CN116814754A - Multi-group chemical detection analysis system and method for detecting transcriptome and histone modification distribution in single cell in cooperation mode - Google Patents

Abstract

The application relates to the technical field of high-flux single-cell multiunit science, in particular to a multiunit science detection analysis system and method for detecting transcriptome and histone modification distribution in a single cell in a cooperative manner. The technical scheme finally realizes the aim of synergetically detecting gene expression and histone modification distribution by using single-cell initiation experiments by subvertedly improving the traditional ChIP-seq method and combining with Smart-seq2 technology, and is named scHT-seq. The scheme solves the technical problems of large amount of cell raw materials, low genome coverage rate, easy occurrence of false positive and the like required by ChIP-seq and the like in the prior art. The scheme has the advantages of less loss of experimental samples, high sensitivity, remarkable improvement of genome coverage rate and the like. The number of the unique mapping reads captured in a single cell can reach millions, which is improved by hundreds of times compared with the similar technology, and the method has no false positive worry of an open chromatin region and has ideal application prospect.

Description

Multi-group chemical detection analysis system and method for detecting transcriptome and histone modification distribution in single cell in cooperation mode

Technical Field

The application relates to the technical field of high-flux single-cell multiunit science, in particular to a multiunit science detection analysis system and method for detecting transcriptome and histone modification distribution in a single cell in a cooperative manner.

Background

The rapid development of high-throughput single-cell multiunit technology greatly promotes the research of molecular characteristics, gene regulation networks and the like of specific cells in complex cell groups. However, the conventional single-cell multi-group technology has the defects of large cell demand, high loss, specific instrument requirement, sparse data captured on genome and the like, and severely restricts the application of the single-cell multi-group technology in a system with precious samples and sparse cell numbers. In addition, methods that internationally allow simultaneous detection of transcriptome and histone modification profiles in a single cell remain very rare to date. Chromatin co-immunoprecipitation combined sequencing technology (Chromatin Immunoprecipitation with sequencing, chIP-seq) is a method of analyzing histone modifications and transcription factor binding sites. The ChIP-seq technique relies on a number of steps such as sonication, enzymatic digestion to break chromatin and co-immunoprecipitation, using only a large number of cells or tissues as starting materials, which yields an average level of chromatin characteristics of a population of cells, and does not reflect the true state of histone modification in individual cells. Chromatin immune cleavage binding sequencing technology (chromatin immunocleavage with sequencing, chIC-seq) is a method based on the in situ transposition of an antibody directed protein a/G-Tn5 fusion protein to cleave chromatin and simultaneously carry a tag sequence at a target region. The ChIC-seq method is independent of steps such as ultrasonic fracture and co-immunoprecipitation, is simple and rapid, can be used for analyzing the chromatin apparent modification characteristics of single cells, and is a good substitution technology of ChIP-seq. However, the only two methods currently available for simultaneous detection of transcriptome and histone modifications have the following major drawbacks: 1) Thousands of cells are required as starting materials and the histone modification data captured in a single cell is extremely rare, with a unique mapping read (unique mapped reads) number of about one thousand to ten thousand; 2) All are Tn5 transposase-based ChIC-seq technologies, and Tn5 has the preference of opening chromatin regions, which easily leads to false positive signals of data.

Although the experimental methods for detecting transcriptomes in single cells are diverse, the international scope of methods capable of detecting single cell histone modification profile based on ChIP-seq remains blank. There is a need for improvements in the traditional ChIP-seq method to ultimately achieve the objective of synergistically detecting gene expression and histone modification profiles by single cell initiation experiments.

Disclosure of Invention

The application aims to provide a multi-group detection analysis method for detecting transcriptome and histone modification distribution in a single cell in a coordinated manner, so as to solve the technical problems of large quantity of required cell raw materials, low genome coverage rate, easy occurrence of false positive and the like in the prior art, such as ChIP-seq, chIC-seq and the like.

