An automated single-cell Hi-C library construction method based on a digital microfluidic chip

Abstract

This invention discloses an automated single-cell Hi-C library construction method based on a digital microfluidic chip. The digital microfluidic chip has at least one droplet separation reaction unit, which includes a first loading region, a second loading region, a mixing reaction region, and two hydrophilic sites. The first loading region has a first loading electrode, the second loading region has a second loading electrode, and the mixing reaction region has multiple mixing reaction electrodes. The first loading region is connected to the mixing reaction region via several first delivery electrodes, and the second loading region is connected to the mixing reaction region via several second delivery electrodes. Larger droplets are formed by switching the first loading electrode and the first delivery electrode on and off, and smaller droplets are formed by switching the second loading electrode and the second delivery electrode on and off. This invention achieves single-cell Hi-C sequencing based on digital microfluidics, offering advantages such as high automation, minimal cell damage, and low cost, solving the problems of cumbersome operation and significant cell disturbance in existing single-cell technologies.

Description

Technical Field

[0001] This invention belongs to the field of digital microfluidics technology, specifically relating to an automated single-cell Hi-C (High-throughput Chromosome Conformation Capture) library construction method based on digital microfluidic chips. Background Technology

[0002] Hi-C technology can capture chromatin-genome-wide interactions at the million-cell level and create averaged three-dimensional genome maps, but it masks the differences in chromosome conformation between single cells. To overcome this bottleneck, it is necessary to develop single-cell Hi-C technology: completing cross-linking, enzyme digestion, ligation, amplification, and sequencing within a single cell, directly reading the unique spatial conformation of each chromosome, revealing how three-dimensional structures such as topological domains, chromatin loops / topological association domain boundary loops, and A / B compartments undergo random remodeling between cells, and pinpointing the spatial relationship between active gene domains and chromosome boundaries to the individual nucleus. Only by obtaining single-cell resolution three-dimensional chromosome maps can the population-averaged "static model" be transformed into a "dynamic probability map" characterizing cellular heterogeneity, thereby truly understanding how genome spatial structure regulates gene expression, replication, and repair, and guiding precise interventions in developmental and disease mechanisms.

[0003] Single-cell Hi-C sequencing overcomes the drawback of gene expression averaging in traditional population analyses. By screening individual cells, it reveals the three-dimensional structural information and gene interaction maps of individual cells, reflecting the heterogeneity of individual cells and achieving higher-precision research. Existing single-cell Hi-C technologies are mainly divided into three categories, but all have significant bottlenecks. Traditional direct separation methods, represented by scHi-C, rely on separating single cell nuclei one by one under a microscope. Although the data quality is acceptable, the throughput is extremely low (only a few dozen cells per run), the operation is cumbersome and time-consuming, and the experimental cycle is long, completely failing to meet the needs of large-scale research. Combinatorial splitting high-throughput methods, represented by sci-Hi-C, use a multi-round barcode strategy to increase throughput, but this results in extremely sparse data (only a few thousand contact points per cell), far lower than traditional methods; moreover, the experimental steps are complex, requiring precise control of cell dilution ratios to avoid tag collisions, and the data sparsity makes it difficult to achieve high-resolution three-dimensional structural reconstruction and allele analysis. In situ reaction high efficiency methods include snHi-C. snHi-C is only effective for samples with large nuclear volume and loose chromatin (such as oocytes). Its applicability to routine somatic cells has not been verified, and the nuclear extraction process is prone to mechanical damage.

[0004] In summary, current single-cell Hi-C technology struggles to achieve a balance between throughput, data quality, operational complexity, and sample applicability, severely hindering the development of large-scale single-cell three-dimensional genome studies. Summary of the Invention

[0005] The purpose of this invention is to overcome at least one of the defects of the existing technology and provide an automated single-cell Hi-C library construction method based on a digital microfluidic chip, which utilizes the dielectric wetting principle and hydrophilic sites to realize single-cell capture and reaction of the digital microfluidic chip.

