Micro-fluidic chip-based high-throughput approximate single cell space transcriptional library building method

Abstract

The invention relates to the technical field of microfluidics, in particular to a high-flux approximate single cell space transcriptional library building method based on a micro-fluidic chip, which comprises the following steps: carrying out earlier stage treatment on a tissue slice in a single micropore, including fixing, permeating and carrying out reverse transcription on the tissue in situ to obtain DNA and RNA molecular hybrid chains; the method comprises the following steps: pasting a micro-fluidic chip on a tissue slice to capture mRNA at a high throughput in a spatial dimension, so that an mRNA molecule of a single pixel site carries an identification code capable of identifying the identity of the mRNA molecule; carrying out chain conversion on the hybrid chain to obtain double-chain cDNA, and then carrying out a round of amplification on the cDNA to enlarge the molecular weight of the cDNA; and then carrying out Tn5 interruption on the quantified cDNA, carrying out two-round amplification, and finally purifying the library with the sequencing linker and then sending the sample for sequencing. According to the present invention, the sufficient gene number and the sufficient mRNA category number for subsequent analysis can be captured under the high throughput in space and the high resolution of the similar single cell.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic technology, and in particular to a method for building a library of high-throughput single-cell spatial transcriptomes based on a microfluidic chip. Background Art

[0002] Almost all of biology, seemingly infinitely complex, occurs in three dimensions. In living organisms, cells must interact and combine in three-dimensional tissues, and the position of each cell is as important as its intrinsic properties, determining the function of the tissue or dysfunction in disease. Therefore, even when studying the structure of simple organisms or a single tissue, it is necessary not only to decipher the molecular profiles of thousands of cells, but also to understand how and what the spatial environment affects them. In recent years, with the improvement of gene sequencing technology and the successive implementation of major international cooperation projects, genome research has developed faster and faster.

[0003] Spatial omics is another hot topic in biotechnology research after single-cell sequencing technology. It can make up for the defect that single-cell sequencing technology cannot obtain information on the spatial distribution of cells. Spatial omics technology mainly studies the relative position relationship of cells in tissue samples, which is used to reveal the impact of the spatial distribution relationship of cells on diseases, and mainly detects mRNA or its epigenetic information. At present, the most common spatial transcriptome technologies are ST, Slide-seq, and HDST. Among them, each capture area of ​​the 10X Genomics Visium chip contains 5,000 barcode-marked points, and the diameter of each point is 55μm, but there is a distance of 45μm between each point that is not covered on the tissue. This method has low resolution, and nearly 1 / 2 of the tissue is not captured, which largely loses useful spatial information. In addition, the Visium chip is expensive and cannot be reused, which increases the cost of scientific research. In 2020, Fan Rong's team improved the spatial transcriptome technology and used microfluidics technology to realize spatial transcriptome research on tissue sections, namely DBiT-seq. This technology achieves high-precision detection at a resolution of up to 10μm, reducing the distance between coding points to 10μm. However, this solution still has some disadvantages. The first is that the number of codes is insufficient and cannot achieve detection of larger areas. The second is that the method process is time-consuming and labor-intensive, which increases labor costs. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for building a high-throughput single-cell spatial transcriptome library based on a microfluidic chip.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] A method for preparing a high-throughput single-cell spatial transcriptome library based on a microfluidic chip is provided, which comprises the following steps:

[0007] S1. Pre-processing the tissue sections in a single microwell, including fixation, permeabilization, and reverse transcription in situ to obtain hybrid chains of DNA and RNA molecules;

[0008] S2, attaching the microfluidic chip to the tissue slice to capture mRNA molecules with high throughput in the spatial dimension, so that the mRNA molecules at a single pixel site carry an identification code that can identify their own identity;

[0009] S3. Convert the hybrid chains of DNA and RNA molecules into double-stranded cDNA, and then perform a round of amplification on the cDNA to expand its molecular weight; then, the quantified cDNA is sheared by Tn5, and then a second round of amplification is performed. Finally, the library with sequencing adapters is purified and sent for sequencing.

[0010] Furthermore, the microfluidic chip is made of polydimethylsiloxane material.

[0011] Furthermore, there are two types of microfluidic chips, horizontal chip A and vertical chip B; the difference between the two types of chips is that after their channels cover the tissue slices, the reaction solutions in the channels flow through the tissue in different directions. During the entire reaction process, after the up, down, left, and right directions of the tissue are given, the reaction solutions in the channels of the horizontal chip A and the vertical chip B flow through the tissue from left to right and from top to bottom, respectively. The horizontal chip A and the vertical chip B are covered on the tissue one after another, and the identification code flows through the tissue through the channel of the chip and is incubated. After two rounds of identification code marking, the mRNA molecules at a single pixel site carry an identification code that can identify their own identity.

