CN111705111A

CN111705111A - Improved method for detecting gastric cancer frozen tissue open chromatin

Info

Publication number: CN111705111A
Application number: CN202010260336.7A
Authority: CN
Inventors: 郑晓斌; 姜昊; 陈海洋; 冯宇亮; 冯通; 揭敏文
Original assignee: Suzhou Ruimaiying Genetic Testing Technology Co ltd
Current assignee: Suzhou Ruimaiying Genetic Testing Technology Co ltd
Priority date: 2020-04-03
Filing date: 2020-04-03
Publication date: 2020-09-25

Abstract

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.

Description

Improved method for detecting gastric cancer frozen tissue open chromatin

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％Tween 20。

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.

Example 1 method for detecting open chromatin in frozen tissue of gastric cancer

Method and device

1. 10mg of gastric carcinoma tissue was removed from liquid nitrogen or-80 freezer and transferred to a 1 × precooled StableBuffer glass homogenizer containing 2mL and homogenized gently at 20 deg.C. The specific formula of the Stable Buffer is shown in Table 1:

TABLE 1 Stable Buffer

Reagent	Final concentration	Per 100mL of required volume
			1M CaCl₂	5mM	500μL
1M Mg(Ac)₂	3mM	300μL
			1M Tris pH 7.8	10mM	1mL
100mM PMSF	0.02mM	20μL
			14.3Mβ-mercaptoethanol	0.2mM	1.4μL
1M Sucrose	320mM	32mL
			500mM EDTA	0.1mM	20μL
10％NP40	0.10％	1mL
			DDW		65.16mL

2. The homogenized sample was filtered through a cell strainer (cell filter) with a 70 μm filter (in a 50mL tube); the filtrate was collected and added back to the glass homogenizer and homogenized gently 15 times.

3. The sample was then filtered through a 40 μm filter in a cellstrainer and the filtrate was transferred to a 2mL EP tube, centrifuged at 350g for 5min at 4 ℃.

4. The supernatant was discarded and 400. mu.L of 1 × precooled Stable Buffer was added.

5. 400 μ L of 50% Iodixanol Solution was added and mixed well with resuspension to a final concentration of 25%.

6. Add 600. mu.L of 30% Iodixanol Solution carefully below the 25% Solution layer to avoid mixing the two Solution layers.

7. Add 600. mu.L of 40% Iodixanol Solution carefully below the 30% Solution layer to avoid mixing the two Solution layers.

8. Centrifuge at 4 ℃ for 20min at 3000 g.

9. Pipette 200. mu.L of the cell nucleus layer solution into a new 1.5mL EP tube. Add 500. mu.L of precooled Restable Buffer-Tween for dilution, resuspend and mix well. The recipe of the Restable Buffer-Tween is shown in Table 2:

TABLE 2 Restable Buffer-Tween

10. Counting nuclei with trypan blue (trypan blue);

11. 50,000 nuclei were transferred to 1.5mL EP, the liquid was made up to 50. mu.L using Restable Buffer-Tween, and then centrifuged at 500g for 10 minutes at 4 ℃;

12. after removing the supernatant, 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 LPBS, 0.5. mu.L of 1% digitonin, 0.5. mu.L of 10% Tween-20 were added and transferred to a homomixer at 1000rpm, 37 ℃ for 30 min.

