CN108315387B - Micro cell ChIP method - Google Patents

Abstract

The invention relates to a micro cell ChIP method. The method comprises the following steps: 1) dividing the cell sample to be detected into n groups, wherein n is a non-0 natural number; 2) cross-linking and fixing the nth group of samples; 3) treating said nth set of samples with a cell membrane perforating agent; 4) digesting and breaking chromatin fragments of the nth group of samples by using Tn5 transposase, and connecting barcode sequences and primer sequences at two ends of product fragment DNA; when n is more than or equal to 2, the barcode sequences and/or primer sequences used in each group in the step 4) are different; 5) when n is more than or equal to 2, combining the samples of each group; 6) enriching the DNA fragments binding to the target protein, and using the primers as index to create a library, sequencing analysis. The method has high library building efficiency during sequencing, can realize the capture of the transcription factor sites of the ultra-trace cells, and ensures better efficiency, stability and accuracy.

Description

Micro cell ChIP method

Technical Field

The invention relates to the technical field of molecular biology experiments, in particular to a micro cell ChIP method.

Background

Gene regulation and expression of organisms is an extremely complex but ordered process, with genomic DNA of an organism existing in the form of chromatin in cells, and protein and DNA interactions are important bases for the functioning of cells. Therefore, studying the interaction of proteins with DNA in the chromatin environment allows a further understanding of gene expression and its regulation pattern.

Chromatin Immunoprecipitation (ChIP) is a standard method for studying DNA-protein interactions in vivo. Chromatin co-immunoprecipitation technology can locate and analyze action sites of proteins and DNA in vivo, ChIP technology is combined with other methods, such as high density ChIP (microarray), sequencing, in vivo footprint method and other technologies, so that a single transcription factor distribution map and trans-factor in vivo binding sites of the whole genome level can be obtained, and epigenetic genetic mechanisms such as nucleosome location and histone modification are systematically disclosed.

The general technical process of the ChIP technology is as follows: firstly, formaldehyde crosslinking is carried out on cells or tissues at different time, cell membranes and cell nuclei are respectively cracked, and chromatin is smashed into segments with a certain length range in an ultrasonic mode. The antibody is added to be specifically combined with the target protein, and then the target protein and the DNA fragments combined with the target protein are enriched through the interaction of the magnetic beads and the antibody. After the proteins are uncrosslinked and digested, the captured DNA fragments are pooled and subjected to second generation sequencing. According to the enrichment condition of the data fragments on the genome, a map of the interaction between the specific protein and the DNA at the whole genome level is obtained.

However, the existing ChIP-seq technology mainly has the following problems:

1. low efficiency of establishing bank, not suitable for small amount of cell experiments

The DNA-chromatin complex with proper fragment size obtained by using proper ultrasonic condition is the precondition of successful whole chromatin co-immunoprecipitation experiment, however, the ChIP-seq technology interrupted by the ultrasonic method has certain requirements on the number of cells, and is suitable for the cell treatment of tens of thousands to millions of orders of magnitude. The main reason is that the adaptor in the traditional TruSeq library construction strategy of Illumina is connected with a DNA fragment with low efficiency, so that many fragments are not enriched and lost in the subsequent amplification set. Especially when the research problem needs to be addressed to a small number of cell populations, the serious information loss makes the experimenter unable to obtain more accurate real information.

2. The method for breaking chromatin fragments by ultrasonic method is not beneficial to research of transcription factors indirectly combined with DNA

In the genome-wide range, many transcription factors are not directly bound to genomic DNA, but indirectly bound to genomic DNA after being bound to other transcription factors. Even in the case of formaldehyde-crosslinked samples, this indirect bond is relatively easily broken. The ultrasonic process has a certain degree of physical breaking strength, and the stability of the combination of the indirect combination target protein and DNA is easily interfered, so that the subsequent information is lost.

