Rapid chromatin co-immunoprecipitation sequencing method based on FFPE sample

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a rapid chromatin immunoprecipitation sequencing (ChIP-seq) method based on an FFPE sample. In order to overcome the defects in the prior art, the invention provides a novel method for performing ChIP-seq through a fixed slice tissue sample (FFPE), which comprises the following steps: dewaxing and hydrating tissues; homogenizing the tissues; dNA (deoxyribonucleic acid) extraction and quantification; magnetic bead pretreatment; co-immunoprecipitation is carried out; and washing and extracting ChIP for reaction. High-quality ChIP-seq data are obtained in a simple, rapid and reliable mode, a new mode is provided for related research on gene regulation, and the feasibility of analysis and research on fixed tissue samples is improved.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to a method for rapid chromatin immunoprecipitation sequencing (ChIP-seq) based on FFPE samples. Background Art

[0002] The prior art discloses that ChIP-seq (chromatin immunoprecipitation sequencing), as an important epigenetics analysis technology, is a technology for identifying protein-DNA interactions in tissue samples. By combining with high-throughput whole-genome sequencing, it can help reveal the deep mechanisms of gene expression regulation. The application scope of this technology is extensive, including fields such as pathway research, disease diagnosis and treatment, etc. It has important significance in biological and medical research, including:

[0003] (1) Revealing transcriptional regulation mechanisms: ChIP-seq can help researchers identify and locate the sites where transcription factors bind to DNA. Transcription factors are a class of key proteins that regulate gene expression. Through the ChIP-seq technology, it is possible to understand which genes' regulation is related to specific transcription factors, thereby revealing the transcriptional regulation mechanisms of genes; (2) Predicting gene functions: Through the ChIP-seq technology, researchers can determine the binding sites of specific transcription factors to a certain gene, thereby inferring the function of this gene, which is of great significance for understanding the role of genes in biological processes and the research on the mechanisms of disease occurrence; (3) Studying epigenetics: ChIP-seq can be used to study epigenetic modifications, such as histone modifications and DNA methylation. By analyzing the distribution of these modifications on the genome, it is possible to understand their roles in processes such as gene expression and cell differentiation, as well as their association with disease occurrence; (4) Discovering new gene regulatory elements: The ChIP-seq technology can help discover new gene regulatory elements, such as enhancers and promoters. By analyzing the sites where transcription factors bind to DNA, new regulatory sequences can be identified and their functions in the gene regulatory network can be further studied; and so on. However, the current ChIP-seq technology usually requires the use of fresh tissue samples, which limits the analysis and research of retrospective cohorts.

[0004] FFPE (Formalin-fixed paraffin-embedding) samples are common biological materials in the medical field. Such samples have been widely used in research fields such as high-throughput sequencing, in situ hybridization, immunohistochemistry, etc. Since it is a common preservation form for pathological specimens, it is particularly suitable for retrospective studies of large sample size cohorts. However, due to the limited amount of FFPE samples and the antigen cross-linking caused by paraformaldehyde-fixed samples, the sites recognized by antibodies are masked.

[0005] Currently, only Active Motif has launched kits for ChIP-seq detection of FFPE samples on the market, including the FFPE Sample Preparation Kit (catNo.53031) and the ChIP-seq Reaction Kit (cat No.53047). This kit is required to be used with Active Motif's ChIP-seq reaction kit. The detection process takes a long time, exceeding 3 days from dewaxing to DNA extraction, and uses agarose beads that need to be separated by centrifugation, resulting in relatively high noise.

[0006] Based on the current situation of the existing technology, the inventors of the present application aim to overcome the obstacles in the application of ChIP-seq technology in FFPE samples and propose a rapid chromatin immunoprecipitation sequencing (ChIP-seq) method based on FFPE samples. In this method, through a suitable lysis system, while obtaining soluble chromosomes, the degradation of protein components therein is reduced, and through a suitable blocking reagent, the background signal is reduced, providing a sample processing solution for obtaining high-quality sequencing data subsequently. Summary of the Invention

[0007] The object of the present invention is, based on the current situation of the existing technology, to solve the deficiencies in the existing technology and provide a new method for performing ChIP-seq on fixed section tissue samples (FFPE), specifically a rapid chromatin immunoprecipitation sequencing (ChIP-seq) method based on FFPE samples. This method obtains high-quality ChIP-seq data in a simple, rapid, and reliable manner, providing a new way for researchers to study gene regulation and improving the feasibility of analyzing and researching fixed tissue samples.

