Chromatin conformation capturing method of high-lipid-droplet-content single cell based on low-melting-point agar immobilization

Abstract

The invention provides a chromatin conformation capturing method of high-lipid-droplet-content single cells based on low-melting-point agar immobilization, and relates to the technical field of chromatin conformation capture.The chromatin conformation capturing method comprises the steps that permeabilized single cells are placed in a buffer solution, and an isometric low-melting-point agarose solution is added to immobilize the cells; the mass concentration of the low-melting-point agarose solution is 0.5% w / v. According to the method, permeabilized cells are fixed by adopting low-melting-point agarose, the problem that cell nucleuses of high-lipid-droplet-content cells are not easy to track in chromatin conformation capture of the cells is solved, the influence of lipid droplets on enzyme digestion of the cell nucleuses is reduced by adopting a 0.5% w / v low-melting-point agarose solution, and the total chromatin interaction number obtained by data is superior to that obtained by adopting a single-cell Hi-C method in the prior art; compared with the bulk Hi-C data of the same type of embryonic cells, the same variation trend and interaction distribution can be displayed. The technical problem that in the prior art, a common single cell Hi-C technology is not suitable for high-fat-content single cells of pig embryos is solved.

Description

Technical Field

[0001] The invention relates to the technical field of chromatin conformation capture, and in particular to a chromatin conformation capture method of single cells with high lipid droplet content based on low melting point agar fixation. Background Art

[0002] The three-dimensional structure of the eukaryotic genome plays a key role in important biological processes such as transcription, DNA replication, and cell division. The development of chromatin conformation capture technologies (such as Hi-C and ChIA-PET) has accelerated our understanding of the three-dimensional structure of the genome. However, traditional sequencing methods based on cell populations detect population averages and it is difficult to distinguish differences between individual cells. Single-cell chromatin conformation capture technology provides new research perspectives and opportunities to address these limitations. Single-cell Hi-C technology can identify cell subpopulations in mixed samples, especially the chromosome folding of rare cell types (such as stem cells, tumor precursor cells, oocytes, etc.), thereby bringing new opportunities for the study of development and disease mechanisms.

[0003] At present, single-cell Hi-C technology is mainly used to study model organisms such as mice and fruit flies, as well as some human tissues and cells. However, there are no reports on the application of single-cell Hi-C technology in large non-human mammals (such as pigs). As an important biomedical model, pigs occupy an important position in the fields of embryonic development and drug development. However, due to the high fat content of pig embryos, ordinary single-cell Hi-C technology is not applicable.

[0004] Characteristics of pig embryonic cells: difficult to obtain, high lipid droplet content (Reference: Species and embryo genome origin affect lipid droplets in preimplantation embryos). Currently, no research methods are used for this type of cell line. In addition, the test results of other methods show that other single-cell Hi-C methods are not suitable for this cell.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a low melting point agar single cell fixation method to solve the technical problem that the common single cell Hi-C technology in the prior art is not suitable for single cells with high fat content in pig embryos.

[0007] The second object of the present invention is to provide the application of the above-mentioned low melting point agar single cell fixation method in the capture of chromatin conformation of single cells with high lipid droplet content.

[0008] The third object of the present invention is to provide a method for capturing the chromatin conformation of single cells with high lipid droplet content.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] In a first aspect, the present invention provides a method for fixing single cells in low melting point agar, comprising placing permeabilized single cells in a buffer solution, and adding an equal volume of low melting point agarose solution to fix the cells;

[0011] The concentration of the low melting point agarose solution is 0.5% w / v.

[0012] Furthermore, after adding an equal volume of low melting point agarose solution, the method further comprises standing at room temperature until solidification.

[0013] Furthermore, the amount of the buffer solution and the low melting point agarose solution added is 5 μl / single cell.

[0014] Furthermore, the method for preparing the permeabilized single cell comprises placing the single cell fixed with 1% formaldehyde solution in a lysis buffer and permeabilizing the cell on ice.

[0015] Furthermore, before the permeabilized single cells are placed in a buffer, the process further comprises eluting at least once with a buffer containing 0.1% BSA;

[0016] Preferably, the buffer is 1×NEBuffer 3.

[0017] In a second aspect, the present invention provides the application of the above-mentioned low melting point agar single cell fixation method in the capture of chromatin conformation of single cells with high lipid droplet content.

