CN118048436A

CN118048436A - Targeting chromatin interaction capturing ULI-eHiChIP library construction method for micro cells and application

Info

Publication number: CN118048436A
Application number: CN202410455129.5A
Authority: CN
Inventors: 孔思远; 田蕴涵; 常荣荣; 孔大帅; 张玉波
Original assignee: Agricultural Genomics Institute at Shenzhen of CAAS
Current assignee: Agricultural Genomics Institute at Shenzhen of CAAS
Priority date: 2024-04-16
Filing date: 2024-04-16
Publication date: 2024-05-17

Abstract

The invention discloses a targeted chromatin interaction capturing ULI-eHiChIP library building method for micro cells, by which targeted chromatin interaction capturing ULI-eHiChIP library building of micro cell initial quantity can be realized, the cell initial quantity can be 100 cells at the minimum, and the requirement of the existing HiChIP technology on the cell sample initial quantity is far lower (at least 10-1500 ten thousand cells are needed in the prior art). When the method is used for preparing the library, the cell requirement is small, the method has application potential for rare cells and trace medical test cells, the difficulty and time for collecting cell materials are saved, the cell culture cost is reduced, and meanwhile, the trace test reagent operation system reduces the cell loss and saves the reagent cost for technology.

Description

Targeting chromatin interaction capturing ULI-eHiChIP library construction method for micro cells and application

Technical Field

The invention relates to the technical field of genome sequencing, in particular to a targeted chromatin interaction capturing ULI-eHiChIP library building method for micro cells and application thereof.

Background

Gene expression is regulated by a number of factors, of which histone modifications and transcription factors play an important role in gene expression regulation, mESC 0.1 million (10 ten thousand) (Chen Guifang et al, 2021, biotechnology report). The analysis of the mechanism of histone modification and transcription factor on gene expression regulation is helpful to the clear understanding of organisms in terms of disease production principle, growth and development epigenetic character and the like. Chromatin co-immunoprecipitation sequencing (ChIP-seq) is a technique in which a target protein-bound DNA fragment is specifically enriched using a chromatin co-immunoprecipitation technique (Chromatin Immunoprecipitation, chIP) and then sequenced. ChIP-seq can map one-dimensional landscape of specific protein factors binding to the genome. However, it does not provide an understanding of chromatin 3D features (e.g., chromatin interactions), and current related methods are rare. Since the advent of chromosome conformation capture technology, methods for detecting long-range interactions of chromatin across the genome have evolved greatly, mainly the genome-wide chromosome conformation capture sequencing technology (Hi-C) and its derivative techniques and methods. Hi-C technology attempts to capture all proximal and distal interactions across the genome. But there are still limitations in that resolution is low and specific interaction gripping is not comprehensive. To meet resolution requirements, high depth sequencing is required to more completely recognize structural features of chromatin (Mumbach MR et al, 2016, nature Methods). The advent of ChIA-PET, PLAC-seq and HiChIP technologies enriched for chromatin interactions mediated by specific protein factors (Zelenka T et al, 2021, methods and Protocols). Higher resolution can be achieved to observe specific protein factor mediated chromatin interactions with low depth sequencing, hiChIP is a method of resolving chromatin conformation in combination with Hi-C and co-immunoprecipitation ChIP. Can be used for conformational analysis of specific protein-mediated chromatin interaction, research on the action mechanism of transcription factors and research on the mechanism of gene regulation by apparent modification. However, the technology has the defects of limited application range, large required cell quantity, incomplete chromatin digestion, low purification efficiency of chromatin antibody precipitation DNA and the like.

Currently HiChIP requires a high cell count (typically 10 tens of thousands of+ cells, and when the cell count is less than 10 tens of thousands, no corresponding library can be prepared), and therefore, based on this technical limitation, development of an efficient HiChIP sequencing technique based on the initial level of the cell count is highly desirable.

Disclosure of Invention

The invention aims to provide a novel targeted chromatin interaction capturing ULI-eHiChIP library building method and application for micro cells so as to solve the problems.

According to a first aspect of the present invention there is provided a method of constructing a pool of targeted chromatin interaction capture ULI-eHiChIP for a small number of cells, wherein the small number of cells is less than 100000, the method comprising the steps of:

S1: collecting target cells;

s2: formaldehyde was then added to the petri dishes containing the collected cells for formaldehyde crosslinking;

S3: lysing the cells after formaldehyde crosslinking to fix chromatin, and performing permeabilization lysis of the cells by using a permeabilization buffer containing 1% PI and 0.2% Igepal CA 630;

S4: double enzyme digestion is carried out on permeabilized and lysed cells by adopting a restriction double endonuclease combination, wherein the restriction double endonuclease combination is any one of three combinations of DpnII and HpyCH4IV, mboI and HpyCh4IV, sau3AI and HpyCh4 IV;

s5: and (3) performing RE-QC quality control on the product obtained after the enzyme digestion in the step S4: the RE-QC quality control method comprises the steps of carrying out electrophoresis detection on the product after enzyme digestion, wherein an electrophoresis band is in a dispersion shape at 100-12000 bp, and a 12kb band is absent, so that the quality control is qualified;

S6: after the quality control of enzyme digestion is qualified, carrying out terminal biotin labeling on the digested DNA fragment, and then carrying out ortho-position connection and ultrasonic disruption to obtain a protein factor-DNA interaction small body in a free single-molecule state;

s7: the Protein factor-DNA interaction small body in the step S6 is subjected to antibody immunoprecipitation, protein A magnetic bead capturing and rinsing to obtain antibody precipitation magnetic beads capturing chromatin interaction small body;

S8: dissolving and purifying the magnetic beads for capturing the chromatin antibody precipitation DNA in the step S7 by a column to obtain purified chromatin antibody precipitation DNA;

S9: capturing the biotin-labeled interactive DNA of the DNA purified in the step S8 by using an immunomagnetic bead, and preparing a ULI-eHiChIP library by using a Tn5 transposase one-step library construction method or a FEA Enzym enzyme slice segmentation method: breaking the interactive DNA by using Tn5 transposase or FEA Enzym enzyme, performing PCR amplification after breaking, performing E-gel electrophoresis on the PCR product, and finally performing gel cutting recovery to prepare a ULI-eHiChIP library, wherein the fragments recovered by the gel cutting are 150-1000 bp.

