CN110066863B - Identification method of BPDE addition gene - Google Patents
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Abstract
The invention relates to the field of identification of BPDE addition genes, in particular to an identification method of BPDE addition genes. Based on the principle of Chromatin Immunoprecipitation (ChIP), the enriched BPDE-DNA adduct is subjected to high-throughput sequencing by Anti-BPDE antibody to construct the precise location technology of the target locus of BaP adduct. In the method, the detected BPDE addition target gene is directly acted between BPDE and the gene, and in the detection process, chemical modification on small molecules is not needed, the original activity of the small molecules is not influenced, the method is not limited by any cell and tissue types, pure DNA is not required, even a whole cell lysate can be used, and the method can be widely applied to the identification of the small molecule addition target gene.
Description
Technical Field
The invention relates to the field of identification of BPDE addition genes, in particular to an identification method of BPDE addition genes. The method can directly identify target genes capable of being adducted with BPDE in chromatin, the previously reported effect of BaP or metabolite BPDE on genome level is mainly based on the presumption that some gene or some gene expression difference may exert some biological function on its related upstream or downstream gene.
Background
Polycyclic Aromatic Hydrocarbons (PAHs) are the most widely distributed and most harmful organic pollutants in the urban atmospheric environment in China, and gaseous PAHs discharged from pollution sources are easy to form particles after being cooled or are enriched on the particles due to adsorption, so the Polycyclic Aromatic Hydrocarbons (PAHs) become one of the main components of fine particles PM 2.5. Benzo [ a ] pyrene (Ba P) is generally regarded as a representative of polycyclic aromatic hydrocarbons and atmospheric pathogenic substances, which are widely present in tobacco, smoked foods, coal tar, automobile exhaust and industrial exhaust gases, and there is sufficient evidence that the incidence of tumors is positively correlated with the levels of Ba P in food and air based on animal experiments and epidemiological investigations.
The harmful effect of BaP on human health is closely related to the genetic background difference of different people. BaP is a high-activity indirect carcinogen which is metabolized to form an active metabolite (final carcinogen: 7,8-dihydrodiol-9,10-epoxybenzo [ a ] pyrene (7,8-dihydrodiol-9,10-epoxyebenza [ a ] pyrene, BPDE) after entering a human body, BPDE has electrophilicity and can be covalently bound with DNA nucleophilic sites to form an adduct, so that irreversible damage to DNA occurs, wherein the damage is an early event of cell mutation and malignant transformation and is closely related to the occurrence of tumor, after DNA damage in cells is formed, a signal cascade (the damage is recognized by a sensor protein and signals are conducted to an effector), the damage repair of the DNA is carried out, so that the cell cycle is blocked.
The toxicological genomics research carried out recently shows that the carcinogenic process of BaP is a complex process involving multiple genes, multiple regulatory mechanisms and multiple signal pathways. The expression level of more than two thousand genes can be influenced by BaP. However, all the research methods for researching the toxicity mechanism of BaP to date, whether phenotypic analysis or new generation sequencing technology, only indirectly supposes the possible genome action site of the metabolite BPDE; the damage of target genes directly caused by the addition of BPDE and nucleophilic sites, and other changes and interrelations on the genome and transcriptome initiated by the damage are still unknown.
Researchers carry out high-throughput sequencing on the enriched BPDE-DNA adduct through Anti-BPDE antibody based on the principle of Chromatin Immunoprecipitation (ChIP) to construct a precise positioning technology of the target locus of the BaP adduct, directly position the damage target of the final carcinogen BPDE on the whole genome of human, excavate the specific genetic marker of the early event of the BaP damage DNA, and screen high risk groups; comprehensively analyzing the relationship between the located target gene function and phenotype (carcinogenesis) by bioinformatics technology, and analyzing the carcinogenesis mechanism; and according to the established BaP damage specificity gene list (gene library), the prediction index and treatment target point of BaP carcinogenesis are provided, so that an important basis is provided for guiding the research of 'accurate' individual prevention and clinical diagnosis and treatment and carcinogenesis mechanisms.
In the technical method, the condition difference of cell culture and the condition difference of cells after infection cannot ensure that the same fragment size is obtained every time when each sample is subjected to ultrasonic fragmentation, and the genes of partial binding sites are interrupted during ultrasonic fragmentation to cause information loss, and in addition, a part of false positive results exist in high-throughput sequencing.
Disclosure of Invention
The present invention provides a method for identifying a BPDE addition gene in order to screen a target gene capable of adding BPDE as many as possible. Because any method of target gene information difference caused by the experiment per se is difficult to avoid, the aim of screening BPDE addition genes as much as possible is not influenced.
