Whole genome multi-recombinant protein modification labeling method based on transposition reaction and fluorescence in-situ hybridization and application of whole genome multi-recombinant protein modification labeling method

Abstract

The invention relates to a whole genome multi-recombinant protein modification labeling method based on transposition reaction and fluorescence in situ hybridization and application thereof, and belongs to the technical field of cell molecular markers. The invention relates to a marking method modified by a whole genome multi-recombinant protein. The marking method comprises the following steps: cleaning cells carrying one or more special sequences for coding oligonucleotides; and mixing the cleaned cells, an EC buffer solution and an aqueous solution of the oligonucleotide fluorescent probe, carrying out room temperature incubation, discarding the liquid, cleaning, and carrying out fluorescence imaging. According to the labeling method, fluorescence in-situ labeling can be carried out on different types of fine histone modifications in the same cell sample, and the labeling method is suitable for an ultrahigh-resolution microscopic imaging platform or a common confocal imaging system. The marker has extremely high specificity and sensitivity, and spatial distribution and co-localization information of various histone modifications in a cell nucleus can be obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell molecular marker, and particularly relates to in-situ labeling of single-cell whole-genome multiple recombination protein modification. BACKGROUND

[0002] As one of the core mechanisms of epigenetic regulation, histone modification regulates the structure and openness of chromatin by specific chemical modification of amino acid residues at the end of histone, and directly affects the spatiotemporal specificity of gene expression. At present, the detection method of histone modification site in whole genome mainly uses chromatin immunoprecipitation technology (ChIP-seq) and targeted cleavage and transposition (CUT&Tag) technology based on second-generation sequencing. The basic experimental steps of ChIP-seq include: 1) cross-linking and fixing the DNA and protein in the cell by formaldehyde; 2) ultrasonic lysis of the cell and breaking the genomic DNA into 150-500 bp fragments; 3) using histone modification antibody to specifically bind the DNA fragments cross-linked and combined with the target protein, and using protein A magnetic beads that can bind to the Fc fragment of the antibody to capture the antibody-protein-DNA complex; 4) digesting the protein in the complex, releasing and enriching the target DNA fragments, and performing sequencing library construction and analysis. Due to the influence of factors such as formaldehyde cross-linking, complexity of chromatin fragmentation, large cell loss in immunoprecipitation, GC bias in the process of lysis and sequencing, etc., ChIP-seq has low signal, high background and poor repeatability, and requires the use of millions of cells for experiments.

[0003] CUT&Tag technology does not rely on formaldehyde cross-linking and ultrasonic fragmentation of genomic DNA, but after cell permeation, it incubates specific histone modification antibodies, and then adds pA-Tn5 transposase fusion protein. Tn5 transposase can cut the genomic DNA in situ while adding exogenous linker primer sequence, which can be directly amplified by PCR. Since only the sequence near the target histone modification is cut and released, and the end repair and primer ligation steps in the library construction are omitted, CUT&Tag not only can be detected at the single-cell level, but also can obtain very low background signal, greatly improving the sensitivity and specificity of detecting histone modification sites. However, neither ChIP-seq nor CUT&Tag can detect multiple histone modifications in the same sample. At the same time, although these second-generation sequencing-based technologies can obtain the positioning information of histone modification on one-dimensional genomic sequence, the spatial information in the cell nucleus will be lost in the process of sequencing, so these technologies cannot capture the real three-dimensional spatial distribution characteristics of histone modification.