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

a multi-group chemical detection analysis method for detecting transcriptome and histone modification distribution in a single cell comprises the following steps in sequence:

s1: lysing the single cells using Triton X-100 solution, and centrifuging to obtain a first cell lysate; part of the supernatant of the first cell lysate was used for Smart-seq2 library construction; the remainder of the first cell lysate is used to extract the chromatin component;

s2: preparing a buffer solution I containing a protease inhibitor, then adding the buffer solution I into the rest part of the first cell lysate, and obtaining a second cell lysate by blowing;

s3: digesting the second cell lysate with MNase to obtain digested DNA fragments;

s4: incubating the antibody and the digested DNA fragment together to obtain a DNA fragment combined with the antibody;

s5: incubating the DNA fragment combined with the antibody with protein A/G magnetic beads to obtain a magnetic bead-DNA complex;

s6: washing the magnetic bead-DNA complex, and then using proteinase K to digest the magnetic bead-DNA complex, thus constructing a DNA library;

s7: the DNA library was sequenced.

The technical scheme also provides a multiunit chemical detection analysis system for detecting transcriptome and histone modification distribution in a single cell in a cooperative way, which comprises a pretreatment unit, a DNA library construction unit and a sequencing unit; the pretreatment unit comprises a cell lysis reagent, a DNA digestion reagent, an antibody binding reagent, an immune coprecipitation reagent and a protein digestion reagent.

Further, in S2, the formula of buffer I is: 0.75-1.5% Tween 20, 0.75-1.5% NP-40, 0.15-0.3% SDS.

Further, in S3, the method of digesting the second cell lysate using MNase is: diluting MNase with buffer II to obtain MNase diluent, mixing MNase diluent with the second cell lysate, and incubating to obtain digested DNA fragment.

Further, in S3, buffer II was formulated as 100-200mM Tris-HCl pH 8.0,0.2-20mM CaCl ₂ The method comprises the steps of carrying out a first treatment on the surface of the In the MNase dilution, the volume ratio of MNase to buffer II was 5 μl:1ml.

Further, in S4, the method of co-incubating the antibody and the digested DNA fragment is: adding buffer solution III, protease inhibitor and antibody into the digested DNA fragment, and incubating at 4 ℃ for 8-12h to obtain the DNA fragment combined with the antibody.

Further, in S4, the formula of buffer III is: 15-30mM Tris-HCl pH 8.0, 210-420mM NaCl, 3.5-7mM EDTA, 3-6mM EGTA, 1-2% Triton X-100, 0.15-0.3% SDS, 0.15-0.3% sodium deoxycholate.

Further, in S6, the method of washing the magnetic bead-DNA complex is: washing the magnetic bead-DNA complex using buffer IV containing a protease inhibitor; the formula of buffer IV is: 10-20mM Tris-HCl pH 8.0, 1-2mM EDTA, 140-280mM NaCl, 1-2% Triton X-100, 0.1-0.3% SDS, 0.1-0.3% sodium deoxycholate; the working temperature of proteinase K was 55deg.C, the working concentration was 0.2. Mu.l/6. Mu.l, and the digestion time was 1.5h.

Further, the cell lysis reagent comprises Triton X-100 solution containing RNase inhibitor and buffer solution I containing protease inhibitor; the DNA digestion reagent comprises a buffer II containing MNase; antibody binding reagents include buffer III, protease inhibitors, and antibodies; the co-immunoprecipitation reagent includes buffer IV containing protease inhibitor using protein A/G magnetic beads; the protein digestion reagent includes water, ex-Taq buffer and proteinase K.

Further, the formula of buffer I is: 0.75% Tween 20, 0.75% NP-40, 0.3% SDS; buffer II was formulated as 100mM Tris-HCl pH 8.0,20mM CaCl ₂ The method comprises the steps of carrying out a first treatment on the surface of the The formula of the buffer III is as follows: 15mM Tris-HCl pH 8.0, 210mM NaCl, 3.5mM EDTA, 3mM EGTA, 1% Triton X-100, 0.3% SDS, 0.3% sodium deoxycholate; the formula of buffer IV is: 10mM Tris-HCl pH 8.0, 1mM EDTA. 140mM NaCl, 1% Triton X-100, 0.3% SDS, 0.3% sodium deoxycholate.