[0008] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0009] A rapid ChIP-seq method based on FFPE samples is provided, including the following steps:

[0010] The first step: Tissue dewaxing and hydration;

[0011] The second step: Tissue homogenization;

[0012] The third step: DNA extraction and quantification;

[0013] The fourth step: Magnetic bead pretreatment;

[0014] The fifth step: Immunoprecipitation;

[0015] The sixth step: Washing and extracting the ChIP reaction.

[0016] Furthermore, the specific method of the first step is:

[0017] (1) Place 5 - 10 paraffin sections into a 1.5 mL EP tube, add 1 mL of xylene or xylene substitute, place the EP tube in a 55 °C water bath, take out the EP tube every 2 minutes and invert it up and down to mix well. After 10 minutes, take out the EP tube, centrifuge at 12000 rpm at room temperature for 5 minutes, and gently discard the upper layer of liquid with a pipette; repeat this step until the paraffin is completely removed.

[0018] (2) Add 1 mL of absolute ethanol, invert it up and down to mix well, incubate at room temperature for 10 minutes, centrifuge at 12000 rpm at room temperature for 5 minutes, and discard the upper layer of liquid with a pipette; gradually reduce the concentration of the ethanol solution and repeat this step, and finally use deionized ultrapure water for hydration.

[0019] (3) Discard the upper layer of water, add 300 μL of Low - salt ChIP buffer, and immediately proceed to the second step.

[0020] Furthermore, in the first step (2), the concentrations of the ethanol solutions are 90%, 70%, 50%, and 20% in sequence.

[0021] Or, in the first step (3), the ChIP buffer contains 3 μL of protease inhibitor cocktail and 3 μL of PMSF.

[0022] Furthermore, the specific method of the second step is as follows:

[0023] (1) Add 500 μL of sodium citrate antigen retrieval solution (pH = 6.0), add 5 μL of protease inhibitor and 5 μL of deacetylase inhibitor, and use a water bath at 85 - 90 °C for 20 minutes.

[0024] (2) Centrifuge at the highest speed at 4 °C for 15 minutes, discard the supernatant, add 200 μL of Low - salt ChIP buffer, add 2 μL of protease inhibitor and 2 μL of deacetylase inhibitor, rotate at a rate of 850 rpm in a 50 °C horizontal shaker for 30 minutes, and then place it on ice.

[0025] (3) Centrifuge at the highest speed at 4 °C for 15 minutes, discard the supernatant, add 200 μL of Low - salt ChIP buffer, and use a non - contact ultrasonic instrument to ultrasonicate in an ice - water bath or at 4 °C for 30 minutes.

[0026] (4) Centrifuge at the highest speed at 4 °C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube, place it on ice, then add 200 μL of High - salt ChIP buffer to the original tube, and ultrasonicate in an ice - water bath or at 4 °C for 30 minutes.

[0027] (5) Use the maximum rotation speed of the centrifuge to centrifuge for 15 minutes at 4°C, transfer the supernatant to the original EP tube, mix well, and the supernatant is the soluble chromatin;

[0028] (6) Pipette 20 μL of the liquid, add 80 μL of TE buffer to dilute it into a 100 μL dilution solution, and store the remaining liquid in a -80°C refrigerator.

[0029] Further, in the second step (2), the inhibitor is 3 μL of protease inhibitor cocktail and 3 μL of deacetylase inhibitor;

[0030] Or, in the second step (4), the ChIP buffer contains 2 μL of protease inhibitor cocktail and 2 μL of PMSF.