The invention is realized by the following technical scheme: a method for identifying a BPDE addition gene, comprising the steps of:
1) The cell fusion degree in the culture medium is observed to be 70-80% under a mirror, and the cell state is good at the moment;
2) Adding formaldehyde into the culture medium to fix the cells in the formaldehyde, uniformly mixing the formaldehyde in the culture medium by a vortex culture dish, and standing for 10min at room temperature;
3) Adding 10 XGlycine into each culture dish, uniformly mixing in a rotating way, incubating for 5min at room temperature, and quenching unreacted formaldehyde;
4) Discarding the culture medium, adding a PBS (phosphate buffer solution) precooled at 0-4 ℃ into a culture dish, washing cells, repeatedly rinsing twice, carefully sucking each time, removing the PBS as much as possible without disturbing the cells;
5) Adding 1 XPICII-containing PBS buffer solution precooled at 0-4 ℃ into each culture dish, collecting cells from each culture dish by using a sterile cell scraper, and collecting the cells into a centrifuge tube;
6) Cells were pelleted by centrifugation at 800 Xg for 5min at 4 ℃ and the supernatant carefully removed;
7) Resuspending the cells in cell lysis buffer containing 1 XPIC II, incubating on ice for 15min, and mixing by vortex once every 5min during incubation;
8) Centrifuging at 4 deg.C at 800 × g for 5min, removing supernatant, and resuspending cell precipitate in cell lysis buffer containing 1 × PIC II to obtain lysis product;
9) Placing two aliquots of the lysate into EP tubes, adding one part of the lysate into DMSO solution of BPDE as an experimental group, adding the other part of the lysate into DMSO solution with the same volume as a control group, and incubating at 37 ℃ overnight;
10 Sonication using a sonicator;
11 Centrifuged at 12000 Xg for 10min at 4 ℃ to remove insoluble material and the sheared chromatins of the experimental and control groups were transferred to new EP tubes, respectively;
12 ChIP dilution buffer solution containing 1 xPIC II is respectively added into the sheared chromatin, 2vol% of supernatant is respectively taken from an experimental group and a control group as input, and the input is preserved at 4 ℃;
13 Anti-BPDE Monoclonal Antibody was added to the remaining experimental and control groups and incubated overnight at 4 ℃ in a constant temperature shaker;
14 Mixing ChIP-Grade Protein A/G magnetic beads uniformly to enable the magnetic beads to be suspended completely, adding the magnetic beads into an experimental group, a control group and two input, sealing a sealing membrane, and incubating for 2 hours at 4 ℃;
15 Experimental group, control group and two input EP tubes are placed on a magnetic separation frame, chIP-Grade Protein A/G magnetic beads are adsorbed to the tube wall, the mixture is kept stand for 3min, and the supernatant is removed;
16 In the order listed below), the magnetic beads were suspended in each of the buffers precooled at 0-4 ℃ and incubated on a constant temperature shaker at 4 ℃ for 5min, respectively, followed by magnetic separation, removal of the supernatant, and washing of the Grade Protein A/G magnetic bead-chromatin complexes:
a. washing once with a low-salt washing buffer solution;
b. washing once by using a high-salt washing buffer solution;
LiCl washing buffer solution, washing once;
TE buffer solution, washing once;
17 Respectively adding ChIP Elution buffers into the EP tubes of an experimental group, a control group and two input;
18 The experimental group, the control group and two input EP tubes are incubated for 2h at 62 ℃ in a water bath with shaking and are cultured for 10min at 95 ℃, and chromatin of the experimental group and the control group is eluted from the ChIP-Grade Protein A/G magnetic beads and is cooled to room temperature;
19 The EP tubes of the experimental group and the control group are placed on a magnetic separation frame, the mixture is kept stand for 3min, the ChIP-Grade Protein A/G magnetic beads are adsorbed to the tube wall, and the supernatant is transferred to a new EP tube and is respectively marked;
20 Adding DNA Binding Buffer into the EP tubes of the experimental group, the control group and two input groups respectively, and mixing evenly by vortex;
21 Transferring to a centrifugal filter with a receiving tube, centrifuging at 12000 Xg for 30s, taking out the centrifugal filter from the receiving tube, reserving the receiving tube, discarding the liquid, and returning the centrifugal filter to the same receiving tube;
22 Adding Wash Buffer,12000 Xg centrifugation for 30s, taking out the centrifugal filter from the receiving tube, reserving the receiving tube, discarding the liquid, and putting the centrifugal filter back into the same receiving tube; centrifuging at 12000 Xg for 30s again, and discarding the receiving tube and liquid;
23 Put into a new receiving tube, add DNA Elution Buffer directly to the center of the white centrifugal filter membrane, centrifuge for 30s at 12000 Xg, take out and discard the centrifugal filter, the Elution Buffer at this moment is purified DNA;
24 Detecting the concentration and purity of purified DNA of an experimental group, a control group and two inputs, performing high-throughput sequencing, and if the concentration of the DNA is not detected by the control group, removing the background value of the input corresponding to the experimental group by the experimental group to obtain a gene, namely a BPDE addition gene; if the control group detects the DNA concentration, the gene obtained after the experimental group removes the control group, namely the BPDE addition gene.