[0018] In a third aspect, the present invention provides a method for capturing chromatin conformation of a single cell with high lipid droplet content, comprising the following steps:

[0019] A. Use the above-mentioned low melting point agar single cell fixation method to fix the permeabilized single cells with high lipid droplet content;

[0020] B. Processing the cells obtained in step A;

[0021] C. Biotin capture and library construction.

[0022] Furthermore, the step B includes subsequent MboI digestion, biotin filling labeling, DNA proximity ligation and cross-linking removal operations in single cells fixed with low melting point agarose.

[0023] Furthermore, step C includes melting low melting point agar at 65°C to release nuclear DNA, then capturing biotin-labeled DNA fragments by streptavidin magnetic beads at 37°C, and finally constructing a library by Tn5 transposase digestion. After fragment screening and purification, the library is sequenced by Illumina platform to obtain the final data.

[0024] Furthermore, the method further comprises step D, data analysis.

[0025] The present invention provides a low melting point agar single cell fixation method, which uses low melting point agarose to fix permeabilized cells, effectively solves the problem that the nucleus of cells with high lipid droplet content is difficult to track in the chromatin conformation capture of cells, and 0.5% w / v low melting point agarose solution reduces the influence of lipid droplets on nucleus enzyme cutting, and the total number of chromatin interactions obtained by data is better than that of the single cell Hi-C method in the prior art; compared with bulk Hi-C data of embryonic cells of the same type, the same change trend and interaction distribution can be shown, indicating that the method is reliable.

[0026] The low melting point agar single cell fixation method can fix single cells with high lipid content, which can make the subsequent detection of chromatin interaction information in cells with high lipid content simpler, more accurate and efficient, which is of great significance for the study of pig embryo development and embryo breeding. On the other hand, a chromatin conformation capture method for single cells with high lipid droplet content is provided. By using low melting point agarose to fix permeabilized cells, it is possible to ensure that embryonic cell information is not lost during the experiment and to obtain high-quality experimental data, thereby achieving the capture of chromatin conformation of single cells with high lipid droplet content. This method is of great significance for studying the dynamic changes of three-dimensional structures during the development of difficult-to-obtain embryonic cells and pig embryo breeding research. It solves the technical problem that ordinary single cell Hi-C technology in the prior art is not suitable for single cells with high fat content in pig embryos. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The heat map of the whole genome interactions of single cells treated with different concentrations of low melting point agarose provided in Experiment 1 of the present invention;

[0029] Figure 2 Agarose gel electrophoresis diagram of enzyme-cut fragments treated with low-melting-point agarose of different concentrations and amounts provided in Experiment 2 of the present invention;

[0030] Figure 3 A data quality comparison diagram of the scAHi-C and single-cell Hi-C methods provided in Experiment 3 of the present invention;

[0031] Figure 4 The data distribution diagram of scAHi-C provided for Experiment 4 of the present invention;

[0032] Figure 5 Pseudo-mixed single-cell Hi-C data provided for Experiment 5 of the present invention are compared with bulk Hi-C. DETAILED DESCRIPTION

[0033] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0034] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0035] On one hand, the present invention provides a method for fixing single cells with low melting point agar, comprising placing permeabilized single cells in a buffer solution, and adding an equal volume of low melting point agarose solution to fix the cells; the concentration of the low melting point agarose solution is 0.5% w / v.

[0036] By using low melting point agarose to fix permeabilized cells, the problem of difficulty in tracking the nuclei of cells with high lipid droplet content in chromatin conformation capture was effectively solved. The 0.5% w / v low melting point agarose solution reduced the effect of lipid droplets on nucleus enzyme digestion, and the total number of chromatin interactions obtained from the data was better than the single-cell Hi-C method in the existing technology; compared with the bulk Hi-C data of embryonic cells of the same type, it can show the same change trend and interaction distribution, indicating that the method is reliable. Therefore, the use of this low melting point agar single cell fixation method to fix single cells with high lipid content can make the subsequent detection of chromatin interaction information in cells with high lipid content simpler, more accurate and efficient, which is of great significance for the study of pig embryo development and embryo breeding.

[0037] In some specific embodiments, the adding of an equal volume of low melting point agarose solution further comprises standing at room temperature until solidification.

[0038] In some specific embodiments, the amount of the buffer and the low melting point agarose solution added is 5 μl / single cell.