Therefore, through the method, the ULI-eHiChIP library establishment of a microscale cell initiation amount chromatin interaction capture technology can be realized, the cell initiation amount can be 100 cells at the minimum, and the requirement of the existing HiChIP technology on the cell sample initiation amount is far lower than that of the existing HiChIP technology (more than 10 ten thousand cells are needed). When the method is used for preparing the library, the cell requirement is small, the method has application potential for rare cells and trace medical test cells, the difficulty and time for collecting cell materials are saved, the cell culture cost is reduced, and meanwhile, the trace test reagent operation system reduces the cell loss and saves the reagent cost for technology.

In certain embodiments, the minicells refer to a cell number of 100-100000. Therefore, the method can realize the construction of the initial amount of the micro cells of the ULI-eHiChIP library, and can obtain the ULI-eHiChIP library with good quality by designing 100, 1000, 10000 and 100000 cells for verification.

In certain embodiments, in addition to single crosslinking with formaldehyde in step S2, double crosslinking can be performed by adding formaldehyde and EGS to the petri dish containing the collected cells. Therefore, the crosslinking efficiency and the crosslinking fixing quality can be greatly improved.

In certain embodiments, the method further comprises subjecting the prepared ULI-eHiChIP library to E-gel electrophoresis and gel cutting recovery again to prepare a pure ULI-eHiChIP library in step S9. Therefore, the initial library containing different 'bar code' joints is mixed into a large library, and then the large library is cut into glue and purified again according to the consistent size range, so that a pure library without joint pollution can be obtained, the joint pollution of the library can be greatly reduced, and the data output is improved.

In certain embodiments, in step S6 of the above method, the end biotin labeling of the digested DNA fragment is performed using a base reagent Fill-in Master Mix mixed with biotin label, wherein the amount of the Fill-in Master Mix used is from 100 to 100000 cells and 4.7. Mu.L. In the prior art, the usage amount of the Fill-in Master Mix is 50 mu L, so that the use of biotin can be greatly reduced, the cost is saved, and the subsequent difficulties of elution, recovery and purification are reduced.

In certain embodiments, in step S7 of the above method, the amount of protein A magnetic beads used is 10. Mu.L, whereas in the prior art 60. Mu.L of protein A magnetic beads are required for every 10 Million cells. Therefore, the consumption of the protein A magnetic beads is reduced, on one hand, the cost of detection reagents can be reduced, and on the other hand, the library construction step can be optimized, and the library construction efficiency is improved.

In certain embodiments, in step S6 of the above method, the ligation reaction used for the ortho-ligation comprises NEB T4 DNA LIGASE buffer, triton X-100, recombinant albumin Recombinant Albumin, T4 DNA LIGASE and water. Thus, the use of recombinant albumin Recombinant Albumin instead of BSA in the conventional art can improve ligation efficiency.

In certain embodiments, step S6 of the above method further comprises subjecting the biotin-labeled and ortholinked protein factor-DNA interaction partner to background elimination with Lambda Exonuclease, recJf exonuclease, followed by ultrasound disruption and subsequent manipulation. Therefore, through the actions of the exonucleases Lambda Exonuclease and RecJF, the noise of the library can be effectively eliminated, the signal to noise ratio is improved, and finally the library quality is improved.

According to a second aspect of the present invention there is provided the use of the above method for the construction of a library of cell ULI-eHiChIP in a minimal cell starting amount, wherein the minimal cell starting amount is 100-100000.

According to a third aspect of the present invention there is provided a library of minicell ULI-eHiChIP prepared by the method described above.

According to a fourth aspect of the present invention there is provided the use of a ULI-eHiChIP library in high throughput sequencing of minicells.

According to a fifth aspect of the present invention there is provided the use of a ULI-eHiChIP library in three-dimensional genomics or functional genomics research against minicells.

The invention has the beneficial effects that:

1. The ULI-eHiChIP library construction method for the chromatin interaction capture technology of the micro cells can realize the ULI-eHiChIP library construction of the chromatin interaction capture technology of the micro cells, the initial cell quantity can be 100 cells at the minimum, and the requirement of the existing HiChIP technology on the initial cell sample quantity is far lower than that of the existing HiChIP technology (more than 10 ten thousand cells are needed). The reason why a low cell initiation amount and high quality library construction can be achieved is as follows: 1) By carrying out quality control RE-QC on the enzyme digestion product, the enzyme digestion quality can be monitored in time, the enzyme digestion strip is ensured to be dispersive at 100-12000 bp, and 12kb has no strip, so that incomplete enzyme digestion is prevented, and the problem that large fragments cannot be recovered after enzyme digestion in the prior art is overcome; 2) By adopting E-gel electrophoresis, the sample loading quantity can be reduced, and the resolution ratio and the gel cutting recovery rate are high. 3) The reaction system is reduced, the reagent dosage is reduced, and the method is suitable for the sufficient reaction of micro cells in a microenvironment. Therefore, the method has the advantages of small cell requirement when being used for preparing the library, application potential for rare cells and trace medical test cells, difficulty and time for collecting cell materials, reduction of cell culture cost, and trace test reagent operation system, reduction of cell loss and saving of reagent cost for technology.

2. And by carrying out Lambda Exonuclease and RecJF exonuclease treatment on the cell nucleus groups subjected to ortho-position connection, the noise of the library can be effectively eliminated, the signal to noise ratio is improved, and the quality of the library is finally improved.

3. And combining the obtained initial libraries, then carrying out electrophoresis and gel cutting recovery again, and performing gel cutting purification according to the range of consistent size, so that a pure library without joint pollution can be obtained, joint pollution can be greatly reduced, and data output is improved.

4. The application also carries out verification of different cell types and different cell numbers, and the ULI-eHiChIP library construction method is proved to be efficient and accurate.

5. The application also provides a Tn5 transposase one-step library construction method and a FEA Enzym enzyme digestion fragmentation method.

6. The application also discloses a micro-cell ULI-eHiChIP library constructed by the method and application of the library in high-throughput sequencing of micro-cells and three-dimensional genomics or functional genomics research aiming at the micro-cells.