As a further improvement of the technical scheme of the identification method, after the step 9) is completed, partial cleavage products of the experimental group and the control group are respectively taken for DNA agarose gel analysis, after the step 11) is completed, the sheared chromatin of the experimental group and the control group is respectively taken for DNA agarose gel analysis, a band is observed under a gel imaging system, and if the width of the band is 100-500bp, the step 12) is continuously carried out.
As a further improvement of the technical scheme of the identification method of the invention, after the step 11) is completed, the DNA concentrations of the ultrasonic-treated cleavage products of the experimental group and the control group are respectively determined, and if the DNA concentration is more than 50 mug/mL, the step 12) is continued.
As a further improvement of the technical scheme of the identification method of the invention, in step 11), after removing insoluble substances, the sheared chromatins of the experimental group and the control group are respectively transferred to three new EP tubes.
As a further improvement of the technical scheme of the identification method, the three new EP tubes of the experimental group are respectively an experimental group test tube, an experimental group negative control tube and an experimental group positive control tube, and the three new EP tubes of the control group are respectively a control group test tube, a control group negative control tube and a control group positive control tube; in step 12), two inputs are taken from the test tube of the experimental group and the test tube of the control group, respectively.
As a further improvement of the technical scheme of the identification method, anti-BPDE Monoclonal Antibody is respectively added into an experimental group test tube and a control group test tube, normal Mouse IgG is respectively added into the experimental group negative control tube and the control group negative control tube, anti-RNA polymerase II Antibody is respectively added into the experimental group positive control tube and the control group positive control tube, the tubes are both incubated overnight at the constant temperature of 4 ℃ by a shaker, and then steps 14) to 23) are carried out.
As a further improvement of the technical scheme of the identification method, the PCR amplification is carried out on part of purified DNA in the experiment group negative control tube, the experiment group positive control tube, the input corresponding to the experiment group, the control group negative control tube, the control group positive control tube and the input corresponding to the control group, if a band is obtained at 166bp, the ChIP quality control is good, and the step 24) is continued.
The major drawbacks in the traditional BPDE adduction gene identification:
1. chemical modification is required to be carried out on the small molecular compound, and labels such as biotin or fluorescein and the like are added. Some small molecule compounds cannot be biotinylated (e.g., BPDE in this study).
2. Modification processes may alter the activity of small molecules.
3. The number of molecular targets that can be identified is limited.
However, the method for identifying the BPDE addition gene has the following beneficial effects compared with the traditional method:
1. does not need any labeled ligand and is not limited by synthetic chemistry.
2. The medicine does not need to be modified, and the system is not influenced by the medicine.
3. The target adducted genes of small molecules with antibodies can be identified without restriction by any cell or tissue type.
4. Large amounts of pure chromatin are not required and even whole cell lysates can be used, only antibodies specific for the small molecule compound.
5. Omics-based high-throughput screening method capable of identifying hundreds of genes
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the results of DNA agarose gel analysis of lysates that were sonicated and not sonicated. In the figure, E represents the experimental group, and C represents the control group.
FIG. 2 is a graph showing comparison of the amplification products of the experimental group, the control group and the input. In the figure, E represents the experimental group, and C represents the control group.
FIG. 3 is a detailed flow chart of the identification method of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The technical scheme of the invention is explained in detail in the following with reference to the attached drawings.
In this example, the human lung bronchial epithelial cell 16HBE was used in the practice, and the method of the present invention is by no means exclusively applicable to this cell, and any cell in which the DNA nucleophilic site is capable of covalently binding BDPE to form an adduct is suitable for use in the method of the present invention. One skilled in the art will also recognize that the methods of the invention are applicable to BPDE addition genes in other cells. Other non-exemplified cells of the invention are only changes or substitutions to the cells of the examples, and do not adversely affect the end result.
In the examples, the reagents, instruments and biological materials are commercially available unless otherwise specified. This example provides formulations and purchasing access to a portion of reagents, instruments, and biomaterials. The solvents used in the following reagents were deionized water unless otherwise specified.
