阅读说明：本技术 一种利用微量临床穿刺样本进行染色质免疫共沉淀文库制备的方法及其应用 (Method for preparing chromatin co-immunoprecipitation library by using trace clinical puncture sample and application thereof ) 是由 薛愿超 苏瑞宝 王磊 于 2019-01-16 设计创作，主要内容包括：本发明公开了一种利用微量临床穿刺样本进行染色质免疫共沉淀文库制备的方法及其应用。本发明利用高敏型Tn5转座酶成功开发了一种基于微量临床穿刺样本的染色质免疫共沉淀测序文库制备方法,可以对微量的穿刺组织样本有效快速进行ChIP-seq文库构建。相比于传统的ChIP-seq建库方式,该方法大大降低样本起始量(5-50mg),有效缩短了建库时间(约1.5天),满足了测序市场对微量样本和大规模样本处理速度的需求。(The invention discloses a method for preparing a chromatin co-immunoprecipitation library by using a trace clinical puncture sample and application thereof. The invention successfully develops a chromatin co-immunoprecipitation sequencing library preparation method based on a trace clinical puncture sample by using a high-sensitivity Tn5 transposase, and can effectively and rapidly construct a ChIP-seq library on the trace puncture tissue sample. Compared with the traditional ChIP-seq library construction method, the method greatly reduces the initial amount (5-50mg) of the sample, effectively shortens the library construction time (about 1.5 days), and meets the requirements of the sequencing market on the processing speed of the micro sample and the large-scale sample.)

The application of Tn5 transposition system in constructing chromatin co-immunoprecipitation library;

or, Tn5 transposition system in preparing products for constructing chromatin co-immunoprecipitation library;

or, the Tn5 transposition system is applied to the construction of the chromatin co-immunoprecipitation library based on the micro tissue sample;

or, the Tn5 transposition system is applied to preparing products for constructing chromatin co-immunoprecipitation libraries based on micro tissue samples.

2. Use according to claim 1, characterized in that: the minimum starting amount of the microtissue sample is 5 mg;

alternatively, the tissue sample is a punctured tissue sample.

3. A method for constructing a chromatin co-immunoprecipitation library, comprising the steps of:

1) sequentially carrying out formaldehyde treatment and chromatin DNA fragmentation on a tissue sample, and then incubating with a specific target protein antibody to obtain a target protein-DNA compound;

2) fragmenting, end repairing and connecting a joint of a DNA fragment in the target protein-DNA complex by using a Tn5 transposition system, and digesting the target protein by using protease to obtain a DNA product;

3) and carrying out PCR amplification by taking the DNA product as a template to obtain a chromatin co-immunoprecipitation library.

4. The method of claim 3, wherein: said 1), the lowest starting amount of said tissue sample is 5 mg;

alternatively, the tissue sample is a punctured tissue sample.

5. The method according to claim 3 or 4, characterized in that: the method comprises the following steps of 1):

1-1) adding a formaldehyde solution into a tissue sample for crosslinking reaction to obtain a crosslinked product;

1-2) homogenizing and crushing the crosslinking product to obtain tissue fragments;

1-3) adding cell lysate into the tissue fragments to obtain lysed cells, centrifuging, and collecting precipitates to obtain cell nuclei;

1-4) fragmenting chromatin DNA in the cell nucleus to obtain fragmented DNA fragments;

1-5) then incubating the fragmented DNA fragments with specific target protein antibodies, and enriching the target protein and the DNA fragments combined with the target protein to obtain the target protein-DNA complex.

6. The method of claim 5, wherein: in the 1-4), chromatin DNA in the cell nucleus is fragmented by using a method of ultrasonic disruption.

7. The method according to any one of claims 3-6, wherein: the 2) comprises the following steps: adding a Tn5 transposition system into the target protein-DNA complex, and reacting to obtain a reaction product; and adding proteinase K into the reaction product to perform a crosslinking reaction, and removing the target protein in the reaction product to obtain a DNA product.