Drawings

FIG. 1 is a flow chart showing the main steps of construction of a library of the initial cell amount ULI-eHiChIP of the micro cells of examples 1-3;

FIG. 2 is a graph of RE-QC results in examples 1 and 3: wherein the left graph is the RE-QC result graph of example 1, and the right graph is the RE-QC graph of example 3;

FIG. 3 shows the result of PCR QC pre-test in example 1: lanes are M, E-GEL DNA LADDER in order from left to right; three replicates of 0.1 million (10 ten thousand) cell mass; three replicates of 1 ten thousand cell mass; three replicates of 1000 cell amounts; three replicates of 100 cell mass with cycle numbers 18, 20, 22, 24, respectively;

FIG. 4 is a PCR-QC negative control system without template in example 1: lanes are M, E-GEL DNA LADDER in order from left to right; repeating the 12-cycle number of two template-free steps; repeating the two template-free steps of 22 cycle numbers;

FIG. 5 shows the results of the actual PCR test in example 1: the left image is before the glue is cut, and the right image is after the glue is cut; lanes are M, E-GEL DNA LADDER in order from left to right; three replicates of 10 thousand cell mass; three replicates of 1 ten thousand cell mass; three replicates of 1000 cell amounts; three replicates of 100 cell mass with cycle numbers of 13, 15, 18, 20, respectively;

FIG. 6 is a chart of a LHCP0-NGS3K library quality assurance in example 1;

FIG. 7 is a map of quality control of LHCPC-NGS 3K library of example 2;

FIG. 8 is a map of quality control of LHCPT-NGS 3K library of example 3;

FIG. 9 is an evaluation chart of LHCP 0-sequencing results in example 1;

FIG. 10 is a diagram showing the evaluation of LHCPC (raw) -sequencing results in example 2;

FIG. 11 is a diagram showing evaluation of LHCPT (raw) -sequencing results in example 3;

FIG. 12 is a map of quality control of LHCPC-NGS 3K library of example 4;

FIG. 13 is a diagram showing evaluation of LHCPC (raw) -sequencing results in example 4;

FIG. 14 shows the result of the PCR QC pre-test in example 5;

FIG. 15 is a map of quality control of LHCPC-NGS 3K library of example 4;

FIG. 16 is a diagram showing the evaluation of LHCPC (raw) -sequencing results in example 4;

FIG. 17 is a flow chart showing the main steps in the construction of a library of the initial cell amount ULI-eHiChIP in example 4 or 5;

FIG. 18A library quality control plot for the detection of the presence of "linker contamination" in example 2.

Detailed Description

The invention is described in further detail below with reference to the accompanying drawings.

The method for capturing chromatin interaction mediated by specific protein factors in the initial cell quantity of a micro-cell, namely Ultra-low input eHiChIP, is hereinafter referred to as 'ULI-eHiChIP', and the main step flow is shown in figure 1 or figure 17.

Example 1

The main steps in example 1 are shown in FIG. 1.

1. Cell collection and cross-linking immobilization

1. Mouse embryonic stem cells mESC were cultured in 60 mm dishes (containing 4 mL medium) and formaldehyde cross-linked when the cell confluency in the dishes reached 70-90%. At the same time, a cell count experiment was performed.

2. 37% Formaldehyde (HCHO) was added to the petri dish to a final concentration of 1%, which was incubated in an incubator at 37℃for 10 min.

3. 356. Mu.L of 2.5M glycine aqueous solution (final concentration 0.2M) was added to the medium, crosslinking was stopped, and 5min was shaken at room temperature on a rotator.

4. The culture medium was removed and the cells were washed twice with 4 mL pre-chilled 1 XPBS.

5. PBS was removed, 0.5 mL PBS was left in the plate, the cells were scraped off with a cell spatula, the suspended cells were aspirated into a new 2 mL centrifuge tube, placed on ice, the cell culture dish was rinsed with 1 mL mass% 0.5% BSA/PBS, and the remaining cells were collected into the centrifuge tube.

6. Centrifuging at 1000g and 4℃for 5min. The supernatant was discarded and the crosslinked cells were either cold stored at-80℃or directly subjected to the next step.

2. Cell lysis

1. The crosslinked cells were thawed and placed on ice.

2. Cell amounts were divided into 100, 1000, 10000, 0.1 million (10 ten thousand) (three replicates each) and placed in 1.5 mL centrifuge tubes, respectively. Adding pre-cooled 150 μl of HICHIP LYSIS Buffer cell lysate (1% PI by mass, 0.2% Igepal CA630, 10mM NaCl,10 mM pH8.0 Tris-HCl by volume), placing on ice, placing on a shaking table, and shaking 30 min;2500 Centrifugation at 5min at g 4℃and the supernatant discarded. The permeabilization buffer solution containing 1% PI and 0.2% Igepal CA630 is used for permeabilization and lysis of cells, so that the permeabilization and lysis of cell membranes and nuclear membranes can be efficiently carried out, the lysis efficiency and the subsequent enzyme digestion efficiency can be improved, and the sample loss can be reduced.

3. The cell pellet was rinsed with 150. Mu.L of pre-chilled HICHIP LYSIS Buffer, centrifuged at 2500 g at 4℃for 5: 5min, and the supernatant discarded.

4. 18.75 Mu L of SDS with the volume percentage of 0.5% is added into a centrifuge tube containing cell clusters, the mixture is placed on a metal bath and incubated for 10 min at the temperature of 62 ℃, 54.38 mu L of ddH ₂ O and 9.38 mu L of Triton X-100 with the volume percentage of 10% are added, and after uniform mixing, the mixture is incubated for 15 min in a water bath pot at the temperature of 37 ℃ to obtain a treated cell solution.

3. Enzymatic cleavage of chromatin

To the above cell solution, 15.38. Mu.L of 10 XNEBuffer DpnII, 1. Mu.L of DpnII (10U/ul NEB, R0543S) and 1. Mu.L of HpyCH4IV (10U/ul, NEB, R0619L) were added, and after being mixed by blowing, they were placed in a 37℃water bath to be digested with shaking overnight to obtain digested DNA.

4. Enzyme digestion quality control RE-QC

1. Taking 1/7.5 volume of about 12 [ mu ] L of the sample solution of 0.1 million (10 ten thousand) subjected to Restriction Enzyme (RE) treatment, adding the sample solution into a new 1.5 ml EP centrifuge tube, and then using the centrifuge tube for Quality Control (Quality Control); the remaining samples were incubated in a metal bath 20min at 62℃and left at 4℃after enzyme inactivation.

2. And adding a proper volume (10 ul) of 1 XNEBuffer enzyme digestion solution into the RE-QC sample to make the volume of the system be 22 [ mu ] l. Then adding 3.7 mu L of 10% SDS to make the final concentration be 1.5%, namely adding 4 mu L of protease K (20 mg/mL), uniformly mixing, and incubating for 1h and 20 min at 65 ℃ (metal bath).

3. VAHTS DNA CLEAN Beads (N411-02) were mixed well in advance and placed at room temperature 30: 30 min. And adding about 54 mu L DNA CLEAN Beads in 1.8 times of the volume of each QC sample, blowing and mixing uniformly, and standing at room temperature for 5min.