The principle of the technical scheme and the beneficial effects are that:

according to the technical scheme, single cells are subjected to lysis treatment, RNA and chromatin are separated after centrifugation, the RNA is used for building and sequencing a Smart-seq2 library, and a lower chromatin part after centrifugation is used for a ChIP experiment. The chromatin fraction obtained in the above was subjected to lysis treatment using a protease inhibitor-containing buffer I to obtain a cell lysate. The cell lysate was digested with MNase to obtain digested DNA fragments. Antibodies were then used to incubate binding to the digested DNA fragments and were selected as desired for the study (e.g., antibodies to methylated histones and acetylated histones). And enriching DNA combined with the antibody through co-immunoprecipitation, and finally digesting the DNA by using proteinase K, wherein the obtained DNA is used for constructing a library and carrying out conventional high-throughput gene sequencing. Millions of obtained sequence tags are precisely positioned on a genome, so that DNA segment information which is interacted with histones, transcription factors and the like in a whole genome range is obtained.

The inventor finally realizes the aim of detecting gene expression and histone modification distribution cooperatively by single cell initiation experiment by subversion improvement of the traditional ChIP-seq method and combination of Smart-seq2 technology, and the aim is named scHT-seq (single cell jointly profiling of histone modification and transcription by sequencing).

scHT-seq has the advantages of less loss of experimental samples, high sensitivity, remarkable improvement of genome coverage rate and the like. The number of unique mapping reads captured in a single cell (unique mapped reads) can reach millions, which is hundreds of times higher than that of similar technologies based on ChIP-seq and ChIC-seq, and the technical scheme does not depend on Tn5 transposase and has no false positive concern in an open chromatin region.

Drawings

Fig. 1 is a diagram of the unique map read statistics and the distribution of H3K27me3 of example 2.

FIG. 2 is a distribution plot of H3K27ac and a plot of Smart-seq2 sequencing results of example 2.

Detailed Description

The present application will be described in further detail with reference to examples, but embodiments of the present application are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.

Example 1:

(1) Buffer solution preparation

Buffer I (for lysing cells): 0.75% Tween 20, 0.75% NP-40 (Nonidet P40, ethylphenyl polyethylene glycol), 0.3% SDS (sodium dodecyl sulfate);

buffer II (for MNase (micrococcus nuclease) binding): 100mM Tris-HCl pH 8.0,2mM CaCl ₂ ；

Buffer III (for antibody binding): 15mM Tris-HCl pH 8.0, 210mM NaCl, 3.5mM EDTA (ethylenediamine tetraacetic acid), 3mM EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid), 1% Triton X-100, 0.3% SDS, 0.3% sodium Deoxycholate (Na-Deoxycholate);

buffer IV (for washing magnetic beads): 10mM Tris-HCl pH 8.0, 1mM EDTA, 140mM NaCl, 1% Triton X-100, 0.3% SDS, 0.3% sodium deoxycholate.

(2) Detection process

The first day:

①Smart-seq2

RNA-seq lysis buffer (0.2% (vol/vol) Triton X-100) was prepared, and RNase inhibitor (volume ratio 19. Mu.l: 1. Mu.l) was added to the buffer to obtain a lysis buffer mixture. Single cells (preferably less than or equal to 0.5 mu l in volume, obtained by manual selection under a stereoscopic microscope or sorting by a flow cytometer) are placed in a 0.2ml PCR tube (thin wall) containing 7 mu l of lysis buffer mixture, vortexed and placed on ice for more than 30 min. Next, the PCR tube is centrifuged (centrifugation parameters can be 1000-3000rpm,1-3 min), 4. Mu.l of the cell lysate in the PCR tube is transferred to a low-adsorption microcentrifuge tube (low-binding tube), and the fraction is stored at-20℃for library construction according to Smart-seq2 (Picelli et al., nature protocols, 2014); the remainder of the PCR tube was used to perform the ChIP assay (extraction of chromatin components for ChIP assay). This step can extract RNA from individual cells for Smart-seq2 sequencing, and the remainder of the PCR tube for subsequent extraction of chromatin components.

(2) Cell resuspension

The protease inhibitor was added to the buffer I to obtain a buffer I containing the protease inhibitor (the ratio of the buffer I to the 50 Xprotease inhibitor was 48. Mu.l: 2. Mu.l). Next, the individual cells (i.e., the "remaining part of PCR tube" in (1)) were resuspended in 15. Mu.l of buffer I containing protease inhibitor, and immediately the cells were gently blown up and down with a gun head several times to lyse the cells, to obtain a cell lysate.