[0031] Further, the specific method of the third step is as follows:

[0032] (1) Transfer the dilution solution obtained in the second step to a 250 μL PCR tube, add 2 μL of RNase A, mix well by oscillation, and incubate at a constant temperature of 37°C in a PCR instrument for 30 minutes;

[0033] (2) Add 2 μL of proteinase K and 5 μL of 5M NaCl, and incubate at 65°C in a PCR instrument for 5 hours;

[0034] (3) Transfer the system to a 1.5 ml EP tube, add 200 μL of DNA extraction solution, invert and mix well, centrifuge at 9000 rpm at 4°C for 15 min;

[0035] (4) Take the supernatant, add double the volume of isopropanol, invert and mix gently, let it stand on ice for 10 min, centrifuge at 12000 rpm at 4°C for 10 min, discard the supernatant, and leave the white precipitate;

[0036] (5) Add 1 ml of pre-cooled 70% ethanol, invert and mix gently, centrifuge at 12000 rpm at 4°C for 5 min, pipette the upper layer of 70% ethanol, discard the supernatant, and repeat this step;

[0037] (6) Use the maximum rotation speed of the centrifuge to centrifuge at 4°C for 5 min, pipette the supernatant and discard it;

[0038] (7) Air dry at room temperature, add 40 μL of Low-EDTA TE buffer to resuspend the DNA precipitate, mix well by pipetting, and use Qubit fluorometer to quantify the DNA to determine the chromosome volume required for the final IP system.

[0039] Further, the specific method of the fourth step is as follows:

[0040] (1) Divide the magnetic bead suspension evenly into each EP tube. Each reaction requires 28 - 32 μL of magnetic beads. Add 200 μL of TE buffer to each tube, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, and discard the supernatant.

[0041] (2) Add 100 μL of Blocking buffer, place it in a rotating shaker at 4 °C for incubation until the pre - treated magnetic beads are used in the next step.

[0042] Further, the specific method for the fifth step is as follows:

[0043] (1) Take out the soluble chromatin supernatant from the third step. According to the final required mass, add an appropriate amount of ChIP buffer to make a 50 - μL reaction system. According to the experimental design, prepare several tubes and add 2 μg of antibody and its negative control to configure the ChIP experiment reaction system. Incubate at room temperature on a rotating shaker for 3 hours or overnight at 4 °C.

[0044] (2) Take out the pre - treated magnetic bead suspension from the fourth step, centrifuge at 3000 rpm and 4 °C for 3 minutes, and discard the supernatant.

[0045] (3) Add the ChIP experiment reaction system to the magnetic beads, and place it in a rotating shaker at room temperature for incubation for 3 hours.

[0046] (4) Separate the incubated mixture on a magnetic bead separation rack, aspirate the supernatant, and do not disturb the magnetic bead pellet.

[0047] Further, the specific method for the sixth step is as follows:

[0048] (1) Add 500 μL of ChIP buffer, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, aspirate the supernatant, and do not disturb the magnetic bead pellet, and repeat this step.

[0049] (2) Add 500 μL of Wash buffer, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, aspirate the supernatant, and do not disturb the magnetic bead pellet, and repeat this step.

[0050] (3) Add 500 μL of TE buffer, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, aspirate the supernatant, and do not disturb the magnetic bead pellet, and repeat this step.

[0051] (4) Add 100 μL of pre - heated Elution buffer at 37 °C to the magnetic beads, incubate in a 37 °C metal bath for 5 minutes, separate the magnetic bead suspension on a magnetic bead separation rack, and aspirate the supernatant, which is the ChIP DNA.

[0052] Further, it also includes reverse cross - linking to extract DNA, and its specific method is as follows:

[0053] (1) Transfer the ChIP DNA into a 250 μL PCR tube, add 2 μL of RNase A, mix well by oscillation, and incubate at a constant temperature of 37 °C in a PCR instrument for 30 minutes;

[0054] (2) Then add 2 μL of proteinase K and 5 μL of 5 M NaCl, and incubate at 65 °C in a PCR instrument for 5 hours;

[0055] (3) Transfer the system to a 1.5 mL EP tube, add 200 μL of DNA extraction solution, invert and mix well, centrifuge at 9000 rpm at 4 °C for 15 min;

[0056] (4) Take the supernatant, add twice the volume of isopropanol, invert and mix gently, let stand on ice for 10 min, centrifuge at 12000 rpm at 4 °C for 10 min, discard the supernatant, and leave the white precipitate;

[0057] (5) Add 1 ml of pre-cooled 70% ethanol, invert and mix gently, centrifuge at 12000 rpm at 4 °C for 5 min, aspirate the upper layer of 70% ethanol, be careful not to disturb the white precipitate, discard the supernatant, and repeat this step;

[0058] (6) Centrifuge at the maximum speed of the centrifuge at 4 °C for 5 min, aspirate the supernatant and discard it;

[0059] (7) Add 40 μL of Low-EDTA TE buffer to resuspend the DNA precipitate;

[0060] The DNA extraction solution is composed of phenol:chloroform:isoamyl alcohol in a mass ratio of 25:24:1.