10 × Glycine: merck Corp.
PBS buffer: 8gNaCl,0.2gKCl,1.44g Na 2 HPO 4 ，0.24gKH 2 PO 4 Dissolving in 800mL deionized water, adjusting the concentration to 7.4 by using HCl, and adding deionized water to constant volume to 1L.
1 × PIC II, also known as protease inhibitor cocktail II: beijing Congregation American Biotechnology Ltd.
Cell lysis buffer: 10mM HEPES (hydroxyethylpiperazine ethanethiosulfonic acid), 0.5% IGEPAL-CA630 (Okoku Biotechnology Co., ltd., beijing century), 1.5mM MgCl 2 ，0mM KCl，pH7.9。
Cell nucleus lysis buffer: 1% SDS,10mM EDTA,50mM Tris, pH8.1 (Merck).
An ultrasonic crusher: ningbo Xinzhi biological Co., ltd, SCIENTZ-IID type.
Rnase a (ribonuclease a): beijing Sorley technologies, inc.
Proteinase K, beijing Solebao science and technology, inc.
ChIP elution buffer: 2mM Tris,200mM NaCl,1% SDS, pH7.4.
ChIP dilution buffer: 0.01% of SDS,1.1% by weight of Triton X-100 (Beijing Boototta technologies, ltd.), 1.2mM EDTA,16.7mM Tris-HCl (Beijing Sorboard technologies, ltd.), 167mM NaCl, pH8.1.
ChIP-Grade Protein A/G magnetic bead: merck Corp.
Low-salt wash buffer: 0.1% SDS,1% Triton X-100,2mM EDTA,20mM Tris-HCl,150mM NaCl, pH8.1.
High-salt wash buffer: 0.1% SDS,1% Triton X-100,2mM EDTA, 2mM Tris-HCl,500mM NaCl, pH8.1.
LiCl wash buffer: 0.25M LiCl,1% IGEPAL-CA630,1% deoxyglycolic acid (sodium salt), 1mM EDTA, 1mM Tris, pH8.1.
TE buffer solution: 10mM Tris-HCl,1mM EDTA, pH 8.0.
DNA Binding buffer: QIAGEN Corp.
DNA Elution Buffer: QIAGEN Corp.
Wash Buffer:2mL TE buffer, 8mL ethanol.
Anti-BPDE Monoclonal Antibody: aimeiji science Ltd, clone 8E11.
Normal Mouse IgG: merck Corp.
Anti-RNA polymerase II Antibody: merck, clone CTD4H8.
Examples
A method for identifying a BPDE addition gene, comprising the steps of:
1) The cell confluence in the culture medium is observed to be 70% -80% under the microscope, and the cell state is good.
In this step, the degree of fusion refers to an index that measures the cell density in the culture dish. This index is a concept well known in the art. Only when the cell fusion length was 70% -80%, indicating that the cell condition was good, the following procedure was performed.
2) Adding formaldehyde into the culture medium to fix the cells in the formaldehyde, vortexing the culture dish to uniformly mix the formaldehyde in the culture medium, and standing at room temperature for 10min.
In this embodiment, the preferred manner of adding formaldehyde is to add methanol solution (solvent is deionized water), and the preferred addition concentration of formaldehyde solution is: 275. Mu.L of 37wt% formaldehyde solution was added per 10mL of medium. Cell disruption was avoided as much as possible during vortexing. The mode of vortex can be manual rotatory the even mixing that realizes formaldehyde in the culture medium, also can adopt the slight rotation of shaking table to realize the even mixing of formaldehyde in the culture medium.
3) Add 10 × Glycine to each dish, spin mix, incubate for 5min at room temperature, and quench unreacted formaldehyde.
In this example, taking 10mL of medium per dish as an example, 1mL of 10 × Glycine is preferably added per dish.
4) After discarding the medium, the culture dish was added with PBS buffer pre-cooled at 0-4 deg.C, the cells were washed and rinsed twice, each time carefully aspirated, removing as much PBS buffer as possible without disturbing the cells.
5) 1 XPICII in 0-4 ℃ pre-cooled PBS buffer was added to each dish, and cells were collected from each dish using a sterile cell scraper and collected into a centrifuge tube.
In this example, a preferred volume of 1 XPICII in 2mL of 0-4 ℃ pre-chilled PBS buffer is used with 10mL of media per dish. The size of the centrifuge tube used in this step was 10mL.
6) The cells were pelleted by centrifugation at 800 Xg (acceleration of gravity) for 5min at 4 ℃ and the supernatant carefully removed.