Prior art ChIP-seq techniques require formaldehyde to crosslink the entire cell line (tissue) prior to cell lysis, i.e., to link the target protein to the chromatin, avoiding separation of the target protein from the chromatin without subsequent ChIP steps. However, in the present technical solution, the inventors adopted a solution of using RNA-seq lysis buffer to lyse cells to obtain RNA, and then a solution of using buffer I of protease inhibitor to lyse cells, and according to the above-described operation procedure, formaldehyde crosslinking and other processes may not be performed, and a desired number of unique mapping reads may still be obtained in the subsequent sequencing process.

The inventors have also made a number of attempts to address the two-step cleavage process of (1) and (2). How to separate RNA from DNA within a single cell without disrupting the cell structure is a major difficulty in this approach. If only the cells are (1) lysed, the histone modification information on the DNA level is lost and the chromatin component is not isolated. If the cells were lysed with protease inhibitor-containing buffer I of (2) alone, RNA could not be isolated for Smart-seq2 sequencing. The inventors finally determined the reaction volume and the lysis time by synergistically adjusting the formulation of the RNA lysate (RNA-seq lysis buffer) and the buffer I, aided by observing the cell morphology under a microscope. The RNA lysate needs to be lysed for 30min, so that RNA is fully released into the supernatant, and the total amount of the obtained RNA is obviously reduced below the time. Then using buffer solution I to crack for 1min, quickly and completely cracking cells, and protecting the binding information of histone modification on DNA to prevent degradation. According to the technical scheme, the RNA and the chromatin components are fully separated through two-time pyrolysis, and the RNA and the chromatin components are respectively used for Smart-seq2 sequencing or ChIP experiments, so that genetic information in one cell is fully utilized. The operation mode of performing Smart-seq2 sequencing and ChIP experiments on the same cell has not been presented in the prior art, and one main reason is that: the separation of RNA and chromatin components from the same cell is performed without destroying the binding information of histone modifications on DNA, which is difficult. According to the technical scheme, the two-time pyrolysis modes of different pyrolysis solutions are used, so that the two processes can be performed on the same cell.

(3) Dilution of Mnase

Mu.l MNase (Sigma N3755 or Sigma N5386) was added to 1ml of buffer II to obtain a diluted MNase solution, and placed on ice.

(4) Mnase digestion

Mu.l of the diluted MNase solution was added to the aforementioned cell lysate, and then left at 37℃for 5min (precision control), and digested DNA fragments were obtained by MNase enzyme treatment.

(5) Antibody binding

Then, 19. Mu.l of buffer III, 1. Mu.l of protease inhibitor and 1. Mu.g of antibody were added, and incubated overnight at 4℃under slow rotation (8-12 h, the subsequent experiments specifically using incubation conditions of about 10 h) to obtain a sample to be IP-treated (antibody-binding DNA fragment).

The following day:

(6) Co-Immunoprecipitation (IP) using protein A/G beads

Dynabeads protein A, twice surface-conjugated protein A using 200. Mu.l buffer IVMost mammalian immunoglobulins (Ig) can be isolated. Mu.l of magnetic beads were used for each sample to be IP-treated, and incubated at 4℃for 2-3 hours with slow rotation to obtain a sample after incubation with magnetic beads.

(7) Magnetic bead washing

Protease inhibitors were added to buffer IV such that the final concentration of protease inhibitor was 0.1×andplaced on ice. Samples incubated with 100. Mu.l of buffer IV containing protease inhibitor were washed 3 times and the beads were collected during washing under magnetic field conditions. If the beads are stuck on the centrifuge tube lid, they can be transferred to the tube by centrifugation. The protease inhibitor was Phosphatase and Protease Inhibitor Cocktails (Roche, 11697498001) in small pieces, each of which was dissolved in 50mL double distilled water, i.e., 1 Xprotease inhibitor solution.

(8) Proteinase K digestion (PK digestion)

After completion of washing, 5.6. Mu.l of ddH was added to the magnetic bead pellet ₂ O, 0.2. Mu.l of 10 XEx-Taq buffer (TaKaRa, RR 001Q) and 0.2. Mu.l of protease K solution (proteinase K, roche, 10910000), the beads were resuspended and then incubated in 55℃for 1.5h. After completion of digestion, the samples were transferred to a new 200. Mu.l centrifuge tube and left at 72℃for 40min to inactivate proteinase K, obtaining DNA for constructing the library.