[0012] Furthermore, the microfluidic chip includes a chip body, and a plurality of injection holes and outlet holes are respectively arranged at the left and right ends of the chip body; the injection hole is connected to the first flow channel, the outlet hole is connected to the second flow channel, and the first flow channel and the second flow channel are connected to the third flow channel. The first flow channel is used to drain the chemical reagents in the injection hole to the third flow channel, and the third flow channel is used to drain the chemical reagents in the first flow channel through the target tissue to perform biochemical reactions. It is a functional area for capturing omics information, and the second flow channel is used to drain the chemical reagents in the third flow channel to the outlet hole to prevent cross-talk of different reaction reagents between channels.

[0013] Furthermore, the width of the first flow channel and the second flow channel is 50 μm, and the width of the third flow channel is narrower, with specifications of 10 μm / 20 μm respectively according to the characteristics of different cell sizes of the sample.

[0014] The beneficial effects of the present invention are:

[0015] 1. The number of codes has been increased to enable detection in a larger area;

[0016] 2. Reduce the experimental cost, and the experimental equipment is easy to carry;

[0017] 3. Simplified the tedious sample addition steps in the experimental process and shortened the entire experimental time. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the microfluidic chip structure in Example 1 Figure 1 ;

[0019] Wherein: 1. chip body; 11. injection hole; 12. outlet hole; 13. first flow channel; 14. second flow channel; 15. third flow channel;

[0020] Figure 2 Schematic diagram of the microfluidic chip structure in Example 1 Figure 2 ;

[0021] Figure 3 Schematic diagram of the microfluidic chip structure in Example 1 Figure 3 ;

[0022] Figure 4 Schematic diagram of the microfluidic chip structure in Example 1 Figure 4 ;

[0023] Figure 5 This is a schematic diagram of the structure of the fixture device matched with the microfluidic chip in Example 2;

[0024] Wherein: 2, fixture body; 21, lever; 22, pressure frame;

[0025] Figure 6 This is the spatial distribution diagram of E9_1 mouse embryos in Example 3;

[0026] Figure 7 This is a UMAP (Uniform Manifold Approximation and Projection) diagram of the E9_1 mouse embryo spatial atlas in Example 3; each point represents a pixel, and different colors represent different cell populations;

[0027] Figure 8 This is the spatial distribution diagram of E9_2 mouse embryos in Example 3;

[0028] Fig. 9 This is the UMAP image of the spatial map of the E9_2 mouse embryo in Example 3; each point represents a pixel, and different colors represent different cell populations. DETAILED DESCRIPTION

[0029] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0030] Example 1

[0031] Reference Figure 1 , a microfluidic chip is provided, the chip is made of polydimethylsiloxane (PDMS), dimethylsiloxane (PDMS) is a highly biocompatible material, and dimethylsiloxane (PDMS) can be tightly combined with glass or another dimethylsiloxane (PDMS) layer through simple plasma treatment. It includes a chip body 1, and a plurality of injection holes 11 and output holes 12 are respectively arranged at the left and right ends of the chip body 1; the injection hole 11 is connected to a first flow channel 13, and the output hole 12 is connected to a second flow channel 14, and a third flow channel 15 is connected between the first flow channel 13 and the second flow channel 14, and the first flow channel 13 is used to drain the chemical reagents in the injection hole 11 to the third flow channel 15, and the third flow channel 15 is used to drain the chemical reagents in the first flow channel 13 through the target tissue to perform biochemical reactions, which is a functional area for capturing genomic information, and the second flow channel 14 is used to drain the chemical reagents in the third flow channel 15 to the output hole 12 to prevent the cross-talk of different reaction reagents between channels.

[0032] Preferably, the width of the first flow channel 13 and the second flow channel 14 is 50 μm. The width of the third flow channel 15 is narrower, and the widths of the third flow channel 15 of different specifications correspond to the resolution of spatial omics.

[0033] There are currently 5 mature microfluidic chip products

[0034] Chip 1 parameters

[0035] Number of channels of chip 1: 50

[0036] Resolution, i.e. width of functional flow channel in the middle covering tissue: 20μm

[0037] Channel design: straight and I-shaped

[0038] Chip 2 parameters

[0039] Number of channels on chip 2: 96

[0040] Resolution, i.e. width of functional flow channel in the middle covering tissue: 20μm

[0041] Channel design: straight and I-shaped

[0042] Chip 3 parameters

[0043] Number of channels on chip 3: 96

[0044] Resolution, i.e. width of functional flow channel in the middle covering tissue: 10μm

[0045] Channel design: straight and I-shaped

[0046] Chip 4 parameters

[0047] Number of channels on chip 4: 192

[0048] Resolution, i.e. width of functional flow channel in the middle covering tissue: 20μm

[0049] Channel design: straight and I-shaped

[0050] Chip 5 parameters

[0051] Number of channels of chip 5: 192

[0052] Resolution, i.e. width of functional flow channel in the middle covering tissue: 10μm

[0053] Channel design: straight and I-shaped

[0054] As a preferred method, refer to Figure 1-4 There are two types of microfluidic chips, horizontal and vertical, named chip A and chip B. The difference between the two types of chips is that after their channels cover tissue slices, the reaction solutions in the channels flow through the tissues in different directions. During the entire reaction process, given the up, down, left, and right directions of the tissue, the reaction solutions in the channels of chip A and chip B flow through the tissues from left to right and from top to bottom, respectively.