4. Placing on a magnetic rack 5min to clarify the mixture, and removing the supernatant. Wash twice with 500 μl freshly prepared 80% ethanol: after 80% ethanol was added, the EP tube was placed on a magnetic rack, incubated at room temperature for 30 seconds, then the supernatant was removed, washed twice, and the magnetic beads were left to stand on the magnetic rack at 3 min for air-drying.

5. Add 16 μl ddH ₂ O, mix well by blowing, incubate for 5min at room temperature. The rack was left to stand for 3 min to clarify and 15 μl of supernatant was transferred to a new 1.5ml centrifuge tube.

6. The Qubit DSDNA HIGH SENSITIVITY ASSAY measured DNA concentration as shown in Table 1. 200ng of DNA was taken and subjected to a 0.8% agarose gel running test. As shown in FIG. 2 (left), there is no genomic band at 12kb, and the Smear band range is desirably 200-4000 bp.

TABLE 1 quality control of cleavage in example 1 RE-QC

MESC 0.1 million (10 ten thousand)	①	②	③
				Concentration ng/ul	11.80	8.66	13.1

By carrying out quality control RE-QC on the digestion products, the digestion quality can be timely monitored, incomplete digestion is prevented, if large-fragment chromatin is not digested, large-fragment DNA cannot be recovered by gel, on one hand, the data output of a library can be influenced to reduce the library quality, and on the other hand, the corresponding sample amount also needs to be increased to fill the large-fragment loss. Therefore, by carrying out quality control RE-QC on the enzyme-digested product, the band is ensured to be 100-12000 bp, and no band is left at 12kb, so that the gel-digested recovery amount of a subsequent library can be greatly improved, and the data output of the library is improved; library construction of the initial amount of the micro cells can also be realized (if quality control is not performed, large fragment loss is large, and the library construction data cannot be met when the sample cells are micro).

5. DNA end markers and ortholigation

1. And (3) carrying out enzyme digestion and quality control RE-QC to indicate that subsequent operation is continued after enzyme digestion. 4.7 μL Fill-in Master Mix (37.5 μL 0.4 mM biotin-dCTP,1.5 μL 10 mM dATP,1.5 μL 10 mM dGTP,1.5 μL 10 mM dTTP,8 μL 5U/μL Klenow (NEB, M0210)) and 14.1. Mu.L of 1 XNEBuffer enzyme cutting solution are added into the cell solution remained after enzyme cutting, and after being mixed evenly, the mixture is placed in a water bath kettle at 37 ℃ for shaking incubation for 1.5 h. In the prior art, a Fill-in Master Mix containing biotin is generally used in an amount of 50. Mu.L.

2. Ortho-ligation: to the biotin-labeled solution was added 337.5 μL Ligation Master Mix (120 μL 10×NEB T4 DNA ligase buffer with 10 mM ATP (NEB, B0202),100 μL vol% Triton X-100,6. Mu.L 20 mg/mL Recombinant Albumin (recombinant albumin), 5. Mu.L 400U/. Mu. L T4 DNA LIGASE (NEB, M0202), 669. Mu.L ddH ₂ O, and after mixing, shaking at 300rpm for 4 hours at room temperature. The use of recombinant albumin Recombinant Albumin in place of BSA in the conventional art can improve ligation efficiency.

3. 2500 G of the mixture was centrifuged at 4℃for 5min, and the supernatant was discarded to obtain a nuclear pellet after labeling and interactive ligation.

6. Ultrasonic breaking

To the nuclear pellet was added 880 μl Nuclear Lysis Buffer (50 mM pH 7.5 Tris-HCl,10 mM EDTA, 1% SDS by volume, 1×pi) and mixed by blow-stirring. The mixture was transferred to Covaris millitube (covaries E220) tubes and the DNA on the small bodies was interrupted by programming (Duty Cycle 5%, intensity 140,Cycles per Burst 200,Time 240s) to give the protein factor-DNA small bodies in the free single-molecule state.

7. Immunoprecipitation, IP magnetic bead capture

1. The solution containing the protein factor-DNA interaction corpuscles after cleavage was transferred to a new 1.5 mL EP tube, 16100g, and centrifuged at 4℃for 15min.

2. The supernatant was transferred, the sample in each centrifuge tube was divided into two (about 420. Mu.L of sample in each centrifuge tube), 750. Mu. L ChIP Dilution Buffer (0.01% SDS by volume, 1.1% Triton X-100,1.2 mM EDTA,16.7 mM pH 7.5 Tris-HCl by volume, 167 mM NaCl) was added, and mixed upside down.

3. An empty centrifuge tube was prepared, 240. Mu. L Protein A beads was added, the mixture was placed on a magnetic rack 3 min, the supernatant was discarded, 1.2. 1.2 mL ChIP Dilution Buffer was reconstituted, 50. Mu.L of each of the centrifuge tubes containing the samples was added with each of the magnetic bead solutions, and the mixture was incubated in a 4℃cold room with rotation (apparatus name: QB-210 Multipurpose shaker) for 1 hour.

4. After incubation, the supernatant was transferred to a new centrifuge tube in a magnetic rack 5min, and 0.5 μ L RNApolII antibody (Abcam, ab 26721) was added and incubated overnight with rotation in a cold chamber at 4 ℃.

5. An empty centrifuge tube was prepared, 240. Mu. L Protein A beads was added, the supernatant was discarded, reconstituted with 1.2. mL ChIP Dilution Buffer, 50. Mu.L of each of the magnetic bead solutions was added to each centrifuge tube containing the sample, and incubated 2. h in a4℃cold chamber with rotation.

6. The sample was placed on a magnetic rack 5 min and the supernatant was discarded.

7. 75 Mu L Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100,2 mM EDTA,20 mM pH 7.5 Tris-HCl,150 and mM NaCl) is added into each sample tube, the two corresponding centrifuge tubes are recombined into a tube, the tube is placed on a magnetic frame 3 min after being uniformly mixed, the supernatant is discarded, then 150 mu L HIGH SALT WASH Buffer (0.1% SDS, 1% Triton X-100,2 mM EDTA,20 mM pH 7.5 Tris-HCl,500 mM NaCl)、LiCl Wash Buffer (10 mM pH 7.5 Tris-HCl,250 mM LiCl,% Igepal CA630, 1% sodium deoxycholate, 1 and mM EDTA) is sequentially used for rinsing the magnetic beads for capturing DNA, the tube is placed on the magnetic frame for 3 min, and the supernatant is discarded.

