Whole genome open chromatin in-situ labeling method and application of whole genome open chromatin in detection of whole genome open chromatin spatial distribution

Abstract

The invention discloses a whole genome open chromatin in-situ labeling method and application thereof in detection of whole genome open chromatin spatial distribution, and belongs to the technical field of cell molecular markers. The method comprises the following steps: (1) preparing a Tn5-fluorescent probe transposon storage solution; (2) taking cells, inoculating the cells on a carrier for division growth, cleaning with a buffer solution, fixing, cleaning with the buffer solution, treating with a lysis buffer solution, and cleaning with the buffer solution; (3) buckling the carrier with the cell side treated in the step (2) on a sealing film containing a standard reaction system, and incubating in a sealed wet box; and (4) taking out the incubated carrier with the cells, and washing the carrier with a washing buffer solution to obtain the Tn5-fluorescent dye transposon in-situ labeled cells. The labeling method disclosed by the invention can be used for carrying out fluorescence labeling on open chromatin regions of genomes in different types of cells, and has extremely high sensitivity and short time consumption.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell molecular labeling, and particularly relates to in-situ labeling of cell genome regulatory elements. BACKGROUND

[0002] The genome contains tens of thousands of genes and a large number of regulatory sequences, and the unfolded linear length is more than 2 meters, while the diameter of the cell nucleus is only about 10 microns. In order to accommodate the entire genome in a limited space, the genome is highly twisted and folded in the cell nucleus to form a complex three-dimensional structure. At present, the method of high-throughput sequencing enriches people's understanding of the structure of the genome, but the sequencing method can only provide relevant information of the genome sequence, and cannot provide direct spatial position information of the genome.

[0003] At present, the spatial position research of the genome is mainly based on the labeling technology of fluorescence in-situ hybridization and the traditional optical imaging method, and the measurement range and precision are limited. The labeling method of fluorescence in-situ hybridization is to use specific oligonucleotide sequences with fluorescent dyes as probes, which are hybridized with complementary nucleic acid chains in the genome in-situ, and the purpose of observing specific nucleic acid molecules in-situ in cells is achieved by imaging detection. However, due to the multiple steps of in-situ hybridization labeling, signal loss is easy to cause false negative; only one specific gene sequence can be labeled at a time and the open chromatin region of the whole genome cannot be labeled. There is no method on the market that can specifically label the open chromatin region of the whole genome. SUMMARY

[0004] In view of the defects in the prior art, in order to solve the problem that the open whole genome region cannot be imaged in-situ on a nanoscale, the purpose of the present application is to design to provide a whole genome open chromatin region fluorescence labeling technology based on transposase mediation. This technology can be combined with PALM or STORM and other ultra-high resolution imaging technologies to measure the spatial distribution of the open chromatin of the whole genome of a single cell.

[0005] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0006] On the one hand, the present application provides a whole genome open chromatin in-situ labeling method, comprising the following steps:

[0007] (1) preparing a Tn5-fluorescent probe transposon storage solution;

[0008] (2) taking cells, inoculating on a carrier for division growth, washing with a buffer solution, fixing, washing with a buffer solution, treating with a lysis buffer, and washing with a buffer solution;

[0009] (3) The carrier with the cell side after the step (2) is buckled on the sealing film containing the standard reaction system, and is placed in a sealed wet box for incubation; wherein the standard reaction system contains the Tn5-fluorescent probe transposon storage solution prepared in step (1);

[0010] (4) The carrier with the cell after the incubation is completed is taken out, washed with a washing buffer, and then the Tn5-fluorescent dye transposon in-situ labeled cell is obtained.

[0011] The specific process of the step (1) of the whole genome open chromatin in-situ labeling method is as follows:

[0012] (i) The purified fluorescent probe A, fluorescent probe B and auxiliary probe are dissolved in water, and then the dissolved fluorescent probe A and fluorescent probe B are mixed with the auxiliary probe in the annealing buffer respectively, and then annealing reaction is carried out to obtain the fluorescent probe A / auxiliary probe and fluorescent probe B / auxiliary probe;

[0013] (ii) The fluorescent probe A / auxiliary probe, fluorescent probe B / auxiliary probe, glycerol, permeabilization buffer, Tn5 transposase and sterile water are mixed to react at room temperature to obtain the Tn5-AF647 transposon storage solution.

[0014] The annealing buffer comprises 20mM TrisHCl, 100mM NaCl and 2mM EDTA.