[0006] To achieve the above objectives, one of the technical solutions of the present invention is: an automated single-cell Hi-C library construction method based on a digital microfluidic chip, wherein the digital microfluidic chip has at least one droplet separation reaction unit, the droplet reaction unit having a first loading region, a second loading region, and a mixing reaction region; the mixing reaction region has at least one hydrophilic site; the first loading region has a first loading electrode, the second loading region has a second loading electrode, and the mixing reaction region is provided with multiple mixing reaction electrodes; the first loading region is connected to the mixing reaction region through a plurality of first delivery electrodes, and the second loading region is connected to the mixing reaction region through a plurality of second delivery electrodes; larger droplets are formed by switching the first loading electrode and the first delivery electrode on and off, and smaller droplets are formed by switching the second loading electrode and the second delivery electrode on and off.

[0007] Specifically, the following steps are included:

[0008] (1) After washing the cells, count them under an optical microscope so that each centrifuge tube contains 100,000 cells. Then add cell fixation solution to fix the cells. After the fixation is completed, glycine quenches the cells. After washing with cell washing solution, resuspend the cells to prepare a cell mixture.

[0009] (2) The cell mixture is placed in the first sample loading area, and the first delivery electrode is energized sequentially to split the droplets and form cell mixture droplets. Then the cell mixture is allowed to pass through the hydrophilic site to capture single cell samples at the hydrophilic site.

[0010] (3) Place the membrane rupture premix in the second loading area, and sequentially energize the second delivery electrode to split the droplet and generate a membrane rupture droplet. Then, through the on-off coordination of the second delivery electrode and multiple mixing reaction electrodes, the membrane rupture droplet is transferred to the hydrophilic site and mixed with the cells to carry out the membrane rupture reaction to obtain a single cell nucleus. Then, the liquid at the hydrophilic site is removed to leave the single cell nucleus.

[0011] (4) The permeation premix is ​​placed in the second sample loading area. The droplets are split by sequentially energizing the second delivery electrode to generate permeation droplets. Then, by switching the second delivery electrode and multiple mixing reaction electrodes on and off, the permeation droplets are transferred to the hydrophilic site and mixed with the cell nucleus to carry out the permeation reaction.

[0012] (5) The permeation quenching premixed solution is placed in the second sample loading area. The droplets are split by sequentially energizing the second delivery electrode to generate permeation quenching droplets. Then, by switching the second delivery electrode and multiple mixing reaction electrodes on and off, the permeation quenching droplets are transferred to the hydrophilic site and mixed with the cell nucleus to carry out the quenching reaction.

[0013] (6) Place the enzyme digestion premixed solution in the second loading area, and sequentially energize the second delivery electrode to split the droplet and generate enzyme digestion premixed droplets. Then, through the on-off coordination of the second delivery electrode and multiple mixing reaction electrodes, the enzyme digestion premixed droplets are transferred to the hydrophilic site and mixed with the cell nucleus to carry out the enzyme digestion reaction.

[0014] (7) Place the premixed liquid for connection in the second loading area, and energize the second delivery electrode in sequence to split the droplet and generate a premixed liquid droplet for connection. Then, through the on-off coordination of the second delivery electrode and multiple mixing reaction electrodes, the premixed liquid droplet for connection is transferred to the hydrophilic site and mixed with the cell nucleus to carry out the connection reaction.

[0015] (8) Place the nucleus splitting premixed solution in the first sample loading area, and sequentially energize the first delivery electrode to split the droplet and generate a nucleus splitting premixed droplet. Then, through the on-off coordination of the first delivery electrode and multiple mixing reaction electrodes, the nucleus splitting premixed droplet is transferred to the hydrophilic site and mixed with the above-mentioned cell nucleus to carry out the nucleus splitting reaction.