[0055] Example 2

[0056] Reference Figure 5 A clamp device matching the microfluidic chip provided in Example 1, the clamp device is made of rust-proof steel, and is composed of three parts: a clamp body 2 for loading the chip and the glass slide, a lever 21 for stabilizing the main functional area of ​​the chip, and a pressure frame 22 connected to a suction pump.

[0057] Instructions for use: First, attach the microfluidic chip to the appropriate position of the tissue patch, and load the chip onto the fixture body 2 with the chip facing upwards; then gently place the lever 21 on the main functional area of ​​the chip. In principle, the two screw holes on the lever should be aligned with the two middle screw holes on the fixture body 2, and at the same time, tighten the upper and lower screws of the lever under force (note that the channel in the functional area is in the same state as when it is not under force, and the channel should not be deformed or bent under force); finally, when the vacuum pump is pumping, buckle the pressure frame 22 on the right side of the chip (the side of the sample outlet), and connect the suction port on the pressure frame 22 to the vacuum pump. The pressure of the vacuum pump is adjusted according to the sample thickness, toughness and chip specifications.

[0058] Example 3

[0059] A method for preparing a library based on a high-throughput single-cell spatial transcriptome of a microfluidic chip provided in Example 1, comprising the following steps:

[0060] First, the tissue sections are pre-processed in a single microwell, including fixation, permeabilization, and reverse transcription in situ on the tissue to obtain hybrid chains of deoxyribonucleic acid molecules (DNA) and ribonucleic acid molecules (RNA); then 96*96 identification codes (barcodes) are used to capture messenger RNA molecules (mRNA) in a high-throughput manner in the spatial dimension on the frozen tissue sections. There are two types of microfluidic chips: horizontal and vertical (hereinafter, described as chip A and chip B). The difference between the two types of chips is that after the channels cover the tissue, the reaction solutions in the channels flow through the tissue in different directions. During the entire reaction process, given the up, down, left, and right directions of the tissue, the reaction solutions in the channels of chip A and chip B flow through the tissue from left to right and from top to bottom, respectively. (below), two chips of chip A and chip B are covered on the tissue one after another, and 96*96 identification codes (barcodes) flow through the tissue through the channel of the chip and are incubated. After two rounds of identification codes (barcodes), the messenger RNA molecule (mRNA) at a single pixel site carries an identification code that can identify its own identity; then the hybrid chain is converted into a double-stranded complementary deoxyribonucleic acid (cDNA), and the double-stranded complementary deoxyribonucleic acid is amplified in one round to expand its molecular weight; then the quantified double-stranded complementary deoxyribonucleic acid (cDNA) is interrupted by a transposase complex (with Tn5), and then a second round of amplification is performed, and finally the library with sequencing adapters is purified and sent for sequencing.

[0061] The specific experimental process of this embodiment is as follows:

[0062] Prepare the experimental process:

[0063] The night before:

[0064] 1. Chip preparation: Check the integrity and smoothness of the chip channel and the cleanliness of the chip. After the inspection, put the chip in an oven for use (temperature>65 degrees Celsius);

[0065] 2. Single-hole preparation: Prepare the single-hole reaction and single-hole sample collection of the polydimethylsiloxane chip, put them into sterile freshly opened anhydrous ethanol for ultrasonic cleaning for more than 10 minutes, and after confirming that there is no tissue residue under a microscope, put the single hole into an oven for use (temperature>65 degrees Celsius);

[0066] 3. Tissue preparation: Cryosection the target experimental tissue - day 9 mouse embryos (10 μm thickness) and record the sectioning sequence. Take photos under a microscope and mark the sections with good tissue morphology as samples for the next experiment.

[0067] On the morning of the experiment:

[0068] 1. The fixtures used in the experiment are disinfected and sterilized with alcohol;

[0069] 2. Turn on the ultraviolet sterilization of the clean bench. The sterilization time is more than 30 minutes. The instruments can be put in for sterilization;

[0070] 3. The three thermostats are adjusted to 37 / 42 / 55 degrees Celsius respectively;

[0071] 4. Take out the barcode oligos in the 96-well plate from -20 degrees Celsius and place them at 4 degrees Celsius to melt and prepare for use;

[0072] 5. Make ice, distribute reagents, and prepare the reagents needed in the first few steps of the experiment;

[0073] 6. Prepare a slice placement dish (you can use a regular plastic dish), and take out the experimental slices prepared in advance - 9th day mouse embryo slices - from the -80 degrees Celsius refrigerator.

