阅读说明：本技术 一种精子染色质免疫共沉淀方法 (Sperm chromatin co-immunoprecipitation method ) 是由 茹彦飞 施惠娟 华敏敏 张天成 顾一骅 汤佳楠 王雪梅 于 2021-08-16 设计创作，主要内容包括：本发明公开了一种精子染色质免疫共沉淀方法。属靶向核酸纯化及二代测序建库样本预处理领域。包括精子消化：将精子经多聚甲醛固定、清洗、裂解后,采用微球菌核酸酶消化-超声相结合的方法进行消化得消化液；超声参数：超声30s、间隔45s,10个循环；微球菌核酸酶消化参数：酶加入量200U/10～(6)个精子,酶解温度37℃,酶解时间30min；染色质分离：消化液经初清后得染色质；染色质免疫共沉淀：抗体与protein A agarose形成复合物,再与染色质中相应的抗原结合,之后经清洗、洗脱得免疫共沉淀反应液。本发明把超声与酶消化结合,提高消化效率,增加DNA回收率,有助于降低建库难度,提高精子免疫共沉淀效率。(The invention discloses a sperm chromatin co-immunoprecipitation method. Belongs to the field of target nucleic acid purification and second-generation sequencing library building sample pretreatment. Comprises the steps of sperm digestion: fixing, cleaning and cracking sperms by paraformaldehyde, and digesting by adopting a method combining micrococcus nuclease digestion and ultrasound to obtain a digestive juice; ultrasonic parameters: ultrasonic treatment for 30s at intervals of 45s for 10 cycles; micrococcal nuclease digestion parameters: the enzyme is added in an amount of 200U/10 6 Carrying out enzymolysis on the sperms at 37 ℃ for 30 min; chromatin separation: obtaining chromatin after the digestive juice is primarily cleared; chromatin co-immunoprecipitation: the antibody and protein A agarose form a compound, then the compound is combined with corresponding antigen in chromatin, and then the co-immunoprecipitation reaction solution is obtained by cleaning and eluting. The invention combines ultrasonic and enzyme digestion, improves digestion efficiency, increases DNA recovery rate, and is helpful forThe difficulty of establishing a bank is reduced, and the efficiency of the sperm co-immunoprecipitation is improved.)

1. A sperm chromatin co-immunoprecipitation method, comprising the steps of:

(1) and (3) sperm digestion: fixing, cleaning and cracking sperms by paraformaldehyde, and digesting the sperms by adopting a method combining micrococcus nuclease digestion and ultrasound to obtain a digestive juice; wherein the content of the first and second substances,

ultrasonic parameters: ultrasonic treatment for 30s at intervals of 45s for 10 cycles;

micrococcal nuclease digestion parameters: the enzyme is added in an amount of 200U/10⁶Carrying out enzymolysis on the sperms at 37 ℃ for 30 min;

(2) chromatin separation: obtaining chromatin after the digestive juice is primarily cleaned;

(3) chromatin co-immunoprecipitation: the antibody and protein A agarose form a compound, then the compound is combined with corresponding antigen in chromatin, and then the co-immunoprecipitation reaction solution is obtained after cleaning and elution.

2. The method for chromatin co-immunoprecipitation by sperm according to claim 1, wherein the specific operation of step (1) is:

(11) fixing the sperms by using paraformaldehyde with the mass concentration of 0.5%;

(12) centrifuging at 20-25 ℃ for 10min at 5,000g, and discarding the supernatant;

(13) adding 4 ℃ precooled PBS for washing, then centrifuging for 10min at 20-25 ℃ and 5,000g, removing supernatant and keeping sperm precipitation;

(14) according to 500. mu.l/10⁶Adding lysis solution to sperm, and incubating at 20-25 deg.C for 1hr to obtain mixed solution A;

the lysis solution comprises the following components in mass concentration: 0.5% SDS and 10mM DTT;

(15) adding 1 MN-ethyl maleimide after cracking;

(16) then ultrasonic treatment is adopted, ultrasonic treatment is carried out for 30s at an interval of 45s, and 10 cycles are carried out; after the ultrasonic treatment, the micro-coccus nuclease is used for digestion, and the enzyme adding amount is 200U/10⁶Carrying out enzymolysis on the sperms at 37 ℃ for 30min to obtain a digestive juice.