[0016] (9) The material obtained in step (8) is sequentially subjected to Tn5 transposase fragmentation, PCR pre-amplification to increase product concentration, PCR amplification to add sequencing adapters, fragment sorting, and then Illumina next-generation sequencing.

[0017] In a preferred embodiment of the present invention, the washing in step (1) uses PBS buffer and centrifugation conditions of 4°C, 900 rpm, and 3 min, and is performed twice.

[0018] In a preferred embodiment of the present invention, the cell fixative in step (1) is 1.0% PFA, and the fixation conditions are room temperature rotation incubation for 10 min.

[0019] In a preferred embodiment of the present invention, the final concentration of glycine in step (1) is 125 mM, and the quenching condition is room temperature rotation incubation for 5 min.

[0020] In a preferred embodiment of the present invention, the cell washing solution in step (1) includes Tris-HCl, NaCl, protein inhibit cocktail and DNase / RNase-Free Deionized Water, wherein the volume ratio of Tris-HCl, NaCl, protein inhibit cocktail and DNase / RNase-Free Deionized Water is 1.0:0.2:10.0:88.8, the concentration of Tris-HCl is 1.0M and the pH is 8.0, the concentration of NaCl is 5.0M and the protein inhibitor cocktail is 10x.

[0021] In a preferred embodiment of the present invention, the cell mixture in step (2) includes a cell suspension and F68, wherein the volume ratio of the cell suspension to F68 is 20.0:1.0, and the concentration of F68 is 1.0%.

[0022] In a preferred embodiment of the present invention, the membrane rupture premix in step (3) comprises Tris-HCl, NaCl, Igepal CA 630, protein inhibit cocktail, and DNase / RNase-Free Deionized Water. The volume ratio of Tris-HCl, NaCl, Igepal CA 630, protein inhibit cocktail, and DNase / RNase-Free Deionized Water is 1.0:0.2:2.0:10.0:5.3. The concentration of Tris-HCl is 0.1M, the pH is 8.0, the concentration of NaCl is 0.5M, the concentration of Igepal CA 630 is 10%, the protein inhibit cocktail is 10x, and the reaction condition is 20 min on ice.

[0023] In a preferred embodiment of the present invention, the permeation premix in step (4) comprises SDS and PBS, wherein the volume ratio of SDS to PBS is 3:97, and the concentration of SDS is 10%.

[0024] In a preferred embodiment of the present invention, the permeation quenching premix in step (4) is 2.5% Triton-100.

[0025] In a preferred embodiment of the present invention, the enzyme digestion premix in step (6) includes 6.6x NEBuffer2, MboI, and F68, wherein the volume ratio of 10x NEBuffer2, MboI, and F68 is 5.0:15.0:0.1, the concentration of MboI is 7.5 U / μL, and the concentration of F68 is 1.0%.

[0026] In a preferred embodiment of the present invention, the premixed solution in step (7) comprises 10x T4 ligase Buffer, BSA (bovine serum albumin), T4 DNA ligase, and F68 (polyoxyethylene-polyoxypropylene block copolymer). The volume ratio of the 10x T4 ligase Buffer, BSA, T4 DNA ligase, F68, and DNase / RNase-Free Deionized Water is 5.0:0.5:1.0:0.05:3.5. The concentration of BSA is 20 mg / ml, the concentration of T4 DNA ligase is 70 U / μL, and the concentration of F68 is 1.0%.

[0027] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0028] 1. This invention is the first to realize single-cell Hi-C sequencing based on digital microfluidics, which has the advantages of high automation, minimal cell damage and low cost, and solves the problems of cumbersome operation and large cell disturbance in existing single-cell technology.

[0029] 2. This invention is based on a highly automated and integrated digital microfluidic chip and platform, which can build a fully automated single-cell sequencing platform.

[0030] 3. This invention uses a closed, hydrophobic digital microfluidic chip to construct single-cell Hi-C sequencing libraries, which can significantly reduce the risk of sample contamination by the external environment and reduce sample loss during library construction.

