℃ centrifugation for 15 min.
6. The ATAC-seq method suitable for plants according to claim 1, wherein: in step S2, the lysate includes 10mM Tris-HCl (pH7.4), 10mM NaCl, 3mM MgCl20.1% (v/v) NP-40 and 1 × protease inhibitors.
7. The ATAC-seq method applicable to plants as claimed in claim 1, wherein in step S3, the transposition reaction is specifically operated as follows: immediately, 2.5. mu.L of Tn5 transposase, 25. mu.L of TD transposase buffer and 22.5. mu.L of enzyme-free water were added to the intact nuclei extracted in step S2, and incubated at 37 ℃ for 30 min.
8. The ATAC-seq method applicable to plants as claimed in claim 1, wherein in step S4, the reaction system of the PCR amplification is: 24 μ L of the DNA purified in step S3, 10 μ L of 5 XTAB, 5 μ L of PPM, 5 μ L N5, 5 μ L N7, 1 μ LTAE.
9. The ATAC-seq method applicable to plants as claimed in claim 1, wherein in step S4, the reaction procedure of PCR amplification is as follows: heating the cover at 105 deg.C, and extending at 72 deg.C for 5 min; denaturation at 98 ℃ for 30 s; then amplifying for 11-15 cycles according to the following parameters: 10s at 98 ℃, 30s at 63 ℃ and 1min at 72 ℃; finally, extension is carried out for 5min at 72 ℃.
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