Improved method for detecting gastric cancer frozen tissue open chromatin
阅读说明：本技术 一种改进的胃癌冷冻组织开放染色质检测方法 (Improved method for detecting gastric cancer frozen tissue open chromatin ) 是由 郑晓斌 姜昊 陈海洋 冯宇亮 冯通 揭敏文 于 2020-04-03 设计创作，主要内容包括：本发明公开了一种改进的胃癌冷冻组织样品的开放染色质检测方法,把胃癌冷冻组织加入Stable buffer中研磨,将重悬后的样品过70μm滤膜,再次研磨,过40μm滤膜收集滤液离心,加入Stable buffer混匀,随后加入不同浓度的碘克沙醇溶液梯度离心,得到含细胞核的滤液；然后加入Restable Buffer-Tween稀释,对滤液中的细胞核进行计数,挑取50,000细胞核,离心并去除上清,向沉淀中加入转座体系,进行转座反应；然后对已转座的DNA进行纯化,再进行PCR扩增,最后将PCR产物纯化后测序。所述方法能有效对临床胃癌冷冻组织进行染色质结构检测,该方法可结合患者的临床数据,从而建立基于染色质结构检测与患者预后的模型,对于促进精准医疗的发展,具有重要的意义。(The invention discloses an improved open chromatin detection method of a gastric cancer frozen tissue sample, which comprises the steps of adding a gastric cancer frozen tissue into a Stable buffer for grinding, filtering a resuspended sample through a 70-micron filter membrane, grinding again, filtering through a 40-micron filter membrane, collecting filtrate for centrifugation, adding the Stable buffer for uniform mixing, and then adding iodixanol solutions with different concentrations for gradient centrifugation to obtain filtrate containing cell nuclei; then adding a Restable Buffer-Tween for dilution, counting the cell nucleuses in the filtrate, picking 50,000 cell nucleuses, centrifuging and removing a supernatant, adding a transposition system into the precipitate, and carrying out transposition reaction; then purifying the transposed DNA, performing PCR amplification, and finally purifying and sequencing the PCR product. The method can effectively detect the chromatin structure of the clinical gastric cancer frozen tissue, and can be combined with clinical data of patients, thereby establishing a model based on the chromatin structure detection and the patient prognosis, and having important significance for promoting the development of accurate medical treatment.)
1. An improved method for detecting gastric cancer frozen tissue open chromatin, which is characterized by comprising the following steps:
s1, adding frozen gastric cancer tissues into a Stable buffer for grinding, filtering a resuspended sample through a 70-micron filter membrane, grinding again, filtering through a 40-micron filter membrane, collecting filtrate, centrifuging to remove supernatant, adding the Stable buffer for mixing uniformly, and then adding 50%, 30% and 40% iodixanol solutions with different concentrations in sequence for gradient centrifugation to obtain filtrate containing cell nuclei; the Sable buffer comprises the following components in final concentration: 5mM CaCl2，3mM Mg(Ac)210mM Tris pH7.8, 0.1mM EDTA, 0.10% NP40, 320mM sucrose, 0.02mM PMSF protease inhibitor, 0.2mM β -mercaptoethanol;
s2, taking the filtrate containing the cell nuclei in the step S1, adding Restable Buffer-Tween for dilution, then counting the cell nuclei in the filtrate, picking 50,000 cell nuclei, centrifuging to remove supernatant, adding a transposition system into the precipitate, and carrying out transposition reaction; then purifying the transposed DNA, performing PCR amplification, and finally purifying a PCR product and then sequencing; the Restable Buffer-Tween contains the following components at the final concentrations: 10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2，0.10％Tween 20。
2. The method according to claim 1, wherein the mass-to-volume ratio of the gastric cancer frozen tissue to Stableb buffer in step S1 is 10-20 mg: 1-2 mL.
3. The method of claim 1, wherein the gradient centrifugation solution of step S1 is prepared by adding 50% iodixanol solution, resuspending and mixing to a final concentration of 25%, adding 30% iodixanol solution below the 25% solution layer, adding 40% iodixanol solution below the 30% solution layer, and centrifuging.
4. The method of claim 1, wherein the volume ratio of the filtrate containing cell nuclei and RestableBuffer-Tween in step S2 is 2: 5 to 6.
5. The method of claim 1, wherein the transposome in step S2 is 25 μ L TD buffer, 2.5 μ L DDW, 2.5 μ L TDE1, 16.5 μ L PBS, 0.5 μ L1% digitonin, 0.5 μ L10% Tween-20.