[0074] Formal preparation experimental process (taking a sample as an example):

[0075] First day experiment process

[0076] Tissue processing before chip application

[0077] 1. Restore tissue temperature:

[0078] After the clean bench is sterilized, turn on the ventilation and lighting. Take out the 9th day mouse embryo slices prepared in advance from the -80 degrees Celsius refrigerator and place them in the clean bench at room temperature for 10 minutes;

[0079] 2.Tissue morphology photography (second time):

[0080] When there is one minute left in the first step of waiting time, the mouse embryo slices on the 9th day after returning to room temperature can be photographed and recorded as the tissue morphology photo No. 2 of the mouse embryo slices on the 9th day;

[0081] 3. Cleaning the residual aqueous mixture of polyethylene glycol and polyvinyl alcohol (OCT):

[0082] Add 1 ml of phosphate buffered saline-nuclease inhibitor 1 mixed solution (PBS-RI1) to cover the entire range of the 9th day mouse embryo slices, wash at room temperature for 3 minutes, and use a suction pump to suck away the reaction liquid after washing. This step requires gentle operation to avoid tissue damage;

[0083] Phosphate buffered saline-nuclease inhibitor 1 mixed solution (PBS-RI1) formula table

[0084]

[0085] 4. Tissue Fixation:

[0086] Add 1 ml of 4% paraformaldehyde fixative to cover the entire range of the mouse embryonic slices on the 9th day, incubate at room temperature for 20 minutes, and use a suction pump to suck away the reaction liquid after fixation. This step requires gentle operation to avoid tissue damage;

[0087] 5. Wash and remove residual fixative

[0088] Add 1 ml of phosphate buffered saline-nuclease inhibitor 1 mixed solution (PBS-RI1) to cover the entire range of the 9th day mouse embryo slices, wash at room temperature for 3 minutes, and use a suction pump to suck away the reaction liquid after washing. This step requires gentle operation to avoid tissue damage;

[0089] 6. Wash the whole slice (first time)

[0090] Wash the slices in a disposable plastic cup filled with high-temperature sterilized 0.1% diethylpyrocarbonate-treated water (0.1% DEPC), up and down 5 times, and remove the remaining liquid with a suction pump;

[0091] 7. Tissue morphology photography (3rd time)

[0092] Take a picture of the mouse embryo slice on day 9, record it as the mouse embryo slice tissue morphology photo No. 3 on day 9, and observe the integrity of the tissue to ensure the necessity of subsequent experiments;

[0093] 8. Tissue permeabilization (this step adds a single hole to the slice)

[0094] A single hole was buckled on the target area of ​​the tissue, 100 μl of permeabilization solution was added to the single hole, and incubated at room temperature for 20 minutes. After the reaction was completed, the reaction solution was sucked away by the pipette tip;

[0095] Permeabilization solution recipe

[0096]

[0097] 9. Wash to remove residual permeabilization solution

[0098] Add 100 μl of 0.5x phosphate buffered saline-nuclease inhibitor 1 mixed solution (0.5x PBS-RI1) to each well for washing, incubate at room temperature for 3 minutes, repeat this process twice, and aspirate the washing solution with the pipette tip after washing;

[0099] 0.5x phosphate buffered saline-nuclease inhibitor 1 mixed solution (0.5x PBS-RI1) cleaning formula table

[0100]

[0101] 10. Reverse Transcription

[0102] A mixture of 60 μl of reverse transcription reaction solution and 15 μl of RNA buffer (NIB) solution was added to a single well, incubated at room temperature for 30 minutes, and then incubated at 42 degrees Celsius in a constant temperature incubator for 90 minutes. After the reaction was completed, the reaction solution was aspirated with a pipette tip;

[0103] Pre-NIB RNA Buffer Recipe

[0104]

[0105] RNA Buffer (NIB) Recipe Table

[0106]

[0107]

[0108] Reverse transcription solution formula table

[0109]

[0110] 11. Termination of reverse transcription

[0111] Add 100 μl of the reverse transcription termination reaction solution to each well and incubate at room temperature for 5 minutes;

[0112] Termination Reverse Transcription Solution Recipe Table

[0113]

[0114] 12. Wash the whole slice (second time)

[0115] Wash the slices in a disposable plastic cup filled with high-temperature sterilized 0.1% diethylpyrocarbonate-treated water (0.1% DEPC), up and down 5 times, and remove the remaining liquid with a suction pump;

[0116] 13. Tissue morphology photography (4th time)

[0117] Take a picture of the mouse embryo slice on day 9, record it as the mouse embryo slice tissue morphology photo No. 4 on day 9, and observe the integrity of the tissue to ensure the necessity of subsequent experiments;

[0118] Tissue processing for application chips

[0119] 14. Organizationally corresponding area connection group A identification code

