High-throughput space transcriptome sequencing method based on microfluidic technology

Abstract

The invention relates to the technical field of space transcriptome sequencing, and particularly discloses a high-throughput space transcriptome sequencing method based on a micro-fluidic technology, which comprises the following steps: S1, preparing a micro-fluidic chip; s2, pasting a chip on a positive electric substrate, introducing B0 together with the chip, and introducing 48 barcode-A after incubation; s3, the second chip is replaced to the positive electric substrate, 48 barcode-B and T4DNA ligase connection systems are introduced, and then incubation is carried out; s4, replacing the chip III onto a positive electric substrate, and adding a T4DNA ligase connection system containing 15 barcode-C sequences into the micropores for incubation; s5, attaching the tissue slice to the substrate loaded with the space coding array; s6, synthesizing cDNA (complementary deoxyribonucleic acid) on the substrate; s7, releasing and recovering the RNA / DNA hybrid chain product from the substrate; s8, synthesizing a cDNA double strand and amplifying a cDNA double strand product; s9, library construction and sequencing are carried out; and S10, performing data analysis. The process of tissue sample detection by the micro-fluidic chip is simplified, tissue shedding and RNA diffusion caused when a PDMS chip is pasted and uncovered are avoided, and the quality of space transcriptome sequencing is ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of spatial transcriptome sequencing, and in particular to a high-throughput spatial transcriptome sequencing method based on microfluidics technology. Background Art

[0002] High-throughput single-cell transcriptome sequencing (scRNA-seq) and spatial transcriptome sequencing (spatial RNA-seq) are the most important developments in the field of single-cell analysis in recent years. High-throughput scRNA-seq can quickly obtain gene expression profiles at the transcriptome level of thousands to tens of thousands of single cells at the resolution of single cells, thereby analyzing the cell types and composition in complex biological samples, and even discovering new cell types. However, one of the shortcomings of high-throughput scRNA-seq is that it loses the spatial information of cells, so spatial transcriptomes came into being.

[0003] The spatial coordinates of cells are unique and exclusive cell identifiers. The cells are matched with the spatial positions, that is, the cell code (16-base oligonucleotide sequence) corresponds to the spatial coordinates on the one hand and the cells on the other hand, so that the cell code and the spatial coordinates can be matched. There are two ways to encode cells in space. One is to randomly generate codes in the spatial region so that each position has a different code, and then the spatial coordinates and the code are matched through a "decoding" process. Professor Rong Fan of Yale University proposed a new method of direct spatial coding (DBiT-seq), that is, to directly encode cells at different positions through microfluidic chips and combined coding, without the need for decoding steps, which greatly simplifies the detection process. The obvious defects of DBiT-seq currently include: 1) The detection throughput is limited and difficult to expand. For microfluidic chips, 50 parallel microchannels below 50 microns are close to the limit of microfabrication and experimental operation (corresponding to 2500 coding and detection areas), and it is difficult to increase to 100 or even more to improve the detection throughput. 2) Tissue shedding affects sequencing quality. During the experiment, the first chip needs to be peeled off from the tissue slice (the chip is clamped tightly against the tissue slice to prevent leakage from the microchannel) and then the second chip is attached to the same slice. This operation will cause part of the slice to fall off and the RNA in the cells on the slice to diffuse, thus affecting the quality of the entire spatial transcriptome sequencing. Summary of the invention

[0004] The present invention designs a high-throughput spatial transcriptome sequencing method based on microfluidic technology to solve the problems in the prior art that the operation of using microfluidic chips for tissue sample detection is complicated, and that the tissue is easily detached and RNA is diffused when sticking and peeling the PDMS chip, thereby affecting the quality of spatial transcriptome sequencing.

[0005] To achieve the above object, the present invention provides the following technical solution: a high-throughput spatial transcriptome sequencing method based on microfluidics technology, comprising the following steps:

[0006] S1. Prepare a new type of microfluidic chip for high-throughput spatial encoding. The new microfluidic chip includes microfluidic PDMS chip one and microfluidic PDMS chip two. Among them, there are 5 longitudinal channels on microfluidic PDMS chip one, and 3 transverse channels on microfluidic PDMS chip two. Each microchannel on microfluidic PDMS chip one and microfluidic PDMS chip two contains 48 parallel microchannels, and the width of each microchannel is 30μm. Through the two microfluidic chips, the transverse microchannels intersect with the longitudinal microchannels 15 times, forming 15 areas with position coordinates, thereby greatly improving the encoding flux of the spatial transcriptome.