[0004] The spatial distribution information of histone modification in the nucleus is currently mainly observed by an immunofluorescence staining method, but the method is limited by the selection of fluorescence channels and antibodies, and it is difficult to perform fluorescence imaging of multiple histone modifications in the nucleus, which is not conducive to the in-depth study of the co-localization of multiple histone modifications. At present, there is a lack of a method for specifically labeling multiple histone modifications in the nucleus at the whole genome level on the market. SUMMARY

[0005] In view of the defects in the prior art, in order to solve the problem that the prior art cannot label multiple histone modifications in the same sample at the same time, the purpose of the present application is to design and provide a whole genome multiple histone modification photosensitive fluorescent labeling technology based on the combination of transposase fusion protein mediation and sequential fluorescence hybridization. The present application can be combined with PALM / STORM and other ultra-high resolution imaging technology platforms or ordinary confocal imaging platforms to detect the spatial position information of multiple histone modifications in the nucleus of the same cell sample.

[0006] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0007] On the one hand, the present application provides an antibody-Tn5 transposon, comprising a pA-Tn5 transposon and a histone modification antibody modified and labeled thereon;

[0008] The pA-Tn5 transposon is formed by assembling pA-Tn5 and a special coding oligonucleotide.

[0009] The antibody-Tn5 transposon, the sequence of the special coding oligonucleotide is shown in SEQ ID NO. 1 or SEQ ID NO. 2;

[0010] The histone is H3K27me3, H3K27ac, H3K4me1, H3K4me2, H3K4me3 or H3K36me3.

[0011] The antibody is an antibody that can be used for immunofluorescence.

[0012] On the second aspect, the present application provides a preparation method of the antibody-Tn5 transposon, comprising the following steps:

[0013] (1) mixing a water solution of a special coding oligonucleotide, a water solution of an auxiliary oligonucleotide sequence and 2x annealing buffer to perform annealing reaction, to obtain a special coding oligonucleotide / auxiliary oligonucleotide sequence pair;

[0014] (2) mixing special coding oligonucleotide / auxiliary oligonucleotide sequence pair, glycerol, 2x dialysis buffer, pA-Tn5 and sterile water to form a reaction system, and performing reaction under room temperature by gently shaking to obtain pA-Tn5 transposon;

[0015] (3) mixing histone modification antibody, pA-Tn5 transposon and washing buffer, and placing under room temperature to obtain antibody-Tn5 transposon.

[0016] The preparation method, and the auxiliary oligonucleotide sequence is shown as SEQ ID NO. 3.

[0017] The concentration of the aqueous solution of the special coding oligonucleotide is 200 μM; and the concentration of the aqueous solution of the auxiliary oligonucleotide sequence is 200 μM.

[0018] The special coding oligonucleotide, the auxiliary oligonucleotide sequence and the 2x annealing buffer are mixed in a volume ratio of 1:1:2.

[0019] The annealing reaction is performed under the following conditions: 95℃ for 5 minutes, and then slowly cooling to 25℃ at a rate of -1℃ per minute.

[0020] The volume ratio of the special coding oligonucleotide / auxiliary oligonucleotide sequence pair, glycerol, 2x dialysis buffer, pA-Tn5 and sterile water is 7.5:40:12:21.5:19.

[0021] The mass / volume ratio of the histone modification antibody, pA-Tn5 transposon and washing buffer is 0.5 μg:5.5 μL:5 μL.

[0022] In a third aspect, the present application provides a cell carrying one or more sequences of special coding oligonucleotide, which is obtained by performing transposition reaction on a cell sample with one or more antibody-Tn5 transposons as claimed in claim 1.

[0023] In a fourth aspect, the present application provides a preparation method of the cell carrying special coding oligonucleotide, which comprises the following steps:

[0024] (1) fixing, permeabilizing and blocking the cell sample on a carrier to obtain a carrier with cells;

[0025] (2) mixing the antibody-Tn5 transposon and high-salt concentration washing buffer to form a mixed solution, dropping the mixed solution on a sealing film, and inverting the carrier with cells on the sealing film to make the mixed solution and the cells fully contact and incubate;

[0026] (3) take the carrier, wash the side of the carrier with cells, add the reaction buffer, incubate, discard the liquid, wash, and obtain the cells carrying the special coding oligonucleotide sequence;

[0027] (4) if there are multiple antibody-Tn5 transposons, repeat steps (1)-(3) for each antibody-Tn5 transposon in turn until all antibody-Tn5 transposons complete the transposition reaction, and obtain the cells carrying multiple special coding oligonucleotide sequences.