6. The method as claimed in claim 1, wherein the transposition reaction in step S2 is 1000rpm, 37 ℃ and 30 min.
7. The method of claim 1, wherein the step S2 is performed by purifying the transposed DNA with AMPure beads: adding AMpure beads into the transposed DNA, mixing uniformly, standing, placing on a magnetic separation rack for standing separation, removing the supernatant, washing with 80% ethanol solution, repeating the step, removing the residual ethanol solution, and finally eluting with EB solution or DDW.
8. The method of claim 1, wherein the PCR product is purified in step S2 using ampurebes: adding AMpure beads with the final concentration of 0.65 multiplied by the weight of the PCR reaction product, uniformly mixing, standing, placing on a magnetic separation frame for standing separation, transferring the supernatant into a new tube, adding AMpure beads with the final concentration of 1.8 multiplied by the weight of the PCR reaction product, uniformly mixing, standing, placing on a magnetic separation frame for standing separation, removing the supernatant, washing with 80% ethanol solution, repeating the step, removing the residual ethanol solution, and finally eluting with EB solution or DDW.
9. Use of the method of any one of claims 1 to 8 for the detection of gastric cancer frozen tissue open chromatin.
10. A product for open chromatin detection in frozen tissues of gastric cancer, which comprises Stable Buffer as defined in claim 1, iodixanol solution of different concentrations and Restable Buffer-Tween.
Technical Field
The invention relates to the technical field of biology, in particular to an improved method for detecting open chromatin of frozen tissues of gastric cancer.
Background
In human cell cells, DNA binds to a variety of proteins to form chromatin with a multilayered structure. However, only 1% of chromatin is active and is called active chromatin (open chromatin). The opening and closing of these active chromatin ((e.g., promoters, enhancers), and their location in the nucleus are closely related to the regulation of gene expression and the biological behavior of the cell.
Researchers have used DNase I digestion and high throughput sequencing methods (DNase-Seq) to reveal the linear position of active chromatin in the genome. However, this method requires 107The above-graded number of cells and the large time investment limit their application in clinical biopsy. The occurrence of ATAC-seq (Assay for transpose-accessible chromatographic using sequence) technology reported by professor Greenleaf university and Howard Chang at the end of 2013 breaks through the limitation, provides a completely new means for studying chromatin accessibility (ATAC-seq inserts sequenced adapters into "accessible" regions on the genome through Tn5 transposase to mark the regulatory regions, can obtain a high-quality chromatin open structure map with only 500-50000 cells as the sequencing depth increases (up to 200millions reads), and can even directly see the binding sites of transcription factors (where transcription factors bind, the transcription factors are not cleaved by Tn5 enzyme to form a blot (FOOTPRINT), greatly simplifies the experimental process and sample loss, and Howard Chang who is one of the ATAC-seq technical inventors can track the apparent time of the patient during the HDI (HDI) drug therapy process by the patient, the dynamic regulation and control mechanism of epigenetic genome and key transcription factor to the drug sensitivity of the patient is deeply researched on the time scale for the first time, and the sensitivity of the patient to the HDACI anti-cancer drug is accurately predicted, so that the method is novelProvides a basis for a targeted treatment scheme and also establishes a template for researching epigenetic regulation mechanisms of precise medicine of other diseases. In addition, the DNA copy number of normal, mixed, host, tumor cells was analyzed by ATAC-seq data, the difference of the copy number of human genomic DNA among each cell type was shown using human chromosome map, Howard Chang confirmed a plurality of reported genetic variations such as the increase of chr4q, chr8q and chr17q, the deletion of chr10q and chr17p, etc. in mixed and tumor cells, and found that these variations did not occur in the host cells of normal persons or patients. Interestingly, Howard channel finds that the difference of DNA copy number at the genetic level cannot reflect the sensitivity of a patient to a drug, and on the contrary, a transcription factor regulation and control mechanism at the epigenetic level can accurately predict the response condition of the patient to the drug, so that the significance of the research on the epigenetic genome of the patient is explained again, and the limitation of the conventional gene sequencing in the current precise medical research is also prompted. Meanwhile, the identified transcription factors can well distinguish normal persons from patients in different cancer stages, and based on the fact, the specific transcription factor nucleic acid sequences of the normal persons, the cancer patients and the cancer patients in different stages can be designed into target nucleotide probes on the gene chip to detect whether the patients have cancer or are in the cancer stage, and the new specific probes greatly expand the existing classical detection probes, so that the result of the gene chip is more accurate.