[0061] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0062] 1. The rapid ChIP-seq method based on FFPE samples exemplified by the present invention adds a process of sodium citrate water bath heat repair during tissue homogenization, which is convenient for exposing the antigenic epitopes of histones or transcription factors, enabling the antibody to capture chromosomes more sensitively and specifically, and improving the quality of subsequent data;

[0063] 2. The rapid ChIP-seq method based on FFPE samples exemplified by the present invention uses Blocking buffer to block Protein A / G magnetic beads, which can effectively reduce the non-specific adsorption of magnetic beads, increase the specificity of detection signals, and improve data quality;

[0064] 3. The rapid ChIP-seq method based on FFPE samples in the examples of the present invention simplifies the cumbersome steps of traditional ChIP-seq, shortening it by half compared to the current standard detection process. After dewaxing and rehydrating the paraffin section samples, chromatin extraction is carried out quickly, avoiding subsequent degradation and loss of the samples, suitable for the tissue characteristics of FFPE, and improving the success rate of the experiment;

[0065] 4. The rapid ChIP-seq method based on FFPE samples in the examples of the present invention can obtain high-quality ChIP-seq data when using FFPE samples, and the results are comparable to those using fresh tissue samples. Moreover, the method is simple, reliable, and fast, providing a new way for related research to study gene regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, objectives, and advantages of the present application will become more apparent:

[0067] Figure 1 is a flowchart of the experimental steps;

[0068] Figure 2 is a heat map of FFPE ChIP;

[0069] Figure 3 is a distribution map of H3K27ac antibody-binding peaks on the genome;

[0070] Figure 4 is a diagram of the enrichment of H3K27ac binding site pathways;

[0071] Figure 5 is a distribution map of H3K27me3 antibody-binding peaks on the genome;

[0072] Figure 6 is a diagram of the enrichment of H3K27me3 binding site pathways;

[0073] Figure 7 is a detection comparison diagram of FFPE DNA ChIP-seq. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0074] The present application will be further described in detail below with reference to the drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the related invention and are not intended to limit the invention. Additionally, it should be noted that for the sake of description, only the parts related to the invention are shown in the drawings.

[0075] It should be noted that, without conflict, the embodiments and features in the embodiments of the present application may be combined with each other. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0076] Example 1

[0077] A rapid ChIP-seq method based on FFPE samples, comprising the following steps:

[0078] The first step: Tissue dewaxing and hydration

[0079] 1. Put 5-10 paraffin sections (each section is about 5-10 μm thick) into a 1.5 ml EP tube, add 1 ml of xylene or xylene substitute, put the EP tube into a 55 °C metal bath, take out the EP tube every 2 minutes and invert it up and down to mix evenly. After 10 minutes, take out the EP tube, centrifuge at 12,000 rpm at room temperature for 5 minutes, gently discard the upper layer of liquid with a pipette, and pay attention not to disturb the lower layer of tissue;

[0080] 2. Repeat the operation of the first step 2 times until the paraffin is completely removed;

[0081] 3. Add 1 ml of absolute ethanol, invert it up and down to mix evenly, pay attention not to shake it quickly, incubate at room temperature for 10 minutes, and then centrifuge at 12,000 rpm at room temperature for 5 minutes, and discard the upper layer of liquid with a pipette;

[0082] 4. Add 1 ml of 90% ethanol solution, invert it up and down to mix evenly, pay attention not to shake it quickly, incubate at room temperature for 10 minutes, and then centrifuge at 12,000 rpm at room temperature for 5 minutes, and discard the upper layer of liquid with a pipette;

[0083] 5. Repeat the steps of 4, gradually reduce the concentration of the ethanol solution (70%, 50%, 20%), and finally use deionized ultrapure water for hydration;

[0084] 6. Discard the upper layer of water, add 300 μL of Low-salt ChIP buffer (containing 3 μL of protease inhibitor cocktail and 3 μL of PMSF), and immediately enter the second step.

