Single-cell sequencing microfluidic chip suitable for high-impurity-content samples and large-volume cell samples, and use thereof

Abstract

A single-cell sequencing microfluidic chip suitable for high-impurity-content samples and large-volume cell samples, and a single-cell sequencing method. A microfluidic channel consists of the following components: three sample inlets, one sample collection port, five microchannels, and one shearing port. The components are communicated along a specific path, thereby enabling generation and collection of "water-in-oil" microdroplets. The sequencing method comprises nine steps in total: chip assembly, reagent preparation and formulation, reagent loading, microdroplet generation, reverse transcription and demulsification, library construction, data volume sequencing, and data analysis.

Description

Microfluidic chips for single-cell sequencing suitable for samples with high proportion of impurities and large-volume cell samples and their applications Technical Field

[0001] This invention belongs to the field of combined biological and microfluidic technology, specifically relating to a droplet generation chip and its application and usage in single-cell sequencing. Background Technology

[0002] Single-cell sequencing technology has become a core tool in life science research in recent years, and has had a revolutionary impact on research fields such as immunology, oncology, developmental biology, and evolutionary biology, greatly promoting the development of these disciplines.

[0003] The most commonly used single-cell sequencing process is microfluidic technology based on droplet generation. The principle is to use methods such as photolithography and thermo-pressing to form tiny channels with diameters of tens to hundreds of micrometers on the surface of materials such as PDMS and COC, and to prepare microfluidic chips. With the help of driving forces such as air pressure, the flow of liquids such as cell suspension and buffer solution in the channels is controlled. With the assistance of specific molecular biology and enzymology methods, the genetic information in a single cell can be captured.

[0004] After years of development, single-cell sequencing based on droplet microfluidic chips has become a relatively mature technology, capable of handling most types of biological samples and cell types. However, for some special samples with a lot of impurities such as cell debris, such as brain tissue cell nuclear suspensions, and some special cells with larger volumes, such as neurons and egg cells, single-cell sequencing experiments cannot be effectively carried out due to the limitations of the flow channels. There is an urgent need to develop a new method to solve this problem. Summary of the Invention

[0005] This invention addresses the problem that existing single-cell sequencing chips, with their microfluidic channel designs, are unsuitable for processing samples containing a high proportion of impurities or large cells, thus limiting the widespread application of single-cell sequencing. Based on extensive research and experimentation, this invention provides a single-cell sequencing microfluidic chip suitable for samples with a high proportion of impurities and large cells. Furthermore, this invention also provides applications for single-cell sequencing experiments using this chip. This invention solves the problems in the field of single-cell sequencing, such as the inability to obtain high-quality data from difficult samples and the potential loss of cell types, laying a solid foundation for the wider application of single-cell sequencing and the acquisition of better data and results.

[0006] The single-cell sequencing microfluidic chip proposed in this invention, suitable for samples with a high proportion of impurities and large-volume cell samples, includes: an inlet, a outlet, a microchannel, and a shearing port; wherein,

[0007] The sample inlets include: an oil sample inlet, a microbead sample inlet, and a cell sample inlet; wherein...

[0008] The oil inlet is used to load the oil phase liquid; the microbead inlet is used to load microbeads with nucleic acid sequence tags; the cell inlet is used to load the sample and the corresponding reaction reagents.

[0009] The sample collection port is used to collect "water-in-oil" droplets containing microbeads and cells;

[0010] The microchannels include: oil microchannels, microbead microchannels, cell microchannels, mixing microchannels, and droplet microchannels; wherein...

[0011] The inlet end of the oil microchannel is connected to the oil inlet port, and the outlet end is connected to the shear port.

[0012] The inlet end of the microbead microchannel is connected to the microbead inlet, and the outlet end is connected to the mixing microchannel.

[0013] The inlet end of the cell microchannel is connected to the cell inlet, and the outlet end is connected to the mixing microchannel.

[0014] The inlet end of the hybrid microchannel is connected to the intersection of the microbead microchannel and the cell microchannel, and the outlet end is connected to the shearing port.

[0015] The inlet end of the microdroplet microchannel is connected to the shearing port, and the outlet end is connected to the sample collection port.

[0016] The shearing port is located at the intersection of the mixing microchannel and the oil microchannel, and is connected to the droplet microchannel.