7) The cells were resuspended in 1mL of cell lysis buffer containing 1 XPICI, transferred to a new 1.5mL EP tube, incubated on ice for 15min, and vortexed every 5min during incubation.
Specifically, the mixture is uniformly mixed by vortex every 5min in the incubation process, and is uniformly mixed by vortex three times in the incubation process. In addition, incubation on ice refers to placing the cell-containing container on ice.
8) Centrifugation at 800 Xg for 5min at 4 ℃ removed the supernatant and resuspended cells pellet in nuclear lysis buffer containing 1 XPIC II to obtain the lysate.
In this step, the volume of cell nucleus lysis buffer containing 1 XPIC II was the same as in step 7).
9) Two aliquots of the lysate were placed in EP tubes, one of which was added to a DMSO solution of BPDE as the experimental group and the other was added to an equal volume of DMSO solution as the control group and incubated overnight at 37 ℃.
10 Sonication using a sonicator.
In the case of ultrasonication of the lysate, it is preferred to place two lysates on ice. In this embodiment, the maximum power of the ultrasonic crusher is 300W, the type of the probe is 2mm, the ultrasonic power set during ultrasonic treatment is 105W, the distance from the probe to the bottom of the EP tube is 2-3mm, and the ultrasonic treatment is performed for 5 times (the cumulative ultrasonic time is 75 s) at intervals of 15s for each time on the cracking liquid, so that an ideal shearing effect can be achieved. Of course, the skilled person can select the type of the sonicator and the corresponding setting parameters according to the actual needs to obtain the desired shearing effect of the cleavage product.
11 Centrifuged at 12000 Xg for 10min at 4 ℃ to remove insoluble material, and the sheared chromatins of the experimental and control groups were transferred to new EP tubes, respectively.
And after the step 9) is finished, respectively taking partial cleavage products of the experimental group and the control group to perform DNA agarose gel analysis, after the step 11) is finished, respectively taking the sheared chromatin of the experimental group and the control group to perform DNA agarose gel analysis, observing a band under a gel imaging system, and continuing to perform the step 12) if the width of the band is 100-500 bp.
In specific implementation, the DNA agarose gel analysis adopts the following steps:
(1) 20 μ L of the lysates of the non-sonicated experimental group and the control group and the lysates of the sonicated experimental group and the control group were placed in an EP tube and 30uL of ChIP Elution Buffer (ChIP Elution Buffer) was added, respectively, to ensure that the final volume was 50 μ L.
(2) mu.L of ribonuclease A (10 mg/mL, RNase A) was added to each EP tube and incubated at 37 ℃ for 30min (constant temperature metal bath).
(3) mu.L of proteinase K was added to each EP tube and incubated at 62 ℃ for 2h.
(4) mu.L of each EP tube was subjected to 1% agarose gel electrophoresis (using 100bp DNA Ladder as Marker), electrophoresed in a horizontal electrophoresis tank at constant pressure of 90V for 1h, and the band was observed under a gel imaging system, as shown in FIG. 1. The band is located at 100-500bp, which shows that the ultrasonic shearing effect is ideal. The next step is performed.
Preferably, after step 11) is completed, the DNA concentrations of the sonicated lysates of the experimental group and the control group are determined, respectively, and if the DNA concentration is above 50. Mu.g/mL, the process continues with step 12). If the DNA concentration is less than 50. Mu.g/mL, step 1) is repeated.
Specifically, in this step, after removing insoluble materials, the sheared chromatin of the experimental group and the control group was transferred to three new EP tubes, respectively, in which 50 μ L of sheared chromatin was present in each EP tube. The three new EP tubes of the experimental group are respectively an experimental group test tube, an experimental group negative control tube and an experimental group positive control tube, and the three new EP tubes of the control group are respectively a control group test tube, a control group negative control tube and a control group positive control tube.
12 ChIP dilution buffer containing 1 XPICI II was added to each sheared chromatin, and 2vol% of supernatant (mixture of sheared chromatin and ChIP dilution buffer containing 1 XPICI II) was taken from each of the experimental and control groups as input and stored at 4 ℃.
Specifically, 450. Mu.L of ChIP dilution buffer containing 1 XPICI was added per 50. Mu.L of cleaved chromatin.
When the sheared chromatin from the experimental and control groups were transferred to three new EP tubes containing 50 μ L of sheared chromatin in each EP tube, two inputs were taken from the experimental and control test tubes, respectively.
13 Anti-BPDE Monoclonal Antibody was added to the remaining experimental and control groups and incubated overnight at 4 ℃ in a shaker.