[0085] The second step: Tissue homogenization

[0086] 1. Add 500 μL of sodium citrate antigen retrieval solution (pH = 6.0), add 5 μL of protease inhibitor and 5 μL of deacetylase inhibitor, and use a water bath at 85-90 °C for 20 minutes;

[0087] 2. Centrifuge at the maximum speed for 15 minutes at 4°C, discard the supernatant, add 200 μL of Low-salt ChIP buffer, 2 μL of protease inhibitor and 2 μL of deacetylase inhibitor, rotate at a rate of 850 rpm in a 50°C horizontal shaker for 30 minutes, and then place on ice;

[0088] 3. Centrifuge at the maximum speed for 15 minutes at 4°C, discard the supernatant, add 200 μL of Low-salt ChIP buffer, and sonicate in an ice-water bath or at 4°C for 30 minutes using a non-contact sonicator (program: 3 seconds on, 3 seconds off, power at 50%);

[0089] 4. Use the centrifuge at the maximum speed, centrifuge for 15 minutes at 4°C, transfer the supernatant to a new 1.5 ml EP tube, and place on ice; then add 200 μL of High-salt ChIP buffer (containing 2 μL of protease inhibitor cocktail and 2 μL of PMSF) to the original tube;

[0090] 5. Use a non-contact sonicator to sonicate in an ice-water bath or at 4°C for 30 minutes (program: 3 seconds on, 3 seconds off, power at 70%);

[0091] 6. Use the centrifuge at the maximum speed, centrifuge for 15 minutes at 4°C, transfer the supernatant to the original EP tube, and mix well with a pipette. The supernatant is the soluble chromatin;

[0092] 7. Pipette 20 μL of the liquid, add 80 μL of TE buffer to dilute it into a 100 μL dilution solution, and store the remaining liquid in an -80°C refrigerator.

[0093] Step 3: DNA Extraction and Quantification

[0094] 1. Transfer the dilution solution obtained in the second step to a 250 μL PCR tube, add 2 μL of RNase A (specification: 10 mg / mL), mix well by oscillation, and incubate at a constant temperature of 37°C in a PCR instrument for 30 minutes;

[0095] 2. Then add 2 μL of proteinase K (specification: 20 mg / mL) and 5 μL of 5 M NaCl, and incubate at 65°C in a PCR instrument for 5 hours;

[0096] 3. Transfer the system to a 1.5 mL EP tube;

[0097] 4. Add 200 μL of DNA extraction solution (phenol:chloroform:isoamyl alcohol mass ratio is 25:24:1), invert and mix well, centrifuge at 9000 rpm at 4°C for 15 min;

[0098] 5. Take the supernatant, add isopropanol with double volume, invert gently to mix well, let it stand on ice for 10 min, centrifuge at 12000 rpm at 4 °C for 10 min, discard the supernatant, and leave the white precipitate.

[0099] 6. Add 1 ml of pre-cooled 70% ethanol, invert gently to mix well, centrifuge at 12000 rpm at 4 °C for 5 min, aspirate the upper layer of 70% ethanol, be careful not to disturb the white precipitate, and discard the supernatant.

[0100] 7. Repeat the previous step.

[0101] 8. Centrifuge at the maximum speed of the centrifuge at 4 °C for 5 min, carefully aspirate and discard the supernatant with a 200 μL pipette tip.

[0102] 9. Air dry at room temperature, which takes about 20 - 30 minutes.

[0103] 10. Add 40 μL of Low-EDTA TE buffer to resuspend the DNA precipitate, pipette to mix well, use Qubit fluorometer to quantify the DNA, and determine the volume of chromosomes required for the final IP system.

[0104] Fourth step: Magnetic bead pretreatment

[0105] 1. Divide the magnetic bead suspension evenly into each EP tube. Each reaction requires about 30 μL of magnetic beads. Add 200 μL of TE buffer to each tube, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, and discard the supernatant.

[0106] 2. Add 100 μL of Blocking buffer, place it in a rotary shaker at 4 °C for incubation until the pretreated magnetic beads are used in the next step.

[0107] Fifth step: Immunoprecipitation

[0108] 1. Take out the soluble chromosome supernatant in the third step, add an appropriate amount of ChIP buffer according to the final required mass to make a 50 μL reaction system. According to the experimental design, prepare several tubes and add 2 μg of antibodies (IgG, H3K4me3 antibody, H3K27me3 antibody, H3K27ac antibody, etc.) and their negative controls to configure the ChIP experimental reaction system, and incubate at room temperature on a rotary shaker for 3 hours or overnight at 4 °C.

