CN111705128A

CN111705128A - Improved method for detecting breast cancer frozen tissue open chromatin

Info

Publication number: CN111705128A
Application number: CN202010261335.4A
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 method for detecting open chromatin of a breast cancer frozen tissue sample, which comprises the steps of grinding liquid nitrogen of the breast cancer frozen tissue into powder, adding lysine Buffer for heavy suspension, filtering the resuspended sample through a filter membrane of 40 mu m, collecting filtrate for centrifugation, adding lysine Buffer for uniform mixing, and then adding iodixanol solution with different concentrations for gradient centrifugation to obtain filtrate containing cell nuclei; then adding Dilution Buffer, counting the cell nucleuses in the filtrate, picking 50,000 cell nucleuses, centrifuging and removing the 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 frozen tissue of the clinical breast cancer, and can be combined with 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.

Description

Improved method for detecting breast cancer frozen tissue open chromatin

Technical Field

The invention relates to the technical field of biology, in particular to an improved method for detecting breast cancer frozen tissue open chromatin.

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 the targeted treatment scheme, and also establishes a template for researching epigenetic regulation mechanisms of precise medical treatment 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 breast 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 breast 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 breast cancer frozen tissues). An improved method for detecting the open chromatin of frozen breast cancer tissues is provided, which can effectively detect the chromatin structure of the frozen breast cancer tissues and has the result highly consistent with the result 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 of breast cancer.

The above object of the present invention is achieved by the following technical solutions:

an improved method for detecting breast cancer frozen tissue open chromatin, comprising the steps of:

s1, grinding breast cancer frozen tissues into powder by using liquid nitrogen, adding a lysine Buffer for heavy suspension, filtering the heavy-suspended sample through a filter membrane of 40 mu m, collecting filtrate, centrifuging, adding the lysine Buffer for uniform mixing, and then sequentially adding iodixanol solutions with different concentrations of 50%, 29% and 35% for gradient centrifugation to obtain filtrate containing cell nuclei; the lysine Buffer comprises the following components in final concentration: 5mM KCl₂，3mM MgCl₂10mM Tris pH7.8, 0.1mM EDTA, 0.10% NP40, 320mM sucrose, 0.02mM Cocktail protease inhibitor, 1mM DTT, 0.15mM Spermine;

s2, taking the filtrate containing the cell nuclei in the step S1, adding Dilution Buffer, 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 Dilutionbuffer comprises the following components in final concentrations: 10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl₂，0.10％Tween 20。

The inventor finds that the classical cell nucleus extraction method and the method disclosed in the patent CN 108300774A are not suitable for breast cancer frozen tissues, the number of cell nuclei required by follow-up research cannot be obtained from limited breast 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 breast cancer frozen tissue, and is convenient for subsequent experiments. The method for extracting cell nucleus can extract enough cell nucleus number from the limited breast cancer frozen tissue for subsequent experiments.

Preferably, the breast cancer frozen tissue is liquid nitrogen or tissue preserved at-80 ℃.

Preferably, the mass-to-volume ratio of the breast cancer frozen tissue to the lysine 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 lysine Buffer is as follows: 1M KCl₂，1M MgCl₂1M TrispH7.8, 500mM EDTA, 10% NP40, 1M sucrose, 100mM Cocktail protease inhibitor, 1M DTT, 150mM SPERMINE.

Preferably, all of the agents are pre-chilled in order to ensure the integrity and viability of the nuclei in the tissue.

Specifically, the transposition system for 50,000 nuclei was 25. mu.L TD Buffer, 2.5. mu.L DDW, 2.5. mu.L TDE1(Tn5 enzyme), 16.5. mu.L PBS, 0.5. mu.L 1% digitonin, 0.5. mu.L 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 transposable DNA is purified by VAHTS DNA Clean beads: adding VAHTS DNA Clean beads into the transposed DNA, uniformly mixing, 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 VAHTS DNA Clean 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 VAHTS DNA Clean beads: adding VAHTS DNA Clean beads with the final concentration of 0.50 x into the PCR reaction product, uniformly mixing, standing, placing on a magnetic separation frame for standing separation, transferring the supernatant into a new tube, adding VAHTS DNA Clean beads with the final concentration of 2.0 x, uniformly mixing, standing, placing on a magnetic separation frame for standing separation, removing the supernatant, washing with 80% ethanol solution, repeating the steps, removing the 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 purification and gel electrophoresis method causes more DNA loss, which causes the complexity of the library to be reduced (the DNA obtained after 50000 cell DNA transposition is less), while the invention can reduce the loss of the DNA by using VAHTS DNAclean beads for recovery (about 30%); 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, it is not necessary to retain large fragments of more than 600bp if the experiment is concerned with only open chromatin regions rather than nucleosome localization information (the efficiency of sequencing fragments of more than 600bp by a second generation sequencer is extremely low). Therefore, the VAHTS DNA Clean beads are utilized to remove the adaptor dimer and the large fragment larger than 600bp through two rounds of VAHTS DNA Clean beads screening (by controlling the ratio of different VAHTS DNA Clean beads to a sample), thereby greatly improving the effective sequencing ratio of the library DNA.

Meanwhile, the application of the method in the detection of the breast 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 breast cancer frozen tissue open chromatin.

A product for detecting breast cancer frozen tissue open chromatin comprises the Lysis Buffer, iodixanol solutions with different concentrations and Dilution Buffer.