8. Chromatin antibody precipitation DNA elution and reverse cross-linking

1. Protein A beads of the captured DNA were reconstituted with 100. Mu. L DNA Elution Buffer (50: 50mM NaHCO ₃, 1% SDS by volume).

2. Placing the mixture on a shaking table, shaking the mixture at room temperature for 10min hours, and then placing the mixture in a water bath kettle at 37 ℃ for shaking for 3: 3 min hours.

3. The sample was placed on a magnetic rack 3 min, the supernatant was transferred to a new centrifuge tube, 100 μ L DNA Elution Buffer was added to the centrifuge tube containing the magnetic beads, step 2 was repeated, and the two supernatants were combined in one centrifuge tube.

4.4 Mu L of protease Proteinase K is added to each sample tube, the mixture is incubated for 45min on a metal bath at 55 ℃ under shaking, the temperature is raised to 67 ℃, and the incubation under shaking is continued for 1.5 h.

5. Chromatin antibody precipitation DNA purification.

(1) 5 Volumes (about 1 mL) of ChIP DNA Binding Buffer were added to each sample tube, mixed and transferred to the column.

(2) 10000G of the solution is centrifuged at normal temperature for 30s and the solution is discarded.

(3) 200 Mu L DNA Wash Buffer was added to the column, centrifuged at 10000g for 30s at room temperature, the solution was discarded and repeated once.

(4) 10. Mu.L of ddH ₂ O was added to the column, and 10000g was centrifuged at room temperature for 30s, and the purified chromatin precipitate DNA solution after passing through the column was collected.

9. Isolation of biotin-labeled interaction DNA and preparation of Illumina sequencing

1. STREPTAVIDIN C-1 beads was left at room temperature for half an hour in advance, 60. Mu.L of magnetic beads (pre-mixed 12 sample volumes) and 600. Mu. L TWEEN WASH Buffer (5 mM pH 7.5 Tris-HCl,0.5 mM EDTA,1M NaCl, 0.05% Tween-20 by mass) were added to a new 1.5 mL centrifuge tube, and after being blown and mixed uniformly, the supernatant was removed by a magnet rack 5 min.

2. 120. Mu.L of 2X Biotin Binding Buffer (10 mM pH 7.5 Tris-HCl, 1mM EDTA,2M NaCl) was added to resuspend the beads, 10. Mu.L of the bead solution was added to the DNA solution, the DNA and bead mixed sample was placed on a metal bath and shaken 15 min at 350r room temperature.

3. Placed on a magnetic rack 5 min and the supernatant discarded.

4. Then 150 mu L TWEEN WASH Buffer is added, placed on a metal bath, 350r and 55 ℃ are oscillated for 2min, a magnetic rack 5 min is put on, the supernatant is discarded, and the process is repeated once.

5. Then 100. Mu.L of 1 XD Buffer (10 mM pH 7.5 Tris-HCl, 5mM MgCl ₂, 10% by mass DMF dimethylformamide) was added, and after mixing, the mixture was placed on a magnetic rack 5min, and the supernatant was discarded.

6. The beads were reconstituted with 25. Mu.L of 2 XD Buffer (20 mM pH 7.5 Tris-HCl,10 mM MgCl ₂, 20% by mass DMF dimethylformamide) to give 25. Mu.L of a library solution of beads.

7. Disruption of the interacting DNA with Tn5 transposase and pooling

1) The reaction system was configured as in tables 2 and 3 below.

Table 2: sample reaction system with 10 ten thousand and 1 ten thousand cell quantities

Table 3: sample reaction system with 100 and 1000 cell quantity

2) After mixing, the mixture is put on a metal bath at 55 ℃ for incubation for 10 min, and the mixture is preserved at 10 ℃ for standby.

3) Then placing 3 min on the magnetic release frame, discarding the supernatant, adding 100 [ mu ] L of 1 xTE Buffer for washing, uniformly mixing, placing 3 min on the magnetic release frame, discarding the supernatant, and repeating for one time; and then washing twice with 150 mu L of 1 XB/W Buffer (5 mM pH 7.5 Tris-HCl,0.5 mM EDTA,1M NaCl), discarding the supernatant, airing 3 min, and redissolving with 40 mu L of ddH ₂ O.

10. PCR library amplification and on-machine sequencing

1. The PCR QC mixtures were prepared as in tables 4 and 5 below, mixed well and centrifuged briefly.

Table 4:10 ten thousand cell sample mixture system

Component Component	Volume Vol (mu L)
		dd H₂O	6
DNA sample	1
		5×TAB	3
TAE	0.5
		10×PPM	1.5
N5XX	1.5
		N7XX	1.5
Total	15

Table 5: sample mixture systems with cell amounts of 1 ten thousand, 100 and 1000

Component Component	Volume Vol (mu L)
		dd H₂O	7.5
DNA sample	1
		5×TAB	3
TAE	0.5
		10×PPM	1.5
N5XX	1.5
		N7XX	1.5
Total	15

2. PCR QC was set up as follows. Pre-denaturation: 98 ℃ 30s; 18-24 cycles (denaturation: 98 ℃ C. 15 s; annealing: 60 ℃ C. 30s; extension: 72 ℃ C. 3 min); final extension: 5min at 72 ℃; and (3) preserving: 4 ℃ infinity min. Sample PCR QC cycle numbers of 10 ten thousand, 1 ten thousand, 100 and 1000 cells are 18, 20, 22 and 24 respectively, as shown in Table 6; 4 repeated tests (namely according to a 10-ten thousand cell sample table) with negative control are taken, no template is added, and the cycle numbers are respectively 12, 22 and 22, so that PCR QC products are obtained.

TABLE 6 detailed information Table of PCR QC in example 1

3. Taking 14 mu L of PCR QC product, running E-gel, and observing Smear band to control quality, as shown in figure 3, it can be seen that PCR QC cycle can meet the range of 150-500 bp; no template negative control group was added, as seen in fig. 4, no template negative control was seen without library bands.

4. And (5) performing formal PCR. The final PCR mixture was prepared as shown in tables 7 and 8 below, mixed well and centrifuged briefly. The PCR procedure was the same as that of PCR QC, and the cycle numbers of samples of 10 ten thousand, 1 ten thousand, 100 and 1000 cells were 13, 15, 18 and 20, respectively, as shown in Table 9. The number of cycles set can meet the requirements of library recovery. The product after the final PCR was obtained as shown in FIG. 5 (left).