[0017] In this invention, the oil microchannel has a bifurcated structure, branching at the oil inlet outlet and then converging again at the shearing port; the oil microchannel has a loop-shaped bend structure near the oil inlet and near the shearing port; the microbead microchannel has a loop-shaped bend structure near the microbead inlet and a funnel-shaped structure near the mixing microchannel; the cell microchannel has a loop-shaped bend structure near the cell inlet; and the droplet microchannel has a wave-shaped bend structure.

[0018] In this invention, the oil microchannel has a cross-sectional depth of 150 μm and a width of 100-150 μm. The funnel-shaped structure of the microbead microchannel has a depth of 50 μm and a width of 50-200 μm, wherein the width of the microchannel outlet is 50 μm; the angle between the funnel-shaped structure and the cell microchannel is a right angle; the microchannel in front of the funnel-shaped structure has a cross-sectional depth of 50 μm and a width of 50 μm.

[0019] In this invention, the cellular microchannel has a cross-sectional depth of 150 μm and a width of 100-150 μm; the mixing microchannel has a cross-sectional depth of 150 μm, a width of 100-150 μm, and a length of 100 μm; and the droplet microchannel has a cross-sectional depth of 220 μm and a width of 150 μm.

[0020] In this invention, the waveform bend structure of the microdroplet microchannel has a radius of curvature of 200~300μm.

[0021] Based on the above chip, this invention also proposes a single-cell sequencing method, which includes the following steps:

[0022] Step 1, Assemble the chip: Connect the gas pipeline to each sample inlet and connect the liquid recovery pipeline to the sample collection port;

[0023] Step 2, Prepare reagents: Dissolve the reverse transcription buffer, reducing agent, and reverse transcriptase separately; remove the oligonucleotide chains and microbeads and allow them to return to room temperature.

[0024] Step 3, Prepare reagents: Prepare reaction buffer by mixing the prepared reaction buffer with an appropriate amount of single cell or single cell nucleus suspension to form the loading buffer;

[0025] Step 4, loading reagents: Load the oil into the oil inlet, load the microbeads into the microbead inlet; load the buffer solution into the cell inlet;

[0026] Step 5, generating microdroplets: Activate the pneumatic device to drive each liquid to flow in its respective microchannel to generate and recover microdroplets;

[0027] Step 6, Reverse Transcription and Demulsification: Reverse transcription is performed using PCR, and cDNA is collected after demulsification.

[0028] Step 7: Construct sequencing libraries;

[0029] Step 8: Sequencing 100G of data using a sequencer;

[0030] Step 9: The analysis platform performs the analysis.

[0031] In step 3 of this invention, the reaction buffer contains 8.0 μL of reverse transcriptase, 18.8 μL of reverse transcription buffer, 3.0 μL of oligonucleotide chain, and 4.0 μL of reducing agent. The loading buffer contains 33.8 μL of reaction buffer, 6% (v / v) iodixanol, 4% (v / v) PEG8000, and single-cell suspension or single-cell nucleus suspension diluted in an appropriate ratio; wherein the impurity ratio of the single-cell suspension or single-cell nucleus suspension is higher than 30%, and there are fragments or clumps with a length greater than 100 μm; preferably, 60% iodixanol, 7.5 μL, final concentration 6% (v / v); 40% PEG8000, 7.5 μL, final concentration 4% (v / v); single-cell suspension, 26.2 μL.

[0032] In step 4 of this invention, the loading process is adjusted according to the chip. For example, the PDMS chip can be connected to each injection port through a 200μL pipette tip via a Teflon tube, and the oil, Single Cell 3' Gel Bead, and loading buffer are loaded into the pipette tip connected to the corresponding injection port.

[0033] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0034] First, it fills a technological gap. Currently, there is a lack of single-cell sequencing chips suitable for high-background samples containing a lot of impurities, which severely limits the application of single-cell sequencing technology in samples such as brain and bone marrow. This invention solves this problem by innovating the chip microfluidic channels and adaptively modifying the reagent system, providing a solid foundation for the wider application of single-cell sequencing.

[0035] Secondly, it is easy to operate and significantly improves data quality. The chip structure provided by this invention is applicable to various materials such as PDMS and COC. Combined with a pneumatic device, it enables a flexible and adjustable single-cell sequencing workflow, which greatly promotes the improvement of data quality. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 is a schematic diagram of the microfluidic chip structure of the present invention.