[0039] In some specific embodiments, the method for preparing the permeabilized single cells comprises placing the single cells fixed with 1% formaldehyde solution in a lysis buffer and permeabilizing them on ice.

[0040] In some specific embodiments, before the permeabilized single cells are placed in the buffer, the step further comprises eluting at least once with a buffer containing 0.1% BSA.

[0041] In some specific embodiments, the buffer is 1×NEBuffer 3.

[0042] According to another aspect of the present invention, there is also provided an application of the above-mentioned low melting point agar single cell fixation method in capturing chromatin conformation of single cells with high lipid droplet content.

[0043] According to another aspect of the present invention, a method for capturing chromatin conformation of a single cell with high lipid droplet content is provided, comprising the following steps:

[0044] A. Use the above-mentioned low melting point agar single cell fixation method to fix the permeabilized single cells with high lipid droplet content;

[0045] B. Processing the cells obtained in step A;

[0046] C. Biotin capture and library construction.

[0047] By using low melting point agarose to fix the permeabilized cells, it is possible to ensure that the embryonic cell information is not lost during the experiment, obtain high-quality experimental data, and capture the chromatin conformation of single cells with high lipid droplet content. This method is of great significance for studying the dynamic changes of the three-dimensional structure during the development of difficult-to-obtain embryonic cells and pig embryo breeding research.

[0048] In some specific embodiments, the step B includes performing subsequent MboI digestion, biotin fill-in labeling, DNA proximity ligation and cross-linking removal operations in single cells fixed with low melting point agarose.

[0049] In some specific embodiments, step C includes melting low melting point agar at 65°C to release nuclear DNA, then capturing biotin-labeled DNA fragments by streptavidin magnetic beads at 37°C, and finally constructing a library by Tn5 transposase digestion. After the library is screened and purified, the final data is obtained by sequencing on the Illumina platform.

[0050] The streptavidin magnetic beads are Dynabeads M-280streptavidin.

[0051] In some specific embodiments, step D, data analysis, is also included.

[0052] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] 1. Materials and Reagents

[0054] Materials: Nextera XT DNA Library Preparation Kit (Illumina, catalog number: FC-131-1096).

[0055] Reagents: formaldehyde solution, glycine, NaCl, EDTA, NP-40, Low-melting-point agarose, SDS, Triton X-100, 10×NEBuffer 3, MboI, Klenow DNA polymerase I, dNTP (dATP, dCTP, dGTPand dTTP) set, Biotin-14-dATP, T4 DNA ligase buffer, T4 DNA ligase, BSA, Dynabeads M-280 streptavidin and Tween 20.

[0056] 2. Single cell acquisition: Follicles were collected from the ovaries of young sows in a local slaughterhouse, placed in a 37°C constant temperature and humidity incubator and allowed to settle for 10 minutes, the upper 2 / 3 liquid was discarded, resuspended with preheated PVA-TL-HEPES and allowed to settle for 5 minutes, and the above operation was repeated twice, and qualified cumulus oocyte complexes (COCs) were picked up under a stereomicroscope. Mature oocytes with uniform cytoplasm and expelled the first polar body were selected from COCs cultured in vitro for 42 to 44 hours for subsequent experiments.

[0057] Parthenogenetic (PA) embryo collection method: Place mature oocytes in a fusion tank, which is pre-filled with fusion fluid, and place them in activation culture medium. Use the BTX Electro-cell Manipulator 2000 electrofusion instrument to apply two 1.2kV / cm DC pulses to activate the oocytes for 30 microseconds, with an interval of 1 second for activation. Transfer the electroshock-activated embryos to the porcine embryo culture medium PZM3, wash them three times, and then transfer them to the PZM3 embryo culture droplets that have been pre-balanced for more than 4 hours for continued culture. Collect MII-stage embryos from cells that survive 20 minutes after electrical stimulation, obtain 2-cell-stage embryos after 24 hours, and obtain 4-cell-stage embryos after 48 hours. The embryo samples of each period obtained by culture are treated with pronase to remove the polar bodies and zona pellucida, dissociated into single cells, and set aside.