Preferably, VAHTS DNA Clean beads for DNA and PCR product purification are also included.

Preferably, the product further comprises a 40 μm cell strainer, Illumina tag DNA Buffer, Illumina nexteratag DNA enzyme, 1% digitonin, 10% Tween-20, NEBNext High-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 breast cancer frozen tissues, and the result of the kit can be highly consistent with the detection result of ENCODE tissues.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides an improved method for detecting open chromatin of frozen breast cancer tissues, which can effectively detect the chromatin structure of clinical breast cancer frozen tissues by improving a method for extracting cell nucleuses of the frozen breast cancer tissues and greatly improving the quality of a library by using a VAHTS DNA Clean 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 data from bioanalyzer analysis before and after screening of library fragments using VAHTS DNA Clean beads; the top panel shows the non-VAHTS DNA Clean beads treated library (similar to the ATAC-seq library reported in Nature Methods 2013, showing characteristic nucleosome distribution), and the bottom panel shows the VAHTS DNA Clean beads treated library.

Fig. 3 is a screenshot of a UCSC genome browser for frozen breast tissue and ENCODE breast 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 Breast cancer

Method and device

1. From liquid nitrogen or-80 refrigerator, 10mg of breast cancer tissue was transferred to a mortar containing a small amount of liquid nitrogen and ground into powder, which was transferred to 2mL of 1 × precooled lysine Buffer and gently mixed. The specific formula of the lysine Buffer is shown in Table 1:

TABLE 1 lysine Buffer

Reagent	Final concentration	Per 100mL of required volume
			1M KCl₂	5mM	500μL
1M MgCl₂	3mM	300μL
			1M Tris pH 7.8	10mM	1mL
100mM Cocktail	0.02mM	20μL
			1M DTT	1mM	100μL
1M Sucrose	320mM	32mL
			500mM EDTA	0.1mM	20μL
10％NP40	0.10％	1mL
			150mM Spermine	0.15mM	100μL
DDW		64.96mL

2. The sample was then filtered through a 40 μm filter in a cellstrainer (50 mL tube) and the filtrate was transferred to a 2mL EP tube at 4 ℃ and 350g and centrifuged for 5 min.

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

4. 400 μ L of 50% Iodixanol Solution was added and mixed well with resuspension to a final concentration of 25%.

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

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

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

8. Pipette 200. mu.L of the cell nucleus layer solution into a new 1.5mL EP tube. Add 500. mu.L of precooled Dilution Buffer to dilute, resuspend and mix well. The formulation of the Dilution Buffer is shown in Table 2:

TABLE 2 Dilution Buffer

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

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

11. 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.

12. The transposed DNA was purified using VAHTS DNA Clean beads:

(1) adding 100 mu L VAHTS DNA Clean beads, blowing and beating for 10 times, and standing for 15min at room temperature;

(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;

13. 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

14. 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

15. 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;

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

17. Performing two rounds of VAHTS DNA Clean beads purification on the PCR product;

first round (0.50 XVAHTS DNA Clean beads)

(1) Make up 45 u LPCR reaction product to 50 u L with water, add 25.0 u L VAHTS DNA Clean beads, blow and 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 round (2.0 XVAHTS DNA Clean beads)

(1) Adding 75.0 μ L of 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.

18. Sequencing the purified PCR product.

Second, result in

1. The frozen tissue library of breast cancer of the present invention was treated with two rounds of VAHTS DNA Clean beads, and the adaptor dimer (left red box) and fragments greater than 600bp (right red box) in the library were well removed (as shown in FIG. 2). While the library not treated with VAHTS DNA Clean beads (similar to the ATAC-seq library reported in Nature Methods 2013, exhibiting a characteristic nucleosome distribution).

2. Sequencing of the ATAC-seq library (after AMPure treatment) of frozen breast cancer tissues in this example resulted in the production of about 52024 peaks; the UCSC genome browser shows that the ATAC-seq detection results of the breast cancer frozen tissue and the breast cancer frozen tissue of ENCODE are highly similar (FIG. 3 shows that the UCSC genome browser intercepts the area near the ACTB gene of the housekeeping gene), and repeated tests show that the result of the open chromatin detection method for the breast cancer frozen tissue sample is stable and reliable (FIG. 4).

Claims

1. An improved method for detecting breast cancer frozen tissue open chromatin, comprising the steps of:

2. The method according to claim 1, wherein the mass to volume ratio of the breast cancer frozen tissue to the Lysis 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 29% iodixanol solution below the 25% solution layer, adding 35% iodixanol solution below the 29% solution layer, and centrifuging.

4. The method according to claim 1, wherein the volume ratio of the filtrate containing cell nuclei and the Diluenbuffer in the 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 to purify the transposed DNA by using VAHTS DNA Clean beads: adding VAHTS DNA Clean beads into the transposed DNA, uniformly mixing, 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 or DDW.

8. The method of claim 1, wherein the PCR product purification of step S2 is performed using VAHTSDNAClean beads: adding VAHTSDNA Clean beads with the final concentration of 0.5 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 VAHTSDNA Clean beads with the final concentration of 2.0 multiplied by the weight, 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 frozen tissue open chromatin in breast cancer.

10. A product for detecting breast cancer frozen tissue open chromatin, which comprises the lysine Buffer of claim 1, iodixanol solution of different concentrations and Dilution Buffer.