8. A kit for constructing a chromatin co-immunoprecipitation library, comprising a Tn5 transposition system.

9. The method of any one of claims 3 to 7 or the kit of claim 8 for use in any one of the following (1) to (3):

(1) studying the interaction between the protein of interest and its target DNA;

(2) obtaining sequence information of the DNA fragment bound to the target protein;

(3) identifying or aiding in the identification of binding sites for DNA binding proteins.

10. A method for obtaining sequence information of DNA fragments bound to a protein of interest, comprising the step of performing high throughput sequencing of a chromatin co-immunoprecipitation library constructed according to the method of any one of claims 3 to 7.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for preparing a chromatin co-immunoprecipitation library by using a trace clinical puncture sample and application thereof.

Background

The chromatin immunoprecipitation sequencing (ChIP-Seq) is a revolutionary technology combining chromatin immunoprecipitation (ChIP) with high-throughput sequencing. The technology can identify the binding sites of transcription factors, histone modification, nucleosomes, RNA binding proteins and the like on chromatin in a genome-wide range. Unlike conventional ChIP-based ChIP (chromatography) amplification combined with DNA ligation arrays, ChIP-seq provides a new strategy for studying protein-DNA interactions with high resolution, specificity and coverage. The ChIP-seq technology can be applied to any species with known genome sequence, can study the interaction between any DNA related protein and its target DNA, and can exactly obtain the sequence information of each fragment bound by the protein. With the reduction of sequencing cost, ChIP-seq gradually becomes a common means for researching gene regulation and epigenetic mechanism. The basic principle is as follows: in a physiological state, cells are firstly treated by formaldehyde to fix proteins and interactive DNA thereof; then cracking the cell and separating the chromatin, and fragmenting the chromatin by ultrasound or enzyme; then, enriching DNA fragments combined with the target protein by using a specific antibody; after adding a linker to the DNA fragment, performing library preparation and high-throughput sequencing by using PCR; finally, combining with the means of bioinformatics analysis, the binding sites of DNA binding proteins are identified in the whole genome range. The ChIP-seq can be used for obtaining DNA sequence information interacting with DNA binding proteins such as histone or transcription factors and the like in the whole genome range, and the length of the DNA sequence information is about 200bp, so that the regulation network and the action mechanism of the DNA binding proteins can be deeply researched.

Although the conventional ChIP-seq technology has the advantages of high resolution, low noise, high coverage and the like, the conventional ChIP-seq technology also has some problems: firstly, the requirement of ChIP-seq on the initial amount of a sample is too high at present, and the total amount of a tissue sample needs at least 200 mg; secondly, the preliminary chromatin co-immunoprecipitation experiment has complicated steps, and needs to undergo the steps of formaldehyde crosslinking, cell lysis, ultrasonic disruption, antibody enrichment of target protein and DNA compound, target protein digestion and decrosslinking, purification of target DNA fragment, target DNA fragment end repair, target DNA fragment end adenylation, target DNA fragment connection joint, PCR amplification and the like. The complexity of the steps causes the defects of long library building period, low success rate and the like.

Compared with the traditional library construction mode, the Tn5 transposition system library constructed in recent years can convert multi-step reactions such as DNA fragmentation, end repair, adaptor connection and the like into one-step reactions, greatly shortens the library construction time, improves the working efficiency, and is widely applied to the field of high-throughput sequencing, such as ATAC-Seq, L IANTI (L initial Amplification factor transposition insertion, linear Amplification through transposon), haploid typing, structural detection and the like.

Disclosure of Invention

The technical problem to be solved by the invention is how to simply, rapidly, efficiently and low-cost chromatin co-immunoprecipitation library preparation on rare and trace clinical tissue samples. Through the evaluation and optimization of the system, the following scheme is formed: firstly, processing a trace amount of puncture tissue samples by formaldehyde and homogenizing and crushing the samples; secondly, cell cracking is carried out on the tissue fragments, and cell nucleuses are extracted; then, chromatin DNA was fragmented using a non-contact sonication disruptor; further incubating with a specific antibody, and enriching target proteins and DNA fragments combined with the target proteins; finally, the enriched DNA fragments are further fragmented and connected with library adaptors by using a Tn5 transposition system, and the library is amplified after the target protein is digested by protease.

In order to solve the above technical problems, the present invention firstly provides a novel use of Tn5 transposition system.