13. The transposed DNA was purified using AMPureboads:

(1) adding 100 μ L AMpurebeads, beating for 10 times, and standing at room temperature for 15 min;

(2) then placing the beads on a magneticrack, and standing for 2min at room temperature;

(3) removing the supernatant;

(4) adding 200 μ L of 80% freshly prepared ethanol;

(5) incubating at room temperature for 30sec, and removing supernatant;

(6) repeatedly washing with ethanol once;

(7) after removing the supernatant, performing spin down for a short time, and then putting the EP tube back into the magneticrack to remove the residual ethanol;

(8) air drying at room temperature for 5 min;

(9) removing the EP tube from the magnetic rack;

(10) resuspend beads with 16.5. mu.L EB buffer;

(11) incubating at room temperature for 2 min;

(12) place the EP tube into a magneticrack and carefully aspirate 15. mu.L of the supernatant;

14. the purified transposable DNA was subjected to PCR amplification in the reaction system shown in Table 3 and the PCR procedure shown in Table 4:

TABLE 3

TABLE 4

15. To reduce GC and DNA fragment size bias, one pre-PCR reaction was performed to determine the number of PCR cycles (to avoid supersaturation) for the remaining sample as follows: wherein, the reaction system is shown in table 5, and the PCR program is shown in table 6;

TABLE 5

Components	Volume of
		DNA amplified in the above 5 cycles	5μL
Nextera PCR primer index N7XX (25. mu.M)	0.25μL
		Nextera PCR primer index S5XX (25. mu.M)	0.25μL
NEBNext High-Fidelity 2x PCR Master	5μL
		100x SYBR Green I**	0.06μL
H
	₂0	4.7μL

TABLE 6

16. The optimal number of cycles for the remaining 45. mu. LPCR reaction was calculated (principle shown in FIG. 1):

the method comprises the following steps: measuring the number of cycles corresponding to the maximum fluorescence intensity of each curve 1/4 or 1/3, which is the optimal number of cycles for the remaining 45 μ L of sample reaction;

17. the remaining 45. mu.L was PCR amplified (the procedure was the same as 12 except for the change in cycle number).

18. Performing two rounds of AMPurebes purification on the PCR product;

first wheel (0.65X AMPure beads)

(1) Make up 45 μ LPCR reaction product to 50 μ L with water, add 32.5 μ LAMPurebes, blow-beat 10 times;

(2) standing at room temperature for 15 min;

(3) the beads are then let on a magnetronrack,

(4) standing for 2 min;

(5) transferring the supernatant to a new EP tube;

second wheel (1.8X AMPure beads)

(1) Adding 57.5 μ L AMPure beads into the supernatant, beating for 10 times, and standing at room temperature for 15 min;

(2) placing the beads on a magneticrack, and standing for 2min at room temperature;

(3) removing the supernatant;

(4) adding 200 μ L of 80% freshly prepared ethanol;

(5) incubating at room temperature for 30sec, and removing supernatant;

(6) repeatedly washing with ethanol once;

(7) removing the supernatant, performing spindown for a short time, and then putting the EP tube back into the magneticrack to remove the residual ethanol;

(8) air drying at room temperature for 5 min;

(9) removing the EP tube from the magneticrack;

(10) resuspend beads with 22.5 μ LEBbuffer and incubate for 2min at room temperature;

(11) the beads were placed in a magneticrack and 20. mu.L of the supernatant carefully aspirated.

19. Sequencing the purified PCR product.

Second, result in

1. After the gastric cancer frozen tissue library of the present invention is treated by two cycles of AMPure beads, the adaptor dimer (left red box) and fragments greater than 600bp (right red box) in the library are well removed (as shown in fig. 2). Whereas the library not treated with AMPure beads (similar to the library of ATAC-seq reported in Nature Methods 2013, exhibiting a characteristic nucleosome distribution).

2. Sequencing is carried out on the ATAC-seq library (after AMPure treatment) of the frozen tissues of the gastric cancer in the embodiment, and the result shows that about 83871 peaks are generated; the UCSC genome browser displays that the detection results of the stomach cancer frozen tissue and the ATAC-seq detection result of the stomach cancer tissue of ENCODE are highly similar and have lower noise (figure 3 shows that the UCSC genome browser intercepts the area near the ACTB gene of the housekeeping gene), and repeated tests show that the open chromatin detection method aiming at the stomach cancer frozen tissue sample has stable and reliable results (figure 4).

Claims

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;

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.