[0120] Attach chip A to the slice (note that the middle functional area covers the target tissue area, and ensure that there are no impurities blocking the channel. Take a photo of the embryo covered with the chip. This is the 5th photo, recorded as the 9th day mouse embryo slice tissue morphology photo No. 5). Assemble the chip and the patch on the reaction fixture device, add the prepared A group identification code mixed reaction solution with the corresponding sequence to the corresponding chip A sample wells in a certain order, add 4μl to each well, and vacuum pump for about 2 minutes until all channels are filled with solution. Place the entire device in a wet box and incubate at 37 degrees Celsius for 30 minutes. After the reaction is completed, use the pump and gun tip to suck away the residual reaction solution in the chip inlet and outlet wells;

[0121] Identification code connection solution formula table

[0122]

[0123] * The AB group identification code uses the same connection solution

[0124] A Group Identification Code Mixed Reaction Solution Formula Table

[0125]

[0126] * There are 96 primers in Group A.

[0127] 15. Neutralize the corresponding area of ​​the organization to connect the redundant A group identification code

[0128] Add the prepared A group identification code neutralization reaction solution to the corresponding chip A sample wells, add 4μl to each well, and use the vacuum pump to pump for about 2 minutes until all channels are filled with solution. Place the entire device in a wet box and incubate at 37 degrees Celsius for 30 minutes. After the reaction is completed, use the pump tip to suck away the residual reaction solution in the chip sample inlet and sample outlet wells;

[0129] A Group Identification Code Neutralization Reaction Solution Formula Table

[0130]

[0131] 16. Clean the reaction channel of chip A

[0132] Add the prepared chip channel cleaning solution to the corresponding sample wells of chip A, and use the vacuum pump to pump for about 2 minutes until all channels are filled with the solution. After the end, use the pump tip to suck away the residual chip channel cleaning solution in the chip inlet and outlet wells;

[0133] Chip channel cleaning solution formula table

[0134]

[0135] 17. Remove chip A and clean the entire slice (3rd time)

[0136] Remove chip A and wash the slice in a disposable plastic cup filled with high-temperature sterilized diethyl pyrocarbonate treated water (0.1% DEPC), up and down 5 times, and remove the remaining liquid with a suction pump;

[0137] 18.Tissue morphology photography (6th time)

[0138] Take a picture of the mouse embryo slices on the 9th day, record it as the mouse embryo slice tissue morphology photo No. 6 on the 9th day, and observe the integrity of the tissue to ensure the necessity of subsequent experiments;

[0139] 19. Organizationally corresponding area connection group B identification code

[0140] Attach chip B to the slice (note that the middle functional area covers the target tissue area, and ensure that there are no impurities blocking the channel. Take a photo of the embryo covered with the chip. This is the 7th photo, recorded as the 9th day mouse embryo slice tissue morphology photo No. 7), assemble the chip and the patch on the reaction fixture equipment, add the prepared B group identification code mixed reaction solution with the corresponding sequence to the corresponding chip B sample wells in a certain order, and vacuum pump for about 2 minutes until all channels are filled with solution. Place the entire device in a wet box and incubate at 37 degrees Celsius for 30 minutes. After the reaction is completed, use the pump and gun tip to suck away the residual reaction solution in the chip inlet and outlet holes;

[0141] Group B identification code mixed reaction solution formula table

[0142]

[0143] *There are 96 primers in Group B

[0144] 20. Neutralize the corresponding area of ​​the organization to connect the redundant B group identification code

[0145] Add the prepared B group identification code neutralization reaction solution to the corresponding sample well of chip B, and use the vacuum pump to pump for about 2 minutes until all channels are filled with solution. Place the entire device in a wet box and incubate at 37 degrees Celsius for 30 minutes. After the reaction is completed, use the pump tip to suck away the residual reaction solution in the chip injection and output wells;

[0146] B Group Identification Code Neutralization Reaction Solution Formula Table

[0147]

[0148] 21. Clean the reaction channel of chip B

[0149] Add the prepared chip channel cleaning solution to the corresponding sample wells of chip B, and use the vacuum pump to pump for about 2 minutes until all channels are filled with the solution. After the end, use the pump tip to suck away the residual chip channel cleaning solution in the chip inlet and outlet wells;

[0150] Chip channel cleaning solution formula table

[0151]

[0152] 22. Remove chip B and clean the entire slice (4th time)

[0153] Remove chip B, wash the slice in a disposable plastic cup filled with high-temperature sterilized diethyl pyrocarbonate treated water (0.1% DEPC), up and down 5 times, and remove the remaining liquid with a suction pump;

[0154] 23.Tissue morphology photography (8th time)

[0155] Take a picture of the mouse embryo slices on the 9th day, record it as the mouse embryo slice tissue morphology photo No. 8 on the 9th day, and observe the integrity of the tissue to ensure the necessity of subsequent experiments;

[0156] Tissue processing after chip application

[0157] 24. Tissue Lysis (Single Well Reaction)

[0158] A single hole is buckled on the target area of ​​the tissue, 50 μl of tissue lysis solution is added to the single hole, and incubated at 55 degrees Celsius for 2 hours. After the reaction is completed, the lysis product is recovered with a low-adsorption 200 μl pipette tip. The product is stored in a 1.5 ml low-adsorption centrifuge tube and stored in a -80 degree Celsius refrigerator for up to 2 weeks.