The invention provides an application of a Tn5 transposition system in constructing a chromatin co-immunoprecipitation library.

The invention also provides application of the Tn5 transposition system in preparation of products for constructing chromatin co-immunoprecipitation libraries.

The invention also provides application of the Tn5 transposition system in constructing a chromatin co-immunoprecipitation library based on a micro tissue sample.

The invention also provides application of the Tn5 transposition system in preparation of a product for constructing a chromatin co-immunoprecipitation library based on a micro tissue sample.

In the above application, the minimum initial amount of the tissue sample is 5 mg; further, the starting amount may be (5-50) mg; still further, the starting amount may be (5-15) mg.

In order to solve the technical problems, the invention also provides a method for constructing the chromatin co-immunoprecipitation library.

The method for constructing the chromatin co-immunoprecipitation library provided by the invention comprises the following steps:

1) sequentially carrying out formaldehyde treatment and chromatin DNA fragmentation on a tissue sample, and then incubating with a specific target protein antibody to obtain a target protein-DNA compound;

2) fragmenting, end repairing and connecting a joint of a DNA fragment in the target protein-DNA complex by using a Tn5 transposition system, and digesting the target protein by using protease to obtain a DNA product;

3) and carrying out PCR amplification by taking the DNA product as a template to obtain the chromatin co-immunoprecipitation library.

In the method for constructing the chromatin co-immunoprecipitation library, in the step 1), the minimum initial amount of the tissue sample is 5 mg; further, the starting amount may be (5-50) mg; still further, the starting amount may be (5-15) mg. In a specific embodiment of the invention, the starting amount of the tissue sample is 7.7mg, 11.4mg, 5.5 mg.

The tissue sample may be a punctured tissue sample, an animal tissue sample or a clinical tissue sample, in particular for a clinically small or rare physiological tissue sample or pathological tissue sample, such as cancer tissue. In a specific embodiment of the invention, the tissue sample is a liver puncture tissue sample.

In the above method for constructing a chromatin co-immunoprecipitation library, the step 1) may comprise the steps of:

1-1) adding a formaldehyde solution into a tissue sample for crosslinking reaction to obtain a crosslinked product;

1-2) homogenizing and crushing the crosslinking product to obtain tissue fragments;

1-3) adding cell lysate into the tissue fragments to obtain lysed cells, centrifuging, and collecting precipitates to obtain cell nuclei;

1-4) fragmenting chromatin DNA in the cell nucleus to obtain fragmented DNA fragments;

1-5) incubating the fragmented DNA fragments with specific target protein antibodies, and enriching the target protein and the DNA fragments combined with the target protein to obtain the target protein-DNA complex.

Further, the method 1-1) further comprises a step of washing the tissue sample, and the washing method can be specifically carried out according to the following steps: the tissue sample was washed by adding a PBS solution (pH 7.4) to the tissue sample, and centrifuged at 500g at 4 ℃ for 10 minutes to remove the PBS solution, thereby obtaining a washed tissue sample.

The 1-1) may include the steps of: adding formaldehyde solution into the washed tissue sample, and standing for 5 minutes; then adding glycine solution to stop the reaction, and standing for 3 minutes to obtain a cross-linked product.

The formaldehyde solution can be 1% formaldehyde solution by volume, and the glycine solution can be glycine solution with the concentration of 0.125 mol/L.

In the 1-2), the homogenate disruption method may be centrifugation; the specific method of centrifugation can be carried out as follows: the crosslinked product was centrifuged at 500g at 4 ℃ for 10 minutes, the supernatant was discarded, and the precipitate was collected.

The homogenate further comprises a washing step after being crushed; the washing method can be specifically carried out according to the following steps: adding PBS solution containing PMSF into the precipitate, centrifuging for 3 minutes at 4 deg.C and 500g, and washing for 2 times to obtain washed tissue fragment.

The 1-3) may include the steps of: adding a cell lysis buffer solution into the washed tissue fragments, and carrying out ice bath for 10 minutes to obtain lysed cells; and centrifuging the lysed cells at 4 ℃ for 10 minutes under the condition of 1,000g, removing supernate, and collecting precipitates to obtain cell nuclei.