3. The library has higher non-specific background and low detection resolution

The ultrasonic break method is to break the dyestuffs randomly to obtain fragments in a certain length range. Therefore, there are some DNA sequences specifically bound by non-target proteins or other protein binding sites around the target protein and DNA complex. These non-specific sites are preserved in the subsequent library construction and sequencing process, and become the interference background of target protein map analysis, so that broad peaks are obtained, and the resolution and accuracy of detecting binding sites are reduced.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention relates to a micro cell ChIP method, which comprises the following steps:

1) dividing the cell sample to be detected into n groups, wherein n is a non-0 natural number;

2) cross-linking and fixing the nth group of samples;

3) treating said nth set of samples with a cell membrane perforating agent;

4) digesting and breaking chromatin fragments of the nth group of samples by using Tn5 transposase, and connecting barcode sequences and primer sequences at two ends of product fragment DNA;

when n is more than or equal to 2, the barcode sequences and/or primer sequences used in each group in the step 4) are different;

5) when n is more than or equal to 2, combining the samples of each group;

6) enriching the DNA fragments binding to the target protein, and using the primers as index to create a library, sequencing analysis.

Chromatin Immunoprecipitation (ChIP) is a standard method for studying DNA-protein interactions in vivo, and when there is an interaction between the two, DNA is usually disentangled from histones and does not exist as nucleosomes. Tn5 transposase does not cleave the nucleosome form of DNA fragment when cleaving chromatin fragments, and thus has better specificity than the prior art sonication method.

The barcode sequence is essentially a nucleic acid sequence which is equivalent to adding different tag sequences to the cut DNA fragments, so that when different groups (n ≧ 2) of samples are subjected to subsequent sequencing operations, each group can be distinguished by its respective specific barcode sequence. In the limit, when the number of cells in each group of samples is 1, the method provided by the invention can realize single-cell ChIP.

The complexity of the barcode based on Tn5 transposase (transposase) (i.e., how many unique barcodes are available) is still relatively limited. To ensure the efficiency of tagmentation, the barcode region may not be too long. Meanwhile, in order to avoid false recognition caused by sequencing errors (if one base is accidentally misdetected but is regarded as another barcode), the complexity of the barcode is not as high as the nth power of 4, and a correction mechanism needs to be introduced. In general, only Tn5 is used to make single cells, and only tens to hundreds of single cells can be identified at one time. The present invention adopts a combinatorial indexing method. The setting mode of the label is a barcode sequence + a primer sequence, so that the overall complexity is increased, and the number of single cells which can be captured at one time is increased. When the sample does not need to be distinguished (n is 1), the primer sequence only adopts a universal primer, and the subsequent library building operation is more convenient; when different samples need to be distinguished (n is more than or equal to 2), the primer sequences and/or barcode sequences of each group are different, so that the samples can be effectively distinguished.

Preferably, the manipulation of single cells is performed by methods known to those skilled in the art, such as by encapsulating a single cell suspension sample and Tn5 enzyme hydrogel beads with barcode and/or primer sequences in an oil droplet on a microfluidic chip. After reverse transcription in the oil droplets, DNA fragments cleaved by Tn5 enzyme were uniquely tagged to each single cell. Finally, DNA fragments of all single cells are mixed together, a library is built by the primer sequence for sequencing, and then the barcode is identified by the sequence to distinguish the single cells.

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

(1) the library building efficiency is high, and the histone information capture amount of trace cells and even single cells is obviously improved;

when Tn5 is used for enzyme digestion, primers containing the barcode sequence are connected to two ends of the product fragment DNA, and the efficient primer connection process greatly improves the efficiency of connecting the amplification primers containing the barcode sequence to two ends of the target DNA fragment, so that more information is captured in the subsequent amplification process. The sequencing and data analysis of histone binding sites are realized at the level of trace cells (100) or even single cells, the information obtained by processing a large number of cells by using a traditional method has higher accuracy and precision, and the non-specific background is obviously reduced (as shown in figure 2). In particular, the data at the single cell level are orders of magnitude better than the ChIP data of the single cell alone (Assaf Rotem,2015, Nature Biotechnology). In the data measured in that article, about 800 unique deputytes reads are obtained on average per cell, whereas about 5000 unique deputytes reads are obtained on average per cell using the technical solution provided by the present invention.

(2) The capture of the transcription factor sites of the extremely trace cells is realized, and better efficiency is ensured;

at present, the whole genome locus detection of the transcription factors of trace cells (100) is realized, the capture signal is ideal, and compared with the data obtained by a large number of cells, the method has certain accuracy and precision.

(3) Reducing non-specific background;

in a sequencing result visualization interface, the nonspecific background of the new technology is obviously reduced compared with the background of ChIP data in a traditional ultrasonic mode, and the detected enrichment peak is more specific and more obvious.