When the cleaved chromatin of the experimental group and the control group were transferred to three new EP tubes, respectively, anti-BPDE Monoclonal Antibody was added to the experimental group test tube and the control group test tube, respectively, normal Mouse IgG was added to the experimental group negative control tube and the control group negative control tube, respectively, anti-RNA polymerase II Antibody was added to the experimental group positive control tube and the control group positive control tube, respectively, and both were incubated overnight at 4 ℃ with shaking, and then steps 14) to 23) were performed, respectively).
Specifically, when the test tube of the experimental group and the test tube of the control group respectively have 490 μ L of supernatant (10 μ L of which is corresponding input), the negative control tube of the experimental group, the negative control tube of the control group, the positive control tube of the experimental group and the positive control group respectively have 500 μ L of supernatant. The amount of Anti-BPDE Monoclonal Antibody added was 4. Mu.g, the amount of Normal Mouse IgG added was 1 to 2. Mu.g, and the amount of Anti-RNA polymerase II Antibody added was 1 to 2. Mu.g.
14 ChIP-Grade Protein A/G magnetic beads are mixed evenly to completely suspend the magnetic beads, the magnetic beads are added into an experimental group, a control group and two input portions, a sealing membrane is sealed, and the mixture is incubated for 2 hours at 4 ℃.
Specifically, 20. Mu.L of ChIP-Grade Protein A/G magnetic beads was added to each EP tube. The experimental group comprises an experimental group test tube, an experimental group negative control tube and an experimental group positive control tube, and the control group comprises a control group test tube, a control group negative control tube and a control group positive control tube.
15 Experimental group (three tubes), control group (three tubes) and two input EP tubes were placed on a magnetic separation rack, chIP-Grade Protein a/G magnetic beads were adsorbed to the tube wall, left to stand for 3min, and the supernatant was removed.
16 In the order listed below), the magnetic beads were suspended in each of the buffers precooled at 0-4 ℃ and incubated on a constant temperature shaker at 4 ℃ for 5min, respectively, followed by magnetic separation, removal of the supernatant, and washing of the Grade Protein A/G magnetic bead-chromatin complexes:
a. washing once with a low-salt washing buffer solution;
b. washing once with a high-salt washing buffer solution;
LiCl washing buffer solution, washing once;
TE buffer, wash once.
In the above procedure, the experimental group, the control group and the two inputs were washed with four buffers under the above conditions.
17 ChIP Elution Buffer was added to each of the EP tubes of the experimental group, the control group and the two inputs.
Preferably, the ChIP Elution Buffer is prepared from 100 mu L of ChIP Elution Buffer and 1 mu L of proteinase K.
18 The experimental group, the control group and two input EP tubes were incubated at 62 ℃ for 2h with shaking in a water bath, and cultured at 95 ℃ for 10min, and the chromatin of the experimental group and the control group was eluted from the ChIP-Grade Protein A/G magnetic beads and cooled to room temperature.
19 The EP tubes of the experimental group and the control group are placed on a magnetic separation rack, and are kept still for 3min, and ChIP-Grade Protein A/G magnetic beads are adsorbed to the tube wall, and the supernatant is transferred to a new EP tube and is respectively marked.
20 Add DNA Binding Buffer to the EP tubes of the experimental group, control group and two inputs, respectively, and mix them by vortexing.
0.5mL of DNA Binding Buffer was added to each of the EP tubes of the experimental group, the control group and the two inputs.
21 Transferring to a centrifugal filter with a receiving tube, centrifuging at 12000 Xg for 30s, taking out the centrifugal filter from the receiving tube, reserving the receiving tube, discarding the liquid, and returning the centrifugal filter to the same receiving tube;
22 Adding Wash Buffer, centrifuging at 12000 Xg for 30s, taking out the centrifugal filter from the receiving tube, reserving the receiving tube, discarding the liquid, and returning the centrifugal filter to the same receiving tube; the cells were centrifuged again at 12000 Xg for 30s, and the receiver tube and liquid were discarded.
23 Put into a new receiving tube, add DNA Elution Buffer directly to the center of the white centrifugal filter membrane, centrifuge at 12000 Xg for 30s, remove and discard the centrifugal filter, the Elution Buffer at this time is purified DNA.
And (3) carrying out PCR amplification on part of purified DNA in the experiment group negative control tube, the experiment group positive control tube, the input corresponding to the experiment group, the control group negative control tube, the control group positive control tube and the input corresponding to the control group, and if a band is obtained at 166bp, indicating that the ChIP quality control is good, and continuing to carry out the step 24).