In the 1-4), chromatin DNA in the cell nucleus is fragmented by using a method of ultrasonic disruption. The ultrasonic crushing method can be specifically carried out according to the following steps: the pellet was resuspended in the sonication tube with sonication buffer and then disrupted on the biodisruption instrument for 10 minutes (set up procedure: work 30 seconds, stop 30 seconds; total 10 cycles) to give the sonicated solution.

The 1-5) may include the steps of: placing an ultrasonic breaking tube containing the solution after ultrasonic treatment into a centrifugal tube, centrifuging at 4 ℃ and 13,000rpm for 5 minutes, transferring the supernatant into a new centrifugal tube, adding the co-immunoprecipitation buffer solution until the total volume is 200 mu l, adding a specific target protein antibody (about 2 mu g) into the centrifugal tube, rotating overnight at 4 ℃, uniformly mixing and incubating to obtain the co-precipitated solution.

In the method for constructing the chromatin co-immunoprecipitation library, a step of purifying with protein A/G magnetic beads may be further included between 1) and 2). The specific method for purifying by using the protein A/G magnetic beads can be carried out according to the following steps: putting 20 mul of protein A/G magnetic beads into a centrifugal tube, putting the centrifugal tube on a magnetic frame until the magnetic beads are completely adsorbed, and removing supernatant; adding 200 μ l PBS solution containing 0.1% (mass percent) BSA, mixing, and rotating and mixing at 4 deg.C overnight to obtain the blocked protein A/G magnetic beads. And adding the sealed protein A/G magnetic beads into the co-precipitation solution, and uniformly mixing for 2 hours at 4 ℃ to obtain a co-incubated solution. Placing the solution after the co-incubation on a magnetic frame until clarification (the subsequent treatment is carried out on ice), removing the supernatant, and then rinsing with 200. mu.l of a rinsing buffer I for 2 times, 200. mu.l of a rinsing buffer II for 1 time, and 200. mu.l of a rinsing buffer III for 1 time; the beads were rinsed 2 times with 200. mu.l of precooled Tris-HCl (pH 8.0) to obtain rinsed beads.

In the above method for constructing a chromatin co-immunoprecipitation library, the step 2) may comprise the steps of: adding a Tn5 transposition system into the target protein-DNA complex, and reacting to obtain a reaction product; and adding proteinase K into the reaction product to perform a crosslinking reaction, and removing the target protein in the reaction product to obtain a DNA product.

Further, the Tn5 transposase system is a complex formed by Tn5 transposase and a DNA adaptor specifically bound thereto, and is a transposase system having double-stranded DNA cleavage activity formed by expressing purified Tn5 transposase bound to a specific double-stranded DNA adaptor. The system can effectively fragment trace DNA and connect the DNA with a connector at two ends of target DNA in one step.

The Tn5 transposition system can be prepared by itself, and the specific preparation method can refer to the literature: the content of the pigment is Picelli S,A K,Reinius B,et al.Tn5transposase and tagmentation procedures formassively scaled sequencing projects.[J]the method of Genome Research,2014,24(12): 2033; the Tn5 transposition system is also commercially available. In a specific embodiment of the invention, the Tn5 transposition system is Nextera from Illumina^TMThe Tn5 transposase in the Tn5 transposase system is a highly sensitive Tn5 transposase (a Tn5 transposase mutant with high activity of specific site mutation, wherein the Tn5 transposase mutant is described in the literature as Picelli S,A K,Reinius B,et al.Tn5transposase andtagmentation procedures for massively scaled sequencing projects.[J]genome research,2014,24(12): 2033).