[0159] 2X Tissue Lysis Solution Recipe Table

[0160]

[0161] 1X Tissue Lysis Solution Recipe

[0162]

[0163] Second day experimental process

[0164] 25.cDNA purification and recovery

[0165] DNA purification and recovery kit (Zymo DNAClean & Concentrator TM -5, catalog number: D4013) to purify and recover cDNA. The final recovery and elution volume of the product is 100 μl, and the product is temporarily stored in a refrigerator at 4 degrees Celsius;

[0166] 26. Superparamagnetic polystyrene microbeads (Dynabeads TM MyOne TM ) Streptavidin C1 preparation

[0167] (1) Return to normal temperature: Preheat the superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Take out streptavidin C1 from 4 degrees Celsius, mix well, aspirate 40 μl of the reaction volume and place it at room temperature for more than half an hour until it returns to room temperature;

[0168] (2) Beads washing: Superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Streptavidin C1 was placed on a magnetic rack, its storage solution was removed, and superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Add 100 μl 1x B&W-T buffer to streptavidin C1, mix well and wash three times in total;

[0169] (3) Superparamagnetic polystyrene microbeads (Dynabeads TM MyOne TM ) Streptavidin C1 resuspended: superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) After washing with streptavidin C1, superparamagnetic polystyrene beads (Dynabeads TM MyOne TM) Add 100 μl of a mixture of pre-2X B&W Buffer and 2 μl of RNase inhibitor 2 to streptavidin C1.

[0170] Pre-2X B&W Buffer recipe

[0171]

[0172] 1x high concentration salt buffer (B&W-T buffer) formula table

[0173]

[0174] 27. Superparamagnetic polystyrene microbeads (Dynabeads TM MyOne TM ) Streptavidin C1 is incubated with the sample

[0175] 100 μl of resuspended superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Streptavidin C1 was added to the sample and incubated at room temperature for 1 hour on a rotating instrument. After the reaction was completed, the tube was removed from the rotating instrument and placed on a magnetic rack to remove the supernatant;

[0176] 28. cDNA second strand synthesis

[0177] (1) After the reaction, superparamagnetic polystyrene microbeads (Dynabeads TM MyOne TM ) Streptavidin C1 high concentration salt wash: Superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Add 500 μl 1x high concentration salt buffer (1x B&W-T buffer) to streptavidin C1, mix well and wash, rotate the instrument to wash for 5 minutes, wash twice in total;

[0178] (2) After the reaction, superparamagnetic polystyrene microbeads (Dynabeads TM MyOne TM ) Streptavidin C1 low concentration salt wash: Superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Add 500 μl of low-concentration salt buffer-nuclease inhibitor mixture (STE+) to streptavidin C1, mix well and wash, rotate the instrument to wash for 5 minutes, wash once;

[0179] (3) Incubation of strand switching primer reaction solution (TSO): Superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Add 220 μl of strand switching primer reaction solution (TSO) to streptavidin C1, mix well, and incubate at room temperature for 30 minutes on a rotating instrument;

[0180] Incubate at 42°C in a metal bath for 90 minutes. After the reaction is complete, place the reaction tube on a magnetic rack and remove the supernatant.

[0181] (4) After incubation with strand switching primer reaction solution (TSO), superparamagnetic polystyrene microbeads (Dynabeads TM MyOne TM ) Streptavidin C1 low concentration salt wash: Superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Add 500 μl of low-concentration salt buffer-nuclease inhibitor mixture (STE+) solution to streptavidin C1 and mix well to wash;

[0182] (5) After incubation with strand switching primer reaction solution (TSO), superparamagnetic polystyrene microbeads (Dynabeads TM MyOne TM ) Streptavidin C1 Nuclease-free Water (NF-H2O) Wash: Superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Add 500 μl of nuclease-free water (NF-H2O) to streptavidin C1 and mix well to wash. Wash multiple times until the superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Streptavidin C1 can be completely bound to the magnetic rack, and the supernatant is clear and transparent;

[0183] Low concentration salt buffer (STE) formula table

[0184]

[0185] Low concentration salt buffer-nuclease inhibitor mixture (STE+) formula table

[0186]

[0187] Strand Switching Primer Reaction Solution (TSO) Formula Table

[0188]

[0189]

[0190] 29.cDNA Amplification

[0191] After washing, superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Add 55 μl cDNA amplification solution to streptavidin C1 and resuspend superparamagnetic polystyrene beads (Dynabeads TM MyOne TM ) Streptavidin C1, put into polymerase chain reaction (PCR) amplification instrument for amplification program, according to the size of mouse embryo slices on day 9, the number of amplification cycles is 10 cycles;

[0192] cDNA Amplification Solution Formula Table

[0193]

[0194] cDNA Amplification Procedure

[0195]

[0196] 30. Rapid detection of amplified fragments

[0197] The size and concentration of the amplified fragments were detected (qualitatively) using the lonza FlashGel DNACass 2.2% fast gel (#Cat. No. 57031). The target fragment size was more than 300 bp, and the molecular quantitative standard was the Tiangen brand 100 bp DNA Ladder (DNA molecular weight standard, #Cat. No. MD109).