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 embodiments or the technical solutions in the prior art will be briefly described below, and it is obvious that the drawings in the following description are one embodiment of the present invention, and other drawings can be obtained according to the drawings without creative efforts for those skilled in the art.

FIG. 1 is a diagram illustrating the location of a barcode sequence in one embodiment of the present invention;

FIG. 2 is a graph showing the results of comparative experiments on the cross-linking time of formaldehyde, the reaction temperature of loose chromatin structure, the digestion temperature and the digestion time in one embodiment of the present invention; FIG. 2A shows the band sizes of the cleavage products under different combinations of conditions: FIG. 2B is a bar graph showing the degree of enrichment of target DNA for a ChIP experiment using a cross-linked 3min sample; (ii) the values of (i) - (iv) represent different treatment conditions (see above the histogram);

FIG. 3 is a graph illustrating an IGV visualization interface comparing ChIP methods provided by one embodiment of the present invention to conventional ultrasound techniques;

FIG. 4 shows ChIP results of comparing single cells, micro cells and macro cells on a visual interface according to an embodiment of the present invention.

Detailed Description

The invention relates to a micro cell ChIP method, which comprises the following steps:

1) dividing the cell sample to be detected into n groups, wherein n is a non-0 natural number;

2) cross-linking and fixing the nth group of samples;

3) treating said nth set of samples with a cell membrane perforating agent;

4) digesting and breaking chromatin fragments of the nth group of samples by using Tn5 transposase, and connecting barcode sequences and primer sequences at two ends of product fragment DNA;

when n is more than or equal to 2, the barcode sequences and/or primer sequences used in each group in the step 4) are different;

5) when n is more than or equal to 2, combining the samples of each group;

6) enriching the DNA fragments binding to the target protein, and using the primers as index to create a library, sequencing analysis.

Preferably, in the method of ChIP for micro cells as described above, the number of cells in the nth group of samples is not less than 1;

in one embodiment, 1 ≦ 1000000 for the number of cells in the nth set of samples;

in one embodiment, 1 ≦ 10000 for the number of cells in the nth set of samples;

in one embodiment, 1 ≦ 5000 for the number of cells in the nth set of samples;

in one embodiment, 1 ≦ 1000 for the number of cells in the nth set of samples.

Preferably, the ChIP method for micro cells as described above further comprises, after step 1) and before step 2):

loosening chromatin in the formaldehyde-crosslinked cell sample using a solution containing SDS;

in one embodiment, the concentration of SDS in the SDS-containing solution is from 0.1 w/v% to 1.0 w/v%; preferably, the concentration of SDS in the SDS-containing solution is 0.1 w/v% to 0.5 w/v%;

in one embodiment, the SDS-containing solution is a hypotonic lysis buffer;

in one embodiment, the SDS-containing solution comprises the following components: 0.1 to 1.0 w/v% SDS, 0.1 to 1.0 v/v% NP-40, 1 to 10 v/v% glycerol, 10 to 50mM KCl and 20 to 100mM HEPES, wherein the pH value is 7.5 to 8.3;

in one embodiment, the SDS-containing solution comprises the following components: 0.1 w/v% -0.5 w/v% SDS, 0.1 v/v% -0.5 v/v% NP-40, 3 v/v% -7 v/v% glycerol, 20 mM-40 mM KCl and 40 mM-80 mM HEPES, wherein the pH value is 7.7-8.1;

in one embodiment, the step of loosening chromatin in a formaldehyde-crosslinked cell sample using a solution comprising SDS specifically comprises:

treating the cell sample by using a solution containing SDS at the temperature of 10-37 ℃ for 5-60 min;

in one embodiment, the step of loosening chromatin in a formaldehyde-crosslinked cell sample using a solution comprising SDS specifically comprises:

treating the cell sample with a solution containing SDS at 20-37 ℃ for 20-40 min; more preferably 3 to 10 min.

Preferably, in the above-mentioned method of ChIP, in step 2), the cross-linking is performed by formaldehyde;

in one embodiment, the concentration of formaldehyde is from 0.5 v/v% to 1.5 v/v%, preferably 1 v/v%;

in one embodiment, the cross-linking is fixed for a time period of 0.5min to 15min, preferably 3min to 10min, and the cross-linking temperature is room temperature.