Further, the 2) may include the steps of:

2-1) resuspending the rinsed magnetic beads in a reaction buffer, adding a Tn5 transposition system and a 5 × PEG8000 buffer (the volume ratio of the Tn5 transposition system to the 5 × PEG8000 buffer can be 3:8), and interrupting the reaction in a constant-temperature rotary reactor at 55 ℃ and 500rpm for 10 minutes to obtain a magnetic bead reaction mixed solution;

2-2) placing the magnetic bead reaction mixed solution on a magnetic frame until the mixed solution is clarified, removing supernatant, rinsing the mixed solution for 2 times by using a rinsing buffer solution I, and rinsing the mixed solution for 2 times by using a rinsing buffer solution IV to obtain rinsed magnetic beads;

2-3) using eluent to re-suspend the rinsed magnetic beads, adding proteinase K solution to react for 1 hour at 55 ℃ and 500rpm, and then performing crosslinking decomposition for 8 hours at 65 ℃ and 500rpm to obtain crosslinked magnetic bead mixed solution; the concentration of the proteinase K solution can be 10 mg/ml;

2-4) placing the magnetic bead mixed solution after the decrosslinking on a magnetic frame until the mixed solution is completely clarified, transferring the supernatant into a collection tube, adding the eluent and the proteinase K solution again, reacting for 1 hour at the temperature of 55 ℃ and the speed of 500rpm, and then collecting the reaction solution in the collection tube again to obtain the eluent (containing DNA products).

In the method for constructing the chromatin co-immunoprecipitation library, a step of purifying the DNA product may be further included between 2) and 3). The purification method can be magnetic bead purification. The specific method for purifying the magnetic beads can be carried out according to the following steps: SPRI AMPure XP magnetic beads (XP magnetic beads for short) are mixed uniformly and balanced for 30 minutes at room temperature in advance. Then, XP magnetic beads are added into the eluent, and the mixture is gently mixed. After standing at room temperature for 5 minutes, the beads were transferred to a magnetic rack and allowed to stand for 5 minutes, the supernatant was removed, and the beads were rinsed 2 times with a fresh 80% ethanol solution. And placing the magnetic beads on a magnetic frame, airing for 2 minutes, adding TE buffer solution to resuspend the magnetic beads, and blowing for 50 times. And standing for 5 minutes at room temperature, then placing the mixture into a magnetic frame for standing for 5 minutes, and collecting supernatant, namely the purified DNA product.

In the method for constructing the chromatin co-immunoprecipitation library, as described in 3), the reaction system for PCR amplification may be 18.7. mu.l of supernatant, 2.5. mu.l of 10 × pfx buffer (Invitrogen), 1. mu.l of each of 10. mu.M upstream and downstream primers, and 50mM MgSO₄Mu.l of the solution, 0.4. mu.l of 25mM dNTP and 0.4. mu.l of pfx enzyme (Invitrogen).

The reaction procedure of the PCR amplification can be as follows: reacting at 94 ℃ for 2 minutes; denaturation at 94 ℃ for 15 seconds, annealing at 62 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and reaction for 24 cycles; the reaction was carried out at 72 ℃ for 10 minutes.

In order to solve the above technical problems, the present invention also provides a method for obtaining sequence information of a DNA fragment bound to a target protein.

The method for obtaining the sequence information of the DNA fragment combined with the target protein comprises the step of sequencing the chromatin co-immunoprecipitation library constructed according to the method.

The sequencing method can be specifically carried out according to the following steps: PE150 paired-end sequencing was performed on the constructed chromatin co-immunoprecipitation library using an illumina Xten sequencer.

In order to solve the above technical problems, the present invention finally provides a kit for constructing a chromatin co-immunoprecipitation library.

The kit for constructing the chromatin co-immunoprecipitation library provided by the invention comprises a Tn5 transposition system.

Further, the kit can also comprise at least one of the following reagents, namely 1% formaldehyde solution by volume, glycine solution with the final concentration of 0.125 mol/L, cell lysis buffer (containing 5 mu l of 100 × PI), ultrasonic buffer, co-immunoprecipitation buffer, 5 × PEG8000 buffer, protease K solution with the concentration of 10mg/ml, SPRI AMPure XP magnetic beads and protein A/G magnetic beads.

Still further, the kit may further comprise at least one of the following reagents: PBS solution (pH 7.4), PBS solution containing PMSF (PMSF concentration of 1 μ M), PBS solution containing BSA (mass percentage of 0.1%), rinsing buffer I, rinsing buffer II, rinsing buffer III, Tris-HCl (pH 8.0), reaction buffer, eluent, 80% ethanol solution and TE buffer.