[0028] In a fifth aspect, the present application provides a labeling method using whole genome multiple recombinant protein modification, comprising the following steps:

[0029] (1) washing the cells carrying one or more special coding oligonucleotide sequences;

[0030] (2) mixing the washed cells, EC buffer and aqueous solution of oligonucleotide fluorescent probe, incubating at room temperature, discarding the liquid, washing with washing buffer, incubating at room temperature, washing with 2×SSC, incubating at room temperature, and performing fluorescence imaging.

[0031] (3) adding 55% washing buffer for washing, incubating at room temperature, adding 2×SSC for washing, incubating at room temperature, eluting the oligonucleotide fluorescent probe in step (2) above, repeating steps (1)-(2) above, and only changing the sequence of the oligonucleotide fluorescent probe to match another special coding oligonucleotide sequence, so that fluorescence labeling imaging can be performed in turn.

[0032] The oligonucleotide fluorescent probe is shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0033] A cell labeled by the whole genome multiple recombinant protein modification labeling method.

[0034] The labeling method is used for labeling one or multiple histone modifications in the same sample.

[0035] The working principle of the technical solution of the present application is as follows:

[0036] 1. By designing a special coding oligonucleotide sequence, assembling it with pA-Tn5 (where pA is the abbreviation of protein A, a surface protein derived from Staphylococcus aureus) and a specific histone modification antibody to form an antibody-Tn5 transposon containing different sequences, such as Figure 1As shown, the antibody-Tn5 transposon containing different special coding sequences is assembled by pA-Tn5, special coding oligonucleotide sequences and different kinds of histone modification antibodies. The antibody-Tn5 transposon includes ① antibody-Tn5 transposon containing special coding sequence No. 1; ② antibody-Tn5 transposon containing special coding sequence No. 2, and so on.

[0037] 2. When the first kind of histone modification in the cell sample is transposon labeled, as shown in Figure 2 , the transposition reaction can insert the oligonucleotide with special coding sequence into the genome near the corresponding target histone modification, and different kinds of histone modifications can be distinguished by the special coding of the inserted sequence. Specifically, by incubating the antibody-Tn5 transposon ① (i.e. antibody-Tn5 transposon containing special coding sequence No. 1) with the cell sample to perform the transposition reaction, the oligonucleotide with special coding sequence No. 1 can be inserted into the genome near the corresponding target histone modification. After the first round of transposon labeling is completed, the second kind of histone modification in the cell is transposon labeled using the antibody-Tn5 transposon ②. In this way, the transposon labeling of all target histone modifications is completed. At this time, the oligonucleotide with special coding sequence is inserted near each labeled histone modification, such as special coding sequence No. 1 near histone modification ①, special coding sequence No. 2 near histone modification ②, and so on.

[0038] 3. A fluorescent probe with a fluorescent group and complementary to the special coding oligonucleotide sequence is designed, and specific fluorescent labeling of the histone modification is achieved by fluorescent hybridization of the fluorescent probe complementary to the special coding oligonucleotide sequence inserted near the histone modification. The process of “fluorescent hybridization-imaging-probe elution” is adopted to achieve specific fluorescent labeling and imaging of multiple histone modifications in the nucleus. As shown in Figure 3 , the oligonucleotide fluorescent probe No. 1 can be complementary to the special coding sequence No. 1 inserted near the histone modification ① to achieve fluorescent labeling and imaging of the first kind of histone modification. After eluting the oligonucleotide fluorescent probe No. 1, the oligonucleotide fluorescent probe No. 2 can be used to be complementary to the special coding sequence No. 2 inserted near the histone modification ② to achieve fluorescent labeling and imaging of the second kind of histone modification. In this way, the fluorescent labeling and imaging of all target histone modifications are completed.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] The labeling method of the present application can fluorescently in situ label different kinds of histone modifications in the same cell sample, and is suitable for an ultra-high resolution microscopic imaging platform or a common confocal imaging system. The labeling has extremely high specificity and sensitivity, and can obtain the spatial distribution and co-localization information of multiple histone modifications in the nucleus. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Assembly of antibody-Tn5 transposon