[0058] In vitro fertilization (IVF) embryo collection method: on the day of egg collection, prepare porcine IVF working solution to make 50 μl fertilization drops, with a final sperm concentration of 5×10 5 / ml. Mature porcine oocytes were selected, washed three times with modified fertilization Tris buffered medium (mTBM), and then transferred into fertilization drops, with 30 to 35 oocytes in each fertilization drop. After the sperm and eggs were incubated together for 4 to 6 hours for fertilization, the oocytes were washed three times with porcine embryo culture medium PZM3. Then they were added to a four-well plate containing PZM3 culture medium, with 50 to 80 oocytes per well, and humidified and statically cultured. Zygotes, 2-cell and 4-cell embryos were collected at 18.5 hours, 24 hours and 48 hours respectively. All embryos were treated with pronase to remove polar bodies, zona pellucida and excess sperm, dissociated into single cells, and set aside.

[0059] Example 1

[0060] A low melting point agar single cell fixation method, taking the separated single cells and operating according to the following steps:

[0061] 1. 0.5% (wt / vol) low melting point agarose preparation: In a 50 ml test tube, add 50 mg of low melting point agarose powder to 10 ml of ultrapure water. Heat in a microwave oven for 15 to 30 seconds until all the agarose powder is dissolved, then incubate at 37°C until needed.

[0062] 2. The single cell fixation process is as follows:

[0063] 1) Add 10 μl of 1% formaldehyde to the microplate, transfer the single cells to the microplate by mouth pipette (transfer twice, the first time is used to wash away the residual culture medium), fix at room temperature for 10 minutes, transfer to 0.125M glycine solution to terminate the reaction. Place on ice for 5 minutes.

[0064] 2) Transfer the cells to 10 μl 1×PBS for elution once, then place in 10 μl lysis buffer on ice for permeabilization for at least 30 minutes. Then transfer the cells to 10 μl 1×NEBuffer 3 containing 0.1% PBS for elution once, then transfer to a PCR tube.

[0065] 3) Add 5 μl of 1×NEBuffer 3 to the PCR tube containing cells and centrifuge briefly. Add 5 μl of low melting point agarose incubated at 37°C (melted in a microwave oven and kept warm at 37°C), flick the PCR tube to mix the liquid, centrifuge briefly to allow the liquid to gather at the bottom of the tube, and let it stand at room temperature for at least 30 minutes to solidify.

[0066] Comparative Example 1

[0067] Different from Example 1, 1% (wt / vol) low melting point agarose was prepared by adding 100 mg of low melting point agarose powder to 10 ml of ultrapure water in a 50 ml test tube. Heat in a microwave oven for 15 to 30 seconds until all the agarose powder is dissolved, and then incubate at 37°C until needed.

[0068] Example 2

[0069] A method for capturing chromatin conformation of single cells with high lipid droplet content (scAHi-C) is described. The isolated single cells are operated according to the following steps:

[0070] 1. Fix single cells according to the steps of Example 1;

[0071] 2. Cell nucleus processing: Continue with the following operations, including enzyme digestion, biotin filling, ligation and cross-linking removal.

[0072] 1) Add 10 μl of 0.3% SDS solution (containing 0.3% (w / v) SDS, 1× NEBuffer 3) to the PCR tube with the single cell fixed, centrifuge briefly, and incubate at 37°C for 1 hour.

[0073] 2) Add 10 μl of 4% Triton X-100 solution (containing 4% Triton X-100, 1× NEBuffer 3) to the tube, gently pipette to mix, centrifuge briefly, and incubate at 37° C. for 1 hour.

[0074] 3) Add 125 U MboI, 2 μl 10×NEBuffer 3, and 13 μl ddH2O to the tube, mix well, centrifuge briefly, and incubate at 37°C for 20 hours.

[0075] 4) Gently remove the upper layer of liquid with a pipette tip, centrifuge slightly, and remove the supernatant with a small pipette tip. Add 6μl of terminal labeled biotin mixture (containing 0.168μl dC / dG / dTTP mix (10mM each), 4.2μl 0.4mM Biotin-14-dATP, 0.6μl 10×NEBuffer 3, 1μl 5U / μl DNA Polymerase I and 0.032μl ddH2O) to the tube, centrifuge slightly, and incubate at 37℃ for 1 hour.

[0076] 5) Add 50μl 1×T4 ligase buffer to the tube, let stand at room temperature for 10 minutes, and then discard the supernatant. Then add 50μl ligase mixture (containing 0.375μl T4 DNA ligase, 0.25μl 20mg / mL BSA, 5μl 10×T4DNA ligase buffer and 44.375μl ddH2O). Centrifuge briefly and incubate at 16℃ for 20 hours.