[0015] The annealing reaction condition is denaturation at 95℃ for 5 minutes, and then slow cooling to 25℃ at a speed of-1℃ per minute.

[0016] The permeabilization buffer comprises 100mM HEPES-KOH, 0.2M NaCl, 0.2mM EDTA, 2mM DTT, 0.2% Triton X-100 and 20% glycerol.

[0017] The volume ratio of the sum of the fluorescent probe A / auxiliary probe and fluorescent probe B / auxiliary probe, glycerol, permeabilization buffer, Tn5 and sterile water is 0.25:0.4:0.12:0.1:0.13.

[0018] The reaction condition at room temperature is gentle shaking for 1-2 hours.

[0019] The nucleic acid sequence of the fluorescent probe A is a fluorescent group-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG.

[0020] The nucleic acid sequence of the fluorescent probe B is Fluorescent group-AGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG; the nucleic acid sequence of the auxiliary probe is CTGTCTCTTATACACATCT;

[0021] Preferably, the fluorescent group is AF647.

[0022] The carrier is a round coverslip coated with collagen in the whole genome open chromatin in situ labeling method.

[0023] The time of the split growth is 1-2 days.

[0024] The buffer is 1x PBS.

[0025] The fixing method in the whole genome open chromatin in situ labeling method is placing in a paraformaldehyde solution for 10-15 min; wherein the concentration of the paraformaldehyde solution is 4%.

[0026] The component group of the lysis buffer is 5M NaCl, 1M Tris HCl, 1M MgCl2, 10% CA-630 NP40, sterile water, and the volume ratio is 0.1:0.5:0.15:2.5:46.75.

[0027] The time of the lysis buffer treatment is 10-15 min.

[0028] The standard reaction system comprises Tn5-fluorescent dye transposon storage solution and TD buffer, and the volume ratio is 1:1 in the whole genome open chromatin in situ labeling method.

[0029] The incubation condition is 37℃ for 1-2 h.

[0030] The washing method of the washing buffer is washing 3 times at 55℃ for 10-15 min each time.

[0031] The composition of the washing buffer comprises PBS buffer containing SDS with a final concentration of 0.01% and 50mM EDTA.

[0032] In the second aspect, the application provides a cell labeled in situ by Tn5-fluorescent dye transposon, which is obtained by the method.

[0033] In the third aspect, the application provides the application of any of the methods in detecting the spatial distribution change of the open region of the genome.

[0034] In a fourth aspect, the present application provides the use of any of the methods in combination with an ultra-high resolution microscopy imaging platform for obtaining direct spatial location information of open chromatin.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] 1、The marking method of the present application can fluorescently label the open chromatin regions in different types of cells, and high-specific in situ labeling of open chromatin has been achieved in mouse embryonic stem cells (ESC) and fibroblasts (MEF). The labeling efficiency is high (the positioning events can reach about 50-100 thousand per nucleus, and ≥90% of the open chromatin sites can be labeled), the time required for labeling is short (the reaction time in fixed cells is usually within 30 minutes), and the imaging method can detect the spatial distribution changes of the open regions of the genome under various physiological and pathological conditions. For example, under the conditions of treatment with histone deacetylase inhibitors or acute depletion of CTCF protein, 3D ATAC-PALM labeling and imaging can accurately detect the spatial rearrangement of the open regions of chromatin, which is manifested as a significant change in the g(r) clustering function or remodeling of the number and structure of ACD (Accessible Chromatin Domain).

[0037] 2、The marking method of the present application is suitable for PALM or STORM ultra-high resolution microscopy imaging platform, and can perform ultra-high resolution in situ imaging on the spatial distribution of open chromatin regions in the nucleus to obtain direct spatial location information of open chromatin. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Figure 1 is a schematic diagram of the 3D ATAC-PALM labeling strategy;

[0039] Figure 2 Figure 3 is a result map of the open chromatin three-dimensional region in the nucleus of wild-type mouse embryonic stem cells obtained by using the 3D ATAC labeling method in combination with PALM microscopy imaging technology, wherein each point represents a single molecule positioning coordinate of a chromatin open site, and the color coding represents the local spatial density (calculated using a 250 nm radius window), and the scale is 2 microns (the visualization software is VISP). DETAILED DESCRIPTION

[0040] The present application will be further described below by means of the accompanying drawings and examples.