3. The method for chromatin co-immunoprecipitation by sperm according to claim 1, wherein the step (2) is specifically performed by:

(21) adding a dilution buffer solution in equal proportion into the digestive juice, wherein the dilution buffer solution comprises the following components in mass concentration: 1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl, pH 8.0;

(22) adding proteinA agarose, shaking at 4 deg.C for 2hr, and performing primary cleaning;

(23) centrifuging at 5,000rpm for 1min, keeping the supernatant as chromatin, taking 1/10 volume as input sample, and performing the subsequent operation on the rest samples.

4. The method of claim 1, wherein the antibody in step (3) is H3K4me2 or H3K27me 3.

5. A sperm chromatin co-immunoprecipitation method as claimed in claim 3, wherein the step (3) is specifically performed by:

(31) adding an antibody into the chromatin, wherein the volume mass ratio of the chromatin to the antibody is 8 mu l:1 mu g, and oscillating overnight at 4 ℃;

(32) adding the chromatin volume 4/5 proteinA agarose, shaking for 2 hr;

(33) centrifuging at 5,000rpm for 1min, and discarding the supernatant;

(34) washing with TSE I, TSE II, buffer solution III and TE buffer solution twice respectively, shaking at 4 deg.C for 2min each time, and removing supernatant to obtain co-immunoprecipitated sample;

the TSEI comprises the following components in mass concentration: 20mM Tris-HCl, 0.5M EDTA, 0.1% SDS, 150mM NaCl and 1% TritonX-100, pH 8.0;

the TSEI comprises the following components in mass concentration: 20mM Tris-HCl, 0.5M EDTA, 0.1% SDS, 500mM NaCl and 1% TritonX-100, pH 8.0;

the buffer solution III comprises the following components in mass concentration: 10mM Tris-HCl, 1mM EDTA, 1% NP-40, and 1% sodium deoxycholate, pH 8.0;

the TE buffer solution comprises the following components in mass concentration: 10mM Tris-HCl and 0.5MEDTA, pH 8.0;

(35) after the co-immunoprecipitation, adding an eluent with the volume 5 times that of the chromatin into a sample for resuspension, carrying out water bath at 65 ℃ for 5min, and then oscillating at 20-25 ℃ for 15 min;

the eluent comprises the following components in mass concentration: 1% SDS and 0.1M NaHCO₃；

(36) Centrifuging at 12,000rpm for 2min, and collecting the supernatant;

(37) repeating 35-36 steps, and merging the supernatant liquid of the two elutions;

(38) adding 0.04 times volume of 5M NaCl and 0.01 times volume of 2mg/ml RNase into the eluate supernatant, and water-bathing at 65 deg.C for 4 hr;

(39) adding 0.02 volume times of 0.5M EDTA, 0.04 volume times of 1.5M Tris-HCl, 0.01 volume times of proteinase K, and water bath at 65 deg.C for 2hr to obtain co-immunoprecipitation solution.

Technical Field

The invention relates to the technical field of target nucleic acid purification and second-generation sequencing library building sample pretreatment, in particular to a sperm chromatin co-immunoprecipitation method.

Background

At present, there are many methods for studying the interaction between proteins, and commonly used methods include yeast two-hybrid elution, co-immunoprecipitation, Pull-down, and the like. Among them, co-immunoprecipitation is a relatively stable, simple and convenient method, and is also a method widely used in the scientific research of proteome.

Co-immunoprecipitation is a technique for studying protein interactions based on specific binding between antibodies and antigens. The non-denaturing agent is mainly used for cracking the whole cells, so that the interaction among a plurality of proteins in the cells is maintained, and the interaction among the proteins is convenient to detect. An antibody against protein A forms an immune complex with A and precipitates, while protein B, which is stably bound to A in cells, is precipitated at the same time. Protein B precipitation is based on physical interaction with a, a technique known as co-immunoprecipitation. The technology utilizes antigen and antibody to form protein compound precipitate, dissolves the protein compound and separates out protein B. The co-immunoprecipitation method is an effective method for determining the interaction of two proteins under cell physiological conditions.

However, the amount of histone contained in the sperm is very small (about 6-15%), so that the co-immunoprecipitation methods related to histone modification of sperm in the prior art are low in efficiency.

In conclusion, how to provide an efficient method for chromatin co-immunoprecipitation by sperm is a problem that needs to be solved by those skilled in the art.

Disclosure of Invention

In view of the above, the present invention provides a sperm chromatin co-immunoprecipitation method.