[0198] 31. Centrifugal

[0199] Place the reaction product in a normal temperature centrifuge and centrifuge at 10,000 g for 1 minute. Place the centrifuged tube on a magnetic rack to remove the supernatant.

[0200] 32. Fragment screening (300bp+)

[0201] (1) Add NF-H2O to the sample to a total volume of 100 μl, then add 80 μl Beckman Coulter brand nucleic acid fragment screening reagent (SPRIselect beads) to the sample and let it stand for 10 minutes. The fragments >300 bp in the product will bind to the nucleic acid fragment screening reagent (SPRIselect beads);

[0202] (2) Place the polymerase chain reaction (PCR) tube containing the product on a magnetic rack for about 2 minutes, then discard the supernatant and wash twice with 80% ethanol to remove impurities such as proteins;

[0203] (3) Use a 200 μl pipette tip to absorb the remaining 80% ethanol and blow it under a clean bench fan for about 5 minutes. Be careful not to let the SPRIselect beads dry out until the SPRIselect beads have a frosted texture.

[0204] (4) Add 36.5 μl NF-H2O to the SPRIselect beads, resuspend the SPRIselect beads, and place for 5 minutes to remove the target DNA on the SPRIselect beads;

[0205] After the fragments are eluted, the polymerase chain reaction (PCR) tube containing the product is placed on a magnetic rack for about 2 minutes, and the eluted product is recovered into the polymerase chain reaction (PCR) tube;

[0206] 33. Concentration determination

[0207] The brand used is Vazyme 1×dsDNAHS Assay Kit (double-stranded DNA (dsDNA) fluorescence quantitative detection kit, # EQ121-01) quantitatively detects the concentration of the target fragment, and extracts a total of 50ng of cDNA for the next experiment;

[0208] 34.cDNA fragmentation and sequencing adapter

[0209] Use 2.5 μl of Vazyme's TruePrep Tagment Enzyme (engineered ultra-high activity transposase, #Cat. No. S601-01, pre-packaged with specific adapters) to shear a total of 50 nanograms of cDNA. The shearing system and shearing procedure are as follows:

[0210] cDNA shearing system

[0211]

[0212] cDNA Shedding Procedure

[0213]

[0214] 35.cDNA interruption and termination

[0215] Add 50 μl of 8-M guanidine hydrochloride (GuHCL) to the sample, then shake and mix to terminate the interruption reaction;

[0216] 36. Purification and recovery after cDNA shearing

[0217] (1) Add nuclease-free water (NF-H2O) to the sample to a total volume of 100 μl, then add 100 μl Beckman Coulter brand nucleic acid fragment screening reagent (SPRIselect beads) to the sample and let it stand for 10 minutes. The fragments <1 kp in the product will bind to the nucleic acid fragment screening reagent (SPRIselect beads);

[0218] (2) Place the polymerase chain reaction (PCR) tube containing the product on a magnetic rack for about 2 minutes, then discard the supernatant and wash twice with 80% ethanol to remove impurities such as proteins;

[0219] (3) Use a 200 μl pipette tip to absorb the remaining 80% ethanol and blow it under a clean bench fan for about 5 minutes. Be careful not to let the SPRIselect beads dry out until the SPRIselect beads have a frosted texture.

[0220] (4) Add 21 μl NF-H2O to the SPRIselect beads, resuspend the SPRIselect beads, and place them for 5 minutes to elute the target DNA fragments on the SPRIselect beads. Place the polymerase chain reaction (PCR) tube containing the product on a magnetic rack for about 2 minutes, and recover the eluted product into the PCR tube.

[0221] 37. cDNA fragmentation and amplification

[0222] The recovered product was amplified before sequencing (5 cycles can reach the concentration standard for sequencing). The amplification system and amplification procedure are as follows:

[0223] cDNA shearing and amplification system

[0224]

[0225] cDNA shearing and amplification procedures

[0226]

[0227] 38. Rapid detection of amplified fragments (400bp+)

[0228] The size and concentration of the amplified fragments (qualitative) were detected using the lonza FlashGel DNACass 2.2% fast gel (#Cat. No. 57031). The target fragment size was above 400 bp. The molecular quantitative standard used was the Tiangen brand 100 bp DNA Ladder (DNA molecular weight standard, #Cat. No. MD109).