[0041] Example 1:

[0042] The present application inserts an oligonucleotide probe containing a fluorescent group into the open chromatin site of a single cell genome using Tn5 transposase to achieve specific in situ labeling of the open chromatin site of the whole genome of fixed cells (or tissues). For example, Figure 1The fluorescent group is attached to the mosaic ends of the Tn5 transposon via a DNA oligonucleotide and recombines with the Tn5 transposase (in cyan) in vitro to form an active transposome complex. After fixation and permeabilization of the cells, open chromatin sites in the genome are selectively labeled by the Tn5-fluorescent group transposome. Details are as follows:

[0043] 1. Preparation of reagents

[0044] (1) Lysis buffer, the composition of which is shown in Table 1 below.

[0045] Table 1 Composition of lysis buffer

[0046] 5M NaCl (500x) 0.1 mL 1M Tris HC1-pH 7.5 (100x) 0.5 mL 1 M MgCl2(333 x) 0.15 mL 10% CA-630 NP40 (20x) 2.5 mL Sterile water 46.75 mL Total 50 mL

[0047] (2) 2x TD buffer, the composition of which is shown in Table 2 below.

[0048] Table 2 Composition of 2x TD buffer

[0049] 1M Tris-HCl pH 7.6 2 mL 1M MgCl2 1 mL Dimethyl Formamide 20 mL Sterile water 77 mL Total 100 mL

[0050] (3) Wash buffer

[0051] PBS buffer containing 0.01% SDS and 50 mM EDTA at a final concentration.

[0052] (4) 2x Permeabilization buffer, the composition of which is shown in Table 3 below.

[0053] Table 3 Composition of 2x Permeabilization buffer

[0054]

[0055] (5) 2x Annealing buffer, the composition of which is shown in Table 4 below.

[0056] Table 4 Composition of 2x Annealing buffer

[0057] 20mM Tris HC1 pH 8.0 100mM NaCl 2mM EDTA

[0058] 2. Assembly and verification of Tn5 transposome (containing Tn5 transposase and oligonucleotide fluorescent probe)

[0059] The three probes used in the labeling were synthesized by a biological company. The key modifications of the adapters are as follows in Table 5:

[0060] Table 5 Key modification sequences of adapters

[0061] Oligonucleotide fluorescent probe-A Fluorophore-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG Oligonucleotide fluorescent probe-B Fluorophore-AGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG Accessory probe 5’-(phos)CTGTCTCTTATACACATCT-3’

[0062] Specifically: HPLC purified oligonucleotide fluorescent probes A, B and helper probes were dissolved in water to 200 mM respectively. Oligonucleotide fluorescent probe A / helper probe pair and oligonucleotide fluorescent probe B / helper probe pair were mixed in 2x annealing buffer at equal molar ratio, denatured at 95℃ for 5 minutes on PCR instrument, and then slowly cooled to 25℃ at a rate of -1℃ per minute. The annealing reaction can be checked on a 5-10% TBE gel, and the annealing efficiency is >90%.

[0063] 0.25 volume of the oligonucleotide fluorescent probe A / helper probe pair + oligonucleotide fluorescent probe B / helper probe pair (50 mM each) obtained by annealing in the above step, 0.4 volume of glycerol (100% solution), 0.12 volume of 2x permeation buffer, 0.1 volume of commercially purchased Tn5 transposase (Shengwo, N608457, 10 U / ul) and 0.13 volume of sterile water were combined together, and the reaction was gently shaken at room temperature for 1 hour (sensitive dyes should be protected from light) to complete the assembly of Tn5 transposase.

[0064] 3. In situ labeling of fixed samples

[0065] The cells were seeded on coated 25 mm round coverslips, the coverslips were washed once with 1x PBS and fixed in 4% paraformaldehyde solution for 10 minutes. The coverslips were washed twice with 1x PBS and treated with lysis buffer for 10 minutes, and then washed twice with 1x PBS.

[0066] Table 6 Preparation of labeling reaction system

[0067] Reaction mix (per sample) 20 μL 2X TD buffer 10 μL Tn5 transposase stock 10 μL

[0068] After the reaction system was prepared as in Table 6, it was added to the sealing film, and the coverslips with the cell side were carefully inverted on it; the reaction in 1.5 was placed in a sealed humid box and incubated at 37℃ for 1 hour, the coverslips were carefully taken out with the sample side up, and the coverslips were placed in a 6-well plate; the sample was washed with washing buffer at 55℃ for 3 times, 10 minutes each time; the sample was stored in 1x PBS and stored at 4℃ in the dark or used for PALM / STORM imaging.