[0109] 2. Take out the pretreated magnetic bead suspension in the fourth step, centrifuge at 3000 rpm at 4 °C for 3 minutes, and discard the supernatant.

[0110] 3. Add the ChIP experimental reaction system to the magnetic beads, and incubate on a rotary shaker at room temperature for 3 hours.

[0111] 4. Separate the incubated mixture on a magnetic bead separation rack, aspirate the supernatant, and do not disturb the magnetic bead pellet.

[0112] Step 6: Wash and extract the ChIP reaction

[0113] 1. Add 500 μL of ChIP buffer, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, aspirate the supernatant, and do not disturb the magnetic bead pellet;

[0114] 2. Repeat step 1;

[0115] 3. Add another 500 μL of Wash buffer, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, aspirate the supernatant, and do not disturb the magnetic bead pellet;

[0116] 4. Repeat step 3;

[0117] 5. Add 500 μL of TE buffer, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, aspirate the supernatant, and do not disturb the magnetic bead pellet;

[0118] 6. Add 100 μL of pre-warmed Elution buffer at 37 °C to the magnetic beads, incubate in a 37 °C metal bath for 5 minutes, separate the magnetic bead suspension on a magnetic bead separation rack, and aspirate the supernatant, which is the ChIP DNA.

[0119] Step 7: Reverse cross-linking and extract DNA

[0120] 1. Transfer the ChIP DNA to a 250 μL PCR tube, add 2 μL of RNase A, mix by vortexing, and incubate at 37 °C for 30 minutes in a PCR machine;

[0121] 2. Add another 2 μL of proteinase K and 5 μL of 5 M NaCl, and incubate at 65 °C for 5 hours in a PCR machine;

[0122] 3. Transfer the system to a 1.5 mL EP tube;

[0123] 4. Add 200 μL of DNA extraction solution, mix well by inverting, centrifuge at 9000 rpm at 4 °C for 15 minutes;

[0124] 5. Take the supernatant, add twice the volume of isopropanol, mix gently by inverting, let stand on ice for 10 minutes, centrifuge at 12000 rpm at 4 °C for 10 minutes, discard the supernatant, and retain the white pellet;

[0125] 6. Add 1 mL of pre-cooled 70% ethanol, mix gently by inverting, centrifuge at 12000 rpm at 4 °C for 5 minutes, aspirate the upper layer of 70% ethanol, being careful not to disturb the white pellet, and discard the supernatant;

[0126] 7. Repeat the previous step;

[0127] 8. Set the centrifuge to its maximum speed and centrifuge at 4°C for 5 minutes. Carefully aspirate the supernatant using a 200 μL pipette tip and discard it.

[0128] 9. Air dry at room temperature, which takes approximately 20 - 30 minutes.

[0129] 10. Resuspend the DNA pellet by adding 40 μL of Low-EDTA TE buffer.

[0130] Step 8: Sequencing on the machine and downstream analysis

[0131] 1. According to the requirements of the sequencing platform, construct a library and perform PCR amplification on the extracted DNA, and then conduct high-throughput sequencing.

[0132] 2. Analyze and interpret the sequencing results, including sequence alignment, peak detection, and annotation, etc. Some common bioinformatics tools such as Bowtie, MACS, HOMER, and IGV can be used.

[0133] The rapid ChIP-seq technology of the examples of the present invention has been tested multiple times and obtained excellent test results. In the case of using FFPE samples, this technology can obtain high-quality ChIP-seq data, and the results are comparable to those using fresh tissue samples. As Figure 2-6 shown, through the testing and comparison of multiple samples, the results show that this technology has high repeatability and accuracy, and can be successfully applied to different types of samples, including cancer tissues and normal tissues. These test results indicate that the rapid ChIP-seq technology of the present invention is a reliable, fast, and simple method that can be used to identify protein-DNA interactions in tissue samples and provides a new way for researchers to study gene regulation.

[0134] The main differences between the rapid ChIP-seq method based on FFPE samples of the examples of the present invention and the kit launched by active motif company for ChIP-seq detection of FFPE samples are as follows:

[0135]

[0136] In addition, for the same batch of samples, the effects of the method of the examples of the present invention and the current standard method were compared respectively, and the comparison results are as Figure 7 shown. The results show that when using this method for the detection of FFPE DNA ChIP-seq, the signal is enhanced in both specificity and sensitivity.