Table 7:10 ten thousand cell sample formal PCR mixture system

Component Component	Volume Vol (mu L)
		DNA sample	18
5×TAB	8
		TAE	1
10×PPM	4
		N5XX	4
N7XX	4
		Total	39

Table 8: sample formal PCR mixture system with cell quantities of 1 ten thousand, 100 and 1000

Component Component	Volume Vol (mu l)
		DNA sample	18
5×TAB8	8
		TAE	1
N5XX	4
		N7XX	4
Water	4
		Total	39

TABLE 9 formal PCR detailed information Table in example 1

5. Transfer the PCR product to a new 1.5 mL centrifuge tube at 1:1, adding DNA Clean Beads (Vazyme, N411), mixing, standing at normal temperature for 5 min, adding a magnetic frame 5, min, discarding supernatant, washing with a freshly prepared 80% ethanol solution twice, air-drying for 3 min, re-dissolving with 20 μl ddH ₂ O, adding a magnetic frame 5, min, and taking 19 μl supernatant, and running E-gel.

6. The gel cutting recovery is carried out, the recovery fragments are that samples with the cell quantity of 10 ten thousand, 1 ten thousand and 1000 are cut into 150-1000 bp, and samples with the cell quantity of 100 are cut into 150-500 bp, as shown in the figure 5 (right). The recovery concentrations are shown in Table 10, and the final library was prepared successfully.

TABLE 10 recovery concentration of gel after formal PCR in example 1

7. And uniformly mixing all samples according to a mass ratio of 1:1 to obtain LHCP0. Real time-qPCR and NGS3K library qualification (FIG. 6) were followed by Illumina Nova-seq PE150 double-ended sequencing, and off-machine data were processed and evaluated by HiC-Pro software (FIG. 9). Effective interactions can be observed at the individual cell number level. The more cells, the higher the library complexity, and the more effective interactions that result. The comparison rate accords with the library rule of chimeric DNA by 40% -70%. The cis-interaction ratio is higher (60% -85%), and the library characteristics similar to Hi-C show that the construction of the ULI-eHiChIP library of the micro mouse embryonic stem cells mESCs (100, 1000, 10000, 100000 initial cell quantity) is successful.

Example 2

The procedure was as in example 1 except for the following procedure modifications.

The cells were mouse C2C12 cells, three samples of MboI & HpyCh4IV (100, 10000, 0.1 million), three samples of Sau3AI & HpyCh4IV (100, 10000, 0.1 million), and the antibodies were RNApolII (Abcam, ab 26721).

Cell lysis (two) step 2, pre-chilled 1000 μl HICHIP LYSIS Buffer cell lysate was added.

Chromatin (III) was digested and 15.38. Mu.l 10X Cutsmart Buffer, 2. Mu.l MboI and 2. Mu.l HpyCh4IV or 2. Mu.l Sau3AI and 2. Mu.l HpyCh4IV were added.

The enzyme digestion quality control RE-QC (IV) has the following results:

TABLE 11 results of quality control of RE-QC concentration in enzyme digestion

Sample of	C2C12-①	C2C12-②
			Concentration ng/ul	2.38	3.40

PCR library amplification and on-machine sequencing (ten), gel cutting range: 150-500bp.

All samples MboI & HpyCh4IV and Sau3AI & HpyCh4IV were mixed together in a 1:1 mass ratio to prepare an initial library LHCPC1. The original library LHCPC was found to have linker contamination, and then the remaining half of the T1 beads were used for re-PCR and gel-cut purification, and after mixing, 1.5 times of the beads were used for re-purification, and the same volume of water as the previous DNA solution was used for re-solubilization, in order to better obtain fragments of 200bp or more, the sample-fed sequencing library was designated LHCPC. After sequencing, LHCPC is found to be contaminated by the linker (as shown in FIG. 18), then the library containing different "bar code" linkers is mixed into a large library, then E-gel electrophoresis is performed again on the gel 14 min, and after gel cutting and recovery are performed according to fragments with the same size, 1.5 times of magnetic beads are used for purification, so that a pure library LHCPC without the linker contamination is finally obtained. Pure library LHCPC2 was subjected to Real time-qPCR and NGS3K library qualification (as in fig. 7) followed by Illumina Nova-seq PE150 double-ended sequencing, and the off-machine data were processed and evaluated by HiC-Pro software as in fig. 10, indicating that the more cells there were cells on average for effective interaction, the higher the library complexity, and the more effective interaction obtained. And the average comparison rate of the library accords with the library rule of chimeric DNA by 30% -60%. The cis-interaction ratio is higher (65% -75%), and similar to the library characteristics of Hi-C, the library characteristics indicate that the construction of the ULI-eHiChIP library of micro mouse C2C12 cells (100, 10000, 100000 initial cell quantity) is successful.

It can be seen that if the library is built by direct excision of the 6 th step in the "PCR library amplification and sequencing on the machine (ten)" step in example one, the library containing the different "barcode" linkers is not pooled into a large library, and then the library is built by excision of the same size. Then the initial library of libraries containing different "barcode" linkers is likely to have linker contamination, as shown in FIG. 18, with a "peak" apparent between 100-200bp in the cross-bar of FIG. 18, indicating the presence of linker contamination in the sequencing result. And combining the initial libraries with joint pollution, then carrying out electrophoresis, cutting glue according to fragments with the same size, and recovering, so that the pure libraries after optimization treatment (the quality inspection result of the pure libraries is shown as figure 7) can obviously eliminate joint pollution, so that the pure libraries without joint pollution can be prepared by cutting glue and purifying after the initial libraries are combined again, the joint pollution can be greatly reduced, and the data output of the sequencing library can be improved.

Example 3

The cells were human 293T cells and three samples of DpnII & HpyCh4IV (1000, 10000, 0.1 million) and the antibodies were RNApolII (Abcam, ab 26721).

Chromatin (III) was digested and 15.38. Mu.l 10 XNEBuffer DpnII, 2. Mu.l DpnII and 2. Mu.l HpyCh4IV were added.

The enzyme digestion quality control RE-QC (IV) has the following results: the concentration was 4.62 ng/ul. Agarose gel electrophoresis was performed, as shown in FIG. 2 (right), with no genomic band at 12kb, and a Smear band range of 200-4000 bp was ideal.

PCR library amplification and on-machine sequencing (ten), gel cutting range: 200-500bp. From examples 1 to 3, library excision was gradually avoided as completely as possible from excision to linker ligation.