Preferably, in the ChIP method for micro cells as described above, in the step 2), the cell membrane perforating agent is specifically a solution containing Triton X-100;

in one embodiment, the concentration of Triton X-100 in the Triton X-100 containing solution is between 0.5 v/v% and 2 v/v%;

in one embodiment, the Triton X-100 containing solution comprises the following components:

0.5v/v％～2v/v％Triton X-100、0.01w/v％～0.05w/v％SDS、2mM～20mM EDTA、100mM～500mM NaCl、10mM～50mM Tris-HCl，pH＝7.5～8.5；

in one embodiment, the Triton X-100 containing solution comprises the following components:

1v/v％～1.5v/v％Triton X-100、0.02w/v％～0.04w/v％SDS、5mM～15mM EDTA、200mM～400mM NaCl、20mM～40mM Tris-HCl，pH＝7.8～8.2；

in one embodiment, the treatment conditions for treating the cells with the cell membrane perforating agent are: incubating for 5-60 min at 10-37 ℃.

Preferably, the ChIP method for micro cells as described above further comprises, after step 2) and before step 3):

sonication to open the chromatin compact structure.

Preferably, the microscale cell ChIP method is as described above, and the ultrasonic treatment conditions are as follows:

140Hz to 180Hz and 10s to 20s of ultrasonic wave;

in one embodiment, the sonication conditions are: 150 Hz-160 Hz and ultrasonic 13 s-17 s.

Preferably, in the above-mentioned minicell ChIP method, in step 4), the Tn5 transposase enzyme digestion reaction system comprises a Tn5 transposase enzyme from group i sample chromatin, connected with the barcode sequence and the primer sequence;

the final concentration of the Tn5 transposase in the reaction system was: 0.01-0.05 μ l/20 μ l.

In one embodiment, the Tn5 transposase is cleaved under the following conditions:

incubating for 5-60 min at 10-37 ℃;

in one embodiment, the Tn5 transposase is cleaved under the following conditions:

incubating for 10-40 min at 25-37 ℃.

Preferably, the ChIP method for micro cells as described above further comprises, after step 5) and before step 6):

centrifuging the sample obtained in the step 4) or 5) and discarding supernatant, resuspending the precipitate by using a dilution buffer solution containing 0.01 w/v% -0.05 w/v% SDS, ultrasonically dispersing the precipitate, incubating and cracking at 0-6 ℃ for 20-60 min, and ultrasonically treating to release the target DNA fragment from cell nucleus;

in one embodiment, the sonication conditions for sonicating the disrupted precipitate are: 140 Hz-180 Hz, 3 s-7 s;

in one embodiment, the sonication conditions for sonicating the disrupted precipitate are: 150 Hz-170 Hz, 4 s-6 s;

in one embodiment, the sonication conditions that release the DNA fragment of interest from the nucleus of the cell are: 140Hz to 180Hz, in each cycle: ON for 10 s-20 s, OFF for 20 s-40 s;

in one embodiment, the sonication conditions that release the DNA fragment of interest from the nucleus of the cell are: 150Hz to 170Hz, and in each cycle: 13s to 17s ON and 25s to 35s OFF.

Preferably, the method for enriching DNA fragments bound to a target protein in step 6) comprises the following steps:

adding an antibody corresponding to a target Protein for incubation, pulling the antibody by using the beads coupled with Protein A, eluting the beads by using an eluent, performing crosslinking treatment on the eluent, and performing digestion treatment by using proteinase K.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

This example provides a ChIP method for micro cells (1-10000)

(1) Cross-linking the cells with 1 v/v% concentration of formaldehyde (room temperature) (cross-linking for 3min), collecting the cells by FACS or mouth picking into 10. mu.l of hypotonic lysis buffer (50mM HEPES (pH 7.9), 30mM KCl, 7 v/v% glycerol, 0.3 v/v% NP-40, 3 w/v% SDS) containing a final concentration of 3 w/v% SDS, and incubating for 3min at 37 ℃ in PCR;

(2) adding Triton X-100 to a final concentration of 1 v/v% (1 v/v% Triton X-100, 0.03 w/v% SDS, 15mM EDTA, 200mM NaCl, 30mM Tris-HCl, pH 7.8), mixing, and incubating at 37 deg.C for 40min in a PCR instrument;

(3) further assisting in opening the compact chromatin structure by using a Q800R non-contact ultrasonic instrument at 160Hz intensity for 15 s;