The PCR comprises the following specific steps: PCR reaction systems were prepared as shown in Table 1, and 18. Mu.L of the reaction system and 2. Mu.L of the corresponding DNA template were added to each PCR tube, and the PCR reaction procedures are shown in Table 2.
TABLE 1 PCR reaction System
TABLE 2 PCR reaction procedure
10 μ L of PCR reaction product was subjected to 2% agarose gel electrophoresis (using 100bp DNA Marker) for analysis, and subjected to electrophoresis for 1 hour in a horizontal electrophoresis tank under a constant voltage of 90V. Positive, input gave a band at 166bp, as shown in FIG. 2. The above results indicate that the ChIP experiment has good quality control.
24 Detecting the concentration and purity of purified DNA of an experimental group, a control group and two inputs, carrying out high-throughput sequencing, and if the concentration of the DNA of the control group is not detected, removing a background value of the input corresponding to the experimental group by the experimental group to obtain a gene, namely a BPDE addition gene; if the control group detects the DNA concentration, the gene obtained after the experimental group removes the control group, namely the BPDE addition gene.
Specifically, an Eppendorf ultraviolet spectrophotometer can be used for detecting the concentration and purity of the experimental group, the control group and the two input parts, and the concentration and purity can be used for subsequent high-throughput sequencing. The concentration and purity of the DNA meet the requirement of chromatin immunoprecipitation-sequencing (ChIP-Seq): the concentration is more than or equal to 1 ng/mu L; the purity OD260/280 is 1.8-2.0; the total amount of DNA is more than or equal to 20ng.
In this example, the undetected DNA concentration of the control group submitted for the same batch is because the control group is not infected with BPDE, the addition of BPDE antibody cannot result in DNA fragments under IP, and even if there is nonspecific binding of a very small amount of antibody, so that the subsequent sequencing can only obtain genes obtained by removing the background value of the Input sample from the IP sample of the infected group, and these genes are the expected difference genes.
A detailed gene list obtained from the study of this example is shown in Table 3.
The potential target genes identified in Table 3 and their distribution on each chromosome
It can thus be seen that: the invention relates to a high-throughput screening method based on omics, which can identify hundreds of genes.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (7)
1. A method for identifying a BPDE addition gene, comprising the steps of:
1) The cell fusion degree in the culture medium is observed to be 70-80% under a mirror, and the cell state is good at the moment;
2) Adding formaldehyde into the culture medium to fix the cells in the formaldehyde, uniformly mixing the formaldehyde in the culture medium by a vortex culture dish, and standing for 10min at room temperature;
3) Adding 10 XGlycine into each culture dish, uniformly mixing in a rotating way, incubating for 5min at room temperature, and quenching unreacted formaldehyde;
4) Discarding the culture medium, adding a PBS (phosphate buffer solution) precooled at 0-4 ℃ into a culture dish, washing cells, repeatedly rinsing twice, carefully sucking each time, removing the PBS as much as possible without disturbing the cells;
5) Adding 1 XPICII-containing PBS buffer solution precooled at 0-4 ℃ into each culture dish, collecting cells from each culture dish by using a sterile cell scraper, and collecting the cells into a centrifuge tube;
6) The cells were pelleted by centrifugation at 800 Xg for 5min at 4 ℃ and the supernatant carefully removed;
7) Resuspending the cells in a cell lysis buffer containing 1 XPIC II, incubating on ice for 15min, and vortexing once every 5min during incubation;
8) Centrifuging at 4 deg.C at 800 × g for 5min, removing supernatant, and resuspending cell precipitate in cell lysis buffer containing 1 × PIC II to obtain lysis product;
9) Placing two aliquots of the lysate into EP tubes, adding one part of the lysate into DMSO solution of BPDE as an experimental group, adding the other part of the lysate into DMSO solution with the same volume as a control group, and incubating at 37 ℃ overnight;
10 Sonication using a sonicator;
11 Centrifugation at 12000 Xg for 10min at 4 ℃ to remove insoluble material, and transfer the sheared chromatins of the experimental and control groups to new EP tubes, respectively;
12 ChIP dilution buffer solution containing 1 XPIC II is respectively added into the sheared chromatin, 2vol% of supernatant is respectively taken from an experimental group and a control group to serve as input, and the input is preserved at 4 ℃;
13 Anti-BPDE Monoclonal Antibody was added to the remaining experimental and control groups and incubated overnight at 4 ℃ in a constant temperature shaker;
14 Mixing ChIP-Grade Protein A/G magnetic beads uniformly to enable the magnetic beads to be suspended completely, adding the magnetic beads into an experimental group, a control group and two input, sealing a sealing membrane, and incubating for 2 hours at 4 ℃;