All samples were mixed at a mass ratio of 1:1 to give LHCPT a 1. After mixing, the mixture was re-purified with 1.5 times of beads and re-dissolved in water in the same volume as the previous DNA solution in order to obtain fragments of 200bp or more better. Real time-qPCR and NGS3K library qualification (as shown in FIG. 8) were performed, illumina Nova-seq PE150 double-ended sequencing was performed, off-machine data were processed and evaluated by HiC-Pro software, and the results are shown in FIG. 11: effective interactions can be observed at the level of each cell number, the more cells, the higher the library complexity, the more effective interactions are obtained. The average comparison rate accords with the library rule of chimeric DNA (deoxyribonucleic acid) 70% -80%. The cis-interaction ratio is higher (80%), and the library characteristics similar to Hi-C show that the construction of the ULI-eHiChIP library of trace human 293T cells (1000, 10000 and 0.1 million cell initial amount) is successful.

Example 4

The procedure was as in example 1, except for the following modifications, and the main procedure is as shown in FIG. 17.

The cells were two samples of mouse myoblasts C2C12, mboI & HpyCh4IV (2400, 100000 cell numbers) and the antibody was H3K27me3 antibody (Abcam).

Cell collection and crosslink fixation step (one) 2, 37% formaldehyde (HCHO) was added to the petri dish to a final concentration of 1%, which was incubated in an incubator at 37 ℃ for 10min; step 3, 3mM EGS 4 ml (freshly prepared) was added at room temperature to crosslink 45 min.

Cell lysis (two) step 2, pre-chilled 500 μl HICHIP LYSIS Buffer cell lysate was added.

Chromatin (III) was digested and 9.61. Mu.L 10X Cutsmart, 2. Mu.L MboI (10U/ul NEB, R0147L) and 2. Mu.L HpyCH4IV (10U/ul, NEB, R0619L) were added.

The enzyme digestion quality control RE-QC (IV) has the following results:

TABLE 12 results of quality control of RE-QC concentration in enzyme digestion

C2C12.1 million (10 ten thousand)	①	②
			Concentration ng/ul	4.82	3.2

DNA end labeling and orthographic ligation (five), 41 μL ddH₂O、5 μL Lambda Exonuclease Reaction Buffer（NEB,B0262S）、1 μL Lambda Exonuclease （NEB, M0262） and 3. Mu.L RecJF (NEB, M0264) were added to the labeled and interacted pellet, and after mixing, the pellet was shaken at 900rpm for 1h at 37 ℃. Through the actions of the exonuclease Lambda Exonuclease and RecJF, the noise of the library can be effectively eliminated, the signal to noise ratio is improved, and the library quality is finally improved.

Immunoprecipitation, IP bead capture (seventh), protein A beads were used in an amount of 10. Mu.L. In the prior art 60. Mu.L of protein A beads per 10 Million cells were required. Therefore, the consumption of the protein A magnetic beads is reduced, on one hand, the cost of detection reagents can be reduced, and on the other hand, the library construction step can be optimized, and the library construction efficiency is improved.

PCR library amplification and on-machine sequencing (ten), gel cutting range: 175-500bp.

All samples were mixed at a 1:1 mass ratio to LHCPC < 3 > (representing only one example). Real time-qPCR and NGS3K library qualification (FIG. 12) were followed by Illumina Nova-seq PE150 double-ended sequencing, and off-machine data were processed and evaluated by HiC-Pro software (FIG. 13). Effective interactions were observed at the individual cell number levels (0.1M group: LHCPC-LHCP 34, LHCPC-LHCP 35, 2400 cell group: LHCPC3-LHCP36, LHCPC3-LHCP 37). The more cells, the higher the library complexity, and the effective interaction ratio is greater than 70% -80%. The comparison rate accords with the library rule of chimeric DNA by 40% -70%. The cis interaction ratio is relatively (55% -65%), and the library characteristics similar to Hi-C show that the construction of the ULI-eHiChIP library of the micro mouse myoblasts C2C12 (2400 and 100000 cells) is successful.

Example 5

The procedure was as in example 4, except for the following steps, which were adjusted, and the main procedure is shown in FIG. 17.

Isolation of biotin-labeled interactive DNA and preparation by Illumina sequencing (nine), library construction using FEA Enzym enzyme digestion was performed as follows:

1. Taking out FEA Buffer V2 FEA Enzyme Mix V2, thawing, mixing, centrifuging to bottom, and placing on ice for use. All the following steps were performed on ice.

2. The following reactions were prepared in an EP tube containing the sample:

TABLE 13 component TABLE

Component (A)	Volume (mu l)
		Input DNA	19
ddH2O	16
		FEA Buffer V2	5
Total	40

3. To each sample was added 10 mu L FEA Enzyme Mix V more 2, and the mixture was blown up with a pipette, the reaction solution was briefly transferred to the bottom of the tube, and immediately placed in a metal bath to carry out the following reaction:

table 14 temperature schedule

Temperature (temperature)	Time of
		37℃	10 min
65℃	30min
		4℃	Hold

4. 251. Mu.l of ddH ₂ O was added to the sample to make the total reaction volume 300. Mu.l.

5. 400 UL 1X TWEEN WASHING Buffer was added to a new 1.5 ml tube, then 25 uL Dynabeads MyOne Streptavidin T1 beads (Life technologies, 10 mg/ml, 65602) were added and mixed well, separated for 3 minutes on a magnetic rack and the solution discarded.

6. After resuspension of the beads with 300. Mu.L of 2 Xbinding Buffer (10 mM Tris-HCl (pH 7.5), 1mM EDTA,2M NaCl), the reaction was added to the bead solution, and after mixing, it was shaken at 350rpm for 15min at room temperature.

7. After 5min in a magnetic rack, the supernatant was discarded.

8. After adding 600 uL 1X TWEEN WASHING Buffer to resuspend the beads, the beads were placed on a metal bath, incubated at 55℃for 2min, then placed on a magnetic rack for 3min, and the supernatant was discarded. The beads were washed repeatedly.

9. Adding 32.5 uL ddH ₂ O re-dissolved magnetic beads, and preserving at 4 ℃ for later use.

10. Library preparation, a mixture was prepared according to table 15 below, mixed well and placed on a metal bath, incubated for 15min at 20℃and stored at 4℃for further use.