The application of the above method or the above kit in any one of the following (1) to (3) also belongs to the protection scope of the present invention:

(1) studying the interaction between the protein of interest and its target DNA;

(2) obtaining sequence information of the DNA fragment bound to the target protein;

(3) identifying or aiding in the identification of binding sites for DNA binding proteins.

The invention has the beneficial effects that: 1. the preparation of a library can be carried out on a trace amount of tissues, so that samples which cannot meet the preparation of the conventional chromosome co-immunoprecipitation library can be effectively researched, and the preparation of the library is specific to the trace amount of samples, so that the cost required by reagents and experiments is effectively saved; 2. the preparation period of the library is shortened. Compared with the 4-5 day library construction period required by the preparation of the conventional chromosome co-immunoprecipitation library, the method only needs about 1.5 days. Thereby greatly saving the time used for the experiment.

According to the characteristics of a Tn5 transposition system, the invention successfully develops a chromatin co-immunoprecipitation sequencing library preparation method based on a trace clinical puncture sample by using a high-sensitivity Tn5 transposase, and can effectively and quickly construct a ChIP-seq library on the trace puncture tissue sample. The method for constructing the ChIP-seq library by utilizing the Tn5 transposition system is efficient and simple, has high success rate, greatly reduces the initial amount (5-50mg) of the sample, effectively shortens the library construction time (about 1.5 days) and meets the requirements of the sequencing market on the processing speed of trace samples and large-scale samples compared with the traditional ChIP-seq library construction mode.

Drawings

FIG. 1 is a scheme of a co-immunoprecipitation based on the Tn5 transposition system.

Fig. 2 is co-immunoprecipitation correlation analysis of clinical liver tissue puncture samples H3K4me3 from different individuals of 3 biological replicates.

FIG. 3 shows the distribution of the H3K4me3 binding DNA fragments in the transcription initiation site region (2000 bp upstream and downstream) of 3 clinical samples.

FIG. 4 is a thermodynamic diagram showing the binding strength of the transcription initiation site region (2000 bp upstream and downstream) of H3K4me3 in 3 samples.

FIG. 5 shows the enrichment of 3 samples of H3K4me3 in the region of approximately 800bp of chromosome 1 in the human genome.

FIG. 6 shows the binding signals of H3K4me3 of gene F2 and HPX specifically expressed in human liver tissue.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The formulations of the solutions used in the following examples are as follows:

PBS solution (pH 7.4), NaCl 137 mmol/L2.7.7 mmol/L, Na₂HPO₄10mmol/L，KH₂PO₄2mmol/L。

Cell lysis buffer 50mM HEPES/KOH (pH 7.4), 140mM NaCl, 1mM EDTA, 0.5mM EGTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 × protease inhibitors.

Ultrasonic buffer solution: 10mM Tris-HCl (pH 7.6), 1mM EDTA, 0.1% SDS.

Co-immunoprecipitation buffer: 20mM HEPES, 0.1% SDS, 1% Triton X-100, 150mM NaCl, 1mM EDTA, 0.5mM EGTA.

Rinsing buffer I: 20mM HEPES, 150mM NaCl, 0.1% SDS, 0.1% DOC, 1% Triton X-100, 1mM EDTA, 0.5mM EGTA.

Rinsing buffer II: 20mM HEPES, 500mM NaCl, 0.1% SDS, 0.1% DOC, 1% Triton X-100, 1mM EDTA, 0.5mM EGTA.

Rinsing buffer III 20mM HEPES, 250mM L iCl, 0.5% DOC, 0.5% NP-40, 1mM EDTA, 0.5mM MEGTA.

Rinsing buffer IV: 20mM HEPES, 1mM EDTA, 0.5mM EGTA.

Eluent: 0.5% SDS, 300mM NaCl, 5mM EDTA, 10mM Tris-HCl (pH 8.0).

TE buffer solution: 10mM Tris-HCl (pH 8.0), 1mM EDTA.

Reaction buffer: 10mM Tris-HCl (pH 8.0), 5mM MgCl₂。

5mM 5 × PEG8000 buffer 50mM TAPS-NaOH (pH 8.5), 25mM MgCl₂，40％PEG8000。