15 Placing the EP tubes of the experimental group, the control group and two inputs on a magnetic separation rack, adsorbing ChIP-Grade Protein A/G magnetic beads to the tube wall, standing for 3min, and removing the supernatant;
16 In the order listed below), the magnetic beads were suspended in each of the buffers precooled at 0-4 ℃ and incubated on a constant temperature shaker at 4 ℃ for 5min, respectively, followed by magnetic separation, removal of the supernatant, and washing of the Grade Protein A/G magnetic bead-chromatin complexes:
a. washing once with a low-salt washing buffer solution;
b. washing once with a high-salt washing buffer solution;
LiCl washing buffer solution, washing once;
TE buffer solution, washing once;
17 Respectively adding ChIP Elution buffers into the EP tubes of an experimental group, a control group and two input;
18 The experimental group, the control group and two input EP tubes are incubated for 2h at 62 ℃ in a water bath with shaking and are cultured for 10min at 95 ℃, and chromatin of the experimental group and the control group is eluted from the ChIP-Grade Protein A/G magnetic beads and is cooled to room temperature;
19 The EP tubes of the experimental group and the control group are placed on a magnetic separation frame, the mixture is kept stand for 3min, the ChIP-Grade Protein A/G magnetic beads are adsorbed to the tube wall, and the supernatant is transferred to a new EP tube and is respectively marked;
20 Add DNA Binding Buffer to the EP tubes of the experimental group, the control group and two inputs respectively, and mix them by vortex;
21 Transferring to a centrifugal filter with a receiving tube, centrifuging at 12000 Xg for 30s, taking out the centrifugal filter from the receiving tube, reserving the receiving tube, discarding the liquid, and returning the centrifugal filter to the same receiving tube;
22 Adding Wash Buffer, centrifuging at 12000 Xg for 30s, taking out the centrifugal filter from the receiving tube, reserving the receiving tube, discarding the liquid, and returning the centrifugal filter to the same receiving tube; centrifuging at 12000 Xg for 30s again, and discarding the receiving tube and liquid;
23 Put into a new receiving tube, add DNA Elution Buffer directly to the center of the white centrifugal filter membrane, centrifuge for 30s at 12000 Xg, take out and discard the centrifugal filter, the Elution Buffer at this moment is purified DNA;
24 Detecting the concentration and purity of purified DNA of an experimental group, a control group and two inputs, performing high-throughput sequencing, and if the concentration of the DNA is not detected by the control group, removing the background value of the input corresponding to the experimental group by the experimental group to obtain a gene, namely a BPDE addition gene; if the control group detects the DNA concentration, the gene obtained after the experimental group removes the control group, namely the BPDE addition gene.
2. The method of claim 1, wherein after step 9) is performed, the cleavage products of the experimental group and the control group are partially separated and analyzed by DNA agarose gel, and after step 11) is performed, the cleaved chromatin of the experimental group and the control group are separated and analyzed by DNA agarose gel, and the band is observed under a gel imaging system, and if the width of the band is 100-500bp, the step 12) is performed.
3. The method of claim 1, wherein the DNA concentration of the sonicated lysates of the experimental group and the control group are determined after step 11) is completed, and if the DNA concentration is above 50 μ g/mL, the step 12) is continued.
4. The method of claim 1, wherein the cleaved chromatin of the experimental group and the control group is transferred to three new EP tubes after removing insoluble materials in step 11).
5. The method of claim 4, wherein the three new EP tubes of the experimental group are the experimental group test tube, the experimental group negative control tube and the experimental group positive control tube, respectively, and the three new EP tubes of the control group are the control group test tube, the control group negative control tube and the control group positive control tube, respectively; in step 12), two inputs are taken from the test tube of the experimental group and the test tube of the control group, respectively.
6. The method of claim 5, wherein Anti-BPDE Monoclonal Antibody is added to the test tubes of the test group and the test tubes of the control group, respectively, normal Mouse IgG is added to the negative control tubes of the test group and the negative control tubes of the control group, respectively, anti-RNA polymerase II Antibody is added to the positive control tubes of the test group and the positive control tubes of the control group, respectively, and the tubes are incubated overnight at 4 ℃ and then steps 14) to 23) are performed.
7. The method of claim 6, wherein the PCR amplification is performed on the partially purified DNA in the experiment group negative control tube, the experiment group positive control tube, the input corresponding to the experiment group, the control group negative control tube, the control group positive control tube and the input corresponding to the control group, and if a band is obtained at 166bp, the control of ChIP is good, and the step 24 is continued).
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