Unfortunately, classical ATAC-seq techniques can only perform transposition experiments for Tn5 in fresh cells, otherwise the quality of the library is very poor. Therefore, the application of ATAC-seq in clinical sample detection is severely limited. Patent CN 108300774a discloses an ATAC-seq method for frozen tissues, but when the method is used for ATAC-seq research of frozen tissues of gastric cancer, the quality of the constructed library is still poor. Therefore, there is a need to provide an improved method for detecting frozen tissue open chromatin in gastric cancer.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defect and the defect that the existing ATAC-seq technology can not be applied to the frozen tissues of the gastric cancer). An improved method for detecting open chromatin of frozen tissue of gastric cancer is provided, which can effectively detect the chromatin structure of frozen tissue of clinical gastric cancer and has the result highly consistent with that of tissue detection in ENCODE. The method can effectively detect the chromatin structure of the tissue in the clinical specimen bank and combine the clinical data of the patient, thereby establishing a model based on the chromatin structure detection and the patient prognosis, and having important significance for promoting the development of accurate medical treatment.
Another object of the present invention is to provide a product for detecting frozen tissue open chromatin from gastric cancer.
The above object of the present invention is achieved by the following technical solutions:
an improved method for detecting gastric cancer frozen tissue open chromatin, which comprises the following steps:
s1, adding frozen gastric cancer tissues into a Stable buffer for grinding, filtering a resuspended sample through a 70-micron filter membrane, grinding again, filtering through a 40-micron filter membrane, collecting filtrate, centrifuging to remove supernatant, adding the Stable buffer for mixing uniformly, and then adding 50%, 30% and 40% iodixanol solutions with different concentrations in sequence for gradient centrifugation to obtain filtrate containing cell nuclei; the Sablebuffer comprises the following components at the final concentrations: 5mM CaCl2，3mM Mg(Ac)210mM Tris pH7.8, 0.1mM EDTA, 0.10% NP40, 320mM sucrose, 0.02mM PMSF protease inhibitor, 0.2mM β -mercaptoethanol;
s2, taking the filtrate containing the cell nuclei in the step S1, adding Restable Buffer-Tween for dilution, then counting the cell nuclei in the filtrate, picking 50,000 cell nuclei, centrifuging to remove supernatant, adding a transposition system into the precipitate, and carrying out transposition reaction; then purifying the transposed DNA, performing PCR amplification, and finally purifying a PCR product and then sequencing; the Restable Buffer-Tween contains the following components at the final concentrations: 10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2，0.10％
The inventor researches and finds that the classical cell nucleus extraction method and the method disclosed in the patent CN 108300774A are not suitable for gastric cancer frozen tissues, the number of cell nuclei required by subsequent researches cannot be obtained from limited gastric cancer frozen tissues, the transposition experiment of Tn5 is difficult to perform, and the quality of the prepared library is extremely poor. Compared with the classic ATAC-seq which counts a certain number of fresh primary cells or cell strains firstly and then carries out lysis by a lysis solution, the method can carry out preliminary lysis on the nuclear membrane of the cell nucleus while extracting the cell nucleus from the frozen tissue of the gastric cancer, and is convenient for subsequent experiments. The method for extracting cell nucleus can extract enough cell nucleus number from the limited frozen tissue of gastric cancer for subsequent experiments.
Preferably, the frozen tissue of the gastric cancer is liquid nitrogen or tissue preserved at-80 ℃.
Preferably, the mass-to-volume ratio of the gastric cancer frozen tissue to the Stable buffer is 10-20 mg: 1-2 mL; the key of ATAC-seq is that the cracking is insufficient, Tn5 cannot go in and the nuclear membrane can be damaged if the cracking is too much, so that a certain requirement is imposed on the ratio of the cracking liquid to the cell tissue.
Preferably, the mother liquor concentration of each component in the Stable buffer is as follows: 1M CaCl2，1M Mg(Ac)21M Tris pH7.8, 500mM EDTA, 10% NP40, 1M sucrose, 100mM PMSF protease inhibitor, 14.3mM β -mercaptoethanol.
Preferably, all of the agents are pre-chilled in order to ensure the integrity and viability of the nuclei in the tissue.
Specifically, the transposable system of 50,000 nuclei was 25. mu.L of TD buffer, 2.5. mu.L of DDW, 2.5. mu.L of TDE1(Tn5 enzyme), 16.5. mu.L of PBS, 0.5. mu.L of 1% digitonin, 0.5. mu.L of 10% Tween-20; the number of nuclei and the amount of TD (Tn5 enzyme) are of critical importance, and together they determine the distribution of DNA fragments produced. The transposable system contained 0.5. mu.L of 1% digitonin, 0.5. mu.L of 10% Tween-20, and the addition of these two detergents greatly reduced the background noise.