In order to achieve the purpose, the invention adopts the following technical scheme:

a sperm chromatin co-immunoprecipitation method comprising the steps of:

(1) and (3) sperm digestion: fixing, cleaning and cracking sperms by paraformaldehyde, and digesting the sperms by adopting a method combining micrococcus nuclease digestion and ultrasound to obtain a digestive juice; wherein the content of the first and second substances,

ultrasonic parameters: ultrasound 30s (selection)Low power of the ultrasound device), 45s apart, 10 cycles;

micrococcal nuclease digestion parameters: the enzyme is added in an amount of 200U/10⁶Carrying out enzymolysis on the sperms at 37 ℃ for 30 min;

(2) chromatin separation: obtaining chromatin after the digestive juice is primarily cleaned;

(3) chromatin co-immunoprecipitation: the antibody and protein A agarose form a compound, then the compound is combined with corresponding antigen in chromatin, and then the co-immunoprecipitation reaction solution is obtained after cleaning and elution.

Further, the specific operation of the step (1) is as follows:

(11) fixing the sperms by using paraformaldehyde with the mass concentration of 0.5%;

(12) centrifuging at 20-25 ℃ for 10min at 5,000g, and discarding the supernatant;

(13) adding 4 ℃ precooled PBS for washing, then centrifuging for 10min at 20-25 ℃ and 5,000g, removing supernatant and keeping sperm precipitation;

(14) according to 500. mu.l/10⁶Adding lysis solution to sperm, and incubating at 20-25 deg.C for 1hr to obtain mixed solution A;

the lysis solution comprises the following components in mass concentration: 0.5% SDS and 10mM DTT;

(15) adding 1M N-ethyl maleimide after cracking;

(16) then ultrasonic treatment is carried out for 30s (selection)Low power of the ultrasound device), 45s apart, 10 cycles; after the ultrasonic treatment, the micro-coccus nuclease is used for digestion, and the enzyme adding amount is 200U/10⁶Carrying out enzymolysis on the sperms at 37 ℃ for 30min to obtain a digestive juice.

The beneficial effects are obtained: allowing full release of chromatin.

Further, the specific operation of the step (2) is as follows:

(21) adding a dilution buffer solution in equal proportion into the digestive juice, wherein the dilution buffer solution comprises the following components in mass concentration: 1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl, pH 8.0;

(22) adding protein A agarose, shaking at 4 deg.C for 2hr, and performing primary cleaning;

(23) centrifuging at 5,000rpm for 1min, and collecting supernatant as chromatin.

The beneficial effects are obtained: sufficient chromatin was obtained.

Further, the antibody in the step (3) is an antibody of H3K4me2 or H3K27me 3.

Further, the specific operation of the step (3) is as follows:

(31) adding an antibody into the chromatin, wherein the volume mass ratio of the chromatin to the antibody is 8 mu l:1 mu g, and oscillating overnight at 4 ℃;

(32) adding protein A agarose of chromatin volume 4/5, and shaking for 2 hr;

(33) centrifuging at 5,000rpm for 1min, and discarding the supernatant;

(34) washing with TSE I, TSE II, buffer solution III and TE buffer solution respectively twice, shaking at 4 ℃ for 2min each time, and removing supernatant to obtain a co-immunoprecipitated sample;

the TSEI comprises the following components in mass concentration: 20mM Tris-HCl, 0.5M EDTA, 0.1% SDS, 150mM NaCl and 1% TritonX-100, pH 8.0;

the TSEI comprises the following components in mass concentration: 20mM Tris-HCl, 0.5M EDTA, 0.1% SDS, 500mM NaCl and 1% TritonX-100, pH 8.0;

the buffer solution III comprises the following components in mass concentration: 10mM Tris-HCl, 1mM EDTA, 1% NP-40, and 1% sodium deoxycholate, pH 8.0;

the TE buffer solution comprises the following components in mass concentration: 10mM Tris-HCl and 0.5M EDTA, pH 8.0;

(35) after the co-immunoprecipitation, adding an eluent with the volume 5 times that of the chromatin into a sample for resuspension, carrying out water bath at 65 ℃ for 5min, and then oscillating at 20-25 ℃ for 15 min;

the eluent comprises the following components in mass concentration: 1% SDS and 0.1M NaHCO₃；

(36) Centrifuging at 12,000rpm for 2min, and collecting the supernatant;

(37) repeating 35-36 steps, and merging the supernatant liquid of the two elutions;

(38) adding 0.04 times volume of 5M NaCl and 0.01 times volume of 2mg/ml RNase into the eluate supernatant, and water-bathing at 65 deg.C for 4 hr;

(39) adding 0.02 volume times of 0.5M EDTA, 0.04 volume times of 1.5M Tris-HCl and 0.01 volume times of proteinase K into the eluate, and heating in 65 deg.C water bath for 2hr to obtain co-immunoprecipitation solution.