[0031] 4. This invention utilizes a digital microfluidic chip with a micro / nanoliter reaction volume for single-cell Hi-C sequencing library construction. The extremely small nanoliter reaction volume within the digital microfluidic chip significantly increases the effective reaction concentration, enhances Hi-C detection capability, overcomes the detection limitations of low abundance in single-cell samples, and effectively reduces reagent consumption, further lowering sequencing costs. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the structure of the digital microfluidic chip in Embodiment 1 of the present invention;

[0033] Figure 2 This is a structural diagram of a layout.

[0034] Figure 3 This is a schematic diagram of cell capture in a droplet.

[0035] Figure 4 This is a flowchart illustrating the construction process of a single-cell Hi-C library in Embodiment 2 of the present invention;

[0036] Figure 5 This refers to the quality of the single-cell Hi-C libraries of K562 and THP-1 in Example 2 of this invention.

[0037] Figure 6 This is a thermogram of side-by-side contact between K562 single cells and bulk Hi-C in Example 2 of the present invention; Detailed Implementation

[0038] Example 1

[0039] A digital microfluidic chip, from bottom to top, includes a fabrication electrode layer, a dielectric layer, and a hydrophobic layer, and is fabricated using photolithography techniques such as mask exposure, wet etching, and lift-off processes.

[0040] See Figures 1 to 3 In this embodiment, the dielectric layer of the digital microfluidic chip has droplet separation reaction units. These units, arranged from left to right, comprise four identical layouts, each with 24 droplet separation reaction zones, totaling 96 droplet separation reaction zones. Specifically, each layout includes a first sample loading zone 11, a second sample loading zone 12, and 22 droplet manipulation zones 13. Fifteen droplet manipulation zones 13 are arranged in three rows and five columns, forming a mixing reaction zone. The second and fourth droplet manipulation zones 13 in the second row have circular hydrophilic sites 14 in between. The remaining droplet separation reaction zones are used to connect the mixing reaction zone and the first sample loading zone 11, or to connect the mixing reaction zone and the second sample loading zone 12.

[0041] The upper surface of all 96 droplet separation reaction zones is provided with a hydrophobic layer, except for the hydrophilic site zone 14, which is provided with a hydrophilic site instead of a hydrophobic layer.

[0042] The electrode layer is distributed as follows: the bottom of the first sample loading area 11 has a first sample loading electrode, the bottom of the second sample loading area 12 has a second sample loading electrode, and the bottom of the mixing reaction area is provided with multiple mixing reaction electrodes; the first sample loading area 11 is connected to the mixing reaction area through several first delivery electrodes, and the second sample loading area 12 is connected to the mixing reaction area through several second delivery electrodes. Larger droplets are formed by switching the first sample loading electrode and the first delivery electrode on and off, and smaller droplets are formed by switching the second sample loading electrode and the second delivery electrode on and off.

[0043] The mask used for photomask exposure of this digital microfluidic chip was drawn using AutoCAD software, specifically as follows: Figure 1 As shown, the pattern has 48 spring-loaded target pads (3 rows, 16 pads per row) at the top and bottom, respectively. Each target pad has a lead wire that connects to the driving electrode in the central droplet manipulation area. Each droplet separation reaction unit contains two loading electrodes: the first loading electrode has a long side of 3.3 mm and a short side of 2.26 mm, and the second loading electrode has a long side of 2.5 mm and a short side of 1.7 mm, serving as the first and second loading areas for generating droplets of the corresponding volume of the reaction system. The remaining electrodes are used to move, merge, and mix droplets. The hydrophilic site with a radius of 0.11 mm is used to capture single cells and the reaction system. The gap between the parallel upper and lower electrodes of the digital microfluidic chip is 120 μm.