(9) Library construction

Using commercial kitsUltra ^TM II DNA Library Prep Kit for(NEB, E7645S), DNA libraries were constructed according to the instructions using the DNA obtained in the above step.

Sequencing on-machine

The DNA library obtained in the last step is sent to a sequencing company, and sequencing is completed by the corresponding company.

Example 2

This example uses mouse GV-stage oocytes (n=237) as test samples, and 237 oocytes were individually tested. The assay was performed as described in example 1, using the scHT-seq technique of the present protocol to detect the H3K27me3 (trimethylation at amino acid 27 of histone H3) profile at the single cell level, corresponding to the use of the H3K27me3 antibody in the "(5) antibody binding" step.

The detection effect of the method of this embodiment is represented by the number of unique mapping reads (unique mapped reads), which represent that the sequencing reads are only aligned to unique positions on the genome, selected in the form of grep in the bam file as follows: 'NH: i:1'. The experimental results of this technical scheme are shown in fig. 1a. By adopting the scheme, the histone modification information captured in a single cell is very rich, and more rich information is provided for the cooperative detection of transcriptome and histone modification distribution. FIG. 1b shows a UCSC browser screen shot showing the distribution of H3K27me3 of ova in a large number of cells, and H3K27me3 in 25 ova detected by scHT-seq. The peak patterns of the scHT-seq for a large number of cells (positive control), the peak patterns of the single cell scHT-seq combined together, and the peak patterns of the 25 single cell scHT-seq are shown in FIG. 1b from top to bottom, and from the results, the detected signal in the single cell scHT-seq falls substantially within the peak pattern of the scHT-seq for a large number of cells, demonstrating the reliability of the single cell scHT-seq technique of the present protocol.

For the Smart-seq2 sequencing referred to above, see FIG. 2b for experimental results. Smart-seq2 technology is RNA extraction followed by library construction and sequencing. The inventors also used the scHT-seq technique of the present protocol to detect the H3K27ac (acetylation at amino acid 27 of histone H3) profile at the single cell level, and used the H3K27ac antibody in the "(5) antibody binding" step, see FIG. 2a (UCSC browser screen shot showing H3K27ac from a large number of ova in cells) for experimental results.

The scHT-seq technique was compared to CoTECH (combined assay of transcriptome and enriched chromatin binding) and the Paired-Tag method. Specific modes of operation of CoTECH can be found in the literature "Xiong Haiqing, single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions, NATURE METHODS,2021". In fig. 1a, the distribution of H3K27me3 and H3K4me3 was detected at single cell level using embryonic stem cells (ESCs, n= 6,993) as samples, and the number of unique mapping reads obtained was far lower than the technical solution of this example. Specific methods of manipulation of the Paired-Tag can be found in the literature: "Cao, j., joint profiling of histone modifications and transcriptome in single cells from mouse brain., chenxu Zhu, nat methods.2021". In fig. 1a, the distribution of H3K27me3 was examined at single cell level using frontal cortex/hippocampal cells (n=558) as a sample, and the number of unique mapping reads obtained was far lower than in the present example.

Experimental example 1: effect of MNase amount on technical Effect

The detection of H3K27me3 at the single cell level was performed as described in example 1, and the concentration of MNAse was examined. The experiment was divided into three groups, the first group was prepared by adding 10. Mu.l of MNase to 1ml of buffer II to obtain a diluted MNase solution (No. 1-3), the second group was prepared by adding 5. Mu.l of MNase to 1ml of buffer II to obtain a diluted MNase solution (No. 4-6), and the third group was prepared by adding 0.5. Mu.l of MNase to 1ml of buffer II to obtain a diluted MNase solution (No. 7-9). MNase was specifically used with Sigma N3755, 3 replicates per group, single cell sequencing results are shown in table 1.