The beneficial effects are obtained: and removing the protein combined with the DNA, thereby facilitating the subsequent purification and detection of the DNA.

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: ultrasonic and enzyme digestion are combined, so that the digestion efficiency is greatly improved, the recovery rate of DNA is obviously increased, the difficulty of building a subsequent library is reduced, and the efficiency of the sperm co-immunoprecipitation is greatly improved.

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 embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the results of agarose gel electrophoresis of a digestion solution in example 1 of the present invention;

FIG. 2 is a diagram showing verification of H3K4me2 positive and negative sites in example 1 of the present invention;

FIG. 3 is a diagram showing verification of the positive and negative sites of H3K27me3 in example 1 of the present invention;

FIG. 4 is a diagram showing verification of H3K4me2 positive and negative sites in example 2 of the present invention;

FIG. 5 is a diagram showing verification of the positive and negative sites of H3K27me3 in example 2 of the present invention;

FIG. 6 is a drawing showing agarose gel electrophoresis of an ultrasonic-optimized digestion solution in example 3 of the present invention, in which S represents the supernatant and P represents the precipitate.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The medicament required by the embodiment of the invention is a conventional experimental medicament purchased from a commercially available channel; the experimental methods not mentioned in the examples are conventional experimental methods, and are not described in detail herein.

Example 1

1. Freezing semen of human (about 2X 10) at-80 deg.C⁷) Paraformaldehyde (PFA) with a mass concentration of 0.5% was added and fixed at 22 ℃ for 10 min.

5,000g at 2.22 ℃ for 10min, and the supernatant was discarded.

3. Washing with 4 deg.C precooled PBS once, washing with 5,000g at 22 deg.C, centrifuging for 10min, and discarding the supernatant.

4. Mu.l of lysate (0.5% SDS, 10mM DTT) was added to the sperm pellet and incubated at 22 ℃ for 1 hr.

5. Then, 3. mu.l of 1M N-ethylmaleimide was added to terminate the DTT effect.

6. Then ultrasonic treatment is carried out for 30s (selection)Low power of the ultrasound device), 45s apart, 10 cycles; after the ultrasonic treatment, the micro-coccus nuclease is used for digestion, and the enzyme adding amount is 200U/10⁶Carrying out enzymolysis on the sperms at 37 ℃ for 30min to obtain a digestive juice.

7. The above digest was centrifuged at 12,000rpm for 15min, and the supernatant was transferred to another 1.5ml EP tube.

8. Adding a dilution buffer to the mixture to make up for 1ml, wherein the dilution buffer comprises the following components in mass concentration: 1% TritonX-100, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl, pH8.0.

9. Add 50. mu.l of protein A agarose and shake at 4 ℃ for 2hr for initial clearing.

10.5,000 rpm for 1min, and the supernatant was transferred to another 1.5ml EP tube.

11. Taking 40 mu l of the sample as an Input sample, and carrying out subsequent operations (12-17) on the rest samples;

the remaining samples were aliquoted into 2 1.5ml Ep tubes, one with 5. mu.g of H3K4me2 antibody or H3K27me3 antibody and the other with 5. mu.g of IgG (control);

shaking overnight at 4 ℃.

12. Add 50. mu.l of proteina agarose to each tube and shake for 2 hr.

13.5,000 rpm for 1min, and the supernatant was discarded.

14. The column was washed twice with TSEI, TSEII, buffer III and TE buffer, each time with shaking at 4 ℃ for 2min (the supernatant was aspirated off each time with a 1ml syringe).

TSEI comprises the following components in mass concentration: 20mM Tris-HCl, 0.5M EDTA, 0.1% SDS, 150mM NaCl and 1% TritonX-100, pH 8.0;

TSEI comprises the following components in mass concentration: 20mM Tris-HCl, 0.5M EDTA, 0.1% SDS, 500mM NaCl and 1% TritonX-100, pH 8.0;

the buffer solution III comprises the following components in mass concentration: 10mM Tris-HCl, 1mM EDTA, 1% NP-40, and 1% sodium deoxycholate, pH 8.0;

the TE buffer comprises the following components in mass concentration: 10mM Tris-HCl and 0.5M EDTA, pH 8.0.