(4) preparing a digestion system for digestion reaction: 20. mu.l total, containing all chromatin samples from minicell processing (approximately 11.2. mu.l), 0.1-1. mu.l Tn5-complex (synthesized by annealing single-stranded DNA containing different barcode sequences to DNA containing 5' -phosphorylated universal single-stranded universal primer sequences, assembled by incubation with Tn5 at room temperature to allow Tn5 to have higher activity), 4. mu.l digestion buffer (cloning buffer), and made up to 20. mu.l with water. Incubating the reaction system for 1h at 37 ℃;

(5) adding a stop solution (50-500mM EDTA) to stop the reaction for a certain time to completely inhibit the activity of Tn 5;

(6) centrifugation and resuspension of the pellet with dilution buffer containing 0.03% SDS (0.03 w/v% SDS, 1 v/v% Triton X-100, 2-20mM EDTA, 10-50mM Tris-HCl (pH 8.0), 100mM NaCl), lysis at 4 ℃ for half an hour, release of chromatin fragments from the nucleus using a non-contact sonicator Q800R (180Hz intensity, 4cycles, 15s ON,30s OFF per cycle);

(7) after collecting the sample liquid, 0.6. mu.g of Millipore 04-745H 3K4me3 antibody was added, and the mixture was incubated overnight with rotation, enriched with magnetic beads (Dynabeads Protein A) the next day, and incubated with rotation at 4 ℃ for 3 hours;

(8) and (3) cleaning magnetic beads: washing the magnetic beads by 150 mu l washing buffer 1(20-200mM NaCl, 1% Triton X-100, 1-5mM EDTA, 10-100mM HEPES and 0.05-0.5% DOC) in a vertically reversed manner, placing the magnetic beads on a magnetic rack after brief centrifugation, adsorbing the magnetic beads, discarding the supernatant, and washing for 3 times in total; then, the beads were washed 1 time upside down using 150. mu.l washing buffer 2(50-500mM NaCl, 0.5-5% Triton X-100, 1-5mM EDTA, 10-100mM HEPES, 0.05-0.5% DOC). After discarding the supernatant, the DNA was eluted with an elusion buffer (20-200mM Tris-HCl (pH 8.0), 5-50mM EDTA, 0.3% -3% SDS), decrosslinked overnight at 70 ℃ and digested with proteinase K at 55 ℃ for at least 4 hours;

(9) after phenol chloroform extraction and ethanol precipitation of DNA, a library-establishing primer containing Nextera-index is directly used for amplifying DNA fragments to obtain a library, and sequencing analysis is carried out.

Example 2

The only difference from example 1 is that in step 1), the formaldehyde crosslinking (fire time) takes 10 min.

Example 3

The only difference from example 1 is that in step 1), the temperature of the SDS treatment (Opening) was 62 ℃.

Example 4

The only difference from example 2 is that in step 1), the temperature of the SDS treatment was 62 ℃.

Example 5

The only difference from example 1 is that in step 4), the reaction conditions for the enzyme digestion (mutagenesis) were replaced by a reaction incubation time of 10min at 55 ℃.

Example 6

The only difference from example 2 is that in step 4), the reaction conditions for the enzyme digestion were replaced by 55 ℃ for 10 min.

Example 7

The only difference from example 3 is that in step 4), the reaction conditions for the enzyme digestion were replaced by 55 ℃ for 10 min.

Example 8

The only difference from example 4 is that in step 4), the reaction conditions for the enzyme digestion were replaced by 55 ℃ for 10 min.

The products obtained in examples 1-8 after enzyme cleavage in step 4) were subjected to electrophoresis, and the results are shown in FIG. 2. FIG. 2A shows the band size of the digestion product under different combinations of conditions, with the ideal band size ranging from 200bp to 1 kb. Insol (insoluble) in the figure represents non-soluble chromatin, which remains in the pellet after centrifugation; sol (soluble) represents soluble chromatin, which was suspended in the supernatant after centrifugation. The conclusion is drawn from fig. 2A: the 3min crosslinking time is better than the 10min crosslinking time. FIG. 2B is a bar graph showing the degree of enrichment of target DNA for ChIP experiments using cross-linked 3min samples. This figure clearly leads to the conclusion that: ideal ChIP enrichment results can be obtained by combining the conditions of loose chromatin structure at 37 ℃ and enzyme digestion at 37 ℃.