[0077] 6) Pipette the supernatant and discard it, add 50 μl 1×PBS (pH 7.4) to the tube, let it stand at room temperature for 10 minutes, and then discard the supernatant. Place the PCR tube in a PCR instrument and decrosslink at 65°C for 16 hours.

[0078] 3. Biotin capture and library construction:

[0079] 1) Add 10 μl of 10 mM Tris-HCl (pH 8.0) to each tube, place in a PCR instrument, incubate at 65°C for 10 minutes, and then incubate at 37°C.

[0080] 2) Take a new PCR tube, add 10μl Dynabead M280, then add 10μl 2×BW buffer, oscillate to mix, centrifuge briefly, place on a magnetic rack until the liquid clarifies, and discard the supernatant; add 20μl 2×BW buffer, oscillate to mix, centrifuge briefly, and incubate the PCR tube at 37℃ for 10 minutes.

[0081] 3) Transfer Dynabead M280 beads to the sample tube. Then place in a shaker preheated to 37°C, shake at 2000 rpm for 30 seconds to mix the sample. Then put it back into the PCR instrument and incubate at 37°C for 1 hour, shaking and resuspending every 15 minutes.

[0082] 4) Place the PCR tube on a magnetic rack and let it stand for 1 minute until the liquid becomes clear, then discard the supernatant.

[0083] 5) Add 20 μl 1×TWB, shake at 2000 rpm for 30 seconds to mix, centrifuge briefly, place on a magnetic rack until the liquid is clear, and discard the supernatant.

[0084] 6) Add 100 μl 10 mM Tris-HCl (pH 8.0), shake at 2000 rpm for 30 seconds to mix, centrifuge briefly, place on a magnetic rack until the liquid is clear, and discard the supernatant. Repeat this step once.

[0085] 7) Add 5 μl 10 mM Tris-HCl (pH 8.0) to the tube to resuspend the magnetic beads, add 10 μl Tagment DNA Buffer and 5 μl Amplicon Tagment Mix, mix gently by pipetting, and incubate in a PCR instrument at 55°C for 10 minutes and maintain at 10°C.

[0086] 8) Immediately after the reaction, add 5 μl Neutralize Tagment Buffer, gently pipette to mix, and let stand at room temperature for 5 minutes. Place the PCR tube on a magnetic rack and let stand for 1 minute until the liquid is clear, and discard the supernatant.

[0087] 9) Add 20 μl 1×TWB, shake at 2000 rpm for 30 seconds to mix, centrifuge briefly, place on a magnetic rack until the liquid is clear, and discard the supernatant.

[0088] 10) Add 100 μl 10 mM Tris-HCl (pH 8.0), shake at 2000 rpm for 30 seconds to mix, centrifuge briefly, place on a magnetic rack until the liquid is clear, and discard the supernatant. Repeat this step once.

[0089] 11) Add 25μl 10mM Tris-HCl to the tube to resuspend the magnetic beads, add 15μl Nextera PCR MasterMix, 5μl I5 primer (10μM) and 5μl I7 primer (10μM). Mix by blowing gently. Centrifuge slightly and place in a PCR instrument for amplification reaction. The PCR reaction conditions are: 72℃ extension and filling for 3 minutes; 95℃ pre-denaturation for 30 seconds; 18 cycles, during which 95℃ denaturation for 10 seconds, 55℃ annealing for 30 seconds, and 72℃ extension for 30 seconds; after the cycle, 72℃ extension for 5 minutes.

[0090] 12) Pipette 35 μl (0.7×) of purified magnetic beads equilibrated at room temperature for 30 minutes and add them to the reaction PCR tube. Mix them by gently pipetting, centrifuge briefly, and incubate at room temperature for 5 minutes.

[0091] 13) Place the tube on a magnetic rack for 1 minute until the liquid becomes clear, discard the supernatant, and use a 10μl pipette to thoroughly discard the residual liquid at the bottom of the centrifuge tube. Add 200μl 80% ethanol and discard after 30 seconds. Repeat the 80% ethanol elution once.

[0092] 14) Dry at room temperature until the ethanol is completely evaporated. Elute the DNA and dissolve it in 21 μl ultrapure water. The final library is obtained. After the library test is completed, sequence it on the Illumina platform.