[0042] Figure 2 Schematic diagram of transpositional labeling of different histone modifications in the nucleus

[0043] Figure 3 Schematic diagram of fluorescent hybridization of transpositional labeled histone modifications

[0044] Figure 4 Imaging map of specific fluorescence of two histone modifications (H3K27me3 and H3K27ac) in the nucleus of mouse embryonic stem cells (the scale is 5 microns). DETAILED DESCRIPTION

[0045] The present application will be further described below by means of the accompanying drawings and examples.

[0046] Example 1: Assembly of antibody-Tn5 transposon

[0047] 1. Assembly of pA-Tn5 and special coding oligonucleotide into pA-Tn5-special coding sequence (i.e. pA-Tn5 transposon)

[0048] (1) Special coding oligonucleotide sequence and related chemical modification were synthesized by Bio Company. The sequence information is shown in Table 1 below:

[0049] Table 1 Sequence information

[0050]

[0051]

[0052] (2) Dissolve the oligonucleotides of special coding No. 1 sequence, special coding No. 2 sequence and auxiliary sequence in water respectively, with a concentration of 200 μM.

[0053] (3) Prepare 2× annealing buffer, with components of 20 mM Tris HCl (pH 8.0), 100 mM NaCl, 2 mM EDTA.

[0054] (4) Mix the dissolved special coding No. 1 sequence, special coding No. 2 sequence, auxiliary sequence and 2× annealing buffer according to the following Table 2, and place them in a PCR instrument set at 95℃ for 5 minutes, and then slowly cool to 25℃ at a rate of -1℃ per minute, to obtain 50 μM of annealed special coding No. 1 sequence / auxiliary sequence pair and special coding No. 2 sequence / auxiliary sequence pair.

[0055] Table 2 Reaction system

[0056] Ingredients 80 μL of reaction dependence system 200 μM of special coding 1st sequence (or special coding 2nd sequence) 20 μL 200 μM of Tn5MERev (i.e. auxiliary sequence) 20 μL 2x annealing buffer 40 μL

[0057] (5) Prepare 2x dialysis buffer with the following components: 100 mM HEPES-KOH (pH 7.2), 0.2 M NaCl, 0.2 mM EDTA, 2 mM DTT, 0.2% Triton X-100, 20% glycerol.

[0058] (6) Combine annealed special code No. 1 sequence / auxiliary sequence pair (or special code No. 2 sequence / auxiliary sequence pair), glycerol, 2x dialysis buffer, pA-Tn5, and sterile water in the proportions shown in Table 3 below, and gently shake at room temperature for 1 hour to obtain assembled pA-Tn5-special code No. 1 sequence (or pA-Tn5-special code No. 2 sequence).

[0059] Table 3 Reaction system

[0060]

[0061] 2. Assembly of antibody-Tn5 transposon

[0062] (1) Prepare a wash buffer with the following components: 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 10 mM sodium butyrate.

[0063] (2) Mix different types of histone modification antibodies with pA-Tn5-special code No. 1 sequence (or pA-Tn5-special code No. 2 sequence) and wash buffer in the proportions shown in Table 4 below, and place at room temperature for 1 hour to obtain assembled antibody-Tn5 transposon 1 and antibody-Tn5 transposon 2 for different types of histone modification markers.

[0064] Table 4 Reaction system proportions

[0065]

[0066] Example 2: Transpositional labeling of different types of histone modifications

[0067] 1. Fixation, permeabilization, and blocking treatment of cell samples

[0068] (1) Prepare a 0.1% BSA-PBS solution: Dissolve 0.05 g of BSA in 1x PBS.