[0044] As droplet 21 passes through hydrophilic site 14, cells within the droplet can be captured, such as... Figure 3 As shown.

[0045] Example 2

[0046] A single-cell Hi-C library construction method based on the digital microfluidic chip of Embodiment 1 includes the following steps:

[0047] (1) After washing the cells, count them under an optical microscope so that each centrifuge tube contains about 100,000 cells. Then add cell fixation solution and incubate at room temperature for 10 min to fix the cells. After that, add glycine at a final concentration of 125 mM and incubate at room temperature for 5 min to quench the cells. Wash the cells with cell washing solution and resuspend them to prepare a cell mixture.

[0048] The formulation of the cell fixation solution used in this step is shown in Table 1 below:

[0049] Table 1

[0050]

[0051] The formulation of the cell washing solution in this step is shown in Table 2 below:

[0052] Table 2

[0053]

[0054] (1) The cell mixture is placed in the first sample loading area, and the first delivery electrode is energized sequentially. The droplets are split to form cell mixture droplets. Then the cell mixture is passed through the hydrophilic site to capture single cell samples. Under the microscope, if there are no single cells at the hydrophilic site, the droplets are controlled to repeatedly pass through the hydrophilic site until only single cells are captured. The specific principle of generating droplets by switching the electrode on and off is as follows: After the chip is energized, the droplets above it will move with it. When the droplets move to the hydrophilic area, the droplets continue to move and a small droplet will be left in the hydrophilic area.

[0055] The formulation of the cell mixture in this step is shown in Table 3 below:

[0056] Table 3

[0057]

[0058] (2) Place the membrane rupture premix in the second loading area, and sequentially energize the second delivery electrode to split the droplets and generate membrane rupture droplets. Then, through the on-off coordination of the second delivery electrode and multiple mixing reaction electrodes, the membrane rupture droplets are transferred to the hydrophilic site and mixed with the cells to carry out the membrane rupture reaction to obtain a single cell nucleus. Then, the single cell nucleus undergoes a programmed reaction at 4℃ for 20min.

[0059] The formulation of the premix solution used in this step is shown in Table 4 below:

[0060] Table 4

[0061]

[0062] (3) The permeation premix is ​​placed in the second sample loading area. The droplets are split by sequentially energizing the second delivery electrode to generate permeation droplets. Then, by switching the second delivery electrode and multiple mixing reaction electrodes on and off, the permeation droplets are transferred to the hydrophilic site and mixed with the cell nucleus to carry out the permeation reaction.

[0063] The formulation of the permeation premix solution for this step is shown in Table 5 below:

[0064] Table 5

[0065]

[0066] The permeation reaction procedure is shown in Table 6 below:

[0067] Table 6

[0068]

[0069] (4) The permeation quenching premixed solution is placed in the second sample loading area. The droplets are split by sequentially energizing the second delivery electrode to generate permeation quenching droplets. Then, by switching the second delivery electrode and multiple mixing reaction electrodes on and off, the permeation quenching droplets are transferred to the hydrophilic site and mixed with the cell nucleus to carry out the quenching reaction.

[0070] The formulation of the permeation quenching premix for this step is shown in Table 7 below:

[0071] Table 7

[0072]

[0073] The quenching reaction procedure is shown in Table 8 below:

[0074] Table 8

[0075]

[0076] (5) Place the enzyme digestion premixed solution in the second loading area, and sequentially energize the second delivery electrode to split the droplet and generate an enzyme digestion premixed droplet. Then, through the on-off coordination of the second delivery electrode and multiple mixing reaction electrodes, the enzyme digestion premixed droplet is transferred to the hydrophilic site and mixed with the cell nucleus to carry out the enzyme digestion reaction.