Table 1: single cell sequencing results Using different MNase levels

Wherein reads refer to sequences generated by a high throughput sequencing platform; total reads refer to the total number of sequences obtained by counting the number of sequences; mapped reads refer to sequences in all reads that align to the reference genome (the reference genome is universal in the world, typically using the mouse mm10 version, human hg19 version); total mapped reads refers to the statistics of mapped reads, the total number of sequences aligned to the reference genome; total mapped% refers to the percentage of sequences aligned to the reference genome, also known as alignment efficiency, which is the most direct manifestation of transcriptome, genomic sequencing data utilization; unique mapped reads refers to the number of sequences aligned to unique positions on the reference genome; unique mapped% refers to the number of sequences aligned to unique positions on the reference genome as a percentage of total reads, which is also a manifestation of transcriptome data utilization. Of the transcriptome sequencing data, only data aligned to the reference genome can be used for subsequent analysis (i.e., mapped Reads). And, the unique mapping Reads in the Mapped Reads can provide more accurate information for subsequent analysis, and the more the number of the unique mapping Reads is, the more accurate information can be provided for subsequent analysis.

As can be seen from the experimental data in Table 1, the number of unique mapped reads obtained for No. 4-6 is the greatest, and the amount of Mnase (5. Mu.l) used for these experimental groups is the most appropriate, yielding as many unique mapped reads as possible for subsequent analysis. MNase, micrococcus nuclease, is a nuclease that degrades the DNA sequence of the nucleosome junction, and digestion of chromatin by MNase releases individual nucleosomes. The amount of MNase generally affects the extent to which chromatin or DNA is digested and digested. In the present technical solution, the MNase usage has a very significant impact on the total reads, the total mapped reads, the unique mapped reads, which is unexpected for the person skilled in the art. The total reads and total mapped reads of No. 4-6 are orders of magnitude different relative to No. 1-3 and No. 7-9; whereas No4-6 is 2 to 15 times the number of uniquely mapped reads as compared to No1-3 and No 7-9. Therefore, the MNase dosage (5 mu l) has the unexpected technical effect, the reading number is greatly improved, more useful information is provided for scHT-seq detection of the scheme, the detection efficiency is improved, and more referenceable data is provided for experimental study in the related field.

Experimental example 2: influence of the antibody incubation time on the technical Effect

The incubation time was adjusted to 37℃for 30min based on No. 1-3 ("5 antibody binding" step), which is No. 10-12. Whereas No. 1-3 was incubated overnight (10 h) at 4 ℃. Single cell sequencing results are shown in table 2.

Table 2: single cell sequencing results using different antibody incubation times

As can be seen from the experimental data in Table 2, more unique mapping reads can be obtained by incubating the antibody at 4 ℃ overnight (10 h), and in repeated experiments, the processing mode of incubating the antibody at 4 ℃ overnight (10 h) can ensure that the number of the obtained unique mapping reads is more stable, and the experimental method has better stability and is more suitable for popularization and application.

Experimental example 3: effects of PK digestion time on technical Effect

On the basis of No. 1-3, in the step of "(8) proteinase K digestion (PK digestion)", the digestion time was changed to 30min, namely No. 13-15. Whereas the digestion time of No. 1-3 was 1.5h. The experimental results are shown in table 3.

Table 3: single cell sequencing results using different PK digestion times

From the data in table 3, proteinase K digestion time set to 1.5h can significantly increase the number of unique mapping reads.

The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims (10)

1. A multi-group chemical detection analysis method for detecting transcriptome and histone modification distribution in a single cell is characterized by comprising the following steps of: the method comprises the following steps of:

s1: lysing the single cells using Triton X-100 solution, and centrifuging to obtain a first cell lysate; part of the supernatant of the first cell lysate was used for smart-seq2 library construction; the remainder of the first cell lysate is used to extract the chromatin component;

s2: preparing a buffer solution I containing a protease inhibitor, then adding the buffer solution I into the rest part of the first cell lysate, and obtaining a second cell lysate by blowing;

s3: digesting the second cell lysate with MNase to obtain digested DNA fragments;

s4: incubating the antibody and the digested DNA fragment together to obtain a DNA fragment combined with the antibody;

s5: incubating the DNA fragment combined with the antibody with protein A/G magnetic beads to obtain a magnetic bead-DNA complex;

s6: washing the magnetic bead-DNA complex, and then using proteinase K to digest the magnetic bead-DNA complex, thus constructing a DNA library;

s7: the DNA library was sequenced.