[0069] (2) Seed cells on 25 mm round coverslips in a six-well plate, and wash once with 0.1% BSA-PBS solution.

[0070] (3) Place the six-well plate on ice, add 3.7% formaldehyde solution to the cell sample for fixation for 10 minutes, then add 1.25M glycine solution and place for 10 minutes.

[0071] (4) Discard all the liquid and wash twice with 0.1% BSA-PBS solution.

[0072] (5) Prepare a normal salt concentration washing buffer, the components of which are 20mM HEPES (pH 7.5), 150mM NaCl, 0.5mM spermidine, 10mM sodium butyrate, 0.01% digitonin, and 0.05% Triton X-100.

[0073] (6) Add the normal salt concentration washing buffer to the cell sample for permeabilization for 5 minutes.

[0074] (7) Discard all the liquid, add 0.01% BSA-PBS solution to the cell sample, and seal for 1 hour at room temperature.

[0075] 2. Transposition reaction of antibody-Tn5 transposon to cell sample

[0076] (1) Prepare a high salt concentration washing buffer (2mM EDTA), the components of which are 20mM HEPES (pH 7.5), 300mM NaCl, 0.5mM spermidine, 10mM sodium butyrate, 0.01% digitonin, 0.05% Triton X-100, and 2mM EDTA.

[0077] (2) Mix 1 unit of assembled antibody-Tn5 transposon ① with the high salt concentration washing buffer (2mM EDTA) to a final total volume of 100μL.

[0078] (3) Drop 100μL of the mixture of antibody-Tn5 transposon ① and high salt concentration washing buffer (2mM EDTA) on a parafilm, and invert the coverslip with cells on the parafilm to allow the mixture to fully contact the cells, and incubate in a humidified box at 37°C for 1 hour.

[0079] (4) Prepare a high salt concentration washing buffer, the components of which are 20mM HEPES (pH 7.5), 300mM NaCl, 0.5mM spermidine, 10mM sodium butyrate, 0.01% digitonin, and 0.05% Triton X-100.

[0080] (5) Hold the coverslip with the cell sample facing upwards, and place it back into the six-well plate, and wash four times with the high salt concentration washing buffer, 5 minutes each time.

[0081] (6) Prepare the reaction buffer, the components are 10 mM TAPS (pH 8.3), 10 mM MgCl2, 10 mM sodium butyrate, 0.01% digitonin.

[0082] (7) Add the reaction buffer to the cell sample and incubate at 37°C for 1 hour.

[0083] (8) Prepare the high-salt concentration washing buffer (5 mM EDTA), the components are 20 mM HEPES (pH 7.5), 300 mM NaCl, 0.5 mM spermidine, 10 mM sodium butyrate, 0.01% digitonin, 0.05% Triton X-100, 5 mM EDTA.

[0084] (9) Discard all the liquid, add the high-salt concentration washing buffer (5 mM EDTA) to the cell sample and wash four times, each time incubate at 37°C for 5 minutes, then the antibody-Tn5 transposon ① transposition reaction of histone modification ① in the cell sample is completed.

[0085] (10) Repeat steps (2)-(9) to transposition mark the histone modification ② by the antibody-Tn5 transposon ② transposition reaction of the cell sample.

[0086] Example 3: Fluorescence in situ hybridization and imaging

[0087] (1) Add 4% formaldehyde solution to the cell sample prepared in the above Example 2 after the transposition reaction (i.e., the cell sample carrying the special coding oligonucleotide sequence) and fix at room temperature for 15 minutes.

[0088] (2) Discard all the liquid, wash with 1xPBS three times, each time for 5 minutes.

[0089] (3) Add 70% ethanol to the cell sample and incubate at room temperature for 3 hours.