The IGV visualization interface compares the experimental results of itChIP and conventional ultrasound techniques provided by the present invention as shown in fig. 3. H3K4me3-itChIP or H3K4me 3-hybridization-ChIP (traditional ultrasound technology) was performed using samples of 1 million (1M) or 1 million (10K) cells, and a panel-based replicate experimental group was designed. In the IGV software, comparing the signal value distributions captured by the two technologies at certain gene sites (e.g., Kank3, Wdr46, Vps52, etc.), it can be seen that the signal values of itChIP are more significant and specific.

Example 9

This example provides a single cell ChIP method

(1) After cross-linking the cells with 1 v/v% concentration of formaldehyde (room temperature) (cross-linking for 3min), the individual cells were mouth picked into 10. mu.l of hypotonic lysis buffer (50mM HEPES (pH 7.9), 30mM KCl, 7 v/v% glycerol, 0.3 v/v% NP-40, 1 w/v% SDS) containing a final concentration of 1 w/v% SDS, and incubated at 37 ℃ for 3min in PCR;

(2) adding Triton X-100 to a final concentration of 0.5 v/v% (0.5 v/v% Triton X-100, 0.03 w/v% SDS, 15mM EDTA, 200mM NaCl, 30mM Tris-HCl, pH 7.8), mixing, and incubating at 27 deg.C in a PCR instrument for 60 min;

(3) Q800R non-contact sonograph slightly loose chromatin structure, 140Hz intensity, ultrasound 20 s;

(4) preparing a restriction enzyme system for restriction enzyme reaction. 20. mu.l in total, containing 11.2. mu.l of chromatin sample, 0.01-0.05. mu.l of Tn5-T5-barcode, 0.01-0.05. mu.l of Tn5-T7-barcode, 4. mu.l of digestion buffer (digestion buffer), were made up to 20. mu.l with water. Adding Tn5 enzyme with different barcode combinations into different cells, and incubating for 5 minutes to 1 hour at 10-37 ℃;

the specific positions of T5-barcode and T7-barcode are shown in FIG. 1, and the primer design mode refers to the literature: sasan Amini, Nature Genetics, 2014;

(5) adding stop solution (50-500mM EDTA) to stop the reaction for a certain time, and fully inhibiting the activity of Tn 5;

(6) the single cell samples were pooled and transferred to a new 1.5ml EP tube using pipette tips, centrifuged at 12,000g for 5min at 4 ℃ and then discarded, the bottom pellet (containing chromatin fragments remaining in the nuclei) was retained, the pellet was resuspended in a dilution buffer (0.02 w/v% SDS, 1 v/v% Triton X-100, 2-20mM EDTA, 10-50mM Tris-HCl (pH 8.0), 100-. After lysis at 4 ℃ for half an hour, fragments were released from the nucleus using a Q800R non-contact sonicator (140Hz intensity, 4cycles, 15s ON,30s OFF per cycle);

(7) after collecting the sample liquid, 0.6. mu.g of Millipore 04-745H 3K4me3 antibody was added, and the mixture was incubated overnight with rotation, enriched with magnetic beads (Dynabeads Protein A) the next day, and incubated with rotation at 4 ℃ for 3 hours;

(8) and (3) cleaning magnetic beads: washing the magnetic beads by using 150 mu l washing buffer 1 (the formula is the same as the example 1) in a way of reversing the top and the bottom, placing the magnetic beads on a magnetic rack after brief centrifugation, adsorbing the magnetic beads, discarding supernatant, and washing for 3 times in total; then, the beads were washed 1 time upside down using 150. mu.l washing buffer 2 (formulation same as example 1). After discarding the supernatant, the DNA was eluted with an elution buffer (formulation same as example 1), crosslinked overnight at 70 ℃ and digested with proteinase K at 55 ℃ for at least 4 hours;

(9) after phenol chloroform extraction and ethanol precipitation of DNA, a library-establishing primer containing Nextera-index is directly used for amplifying DNA fragments to obtain a library, and sequencing analysis is carried out.