[0007] S2. A microfluidic PDMS chip is attached to a positively charged glass slide, and B0 is introduced into the microchannel. B0 is fixed to the positively charged glass slide through the mutual attraction between positive and negative charges. At the same time, B0 contains a sequence that is complementary to the base pairing of barcode-A. After incubation at room temperature for 30 minutes, 48 ​​barcode-As (1-48) are introduced into the channel, wherein barcode-A contains a complementary sequence to B0, an index sequence (8bp) and a complementary sequence to ligation linker1. Barcode-A binds to B0 through complementary base pairing.

[0008] S3, peel off the microfluidic PDMS chip 1, attach the microfluidic PDMS chip 2, and pass 48 barcode-B (1-48) and T4 DNA ligase ligation system into the channel, where barcode-B contains the complementary sequence of ligation linker1, UMI, Index sequence (8bp) and the complementary sequence of ligation linker2. After the microfluidic is passed through the reaction solution, it is placed in a wet box and incubated at 37℃ for 30 minutes. After this step of reaction, barcode-A and barcode-B are connected under the action of T4 DNA ligase.

[0009] S4. Remove the microfluidic PDMS chip 2 and attach the bottomless microporous PDMS chip 3. 15 independent areas can be formed on the chip. Add the T4 DNA ligase ligation system containing the barcode-C sequence (1-15) to different micropores respectively, and incubate at 37°C in a wet box for 30 minutes. The barcode-C sequence contains the complementary sequence of ligation linker2, the Index sequence (8bp) and the poly (T) structure for RNA capture and labeling. After this step of reaction, barcode-B and barcode-C are connected under the action of T4 DNA ligase, thereby forming a triple combination code.

[0010] S5. Peel off the bottomless microporous PDMS chip 3, attach the tissue slice to the glass slide loaded with the spatially encoded array, and then permeabilize the cell membrane to release the mRNA in the cell. The released mRNA is captured by the spatially encoded poly(T) on the surface of the glass slide.

[0011] S6. mRNA synthesizes cDNA on the surface of the glass slide under the catalysis of reverse transcriptase.

[0012] S7, digest the tissue sections, and destroy the base complementary pairing between B0 and barcode-A by adding alkaline solution, so as to release and recover the RNA / DNA hybrid chain product from the glass slide surface.

[0013] S8. Synthesize double-stranded cDNA and amplify double-stranded cDNA products.

[0014] S9, library construction and sequencing.

[0015] S10. Perform data analysis.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present invention relates to a high-throughput spatial transcriptome sequencing method based on microfluidic technology, which performs three-dimensional spatial encoding through multiple rounds of DNA ligation reactions, and then obtains the spatial position and biological information of cells in the corresponding tissue through sequencing, thereby realizing three-dimensional spatial omics analysis of the tissue and greatly simplifying the process of microfluidic chip for tissue sample detection.

[0018] 2. The present invention relates to a high-throughput spatial transcriptome sequencing method based on microfluidic technology, which completely separates the preparation of spatially encoded RNA capture probe arrays from tissue cell RNA release, capture, and reverse transcription. That is, a spatially encoded RNA capture probe array (which can be stored for a long time) is first prepared on a glass slide through a microfluidic chip; when there is a sample, the experimenter sticks the glass slide loaded with the spatially encoded probe array onto the tissue section for RNA release, capture, and reverse transcription. This step is simple to operate, does not require complex equipment, reduces cost investment, and is very suitable for general laboratories and experimenters to operate. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Embodiment 1: The present invention provides a technical solution, a high-throughput spatial transcriptome sequencing method based on microfluidics technology, comprising the following steps:

[0021] S1. Prepare a new type of microfluidic chip for high-throughput spatial encoding. The new microfluidic chip includes microfluidic PDMS chip one and microfluidic PDMS chip two. Among them, there are 5 longitudinal channels on microfluidic PDMS chip one, and 3 transverse channels on microfluidic PDMS chip two. Each microchannel on microfluidic PDMS chip one and microfluidic PDMS chip two contains 48 parallel microchannels, and the width of each microchannel is 30μm. Through the two microfluidic chips, the transverse microchannels intersect with the longitudinal microchannels 15 times, forming 15 areas with position coordinates, thereby greatly improving the encoding flux of the spatial transcriptome.