2. The method for multiplex assay in single cells for the collaborative detection of transcriptome and histone modification profile according to claim 1, wherein: in S2, the formula of buffer I is: 0.75-1.5% Tween 20, 0.75-1.5% NP-40, 0.15-0.3% SDS.

3. The method for multiplex assay in single cells for the collaborative detection of transcriptome and histone modification profile according to claim 1, wherein: in S3, the method of digesting the second cell lysate using MNase is: diluting MNase with buffer II to obtain MNase diluent, mixing MNase diluent with the second cell lysate, and incubating to obtain digested DNA fragment.

4. A method of multiplex assay for the collaborative detection of transcriptome and histone modification profiles in single cells according to claim 3, wherein: in S3, buffer II was formulated as 100-200mM Tris-HCl pH 8.0,0.2-20mM CaCl ₂ The method comprises the steps of carrying out a first treatment on the surface of the In the MNase dilution, the volume ratio of MNase to buffer II was 5 μl:1ml.

5. The method for multiplex assay in single cells for the collaborative detection of transcriptome and histone modification profile according to claim 1, wherein: in S4, the method of co-incubating the antibody with the digested DNA fragment is: adding buffer solution III, protease inhibitor and antibody into the digested DNA fragment, and incubating at 4 ℃ for 8-12h to obtain the DNA fragment combined with the antibody.

6. The method for multiplex assay in single cells for the collaborative detection of transcriptome and histone modification profile according to claim 5, wherein: in S4, the formula of buffer III is: 15-30mM Tris-HCl pH 8.0, 210-420mM NaCl, 3.5-7mM EDTA, 3-6mM EGTA, 1-2% Triton X-100, 0.15-0.3% SDS, 0.15-0.3% sodium deoxycholate.

7. The method for multiplex assay in single cells for the collaborative detection of transcriptome and histone modification profile according to claim 1, wherein: in S6, the method of washing the magnetic bead-DNA complex is: washing the magnetic bead-DNA complex using buffer IV containing a protease inhibitor; the formula of buffer IV is: 10-20mM Tris-HCl pH 8.0, 1-2mM EDTA, 140-280mM NaCl, 1-2% Triton X-100, 0.1-0.3% SDS, 0.1-0.3% sodium deoxycholate; the working temperature of proteinase K was 55deg.C, the working concentration was 0.2. Mu.l/6. Mu.l, and the digestion time was 1.5h.

8. A multiplex chemical detection analysis system for detecting transcriptome and histone modification distribution in a single cell, which is characterized in that: comprises a pretreatment unit, a DNA library construction unit and a sequencing unit; the pretreatment unit comprises a cell lysis reagent, a DNA digestion reagent, an antibody binding reagent, an immune coprecipitation reagent and a protein digestion reagent.

9. The multi-set chemical detection assay system for the collaborative detection of transcriptome and histone modification profiles within a single cell according to claim 8, wherein: the cell lysis reagent comprises Triton X-100 solution containing RNase inhibitor and buffer solution I containing protease inhibitor; the DNA digestion reagent comprises a buffer II containing MNase; antibody binding reagents include buffer III, protease inhibitors, and antibodies; the co-immunoprecipitation reagent includes buffer IV containing protease inhibitor using protein A/G magnetic beads; the protein digestion reagent includes water, ex-Taq buffer and proteinase K.

10. The multi-set chemical detection assay system for the collaborative detection of transcriptome and histone modification profiles within a single cell according to claim 9, wherein: the formula of the buffer solution I is as follows: 0.75% Tween 20, 0.75% NP-40, 0.3% SDS; buffer II was formulated as 100mM Tris-HCl pH 8.0,2mM CaCl ₂ The method comprises the steps of carrying out a first treatment on the surface of the The formula of the buffer III is as follows: 15mM Tris-HCl pH 8.0, 210mM NaCl, 3.5mM EDTA, 3mM EGTA, 1% Triton X-100, 0.3% SDS, 0.3% sodium deoxycholate; the formula of buffer IV is: 10mM Tris-HCl pH 8.0, 1mM EDTA, 140mM NaCl, 1% Triton X-100, 0.3% SDS, 0.3% sodium deoxycholate.