[0137] Those skilled in the art should understand that the scope of the invention involved in this application is not limited to the technical solutions formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the technical solutions formed by mutually replacing the above features with the technical features (but not limited to) disclosed in this application that have similar functions.

Claims

1. A rapid chromatin immunoprecipitation sequencing method based on FFPE samples, characterized in that, It includes the following steps: The first step: Tissue dewaxing and hydration; The second step: Tissue homogenization; The third step: DNA extraction and quantification; The fourth step: Magnetic bead pretreatment; The fifth step: Immunoprecipitation; The sixth step: Washing and extraction of ChIP reaction.

2. The method according to claim 1, characterized in that, Among them, the first step includes: (1) Put 5 - 10 paraffin sections into a 1.5 mL EP tube, add 1 mL of xylene or xylene substitute, place the EP tube in a 55 °C water bath, take out the EP tube every 2 minutes and invert it up and down to mix evenly. After 10 minutes, take out the EP tube, centrifuge at 12000 rpm at room temperature for 5 minutes, and discard the upper liquid with a pipette; repeat this step until the paraffin is completely removed; (2) Add 1 mL of absolute ethanol, invert it up and down to mix evenly, incubate at room temperature for 10 minutes, centrifuge at 12000 rpm at room temperature for 5 minutes, and discard the upper liquid with a pipette; reduce the concentration of the ethanol solution and repeat this step. The last time, use deionized ultrapure water for hydration; (3) Discard the upper water, add 300 μL of Low - salt ChIP buffer, and immediately enter the second step.

3. The method according to claim 2, wherein In the first step (2), the concentrations of the ethanol solutions are 90%, 70%, 50%, and 20% in sequence; Or, in the first step (3), the Low - salt ChIP buffer contains 3 μL of protease inhibitor cocktail and 3 μL of PMSF.

4. The method according to claim 1, wherein Among them, the second step includes: (1) Add 500 μL of L - sodium citrate antigen retrieval solution (pH = 6.0), add 5 μL of protease inhibitor and 5 μL of deacetylase inhibitor, and incubate in a water bath at 85 - 90 °C for 20 minutes; (2) Centrifuge at high speed at 4 °C for 15 minutes, discard the supernatant, add 200 μL of Low - salt ChIP buffer, add 2 μL of protease inhibitor and 2 μL of deacetylase inhibitor, rotate at a rate of 850 rpm in a 50 °C horizontal shaker for 30 minutes, and then place it on ice; (3) Centrifuge at the highest speed at 4 °C for 15 minutes, discard the supernatant, add 200 μL of Low - salt ChIP buffer, and use a non - contact ultrasonic instrument to ultrasonicate in an ice - water bath or at 4 °C for 30 minutes; (4) Centrifuge at the highest speed at 4 °C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube, place it on ice, then add 200 μL of High - salt ChIP buffer to the original tube, and ultrasonicate in an ice - water bath or at 4 °C for 30 minutes; (5) Use the centrifuge at the maximum speed, centrifuge at 4 °C for 15 minutes, transfer the supernatant to the original EP tube, mix evenly, and the supernatant is the soluble chromatin; (6) Pipette 20 μL of the liquid, add 80 μL of TE buffer to dilute it into a 100 μL dilution solution, and store the remaining liquid in a - 80 °C refrigerator.

5. The method according to claim 4, wherein In the second step (2), the inhibitors are 3 μL of protease inhibitor cocktail and 3 μL of deacetylase inhibitor; Or, in the second step (4), the High - slat ChIP buffer contains 2 μL of protease inhibitor cocktail and 2 μL of PMSF.