[0090] (4) Discard all the liquid, wash with 2xSSC twice.

[0091] (5) The fluorescent probe complementary to the special coding oligonucleotide is synthesized by a biological company. The sequence information is as follows in Table 5:

[0092] Table 5 Sequence information

[0093]

[0094] (6) Dissolve the oligonucleotide fluorescent probe No. 1 and the oligonucleotide fluorescent probe No. 2 in water respectively, the concentration is 100 μM.

[0095] (7) Prepare 10% EC buffer, the components are 10% Ethylene carbonate, 2xSSC, 0.1 g / mL Dextran sulfate, 0.02 U / mL SUPERase In RNase Inhibitor.

[0096] (8) Add 2 mL 10% EC buffer and 1 μL oligonucleotide fluorescent probe No. 1 into the cell sample, and incubate at room temperature for 20 minutes.

[0097] (9) Prepare 10% washing buffer, the components are 2xSSC, 10% Formamide (v / v), 0.1% Triton X-100.

[0098] (10) Discard all the liquid, and wash once with 10% washing buffer, and incubate at room temperature for 5 minutes.

[0099] (11) Wash twice with 2xSSC, each time incubate at room temperature for 1 minute, and the fluorescent in situ labeling of histone modification ① is completed, and fluorescence imaging can be performed.

[0100] (12) Prepare 55% washing buffer, the components are 2xSSC, 55% Formamide (v / v), 0.1% Triton X-100.

[0101] (13) Add 55% washing buffer into the cell sample, and incubate at room temperature for 5 minutes. Wash once with 2xSSC, and incubate at room temperature for 1 minute to elute oligonucleotide fluorescent probe No. 1.

[0102] (14) Perform fluorescent in situ labeling of histone modification ②, and use oligonucleotide fluorescent probe No. 2, and repeat steps (5)-(11) to sequentially perform fluorescent labeling of each histone modification.

[0103] According to the above experimental steps, two rounds of transposition labeling, fluorescent in situ hybridization and imaging are performed on two histone modifications (H3K27me3 and H3K27ac) in a mouse embryonic stem cell sample. As shown in FIG. 1, the fluorescent signal of the first round of fluorescent in situ hybridization is distributed in the nucleus, and the nuclear fluorescent signal disappears after the fluorescent probe is eluted. After the second round of fluorescent in situ hybridization, the fluorescent signal appears again, and the distribution is different from the first time, indicating that the present application can specifically label two histone modifications by fluorescence in the same cell sample. Figure 4 As can be seen, the fluorescent signal of the first round of fluorescent in situ hybridization is distributed in the nucleus, and the nuclear fluorescent signal disappears after the fluorescent probe is eluted. After the second round of fluorescent in situ hybridization, the fluorescent signal appears again, and the distribution is different from the first time, indicating that the present application can specifically label two histone modifications by fluorescence in the same cell sample.

Claims

1. An antibody-Tn5 transposon, characterized in that, The antibody of histone modification comprising pA-Tn5 transposon and a special coding oligonucleotide; The pA-Tn5 transposon is assembled by pA-Tn5 and a special coding oligonucleotide.

2. An antibody-Tn5 transposon according to claim 1, wherein, The sequence of the special coding oligonucleotide is a sequence which does not match the genomic sequence in cells, does not form stable secondary structure by itself, has a GC content of 30-70%, does not cross-react with other sequences, and has a length of about 30 nt. Preferably, the sequence of the special coding oligonucleotide is shown in SEQ ID NO. 1 or SEQ ID NO.

2. The histone is H3K27me3, H3K27ac, H3K4me1, H3K4me2, H3K4me3 or H3K36me3. The antibody is an antibody which can be used for immunofluorescence.