[0077] The formulation of the enzyme digestion premix for this step is shown in Table 9 below:

[0078] Table 9

[0079]

[0080] The procedure for the enzyme digestion reaction is shown in Table 10 below:

[0081] Table 10

[0082]

[0083] (6) Place the premixed liquid for connection in the second loading area, and energize the second delivery electrode in sequence to split the droplet and generate a premixed liquid droplet for connection. Then, through the on-off coordination of the second delivery electrode and multiple mixing reaction electrodes, the premixed liquid droplet for connection is transferred to the hydrophilic site and mixed with the cell nucleus to carry out the connection reaction.

[0084] The formulation of the premixed solution used in this step is shown in Table 11 below:

[0085] Table 11

[0086]

[0087] The procedure for the ligation reaction is shown in Table 12 below:

[0088] Table 12

[0089]

[0090] (7) Place the nucleus splitting premixed solution in the first sample loading area, and sequentially energize the first delivery electrode to split the droplet and generate a nucleus splitting premixed droplet. Then, through the on-off coordination of the first delivery electrode and multiple mixing reaction electrodes, the nucleus splitting premixed droplet is transferred to the hydrophilic site and mixed with the above-mentioned cell nucleus to carry out the nucleus splitting reaction.

[0091] The formulation of the premixed solution used in this step is shown in Table 13 below:

[0092] Table 13

[0093]

[0094] The procedure for the ligation reaction is shown in Table 14 below:

[0095] Table 14

[0096]

[0097] (8) The material obtained in step (7) is subjected to Tn5 transposase fragmentation, PCR pre-amplification to increase product concentration, PCR amplification to add sequencing adapters, fragment sorting, and then Illumina next-generation sequencing.

[0098] In this embodiment, 0.8x AMpureXP magnetic beads were used for DNA purification. Tn5 transposase fragmentation and subsequent steps were performed in accordance with the Hi-C treatment method in the sc-CARE method. For details, please refer to Qu, J., Sun, J., Zhao, C., E. et al. Nature Structural & Molecular Biology, 2023, 30(9), 1393–1402.

[0099] Based on the above steps, as follows Figure 2 As shown, this embodiment uses K562 cells and THP-1 cells to construct single-cell Hi-C libraries. First, cells are fixed and permeabilized with paraformaldehyde, then diluted to an appropriate concentration. Next, single cells are captured on a digital microfluidic chip, and cell membrane lysis buffer is introduced for lysis to obtain single cell nuclei. The nuclear membrane is then permeabilized with SDS to facilitate the entry of subsequent reagents. SDS is quenched using Triton X-100. Enzyme digestion reagents are then introduced for digestion, followed by ligation reagents for ligation to achieve spatially adjacent chromatin ligation. Nucleus splitting is then performed to release the proximal-ligated DNA. Subsequently, Tn5 transposase fragmentation and PCR pre-amplification are performed to increase DNA abundance, followed by PCR amplification, addition of sequencing adapters, column purification, fragment sorting, and Illumina next-generation sequencing. The quality of the single-cell Hi-C libraries for both cell types is as follows: Figure 3 As shown, the Hi-C library data obtained in this embodiment has a normal alignment rate, which is better than the contact number and high long-range contact ratio of most current single-cell Hi-C methods, proving that single-cell Hi-C information based on digital microfluidics has accuracy and can obtain high-quality data.

[0100] The side-by-side contact heatmap of single-cell data and bulk level data for K562 is shown below. Figure 4 As shown, it is demonstrated that clear three-dimensional structural information can be obtained from single-cell Hi-C based on digital microfluidics.