[0093] 4. Data analysis:

[0094] For scAHi-C sequencing data, FastQC was used to assess the quality of the sequencing data, and then the Trim Galore software, which can automatically identify adapters, was used to remove the adapter sequences. Reads with an average quality score less than or equal to 30 were removed from the fastq file to ensure that high-quality reads were used for analysis. The scAHi-C data were aligned using HiC-Pro software, with the reference genome being susScr11, and the file configuration conditions were performed according to the default parameters. The data were then used to generate an interaction matrix and corrected using ICE (Iterative Correction and Eigenvector Decomposition) (part of HiC-Pro). The interaction matrix similarity assessment was completed using the hicCorrelate module in the HiCExplorer software, and the Pearson and Spearman correlation coefficients between the two matrices were calculated, respectively.

[0095] Comparative Example 2

[0096] Different from Example 2, single cell fixation was performed according to the steps of Comparative Example 1.

[0097] Experiment 1

[0098] According to the methods of Example 2 and Comparative Example 2, the scHi-C data of single cells at the MII stage after oocyte activation were obtained, and the results were analyzed as follows: Figure 1 The figure shows a single-cell whole-genome interaction heat map, where A is Example 2, the red square represents the maximum range value, and B is Comparative Example 2. It can be seen that the scHi-C interaction heat map of Example 2 shows the characteristics of low noise, obvious diagonal lines, and clear local interactions, which is consistent with the interaction pattern of qualified single-cell Hi-C in the MII stage. However, the comparative example 2 shows high noise, unclear diagonal lines, and too many local interactions, which does not meet the interaction pattern of qualified single-cell Hi-C.

[0099] Experiment 2

[0100] In order to further verify the reason why the scHi-C data obtained in Comparative Example 2 was unqualified, this experiment tested the subsequent enzyme digestion efficiency. The specific steps were as follows:

[0101] (1) Cell fixation and permeabilization

[0102] Take a bottle of K562 cells in the logarithmic growth phase, add a certain amount of 37% formaldehyde solution to the bottle to make the final concentration of 1%, shake the bottle slowly to mix the solution, incubate at room temperature for 10 minutes, immediately add a glycine solution with a final concentration of 0.125M, and incubate at room temperature for 5 minutes to terminate the formaldehyde reaction. Then centrifuge the cross-linked sample at 4°C, 3000×g for 5 minutes. After centrifugation, remove the supernatant, add 100μl pre-cooled Hi-C permeabilization solution (containing 10mM Tris-HCl (pH 8.0), 10mM NaCl and 0.2% Igepal CA630), add 1μl protease inhibitor, and lyse on ice for at least 15 minutes. Centrifuge at 4°C, 3000×g for 5 minutes, and remove the supernatant. Add 100μl 1×NEBuffer 3 buffer to elute once.

[0103] (2) Enzyme digestion test

[0104] Five groups of experiments were designed, including the control group (without low-melting point agarose + 125U MboI, incubated for 20 hours), the agar group (10μl 1% low-melting point agarose + 125U MboI, incubated for 20 hours), the agar group with double enzyme dosage (10μl 1% low-melting point agarose + 250U MboI, incubated for 20 hours), the agar volume halved group (5μl 1% low-melting point agarose + 125U MboI, incubated for 20 hours) and the agar volume and concentration halved group (5μl 0.5% low-melting point agarose + 125U MboI, incubated for 20 hours).

[0105] The specific implementation is as follows: the cell density is measured by a cell counter, about 6000 cells are transferred to a new PCR tube, and 1×NEBuffer 3 solution is added to 5μl or 10μl, and the same volume of agarose solution of different concentrations (1% or 0.5%) is added respectively, mixed and centrifuged briefly, and the agar is allowed to solidify at room temperature. After solidification, the cells are treated with SDS and Triton in step 2 of Example 2, and then the corresponding MboI enzyme is added and incubated at 37°C. The enzyme-cut fragments are recovered by an agarose gel recovery kit. Then, 1% agarose gel electrophoresis is performed on the recovered samples to check the size distribution of DNA fragments.