6. The method according to claim 1, characterized in that, Among them, the third step includes: (1) Transfer the diluted solution obtained in the second step into a 250 μL PCR tube, add 2 μL of RNase A, mix well by oscillation, and incubate at a constant temperature of 37 °C in a PCR instrument for 30 minutes; (2) Add 2 μL of proteinase K and 5 μL of 5 M NaCl, and incubate at 65 °C in a PCR instrument for 5 hours; (3) Transfer the system to a 1.5 ml EP tube, add 200 μL of DNA extraction solution, mix well, centrifuge at 9000 rpm at 4 °C for 15 min; (4) Take the supernatant, add double the volume of isopropanol, mix gently, let stand on ice for 10 min, centrifuge at 12000 rpm at 4 °C for 10 min, discard the supernatant, and leave the white precipitate; (5) Add 1 ml of pre-cooled 70% ethanol, mix gently, centrifuge at 12000 rpm at 4 °C for 5 min, aspirate the upper layer of 70% ethanol, discard the supernatant, and repeat this step; (6) Centrifuge at the maximum speed of the centrifuge at 4 °C for 5 min, aspirate the supernatant and discard it; (7) Air-dry at room temperature, add 40 μL of Low-EDTA TE buffer to resuspend the DNA precipitate, mix well by pipetting, and use Qubit fluorometer to quantify the DNA to determine the chromosome volume required for the final IP system.

7. The method according to claim 1, characterized in that Step 4 includes: (1) Divide the Protein A / G magnetic bead suspension evenly into each EP tube. Each reaction requires 28 - 32 μL of magnetic beads. Add 200 μL of TE buffer to each tube, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, and discard the supernatant; (2) Add 100 μL of Blocking buffer, place it in a rotating shaker at 4 °C for incubation until the pre-treated magnetic beads are used in the next step.

8. The method according to claim 1, wherein Step 5 includes: (1) Take out the soluble chromosome supernatant in the third step, add an appropriate amount of Low-salt ChIP buffer according to the final required mass to make a 50 μL reaction system. Prepare several tubes and add 2 μg of antibody and its negative control to configure the ChIP experiment reaction system, and incubate at room temperature on a rotating shaker for 3 hours or overnight at 4 °C; (2) Take out the pre-treated magnetic bead suspension in the fourth step, centrifuge at 3000 rpm at 4 °C for 3 minutes, and discard the supernatant; (3) Add the ChIP experiment reaction system to the magnetic beads, and incubate on a rotating shaker at room temperature for 3 hours; (4) Separate the incubated mixture on a magnetic bead separation rack, aspirate the supernatant without disturbing the magnetic bead precipitate.

9. The method according to claim 1, wherein Step 6 includes: (1) Add 500 μL of Low-salt ChIP buffer, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, aspirate the supernatant without disturbing the magnetic bead precipitate, and repeat this step; (2) Add 500 μL of Wash buffer, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, aspirate the supernatant without disturbing the magnetic bead precipitate, and repeat this step; (3) Add 500 μL of TE buffer, mix well, separate the magnetic bead suspension on a magnetic bead separation rack, aspirate the supernatant without disturbing the magnetic bead precipitate, and repeat this step; (4) Add 100 μL of pre-warmed Elution buffer at 37 °C to the magnetic beads, incubate in a 37 °C metal bath for 5 minutes, separate the magnetic bead suspension on a magnetic bead separator, and aspirate the supernatant, which is the ChIP DNA.

10. The method according to claim 1, characterized in that, The method described above further includes DNA extraction by reverse cross-linking, which includes: (1) Transfer the ChIP DNA to a 250 μL PCR tube, add 2 μL of RNase A, mix by vortexing, and incubate at a constant temperature of 37 °C in a PCR instrument for 30 minutes; (2) Then add 2 μL of proteinase K and 5 μL of 5 M NaCl, and incubate at 65 °C in a PCR instrument for 5 hours; (3) Transfer the system to a 1.5 mL EP tube, add 200 μL of DNA extraction solution, invert to mix well, centrifuge at 9000 rpm at 4 °C for 15 min; (4) Take the supernatant, add twice the volume of isopropanol, invert gently to mix, let stand on ice for 10 min, centrifuge at 12000 rpm at 4 °C for 10 min, discard the supernatant, and leave the white precipitate; (5) Add 1 ml of pre-cooled 70% ethanol, invert gently to mix, centrifuge at 12000 rpm at 4 °C for 5 min, aspirate the upper layer of 70% ethanol without disturbing the white precipitate, discard the supernatant, and repeat this step; (6) Centrifuge at the maximum speed of the centrifuge at 4 °C for 5 min, aspirate the supernatant and discard it; (7) Add 40 μL of Low-EDTA TE buffer to resuspend the DNA precipitate; The DNA extraction solution is composed of phenol:chloroform:isoamyl alcohol in a mass ratio of 25:24:1.

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