[0101] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. An automated single-cell Hi-C library construction method based on a digital microfluidic chip, characterized in that, The digital microfluidic chip has at least one droplet separation reaction unit, which has a first sample loading area, a second sample loading area, and a mixing reaction area. The mixing reaction area has at least one hydrophilic site. The first sample loading area has a first sample loading electrode, the second sample loading area has a second sample loading electrode, and the mixing reaction area has multiple mixing reaction electrodes. The first sample loading area is connected to the mixing reaction area through a plurality of first delivery electrodes, and the second sample loading area is connected to the mixing reaction area through a plurality of second delivery electrodes. Large droplets are formed by switching the first sample loading electrode and the first delivery electrode on and off, and small droplets are formed by switching the second sample loading electrode and the second delivery electrode on and off. The method includes the following steps: (1) After washing the cells, count them under an optical microscope to ensure that each centrifuge tube contains the predetermined cell concentration. Then, add cell fixation solution to fix the cells. After the fixation is completed, quench the cells with glycine. Wash the cells with cell washing solution and resuspend them to prepare a cell mixture. (2) The cell mixture is placed in the first sample loading area, and the droplets are sequentially energized through the first delivery electrode to split the droplets into cell droplets. Then, the cell droplets are electrically driven to the reaction electrode containing hydrophilic sites to capture single-cell samples at the hydrophilic sites. (3) Place the membrane rupture premix in the second loading area, and sequentially energize the second delivery electrode to split the droplet and generate a membrane rupture droplet. Then, through the on-off coordination of the second delivery electrode and multiple mixing reaction electrodes, the membrane rupture droplet is transferred to the hydrophilic site and mixed with the cells to carry out the membrane rupture reaction to obtain a single cell nucleus. Then, the liquid at the hydrophilic site is removed to leave the single cell nucleus. (4) The permeation premix is ​​placed in the second sample loading area. The droplets are split by sequentially energizing the second delivery electrode to generate permeation droplets. Then, by switching the second delivery electrode and multiple mixing reaction electrodes on and off, the permeation droplets are transferred to the hydrophilic site and mixed with the cell nucleus to carry out the permeation reaction. (5) The permeation quenching premixed solution is placed in the second sample loading area. The droplets are split by sequentially energizing the second delivery electrode to generate permeation quenching droplets. Then, by switching the second delivery electrode and multiple mixing reaction electrodes on and off, the permeation quenching droplets are transferred to the hydrophilic site and mixed with the cell nucleus to carry out the quenching reaction. (6) Place the enzyme digestion premixed solution in the second loading area, and sequentially energize the second delivery electrode to split the droplet and generate enzyme digestion premixed droplets. Then, through the on-off coordination of the second delivery electrode and multiple mixing reaction electrodes, the enzyme digestion premixed droplets are transferred to the hydrophilic site and mixed with the cell nucleus to carry out the enzyme digestion reaction. (7) Place the premixed liquid for connection in the second loading area, and energize the second delivery electrode in sequence to split the droplet and generate a premixed liquid droplet for connection. Then, through the on-off coordination of the second delivery electrode and multiple mixing reaction electrodes, the premixed liquid droplet for connection is transferred to the hydrophilic site and mixed with the cell nucleus to carry out the connection reaction. (8) Place the nucleus splitting premixed solution in the first sample loading area, and sequentially energize the first delivery electrode to split the droplet and generate a nucleus splitting premixed droplet. Then, through the on-off coordination of the first delivery electrode and multiple mixing reaction electrodes, the nucleus splitting premixed droplet is transferred to the hydrophilic site and mixed with the above-mentioned cell nucleus to carry out the nucleus splitting reaction. (9) The material obtained in step (8) is sequentially subjected to Tn5 transposase fragmentation, PCR pre-amplification to increase product concentration, PCR amplification to add sequencing adapters, fragment sorting, and then Illumina next-generation sequencing.

2. The automated single-cell Hi-C library construction method based on a digital microfluidic chip as described in claim 1, characterized in that, The cell washing solution in step (1) includes Tris-HCl, NaCl, protein inhibiting cocktail, and DNase / RNase-Free Deionized Water. The volume ratio of Tris-HCl, NaCl, protein inhibiting cocktail, and DNase / RNase-Free Deionized Water is 1.0:0.2:10.0:88.