[0038] Figure 2 shows the microscopic examination results of single-cell nucleus suspension of mouse brain tissue according to the present invention. Detailed Implementation

[0039] The invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the invention are all common knowledge and general knowledge in the art, and the invention does not have any particular limitations.

[0040] In Figure 1, 1-oil inlet; 2-microbead inlet; 3-cell inlet; 4-sample collection port; 5-oil microchannel; 6-microbead microchannel; 7-cell microchannel; 8-mixing microchannel; 9-droplet microchannel; 10-shearing port.

[0041] The single-cell sequencing microfluidic chip proposed in this invention is suitable for samples with a high proportion of impurities and large-volume cell samples. Its microfluidic channel consists of the following parts: 3 sample inlets, 1 sample outlet, 5 microchannels, and 1 shearing port, wherein:

[0042] The oil inlet, also known as the first inlet (in order from left to right according to the drawing), is used to load the oil phase liquid.

[0043] The microbead inlet, also known as the second inlet, is used to load microbeads with nucleic acid sequence tags.

[0044] The cell inlet, also known as the third inlet, is used to load the sample (single-cell suspension) and the corresponding reaction reagents.

[0045] The collection port is used to collect "water-in-oil" droplets containing microbeads and cells.

[0046] The oil microchannel, also known as the first microchannel (in order from left to right in the drawing), has a bifurcated structure. After branching at the outlet, the two paths converge again at the shear port. Its inlet end connects to the oil injection port, and its outlet end connects to the shear port, used to constrain the flow of the oil phase liquid within it and guide it to the shear port. In its structure, the end near the oil injection port and the end near the shear port include a continuous loop-shaped bend structure to stabilize the pressure within the microchannel.

[0047] The microbead microchannel, also known as the second microchannel, has an inlet end connected to the microbead inlet and an outlet end connected to the mixing microchannel inlet. It is used to constrain the flow of microbeads carrying nucleic acid sequence tags and guide them to the shearing port. Its structure includes a continuous loop-shaped bend near the microbead inlet and a funnel-shaped structure near the microchannel outlet for ordering the microbeads.

[0048] The cell microchannel, also known as the third microchannel, has an inlet end connected to the cell inlet and an outlet end connected to the mixing microchannel inlet. It is used to constrain cell flow within the channel and guide it to the shearing port. Its structure includes a continuous loop-shaped bend near the cell inlet.

[0049] The mixing microchannel, also known as the fourth microchannel, has its inlet end at the intersection of the microbead microchannel and the cell microchannel, and its outlet end connected to the shear port. It is used to constrain the flow of the liquid after the microbeads and cells are mixed and to guide it to the shear port.

[0050] The microdroplet microchannel, also known as the fifth microchannel, has an inlet end connected to a shearing port and an outlet end connected to a sample collection port. It is used to constrain the flow of "water-in-oil" microdroplets encapsulating microbeads and cells. Its structure includes a wavy, curved section to enhance the mixing of liquids within the microdroplets.

[0051] The shearing port is located at the intersection of the mixing microchannel and the oil microchannel, and is connected to the droplet microchannel.

[0052] The oil microchannel has a cross-sectional dimension of 150×(100~150)μm (depth×width), preferably 150×150μm.

[0053] The funnel-shaped structure of the microbead microchannel has a depth of 50 μm and a width of 50–200 μm, with the microchannel outlet having a width of 50 μm. The angle between the outlet and the microchannel is a right angle. The microchannel preceding the funnel-shaped structure has a cross-sectional dimension of 50 × 50 μm.

[0054] The cell microchannel has a cross-sectional size of 150×(100~150)μm, preferably 150×150μm.

[0055] The hybrid microchannel has a cross-sectional dimension of 150×(100~150)μm and a length of 100μm, preferably a cross-sectional dimension of 150×150μm and a length of 100μm.

[0056] The microdroplet microchannel has a cross-sectional dimension of 220×150μm. The wave-shaped bend structure has a radius of curvature of 200~300μm, preferably 220μm.

[0057] In addition, this invention also provides an application of this chip for single-cell sequencing experiments, including the following steps:

[0058] (1) Assemble the chip: Connect the gas pipeline to each sample inlet and connect the liquid pipeline for recovery to the sample collection port.