[0069] Example 2: AF647 transposase labeling of whole genome open chromatin of mouse embryonic stem cells

[0070] The experimental procedure for transposase labeling of whole genome open chromatin of mouse embryonic stem cells with fluorescent dye Alexa Fluor 647 (AF647) is as follows:

[0071] 1. Assembly and verification of Tn5-AF647 transposase

[0072] (1) Tn5 transposase linker was synthesized by Bio Company. The oligonucleotide sequence of the linker and key modifications are as follows:

[0073]

[0074] (2) HPLC purified Tn5ME-A-AF647, Tn5ME-B-AF647, Tn5MErev oligonucleotides were dissolved in water to 200 μM respectively;

[0075] (3) The water-dissolved Tn5ME-A-AF647 or Tn5ME-B-AF647 was mixed with Tn5MErev in 2x annealing buffer at equimolar ratio, denatured at 95°C for 5 minutes on PCR instrument, and then slowly cooled to 25°C at a rate of -1°C per minute; the annealing reaction can be checked on a 5-10% TBE gel, and the annealing efficiency is >90%;

[0076] (4) Tn5 transposase was assembled with AF647 by combining 0.25 volume of Tn5MErev / Tn5ME-A-AF647 + Tn5MErev / Tn5ME-B-AF647 (50 μM each) obtained above, 0.4 volume of glycerol (100% solution), 0.12 volume of 2x permeation buffer, 0.1 volume of Tn5 (10 U / μl) and 0.13 volume of sterile water.

[0077] (5) The reaction was shaken gently at room temperature for 1 hour (sensitive stains should avoid light), and Tn5-AF647 transposon stock solution was obtained.

[0078] 2. In situ labeling

[0079] (1) Mouse embryonic stem cells were seeded on collagen-coated 25 mm round coverslips and allowed to grow and divide for 1-2 days;

[0080] (2) The coverslips with cells were washed once with 1x PBS and fixed in 4% paraformaldehyde solution for 10 minutes;

[0081] (3) The coverslips were washed twice with 1x PBS and treated with lysis buffer for 10 minutes;

[0082] (4) The coverslips were washed twice with 1x PBS;

[0083] (5) The labeling reaction system was prepared, as shown in Table 6, wherein the Tn5 transposon stock solution was the Tn5-AF647 transposon stock solution obtained in the above step.

[0084] After the reaction system was prepared, it was added to the parafilm, and the coverslip with the cell side was carefully inverted on it;

[0085] (6) Put the reaction system in (5) into a sealed wet box and incubate at 37°C for 1 hour;

[0086] (7) Carefully take the coverslip with the sample facing up and place it into a 6-well plate;

[0087] (8) Wash the sample with the washing buffer at 55°C for 3 times, 10 minutes each time;

[0088] (9) Store the sample in 1xPBS at 4°C in the dark, and perform single molecule localization super-resolution imaging combined with the STORM technology.

[0089] Example 3:

[0090] The photoactivation of the fluorescent group by 405 nm laser (hv) can be used for PALM or STORM single molecule localization super-resolution microscopy. The specific process is as follows:

[0091] The sample is treated with the anti-blinking buffer system required for STORM imaging, and is placed under the STORM microscope on a high-power oil lens platform. The dye fluorescent molecules are gradually activated under low-intensity 405 nm wavelength, and the activated fluorescent molecules are excited using a 640 nm wavelength laser. The discrete distributed single molecule signals are collected and positioned, and the activation, excitation, collection and positioning are repeated to accumulate the super-resolution image results. The number of frames collected for each sample is about 20,000 frames, and the total imaging time is controlled within 30-60 minutes.

[0092] The results are shown in Figure 2 By covalently inserting the probe carrying the photoactivatable dye (PA-JF549) into the open region of chromatin using Tn5 transposase, a nanoscale three-dimensional image of the open region of chromatin can be obtained with a transverse positioning accuracy of about 20 nm and an axial positioning accuracy of about 50 nm when using a lattice light sheet microscope for 3D PALM imaging. The experimental results show that this method not only can distinguish the spatial isolation between ACDs, but also can be integrated with imaging techniques such as Oligopaint DNA-FISH and RNA-FISH, to realize the spatial co-localization analysis of chromatin state and gene expression. The inventors further applied ATAC-PALM to observe the formation of YAP protein nuclear phase separation condensate, and verified that the ATAC signal can reveal that the transcription-related active region (such as the super-enhancer region) is enriched in the accessible chromatin region under the super-resolution imaging platform, which verifies that the spatial positioning accuracy of this labeling method far exceeds that of traditional fluorescent labeling techniques, and it also has imaging compatibility and biological sensitivity.