Specifically, the transposition is carried out on a constant-temperature blending instrument at 1000rpm and 37 ℃ for 30 min.
Specifically, the purification of the transposed DNA is carried out by using AMPure beads: adding AMpure beads into the transposed DNA, mixing uniformly, standing, placing on a magnetic separation rack for standing separation, removing supernatant, washing with 80% ethanol solution, repeating the step, removing residual ethanol solution, and finally eluting with EB solution.
Preferably, the volume ratio of the transposed DNA to the AMpure beads is 1: 2 to 3.
Specifically, the PCR amplification method after DNA purification is the same as the classical method.
Specifically, the PCR product was purified using AMPure beads: adding AMpure beads with the final concentration of 0.65 multiplied by the weight into a PCR reaction product, uniformly mixing, standing, placing on a magnetic separation frame for standing separation, transferring a supernatant into a new tube, adding AMpure beads with the final concentration of 1.8 multiplied by the weight, uniformly mixing, standing, placing on the magnetic separation frame for standing separation, removing a supernatant, washing with 80% ethanol solution, repeating the step, removing residual ethanol solution, and finally eluting with EB solution.
The classical ATAC-seq carries out column purification on the transposed DNA through Qiagen nImute kit, and PCR products are purified and sorted through a gel electrophoresis method; the traditional column-passing purification and gel electrophoresis methods cause more DNA loss, which causes the complexity of the library to be reduced (the DNA obtained after 50000 cell DNA transposes is less), while the invention can reduce the loss of the DNA (about 30 percent) by using AMPurebes to recover; removal of invalid sequencing fragments in the library: classical ATAC-seq did not remove the adaptor dimer and fragments larger than 600bp from the library (retaining all nucleosome patterns), however, if the experiment only focused on open chromatin regions rather than nucleosome localization information, it was not necessary to retain large fragments above 600bp (the efficiency of sequencing fragments above 600bp by a second generation sequencer was extremely low). Therefore, the AMPure beads are utilized, and through two rounds of AMpure bead screening (by controlling the proportion of different AMPure beads to a sample), the adaptordimer and a large fragment larger than 600bp are removed, and the effective sequencing proportion of the library DNA is greatly improved.
Meanwhile, the application of the method in the detection of the gastric cancer frozen tissue open chromatin is also within the protection scope of the invention.
The invention also requests to protect the application of the method in preparing a product for detecting the gastric cancer frozen tissue open chromatin.
A product for detecting the open chromatin of frozen tissues of gastric cancer contains said Stableb Buffer, iodixanol solutions with different concentrations and Restable Buffer.
Preferably, AMpure beads for DNA and PCR product purification are also included.
Preferably, the product further comprises a 40 μm cell strainer, a 70 μm cell strainer, an Illumina tag DNAbuffer, Illumina nexteratag DNA enzyme, 1% digitonin, 10% Tween-20, NEBNextHigh-fidelity 2xPCR Master Mix, 100x SYBR Green I.
Preferably, the product is a kit. The kit can be used for performing open chromatin detection on clinical gastric cancer frozen tissues, and the result of the kit can be highly consistent with the ENCODE tissue detection result.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an improved method for detecting open chromatin of frozen tissues of gastric cancer, which can effectively detect the chromatin structure of clinical frozen tissues of gastric cancer by improving a method for extracting nuclei of frozen tissues of gastric cancer and greatly improving the quality of a library by using an AMpure beads technology. The method can effectively detect the chromatin structure of the tissue in the clinical specimen bank and combine the clinical data of the patient, thereby establishing a model based on the chromatin structure detection and the patient prognosis, and having important significance for promoting the development of accurate medical treatment.
Drawings
FIG. 1 is a diagram showing a method for calculating the number of PCR cycles according to the present invention.
FIG. 2 is a graph of bioanalyzer analysis data before and after screening of library fragments using AMPure beads; the top panel shows the library without AMPure beads (which is similar to the ATAC-seq library reported in Nature Methods 2013 and exhibits a characteristic nucleosome distribution), and the bottom panel shows the AMPure beads treated library.
Fig. 3 is a screenshot of UCSC genome browser for frozen breast tissue and ENCODE gastric cancer tissue.
Fig. 4 is a screenshot of the UCSC genome browser for multiple replicates.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.