[0059] (2) Prepare reagents: In accordance with the official instructions of 10x Genomics (number CG000204 Rev C), RT Reagent B (reverse transcription buffer), Reducing Agent B (reducing agent), and RT Enzyme C (reverse transcriptase) were dissolved at 4℃ after being placed at -20℃.

[0060] Remove the Template Switch Oligo (oligonucleotide chain) and Single Cell 3' Gel Bead (microbead, catalog number 1000122) from -80℃ and allow them to return to room temperature.

[0061] (3) Preparation of reagents: Refer to the official instructions of 10x Genomics (number CG000204 Rev C) to prepare the reaction buffer (Master Mix). Mix the prepared reaction buffer with an appropriate amount of single cell or single cell nucleus suspension to form the loading buffer.

[0062] (4) Loading reagents: Load the partitioning oil into the oil inlet. Load the Single Cell 3' Gel Bead into the microbead inlet. Load the loading buffer into the cell inlet.

[0063] (5) Generation of microdroplets: Start the air pressure device to drive each liquid to flow in its own microchannel to generate and recover microdroplets.

[0064] (6) Reverse transcription and demulsification: cDNA was collected by reverse transcription and demulsification using PCR reaction, in accordance with the official instructions of 10x Genomics (number CG000204 Rev C).

[0065] (7) Constructing the library: Construct the sequencing library according to the official instructions of 10x Genomics (number CG000204 Rev C).

[0066] (8) Sequencing: 100G of data was sequenced using an Illumina sequencer.

[0067] (9) Data analysis and result interpretation: The analysis was conducted using the 10x Genomics analytics platform.

[0068] In step (1), the assembly process is carried out according to the chip's material, size, etc. For example, PDMS chips can be connected using Teflon tubing, as detailed in the embodiments.

[0069] In step (3), the reaction buffer (Master Mix) contains 8.0 μL RT Enzyme C (reverse transcriptase), 18.8 μL RT Reagent B (reverse transcription buffer), 3.0 μL Template Switch Oligo (oligonucleotide chain), and 4.0 μL Reducing Agent B (reducing agent).

[0070] In step (3), the buffer solution comprises 33.8 μL of reaction buffer (Master Mix), 6% (v / v) iodixanol, 4% (v / v) PEG8000, and single-cell suspension or single-cell nucleus suspension diluted in an appropriate ratio. The single-cell suspension or single-cell nucleus suspension contains more than 30% impurities (number of impurities / number of cells) and contains fragments or clumps longer than 100 μm.

[0071] In step (4), the loading process is adjusted according to the chip. For example, the PDMS chip can be connected to each injection port through a 200μL pipette tip via a Teflon tube, and the partitioning oil, single cell 3' gel bead, and loading buffer are loaded into the pipette tip connected to the corresponding injection port.

[0072] In step (5), the air pressure device is a commercially available pump that can provide gas pressure and regulate the gas pressure.

[0073] Example 1

[0074] This embodiment describes the application of the microfluidic chip of the present invention in single-cell sequencing of mouse brain tissue (Figure 2). The microfluidic chip in this example is made of PDMS material and is mounted on a glass slide. The cross-sectional dimensions of its oil microchannels, cell microchannels, and mixing microchannels are all 150×150μm (depth×width), and the other parameters are consistent with the description in the invention.

[0075] Assemble chips.

[0076] Cut a 7mm Teflon tube and connect it to the tip of a 200μL pipette. Insert the connected Teflon tube into the wells of the oil inlet, microbead inlet, and cell inlet, respectively. Cut a 15cm length of Teflon tube and insert it into the well of the sample collection port.

[0077] Prepare reagents.

[0078] Following the official instructions from 10x Genomics (product number CG000204 Rev C), RT Reagent B (reverse transcription buffer), Reducing Agent B (reducing agent), and RT Enzyme C (reverse transcriptase) were dissolved at 4°C after being placed at -20°C. Template Switch Oligo (oligonucleotide chains) and Single Cell 3' Gel Beads (microbeads, product number 1000122) were then removed from -80°C and allowed to return to room temperature.

[0079] Prepare reagents.