8. The concentration of Tris-HCl is 1.0M and the pH is 8.

0. The concentration of NaCl is 5.0M and the concentration of protein inhibiting cocktail is 10x.

3. The automated single-cell Hi-C library construction method based on a digital microfluidic chip as described in claim 1, characterized in that, In step (2), the cell mixture includes a cell suspension and F68, with a volume ratio of 20.0:1.0 for the cell suspension and a concentration of 1.0% for F68.

4. The automated single-cell Hi-C library construction method based on a digital microfluidic chip as described in claim 1, characterized in that, The membrane rupture premix in step (3) includes Tris-HCl, NaCl, Igepal CA 630, protein inhibit cocktail, and DNase / RNase-Free Deionized Water. The volume ratio of Tris-HCl, NaCl, Igepal CA 630, protein inhibit cocktail, and DNase / RNase-Free Deionized Water is 1.0:0.2:2.0:10.0:5.

3. The concentration of Tris-HCl is 0.1M, the pH is 8.0, the concentration of NaCl is 0.5M, the concentration of Igepal CA 630 is 10%, and the concentration of protein inhibit cocktail is 10x. The reaction condition is to place the mixture on ice for 20 minutes.

5. The automated single-cell Hi-C library construction method based on a digital microfluidic chip as described in claim 1, characterized in that, The permeation premix in step (4) includes SDS and PBS, with a volume ratio of SDS to PBS of 3:97, and the concentration of SDS is 10%.

6. The automated single-cell Hi-C library construction method based on a digital microfluidic chip as described in claim 1, characterized in that, The permeation reaction procedure in step (4) is 62℃ for 10 min, followed by storage at 4℃; The permeation reaction procedure in step (5) is as follows: 37℃, 15min: then store at 4℃; The procedure for the enzyme digestion reaction in step (6) is as follows: 37℃, 12h; 65℃, 20min; then store at 4℃. The procedure for the ligation reaction in step (7) is as follows: 16℃, 4h; 65℃, 20min; then store at 4℃. The procedure for the nucleus splitting reaction in step (8) is: 50℃, 3h; 70℃, 1h; then store at 4℃.

7. The automated single-cell Hi-C library construction method based on a digital microfluidic chip as described in claim 1, characterized in that, The enzyme digestion premix in step (6) includes 6.6x NEBuffer2, MboI, and F68, with a volume ratio of 5.0:15.0:0.

1. The concentration of MboI is 7.5 U / μL, and the concentration of F68 is 1%.

8. The automated single-cell Hi-C library construction method based on a digital microfluidic chip as described in claim 1, characterized in that, The ligation premix in step (7) includes 10x T4 ligase Buffer, BSA, T4 DNA ligase, and F68. The volume ratio of the 10x T4 ligase Buffer, BSA, T4 DNA ligase, F68, and DNase / RNase-Free Deionized Water is 5.0:0.5:1.0:0.05:3.

5. The concentration of BSA is 20 mg / ml, the concentration of T4 DNA ligase is 70 U / μL, and the concentration of F68 is 1.0%.

9. The automated single-cell Hi-C library construction method based on a digital microfluidic chip as described in claim 1, characterized in that, The nucleus splitting premix in step (8) includes Tris-HCl, NaCl, Triton-100, EDTA, DTT, protease, and DNase / RNase-Free Deionized Water. The volume ratio of Tris-HCl, NaCl, Triton-100, EDTA, DTT, protease, and DNase / RNase-Free Deionized Water is 4.0:2.0:3.0:1.0:5.0:1.0:4.

0. The concentration of Tris-HCl is 100 mM and the pH is 8.

0. The concentration of NaCl is 0.5 M. The concentration of Triton-100 is 1.0%. The concentration of EDTA is 20 mM. The concentration of DTT is 100 mM. The concentration of protease is 10 mg / ml.

10. The application of the automated single-cell Hi-C library preparation method based on digital microfluidic chips as described in any one of claims 1-9 in single-cell Hi-C sequencing.

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