[0106] The experimental results are as follows Figure 2As shown, lane M is a DNA molecular weight marker, from top to bottom: 10kb, 8kb, 7kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb; lanes 1 and 2 in A are enzyme digestion samples under agar fixation conditions, both of which are parallel samples; lanes 3 and 4 are control group samples digested with 25U MboI, both of which are parallel samples; lanes 5 and 6 are samples treated with double the amount of enzyme, both of which are parallel samples. Lane 7 in B is a control group sample digested with 125U MboI, lanes 8 and 9 are samples with 0.5% concentration and 5μl agar fixation conditions, both of which are parallel samples; lane 10 is a sample with 1% concentration and 5μl agar fixation conditions.

[0107] like Figure 2 As shown in A, compared with the control group (lane 3 and lane 4), the fragment distribution of the 1% + 10μl agar system group (lane 1 and lane 2) is significantly larger, suggesting a lower enzyme digestion efficiency. Even if the amount of MboI used (1% + 10μl + enzyme addition (250U MboI)) is increased, the length distribution of the digested fragments (lane 5 and lane 6) is not significantly reduced, especially the digested fragments below 3kb are basically not observed.

[0108] like Figure 2 As shown in B, the combination of 0.5% concentration and 5μl volume of low melting point agarose significantly improves the enzyme digestion effect. Reducing the volume of low melting point agarose (1% + 5μl, lane 10) can improve the enzyme digestion effect to a certain extent. The distribution of fragments below 3kb can be clearly seen, although the main enzyme digestion fragment is still near the 10kb band. Further reducing the agar concentration (0.5% + 5μl, lanes 8 and 9) produced more small fragments (about 1kb) distribution. It can be seen that improving agar fixation conditions, such as volume and concentration, can reduce the impact of agar.

[0109] It can be seen that the enzyme digestion efficiency is relatively high under low concentration and low volume agarose conditions. Therefore, 0.5% agarose concentration and 5 μl volume were determined as the new single cell Hi-C fixation conditions to be developed.

[0110] Experiment 3

[0111] 2019-Tan: Data processed by single-cell Hi-C method in the publicly published "Single-cell chromatin conformation capture ofmouse sensory neurons in visual and olfactory systems by Dip-C".

[0112] 2021-Tan: Data processed by single-cell Hi-C method in the publicly published paper "Changes in genome architecture and transcriptional dynamics progress independently of sensory experience during post-natal brain development".

[0113] 2017-Flyamer: Processed data for the single-cell Hi-C method in the publicly published "Single-nucleus Hi-C reveals uniquechromatin reorganization at oocyte-to-zygote transition".

[0114] 2017-Nagano: Processed data for the single-cell Hi-C method in the publicly published "Cell-cycle dynamics of chromosomal organization at single-cell resolution".

[0115] 2023-Qu: Data processed for the single-cell Hi-C method in the publicly published "Simultaneous profiling of chromatinarchitecture and transcription in single cells".

[0116] 2019-Li: Processed data for the single-cell Hi-C method in the publicly published "Joint profiling of DNA methylation andchromatin architecture in single cells".

[0117] 2017-Ramani: Data processed for the single-cell Hi-C method in the publicly published "Massively multiplex single-cell Hi-C" (2017-Ramani).

[0118] 2013-Nagano: Data processed by the single-cell Hi-C method in the publicly published "Single-cell Hi-C reveals cell-to-cell variability in chromosome structure".

[0119] Compared with the single-cell three-dimensional genome construction technology based on low-melting-point agarose fixation in the method of Example 2 of the present invention, although these methods have certain differences in experimental procedures, cell types or specific conditions, the purpose is to solve the detection problem of high-complexity genome structure at the single-cell level, and from the perspective of comparing the relative capture capabilities of different single-cell chromatin conformation capture methods, they have certain comparability. In addition, there is currently no standard comparison index. Therefore, this experiment uses the published results as a reference, combined with the application results of Example 2 of the present invention in processing single cells in all stages of pig parthenogenetics and IVF to obtain sequencing data, for comparative analysis.

[0120] Analysis indicators: summary of the number of interactions, and the ratio of intra-chromosome and inter-chromosome interactions.

[0121] The results are as follows Figure 3 As shown, the number of cells corresponding to each treatment is shown above the figure. A is a comparison of the number of interactions processed by scAHi-C and other single-cell Hi-C methods. The number of interactions obtained by scAHi-C has a clear advantage and is significantly higher than other single-cell Hi-C methods; B is a comparison of the distribution of cis and trans interactions of chromosomes processed by scAHi-C and other single-cell Hi-C methods. The distribution of the proportion of intra-chromosomal and inter-chromosomal interactions of scAHi-C is close to that of other single-cell Hi-C methods, and the distribution is reasonable.