[0080] To prepare the reaction buffer, add the appropriate volumes of each component sequentially according to the following steps:

[0081] RT Reagent B, 18.8 μL;

[0082] Template Switch Oligo, 3.0μL;

[0083] Reducing Agent B, 4.0 μL;

[0084] RT Enzyme C, 8.0 μL.

[0085] Add the appropriate volumes of each component sequentially according to the following steps to prepare the buffer solution for use:

[0086] Reaction buffer, 33.8 μL;

[0087] 60% iodixanol, 7.5 μL;

[0088] 40% PEG8000, 7.5μL;

[0089] Single-cell nucleus suspension, 26.2 μL, impurity ratio >30%, containing cell nucleus clusters longer than 100 μm (Figure 2).

[0090] Load reagents.

[0091] Load 200 μL of partitioning oil into the pipette tip on the oil inlet. Load 50 μL of a room-temperature Single Cell 3' Gel Bead into the pipette tip on the bead inlet. Load the entire 75 μL of loading buffer into the pipette tip on the cell inlet. Take care to avoid generating air bubbles during the addition of all liquids to the pipette tips.

[0092] Droplets are generated.

[0093] Place the chip in an inverted microscope, activate the air pressure device, and observe the formation of "water-in-oil" droplets at the shear cut. Adjust the pressure in each tube as needed based on the droplet formation.

[0094] Reverse transcription and demulsification.

[0095] According to the official instructions from 10x Genomics (number CG000204 Rev C), cDNA was collected by reverse transcription and demulsification via PCR.

[0096] Build a library.

[0097] The sequencing library was constructed according to the official instructions from 10x Genomics (number CG000204 Rev C).

[0098] Sequencing.

[0099] 100G of data was sequenced using an Illumina sequencer.

[0100] Data analysis and interpretation of results.

[0101] Analysis was performed using the 10x Genomics platform. The results showed that the number of cells captured reached 19056, the median gene count reached 1576, and all quality control indicators were normal (Table 1). This indicates that the microfluidic chip and corresponding technical solution described in this invention can effectively achieve successful loading of high-background single-cell nucleus suspensions and obtain high-quality data.

[0102] Example 2

[0103] This embodiment describes the application of the microfluidic chip of the present invention in single-cell sequencing of mouse brain tissue (Figure 2). The microfluidic chip in this example is made of PDMS material and is mounted on a glass slide. The cross-sectional dimensions of its oil microchannel, cell microchannel, and mixing microchannel are all 150×100μm (depth×width), and the other parameters are consistent with those described in Example 1.

[0104] The results showed that the number of cells captured in this test reached 18,572, the median gene count reached 1,566, and all quality control indicators were normal (Table 1). This indicates that the microfluidic chip and corresponding technical solution described in this invention can effectively achieve the successful loading of high-background single-cell nucleus suspensions and obtain high-quality data.

[0105]

[0106] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0107] As used in this invention, the term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention but does not exclude other aspects.

[0108] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0109] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A single-cell sequencing microfluidic chip suitable for samples with a high proportion of impurities and large-volume cell samples, characterized in that, include: The sample inlet, the sample outlet (4), the microchannel, and the shearing port (10) are all part of the sample inlet. The sample inlets include: an oil inlet (1), a microbead inlet (2), and a cell inlet (3); wherein, The oil inlet (1) is used to load the oil phase liquid; the microbead inlet (2) is used to load microbeads with nucleic acid sequence tags; the cell inlet (3) is used to load the sample and the corresponding reaction reagents; The sample collection port (4) is used to collect "water-in-oil" droplets containing microbeads and cells; The microchannels include: oil microchannels (5), microbead microchannels (6), cell microchannels (7), mixing microchannels (8), and droplet microchannels (9); wherein, The inlet end of the oil microchannel (5) is connected to the oil injection port (1), and the outlet end is connected to the shear port (10); The inlet end of the microbead microchannel (6) is connected to the microbead inlet (2), and the outlet end is connected to the mixing microchannel (8); The inlet end of the cell microchannel (7) is connected to the cell inlet (3), and the outlet end is connected to the mixing microchannel (8); The inlet end of the hybrid microchannel (8) is connected to the intersection of the microbead microchannel (6) and the cell microchannel (7), and the outlet end is connected to the shear port (10). The inlet end of the microdroplet microchannel (9) is connected to the shearing port (10), and the outlet end is connected to the sample receiving port (4); The shearing port (10) is located at the intersection of the mixing microchannel (8) and the oil microchannel (5), and is connected to the droplet microchannel (6).