15. Adding 200 μ l of newly preparedEluent (1% SDS, 0.1M NaHCO)₃) Resuspending, carrying out water bath at 65 ℃ for 5min, and then shaking at 20-25 ℃ for 15 min.

16.12,000 rpm for 2min, the supernatant was retained as the elution supernatant, and the elution supernatant was transferred to another 1.5ml Ep tube.

17. Repeat 15-16 times, combine the two buffered supernatants, about 400. mu.l.

18. Mu.l of 5M NaCl, 4. mu.l of 2mg/ml RNase was added to 400. mu.l of the buffered supernatant;

at the same time, 180. mu.l of eluent eluonbuffer was used to make up the volume of Input sample to 200. mu.l, 8. mu.l of 5M NaCl, 4. mu.l of 2mg/ml RNase were added, and water bath at 65 ℃ was carried out for 4 hr.

19. Add 8. mu.l 0.5M EDTA, 16. mu.l 1.5M Tris-HCl, 4. mu.l proteinase K;

at the same time, 8. mu.l of 1.5M Tris-HCl and 4. mu.l proteinase K were added to the Input sample, and the mixture was incubated at 65 ℃ for 2 hr.

20. Extracting DNA (absolute ethyl alcohol and 70% ethyl alcohol put into a refrigerator at-80 ℃ in advance)

A. Equal volume of phenol was added: chloroform: isoamyl alcohol, mixing.

B.12,000rpm, 5min at 22 ℃.

C. The supernatant was transferred to another 1.5ml Ep tube, and 1/10 volumes of 3M NaAc (pH5.2), 1.5. mu.l glycogen, 2 volumes of absolute ethanol were added, and the mixture was precipitated in a refrigerator at-80 ℃ for 15 min.

D.4 ℃ for 15min at 12,000rpm, the supernatant was discarded.

E.70% ethanol washing, 12,000rpm centrifugation for 10min, abandon the supernatant.

F.1ml absolute ethanol washing to facilitate DNA drying, 12,000rpm centrifugation for 10 min.

G. After air drying, the DNA was dissolved in 100. mu.l of TE buffer.

The digestion solution was subjected to agarose gel electrophoresis, and the results are shown in FIG. 1. The results in FIG. 1 show that it is possible to stably obtain mononucleosomes with uniform and concentrated bands, and that the conditions for performing co-Immunoprecipitation (IP) are provided.

Using the obtained mononucleosomes from human sperm, co-immunoprecipitation was performed using antibodies against H3K4me2 and H3K27me3, respectively. The results of quantitative PCR showed that the sites on human sperm that had H3K27me3 modification (LIF, HOXA9, MYC, H2B1C, GATA6) were significantly enriched, while the sites that had no H3K27me3 modification (NANOG, GDF3, BRDT, IG) were not enriched (figure 2). Sites with H3K4me2 modification (BRDT, TAF8, PGK2) were significantly enriched on human sperm, while sites without H3K4me2 modification (LIF, IG, STRA8) were not enriched (fig. 3).

Furthermore, due to the precious and scarce nature of human sperm samples, we have searched for the minimum number of sperm required to meet ChIP-seq by adjusting the number of sperm and determining the DNA yield after ChIP and have found that 2X 10 sperm samples are used⁷The number of human sperm (20M) (Table 1) was found to satisfy the ChIP-seq requirement (the amount of DNA obtained after ChIP was 10ng or more).

TABLE 1 sperm count and yield of DNA recovered after CHIP

Example 2

The procedure of example 1 was repeated except that the mouse sperm was replaced with the human sperm.

The verification of the positive and negative sites of H3K4me2 and H3K27me3 is shown in fig. 4 and 5.

Example 3

The ultrasonic conditions were optimized to change the ultrasonic cycle numbers to 0, 3, 6, and 10, respectively, and then the supernatant and the pellet of the digestion solution were subjected to agarose gel electrophoresis, and the results are shown in fig. 6.

The supernatant and the precipitate after the digestion and the co-immunoprecipitation under the above conditions were used to calculate the DNA yield, and the results are shown in Table 2.

TABLE 2 DNA yield after optimization of ultrasound conditions

Sample (I)	Concentration (ng/. mu.l)
		0-S	254.4
0-P	342.4
		3-S	361.5
3-P	52.9
		6-S	210.6
6-P	91.2
		10-S	106.2
10-P	48.0

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.