[0229] 39. Fragment screening

[0230] (1) Add NF-H2O to the sample to a total volume of 100 μl, then add 70 μl Beckman Coulter brand nucleic acid fragment screening reagent (SPRIselect beads) to the sample and let it stand for 10 minutes. The fragments >400 bp in the product will bind to the nucleic acid fragment screening reagent (SPRIselect beads);

[0231] (2) Place the polymerase chain reaction (PCR) tube containing the product on a magnetic rack for about 2 minutes, then discard the supernatant and wash twice with 80% ethanol to remove impurities such as proteins;

[0232] (3) Use a 200 μl pipette tip to absorb the remaining 80% ethanol and blow it under a clean bench fan for about 5 minutes. Be careful not to let the SPRIselect beads dry out until the SPRIselect beads have a frosted texture.

[0233] (4) Add 13 μl NF-H2O to the SPRIselect beads, resuspend the SPRIselect beads, and place for 5 minutes to elute the target DNA fragments on the SPRIselect beads. Place the polymerase chain reaction (PCR) tube containing the product on a magnetic rack for about 2 minutes, and recover the eluted product into a 1.5 ml centrifuge tube.

[0234] 40. Send samples for sequencing

[0235] The samples were sent to the company for sequencing, and then the data was analyzed by bioinformatics. This example was repeated 2 times, and the experimental results are shown in the following table. Figure 6-9 As shown in Table 1-2 below.

[0236] Table 1

[0237] sample s period slice Chip Specifications Channel Specifications Cell number Median ΜMI Median gene E9_1 E9.0 mEmbryo_E9_1 96*96 10μm 6,135 5,930 2,614 E9_2 E9.0 mEmbryo_E9_2 96*96 10μm 5,860 6,802 2,994

[0238] Table 2

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251] In summary, the present invention achieves the capture of a sufficient number of genes and messenger RNA molecule (mRNA) types for subsequent analysis at high spatial throughput and high resolution close to that of a single cell.

[0252] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0253] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for high-throughput single-cell spatial transcriptome library preparation based on a microfluidic chip, characterized in that: The following steps are involved: S1. Pre-processing the tissue sections in a single microwell, including fixation, permeabilization, and reverse transcription in situ to obtain hybrid chains of DNA and RNA molecules; S2, attaching the microfluidic chip to the tissue slice to capture mRNA molecules with high throughput in the spatial dimension, so that the mRNA molecules at a single pixel site carry an identification code that can identify their own identity; S3. Convert the hybrid chains of DNA and RNA molecules into double-stranded cDNA, and then perform a round of amplification on the cDNA to expand its molecular weight; then, the quantified cDNA is sheared by Tn5, and then a second round of amplification is performed. Finally, the library with sequencing adapters is purified and sent for sequencing.

2. The method for high-throughput single-cell spatial transcriptome library based on microfluidic chip according to claim 1, characterized in that: The microfluidic chip is made of polydimethylsiloxane material.

3. The method for high-throughput single-cell spatial transcriptome library based on microfluidic chip according to claim 1, characterized in that: There are two types of microfluidic chips, horizontal chip A and vertical chip B. The difference between the two types of chips is that after their channels cover tissue slices, the reaction solutions in the channels flow through the tissue in different directions. During the entire reaction process, after the up, down, left, and right directions of the tissue are given, the reaction solutions in the channels of horizontal chip A and vertical chip B flow through the tissue from left to right and from top to bottom, respectively. The horizontal chip A and vertical chip B are covered on the tissue one after another, and the identification code flows through the tissue through the channel of the chip and is incubated. After two rounds of identification code marking, the mRNA molecules at a single pixel site carry an identification code that can identify their own identity.

4. The method for high-throughput approximate single-cell spatial transcriptome library based on a microfluidic chip according to any one of claims 1 to 3, characterized in that: The microfluidic chip comprises a chip body (1), wherein a plurality of injection holes (11) and output holes (12) are respectively arranged at the left and right ends of the chip body (1); the injection hole (11) is connected to a first flow channel (13), the output hole (12) is connected to a second flow channel (14), a third flow channel (15) is connected between the first flow channel (13) and the second flow channel (14), the first flow channel (13) is used to guide the chemical reagent in the injection hole (11) to the third flow channel (15), the third flow channel (15) is used to guide the chemical reagent in the first flow channel (13) through the target tissue to perform a biochemical reaction, and is a functional area for capturing omics information, and the second flow channel (14) is used to guide the chemical reagent in the third flow channel (15) to the output hole (12) to prevent the cross-talk of different reaction reagents between channels.

5. The method for high-throughput single-cell spatial transcriptome library based on microfluidic chip according to claim 4, characterized in that: The flow channel widths of the first flow channel (13) and the second flow channel (14) are 50 μm, and the flow channel width of the third flow channel (15) is even narrower.

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