2. The microfluidic chip as described in claim 1, characterized in that, The oil microchannel (5) has a bifurcated structure. After bifurcating at the outlet end of the oil inlet (1), the two paths converge again at the shear port (10). The oil microchannel (5) has a loop-shaped bend structure near the oil inlet (1) and near the shear port (10). And / or, The microbead microchannel (6) has a meander structure near the microbead inlet and a funnel-shaped structure near the mixing microchannel (8). And / or, The cell microchannel (7) has a meander structure near one end of the cell inlet (3); And / or, The microdroplet microchannel (9) is provided with a wave-shaped bend structure.

3. The microfluidic chip as described in claim 1, characterized in that, The cross-sectional depth of the oil microchannel (5) is 150 μm and the width is 100~150 μm.

4. The microfluidic chip as described in claim 1, characterized in that, The funnel-shaped structure of the microbead microchannel (6) has a depth of 50 μm and a width of 50~200 μm, wherein the width of the microchannel outlet is 50 μm; And / or, The angle between the funnel-shaped structure and the cell microchannel (7) is a right angle; And / or, The microchannel in front of the funnel-shaped structure has a cross-sectional depth of 50 μm and a width of 50 μm.

5. The microfluidic chip as described in claim 1, characterized in that, The cross-sectional depth of the cell microchannel (7) is 150 μm and the width is 100~150 μm; And / or, The cross-sectional depth of the hybrid microchannel (8) is 150 μm, the width is 100~150 μm, and the length is 100 μm; And / or, The cross-sectional depth of the microdroplet microchannel (9) is 220 μm and the width is 150 μm.

6. The microfluidic chip as described in claim 1, characterized in that, The wave-shaped curved structure of the microdroplet microchannel has a radius of curvature of 200~300μm.

7. A single-cell sequencing method, characterized in that, The method employs a microfluidic chip as described in any one of claims 1-6, and the method includes the following steps: Step 1, Assemble the chip: Connect the gas pipeline to each sample inlet and connect the liquid recovery pipeline to the sample collection port; Step 2, Prepare reagents: Dissolve the reverse transcription buffer, reducing agent, and reverse transcriptase separately; remove the oligonucleotide chains and microbeads and allow them to return to room temperature. Step 3, Prepare reagents: Prepare reaction buffer by mixing the prepared reaction buffer with an appropriate amount of single cell or single cell nucleus suspension to form the loading buffer; Step 4, loading reagents: Load the oil into the oil inlet, load the microbeads into the microbead inlet; load the buffer solution into the cell inlet; Step 5, generating microdroplets: Activate the pneumatic device to drive each liquid to flow in its respective microchannel to generate and recover microdroplets; Step 6, Reverse Transcription and Demulsification: Reverse transcription is performed using PCR, and cDNA is collected after demulsification. Step 7: Construct sequencing libraries; Step 8: Sequencing 100G of data using a sequencer; Step 9: The analysis platform performs the analysis.

8. The method as described in claim 7, characterized in that, In step 3, the reaction buffer contains 8.0 μL of reverse transcriptase, 18.8 μL of reverse transcription buffer, 3.0 μL of oligonucleotide chain, and 4.0 μL of reducing agent.

9. The method as described in claim 7, characterized in that, In step 3, the loading buffer contains 33.8 μL of reaction buffer, 6% (v / v) iodixanol, 4% (v / v) PEG8000, and single-cell suspension or single-cell nucleus suspension diluted in an appropriate ratio; wherein the impurity ratio of the single-cell suspension or single-cell nucleus suspension is higher than 30%, and there are fragments or clumps with a length greater than 100 μm; Preferably, 60% iodixanol, 7.5 μL, final concentration 6% (v / v); 40% PEG8000, 7.5 μL, final concentration 4% (v / v); single cell suspension, 26.2 μL.

10. The method as described in claim 7, characterized in that, In step 4, the loading process is adjusted according to the chip, including connecting the PDMS chip to each injection port through a 200μL pipette tip and a Teflon tube, and loading the oil, Single Cell 3' Gel Bead, and loading buffer into the pipette tip connected to the corresponding injection port.

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