[0122] Experiment 4

[0123] The scAHi-C data obtained from single cells of parthenogenetic (MII stage, 2-cell stage, 4-cell stage after oocyte activation) and IVF (1-cell stage, 2-cell stage, 4-cell stage) embryos processed in Example 2 were directly statistically analyzed to analyze the proportion of long-range interactions within chromosomes.

[0124] The results are as follows Figure 4 As shown. It can be seen that scAHi-C can capture more long-distance interactions between chromosomes. The median value of the proportion of cis long-distance interactions (>20kb) in scAHi-C is 62.92%, which indicates that scAHi-C can capture more long-distance chromatin interactions, and the data of scAHi-C meets the distribution expectations of qualified single-cell Hi-C data.

[0125] Experiment 5

[0126] The method of Example 2 was used to process single cells at the parthenogenetic 4-cell stage, and the pseudo-mixed single-cell data were compared with the in situ Hi-C data at the parthenogenetic 4-cell stage to analyze the consistency of the pseudo-mixed single-cell data with the bulk Hi-C results.

[0127] Pseudo-mixed single-cell data are single-cell data obtained by scAHi-C, which are directly merged data samples.

[0128] Mixing method: directly mix the obtained single-cell interaction matrix.

[0129] The Pearson, Spearman, and hierarchical correlation coefficients (SCC) between the parthenogenetic four-cell mixed sample and the population Hi-C data were calculated. The Pearson and Spearman correlation coefficients were calculated by hicCorrelate software, with values ​​of 0.97 and 0.96, respectively. The SCC was calculated by HiCRep, with a value of 0.91. Figure 5 As shown in the figure, the resolution of the left figure is 250kb; the resolution of the 40-70 Mb region in the right figure is 50kb, and the red square represents the maximum range value. It can be seen that this similarity can still be well maintained even at a larger resolution. The SCC of chromosome 2 (Chr 2) in the parthenogenetic embryo sample still reaches 0.85 at 50kb resolution. The above analysis suggests that the pseudo-population Hi-C of single-cell mixing has a high similarity with the real population Hi-C.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for fixing single cells in low melting point agar, characterized in that: The method includes placing the permeabilized single cells in a buffer solution and adding an equal volume of a low melting point agarose solution to fix the cells; The concentration of the low melting point agarose solution is 0.5% w / v.

2. The low melting point agar single cell fixation method according to claim 1, characterized in that: The step of adding an equal volume of the low melting point agarose solution further comprises standing at room temperature until solidification.

3. The low melting point agar single cell fixation method according to claim 1, characterized in that: The amount of the buffer and low melting point agarose solution added was 5 μl / single cell.

4. The low melting point agar single cell fixation method according to claim 1, characterized in that: The method for preparing permeabilized single cells comprises placing single cells fixed with 1% formaldehyde solution in a lysis buffer and performing permeabilization on ice.

5. The low melting point agar single cell fixation method according to claim 1, characterized in that: The permeabilized single cells are eluted at least once using a buffer containing 0.1% BSA before being placed in the buffer; Preferably, the buffer is 1×NEBuffer 3.

6. Use of the low melting point agar single cell fixation method according to any one of claims 1 to 5 in capturing chromatin conformation of single cells with high lipid droplet content.

7. A method for capturing chromatin conformation of single cells with high lipid droplet content, characterized in that: The following steps are involved: A. Fixing the permeabilized single cells with high lipid droplet content using the low melting point agar single cell fixation method according to any one of claims 1 to 5; B. Processing the nuclei of the cells obtained in step A; C. Biotin capture and library construction.

8. The chromatin conformation capture method according to claim 7, characterized in that: The step B includes performing subsequent MboI digestion, biotin filling labeling, DNA proximity ligation and cross-linking removal operations in a single cell fixed with low melting point agarose.

9. The chromatin conformation capture method according to claim 7, characterized in that: The step C includes melting low melting point agar at 65°C to release nuclear DNA, then capturing biotin-labeled DNA fragments by streptavidin magnetic beads at 37°C, and finally constructing a library by Tn5 transposase digestion. After the library is screened and purified, the final data is obtained by sequencing on the Illumina platform.

10. The chromatin conformation capture method according to any one of claims 7 to 9, characterized in that: Also included is step D, data analysis.

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