3. A method for preparing an antibody-Tn5 transposome according to claim 1 or 2, wherein, The method comprises the following steps: (1) mixing a water solution of the special coding oligonucleotide, a water solution of the auxiliary oligonucleotide sequence, and 2× annealing buffer in a proportion to perform annealing reaction, to obtain a special coding oligonucleotide / auxiliary oligonucleotide sequence pair; (2) mixing the special coding oligonucleotide / auxiliary oligonucleotide sequence pair, glycerol, 2× dialysis buffer, pA-Tn5 and sterile water to form a reaction system, and performing reaction at room temperature by gently shaking, to obtain the pA-Tn5 transposon; (3) mixing the antibody of histone modification, the pA-Tn5 transposon and washing buffer, and placing at room temperature, to obtain the antibody-Tn5 transposon.

4. The production method according to claim 3, wherein The auxiliary oligonucleotide sequence is shown in SEQ ID NO.

3. The concentration of the water solution of the special coding oligonucleotide is 200 μM; and the concentration of the water solution of the auxiliary oligonucleotide sequence is 200 μM. The proportioning mixing is mixing the water solution of the special coding oligonucleotide, the water solution of the auxiliary oligonucleotide sequence, and 2× annealing buffer in a volume ratio of 1:1:

2. The annealing reaction is performed at 95℃ for 5 minutes, and then slowly cooled to 25℃ at a rate of -1℃ per minute. The volume ratio of the special coding oligonucleotide / auxiliary oligonucleotide sequence pair, glycerol, 2× dialysis buffer, pA-Tn5 and sterile water is 7.5:40:12:21.5:

19. The mass / volume ratio of the antibody of histone modification, the pA-Tn5 transposon and washing buffer is 0.5 μg:5.5 μL:5 μL.

5. A cell carrying one or more sequences of specially coded oligonucleotides, characterized in that, The method comprises the following steps:

6. A method for preparing a cell carrying a sequence of special coded oligonucleotides according to claim 5, characterized in that, (1) fixing, permeabilizing and blocking the cell sample on a carrier, to obtain a carrier with cells; (2) mixing the antibody-Tn5 transposon and high-salt concentration washing buffer to form a mixed solution, dropping the mixed solution on a sealing film, and placing the carrier with cells upside down on the sealing film, so that the mixed solution and the cells are in full contact, and incubating; (3) taking the carrier, washing the side of the carrier with cells, adding reaction buffer, incubating, discarding the liquid, and washing, to obtain cells carrying the sequence of the special coding oligonucleotide. ​ (4) If there are multiple antibody-Tn5 transposons, repeat steps (1)-(3) for each antibody-Tn5 transposon in turn until all antibody-Tn5 transposons complete the transposition reaction and obtain cells carrying sequences of multiple special coding oligonucleotides.

7. A method of tagging using whole genome multiplexed recombination protein modification, characterized in that, comprising the following steps: (1) washing the cells carrying one or more special coding oligonucleotide sequences as claimed in claim 5; (2) mixing the washed cells, EC buffer and an aqueous solution of an oligonucleotide fluorescent probe, incubating at room temperature, discarding the liquid, washing with a washing buffer, incubating at room temperature, washing with 2×SSC, incubating at room temperature, and performing fluorescence imaging. (3) adding 55% washing buffer, incubating at room temperature, adding 2×SSC, incubating at room temperature, and eluting the oligonucleotide fluorescent probe of step (2) above, and repeating steps (1)-(2) above, only changing the sequence of the oligonucleotide fluorescent probe to match the sequence of another special coding oligonucleotide, to sequentially perform fluorescence labeling imaging.

8. A marker method using whole genome multiplexed protein modifications as claimed in claim 7, wherein, The oligonucleotide fluorescent probe is shown in SEQ ID NO. 3 or SEQ ID NO.

4.

9. A cell labeled with a whole genome multiplexed recombinome modification, wherein the cell is a cell of claim 1. obtained by the labeling method as claimed in claim 7 or 8.

10. Use of the labeling method as claimed in claim 7 or 8 to label one or multiple histone modifications simultaneously in the same sample.

Images