Table 15 library mixture table

Component (A)	Volume of
		End Preparation Product	32.5 μL
Rapid Ligation buffer 2	12.5 μL
		Rapid DNA ligase	2.5 μL
DNA adapter X	1.5 μL
		Water	1 μL
totel	50 μL

11. Placing the mixture in a magnetic frame for 5min, discarding the supernatant, adding 150 μL of 1 xTE Buffer, mixing under a spring, placing in the magnetic frame for 3min, discarding the supernatant, repeating the steps, washing the beads twice with the same step of B/W Buffer with the same volume, and storing the 40 μL of ddH ₂ O redissolved beads at 4 ℃ for later use.

PCR library amplification and on-machine sequencing (ten), the specific steps are as follows

The PCR QC mixture was prepared as in Table 16 below, mixed well and centrifuged briefly.

Table 16 PCR QC mixture System

Component (A)	Quality control PCR component volume (μL)
		VAHTS HiFi AmpMix	10
PCR Primer Mix 3	1
		Adapter-ligated library	1
Water	8
		Total volume	20

2. PCR QC was set up as follows. Pre-denaturation: 98 ℃ for 1min;18-22 cycles (denaturation: 98 ℃ C. 15 s; annealing: 60 ℃ C. 30s; extension: 72 ℃ C. 3 min); final extension: 5min at 72 ℃; and (3) preserving: 4 ℃ infinity min. The PCR QC cycle number of the 10-ten thousand cell amount sample was 18, and the results are shown in Table 17 below:

Table 17 example 5 PCR QC detailed information

3. Taking 19 mu L of PCR QC product, running E-gel, observing Smear band for quality control, and as shown in FIG. 14, it can be seen that PCR QC cycle can meet the cut gel range of 300-700 bp.

4. And (5) performing formal PCR. The final PCR mixture was prepared as shown in Table 18 below, mixed well and centrifuged briefly. The PCR procedure was the same as that of PCR QC, and the cycle number of the 10-kilocell sample was 14, as shown in Table 19 below. The number of cycles set can meet the requirements of library recovery. The final PCR product was obtained.

TABLE 18 formal PCR Components Table

Component (A)	Formal PCR component volume (μL)
		VAHTS HiFi AmpMix	20
PCR Primer Mix 3	2
		Adapter-ligated library	18
Total volume	40

TABLE 19 formal PCR detailed information

5. Transfer the PCR product to a new 1.5mL centrifuge tube at 1:1, adding DNA Clean Beads (Vazyme, N411), mixing, standing at normal temperature for 5 min, adding a magnetic frame 5 min, discarding supernatant, washing with a freshly prepared 80% ethanol solution twice, airing for 3 min, re-dissolving with 20 μl ddH2O, adding a magnetic frame 5 min, and taking 19 μl supernatant, and running E-gel.

6. And (3) performing gel cutting recovery, wherein the recovered fragments are samples with 10 ten thousand cell quantities, namely 300-700 bp, the recovery concentration is shown in Table 20, and the library preparation is successful.

TABLE 20 recovery concentration of gel after formal PCR

7. All samples were mixed at a 1:1 mass ratio to give LHCPC4. Real time-qPCR and NGS3K library qualification (FIG. 15) were followed by Illumina Nova-seq PE150 double-ended sequencing, and off-machine data were processed and evaluated by HiC-Pro software (FIG. 16). Effective interaction was observed at the individual cell number level (group 0.1M: LHCPC4_LHCP40LHCPC4 _LHCP41). The more cells, the higher the library complexity, and the greater the proportion of effective interactions, all greater than 80%. The comparison rate accords with the library rule of chimeric DNA by 40% -70%. The cis interaction ratio is relatively (55% -65%), and the library characteristics similar to Hi-C show that the construction of the ULI-eHiChIP library of the micro mouse myoblasts C2C12 (2400 and 100000 cells) is successful.

In comparative examples 4 and 5, it can be seen that the Tn5 enzyme and FEA Enzym enzyme can achieve a good library construction effect. The Tn5 enzyme is expensive, can be used for constructing libraries of more trace cells, such as 100, 1000 cells, is suitable for constructing libraries of rare cells and trace medical test cells, and can obtain the highest cost performance. The FEA Enzyme library construction method can be used for constructing libraries of 1 ten thousand and 10 ten thousand cells, is relatively low in price, is more suitable for large-scale cell library construction, and has better economic value.

What has been described above is merely some embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.

Claims

1. A targeted chromatin interaction capture ULI-eHiChIP pooling method for micro cells, wherein the micro cells refer to a cell number below 100000, the method comprising the steps of:

S1: collecting target cells;

S2: formaldehyde is then added to the petri dishes containing the collected cells for cross-linking;

S3: lysing the cells after cross-linking the immobilized chromatin, permeabilizing the cells with a permeabilizing buffer comprising 1% PI and 0.2% Igepal CA 630;

S7: performing antibody immunoprecipitation, protein A magnetic bead capturing and rinsing on the protein factor-DNA interaction small body in the step S6 to obtain antibody precipitation magnetic beads captured with chromatin interaction small body;

2. The method according to claim 1, wherein the micro cells are 100-100000 cells; in addition to the single cross-linking with formaldehyde in step S2, the double cross-linking can be performed by adding formaldehyde and EGS to the petri dish containing the collected cells.

3. The method of claim 1, wherein in step S9, the prepared ULI-eHiChIP library is subjected to E-gel electrophoresis and gel cutting recovery again to prepare a non-linker-contaminated ULI-eHiChIP library.

4. The method according to claim 1, wherein in the step S6, the end biotin labeling of the digested DNA fragment is performed by using a base reagent Fill-in Master Mix mixed with biotin label, wherein the usage amount of the Fill-in Master Mix is 4.7. Mu.L; in the step S7, the amount of the protein A magnetic beads is 10. Mu.L.

5. The method according to claim 1, wherein the ligation reaction used in the ortho-ligation in step S6 comprises NEB T4 DNA LIGASE buffer, triton X-100, recombinant albumin Recombinant Albumin, T4 DNA LIGASE and water.

6. The method according to claim 1, wherein the step S6 further comprises performing background elimination of biotin-labeled and ortholinked protein factor-DNA interaction corpuscles with Lambda Exonuclease, recJf exonuclease, followed by ultrasound disruption and subsequent manipulation.

7. Use of the method according to any one of claims 1-6 for the construction of a library of cell ULI-eHiChIP in a trace amount of cell initiation, wherein the trace amount of cell initiation is 100-100000.

8. A library of minicell ULI-eHiChIP produced by the method of any one of claims 1-6.

9. Use of the ULI-eHiChIP library as claimed in claim 8 for high throughput sequencing of minicells.

10. Use of the ULI-eHiChIP library as described in claim 8 in a three-dimensional genomics or functional genomics study against minicells.