IGV visualization interfaces were used to compare single cells, trace cells and bulk cells itChIP, and the results are shown in fig. 4. ESC cells and MEF cells show the distribution of effective reads obtained from single cells; integrating single cell data of cells of the same kind into a file, namely 'agg ESC' and 'agg MEF', and examining the single cell data from the overall perspective; it can be seen that the signal distribution of the single cell data has very high consistency and specificity with trace cells and a large number of cells, and the signal to noise ratio is high.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (11)

1. A minicell ChIP method, comprising:

1) dividing the cell sample to be detected into n groups, wherein n is a non-0 natural number;

2) cross-linking and fixing the nth group of samples;

3) treating said nth set of samples with a cell membrane perforating agent;

4) digesting and breaking chromatin fragments of the nth group of samples by using Tn5 transposase, and connecting barcode sequences and primer sequences at two ends of product fragment DNA;

when n is more than or equal to 2, the barcode sequences and/or primer sequences used in each group in the step 4) are different;

5) when n is more than or equal to 2, combining the samples of each group;

6) enriching DNA fragments combined with the target protein, using the primer as an index to build a library, and carrying out sequencing analysis;

after step 1) and before step 2), further comprising: loosening chromatin in the formaldehyde-crosslinked cell sample using a solution containing SDS; the concentration of SDS in the solution containing SDS is 0.1 w/v% -1.0 w/v%; the step of loosening chromatin in a formaldehyde-crosslinked cell sample using a solution comprising SDS specifically comprises: treating the cell sample with a solution containing SDS at 37 ℃ for 3 min; crosslinking is carried out by crosslinking cells with formaldehyde at a concentration of 1 v/v% for 3 min;

after step 2) and before step 3), further comprising: sonication to open chromatin compact structures; the ultrasonic treatment conditions are as follows: 140 Hz-180 Hz and 10 s-20 s of ultrasound;

in the step 4), a reaction system cut by the Tn5 transposase comprises a Tn5 transposase from the group i sample chromatin, connected with the barcode sequence and the primer sequence; the final concentration of the Tn5 transposase in the reaction system was: 0.01-0.05 μ l/20 μ l; the reaction conditions of Tn5 transposase enzyme digestion are as follows: incubating at 37 ℃ for 5-60 min.

2. The method for ChIP according to claim 1, wherein the number of cells in the nth sample group is not less than 1.

3. A micro-cell ChIP method according to claim 2, wherein the number of cells in the n-th group of samples is 1 or less and 1000000 or less.

4. A micro-cell ChIP method according to claim 3, wherein the number of cells in the n-th group of samples is 1 or less and 1000 or less.

5. The method for ChIP of micro-cells according to claim 1, wherein in step 2), the cell membrane perforating agent is a solution containing Triton X-100.

6. The micro-cellular ChIP method according to claim 5, wherein the concentration of Triton X-100 in the solution containing Triton X-100 is 0.5 v/v% to 2 v/v%.

7. The method for ChIP according to claim 6, wherein the conditions for treating the cells with the cell membrane-perforating agent are as follows: incubating at 10-37 ℃ for 5-60 min.

8. The method for ChIP of micro-cells according to claim 1, further comprising, after step 5) and before step 6):

centrifuging the sample obtained in the step 4) or 5) and discarding supernatant, resuspending the precipitate with a dilution buffer containing 0.01 w/v% -0.05 w/v% SDS, and incubating and lysing at 0-6 ℃ for 20-60 min; sonication releases the DNA fragment of interest from the nucleus.

9. The method of ChIP as claimed in claim 8, wherein the step of after resuspending the pellet in SDS dilution buffer and before the step of releasing the DNA fragment of interest from the cell nucleus by sonication further comprises: ultrasonically breaking up the precipitate; the ultrasonic conditions for ultrasonically breaking up the precipitate are as follows: 140Hz to 180Hz, 3s to 7 s.

10. The method for ChIP of micro-cells as claimed in claim 8, wherein the ultrasonic conditions for the ultrasonic treatment to release the DNA fragment of interest from the cell nucleus are as follows: 140 Hz-180 Hz, and in each cycle: 10s to 20s ON and 20s to 40s OFF.

11. The method for ChIP trace cell production according to claim 1, wherein the method for enriching DNA fragments binding to the target protein in step 6) comprises:

adding an antibody corresponding to a target Protein for incubation, pulling the antibody by using the beads coupled with Protein A, eluting the beads by using an eluent, performing crosslinking treatment on the eluent, and performing digestion treatment by using proteinase K.

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