Claims

1. A genome-wide open chromatin in situ labeling method, characterized in that, The method comprises the following steps: (1) preparing a Tn5-fluorescent probe transposon storage solution; (2) taking cells, inoculating them on a carrier for split growth, washing them with a buffer solution, fixing them, washing them with a buffer solution, treating them with a lysis buffer solution, and washing them with a buffer solution again; (3) placing the carrier with the cells treated in step (2) on a sealing film containing a standard reaction system, and placing it in a sealed wet box for incubation; the standard reaction system contains the Tn5-fluorescent probe transposon storage solution prepared in step (1); (4) taking out the carrier with the cells after the incubation is completed, washing it with a washing buffer solution, and obtaining the cells labeled in situ by the Tn5-fluorescent dye transposon.

2. A method of whole genome open chromatin in situ labeling according to claim 1, wherein, The specific process of step (1) is as follows: (i) dissolving the purified fluorescent probe A, the fluorescent probe B and the auxiliary probe in water, mixing the dissolved fluorescent probe A and the auxiliary probe in an annealing buffer solution, mixing the dissolved fluorescent probe B and the auxiliary probe in an annealing buffer solution, and performing an annealing reaction to obtain the fluorescent probe A / auxiliary probe and the fluorescent probe B / auxiliary probe; (ii) mixing the fluorescent probe A / auxiliary probe, the fluorescent probe B / auxiliary probe, glycerol, a permeabilization buffer solution, Tn5 transposase and sterile water, and reacting at room temperature to obtain a Tn5-AF647 transposon storage solution.

3. A method of whole genome open chromatin in situ labeling according to claim 2, wherein, The annealing buffer solution comprises 20 mM Tris HCl, 100 mM NaCl and 2 mM EDTA; The annealing reaction is performed on a PCR instrument at 95°C for 5 minutes, and then slowly cooled to 25°C at a rate of -1°C per minute.

4. A method of whole genome open chromatin in situ labeling according to claim 2, wherein, The permeabilization buffer solution comprises 100 mM HEPES-KOH, 0.2 M NaCl, 0.2 mM EDTA, 2 mM DTT, 0.2% Triton X-100 and 20% glycerol; The volume ratio of the sum of the fluorescent probe A / auxiliary probe and the fluorescent probe B / auxiliary probe, glycerol, the permeabilization buffer solution, Tn5 and sterile water is 0.25:0.4:0.12:0.1:0.13; The reaction at room temperature is performed by gently shaking for 1-2 hours.

5. A method of whole genome open chromatin in situ labeling according to claim 2, wherein, The nucleic acid sequence of the fluorescent probe A is a fluorescent group-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG; The nucleic acid sequence of the fluorescent probe B is a fluorescent group-AGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG; and the nucleic acid sequence of the auxiliary probe is CTGTCTCTTATACACATCT. Preferably, the fluorescent group is AF647.

6. A method of whole genome open chromatin in situ labeling according to claim 1, wherein, The carrier is a round cover glass coated with collagen; The split growth time is 1-2 days; The buffer solution is 1×PBS. The fixing method is placing in a paraformaldehyde solution for 10-15 minutes; the concentration of the paraformaldehyde solution is 4%; The components of the lysis buffer solution are 5 M NaCl, 1 M Tris HCl, 1 M MgCl2, 10% CA-630 NP40 and sterile water, and the volume ratio is 0.1:0.5:0.15:2.5:46.

75. The time for treating the lysis buffer is 10-15 min.

7. A method of whole genome open chromatin in situ labeling according to claim 1, wherein, The standard reaction system comprises Tn5-fluorescent dye transposon storage solution and TD buffer, and the volume ratio is 1:

1. The incubation condition is: temperature 37℃, time 1-2 h. The washing mode of the washing buffer is washing 3 times at 55℃, each time for 10-15 min. The composition of the washing buffer comprises PBS buffer containing 0.01% SDS and 50 mM EDTA at a final concentration.

8. A cell labeled in situ with a Tn5-fluorescent dye transposon, characterized by, Obtained by the method according to any one of claims 1-7.

9. The method according to any one of claims 1-7 for use in detecting the spatial distribution of open chromatin genome-wide.

10. The method according to any one of claims 1-7 in combination with an ultra-high resolution microscopy platform for obtaining direct spatial location information of open chromatin.

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