[0022] S2. A microfluidic PDMS chip is attached to a positively charged glass slide, and B0 is introduced into the microchannel. B0 is fixed to the positively charged glass slide through the mutual attraction between positive and negative charges. At the same time, B0 contains a sequence that is complementary to the base pairing of barcode-A. After incubation at room temperature for 30 minutes, 48 ​​barcode-As (1-48) are introduced into the channel, wherein barcode-A contains a complementary sequence to B0, an index sequence (8bp) and a complementary sequence to ligation linker1. Barcode-A binds to B0 through complementary base pairing.

[0023] S3, peel off the longitudinal microfluidic PDMS chip 1, attach the microfluidic PDMS chip 2, and pass 48 barcode-B (1-48) and T4 DNA ligase ligation system into the channel, where barcode-B contains the complementary sequence of ligation linker1, UMI, Index sequence (8bp) and the complementary sequence of ligation linker2. After the microfluidic is passed through the reaction solution, it is placed in a wet box and incubated at 37℃ for 30 minutes. After this step of reaction, barcode-A and barcode-B are connected under the action of T4 DNA ligase.

[0024] S4, peel off the lateral micro-channel PDMS chip 2, and stick on the bottomless micro-hole PDMS chip 3, which can form 15 independent areas on the chip. Add the T4 DNA ligase connection system containing the barcode-C sequence (1-15) to different micro-holes respectively, and incubate at 37°C in a wet box for 30 minutes. The Barcode-C sequence contains the complementary sequence of ligation linker2, the Index sequence (8bp) and the poly (T) structure for RNA capture and labeling. After this step of reaction, barcode-B and barcode-C are connected under the action of T4 DNA ligase, thereby forming a triple combination code.

[0025] S5. Peel off the bottomless microporous PDMS chip 3, attach the tissue slice to the glass slide loaded with the spatially encoded array, and then permeabilize the cell membrane to release the mRNA in the cell. The released mRNA is captured by the spatially encoded poly(T) on the surface of the glass slide.

[0026] S6. mRNA synthesizes cDNA on the surface of the glass slide under the catalysis of reverse transcriptase.

[0027] S7, digest the tissue sections, and destroy the base complementary pairing between B0 and barcode-A by adding alkaline solution, so as to release and recover the RNA / DNA hybrid chain product from the glass slide surface.

[0028] S8. Synthesize double-stranded cDNA and amplify double-stranded cDNA products.

[0029] S9, library construction and sequencing.

[0030] S10. Perform data analysis.

[0031] Embodiment 2: Microfluidic chip design.

[0032] There are 5 longitudinal channels on microfluidic PDMS chip 1 and 3 transverse channels on microfluidic PDMS chip 2. The two chips allow the microfluidic channels to intersect 15 times on the glass slide, forming 15 areas with position coordinates. That is, the chip design can obtain spatial encoding and transcriptome data of 34,560 points (15×48×48) in one detection. It achieves extremely high detection throughput and can be used to detect larger tissue slices.

[0033] Example 3: Design of microfluidic device.

[0034] Each capillary tube is pre-filled with a specific barcode by a syringe and then inserted into the corresponding chip hole. The barcode solution in the microtube is injected into the microchannel by an air pressure device until the entire microchannel is filled.

[0035] Example 4: Microfluidic fluorescence dot matrix imaging experiment.

[0036] First, the non-fluorescently labeled barcode-A was introduced into the microchannel of the microfluidic PDMS chip 1 and adsorbed on the positively charged glass slide by electrostatic adsorption.

[0037] Secondly, a closed barcode is introduced into the microchannel of the second microfluidic PDMS chip to prevent the subsequent introduction of the fluorescently labeled barcode-B from being electrostatically adsorbed on the surface of the glass slide.

[0038] Next, the fluorescently labeled barcode-B is introduced into the microchannel of the second microfluidic PDMS chip for the second time. The fluorescently labeled barcode-B and barcode-A have complementary base pairing sequences and can bind to each other.

[0039] Finally, fluorescence scanning imaging was performed to generate a square fluorescent spot at the intersection of the two microfluidic channels.

[0040] This method can form a fluorescent image of a DNA coordinate barcode array. This experiment proves the feasibility of constructing a high-throughput, high-density spatially coded probe array on a positively charged glass slide using a microfluidic device.

[0041] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0042] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A high-throughput spatial transcriptome sequencing method based on microfluidics technology, characterized in that: The following steps are involved: S1. Preparation of microfluidic chips for high-throughput spatial encoding; The microfluidic chip includes a microfluidic PDMS chip 1 and a microfluidic PDMS chip 2. The microfluidic PDMS chip 1 is provided with 5 longitudinal microchannels, and the microfluidic PDMS chip 2 is provided with 3 transverse microchannels. When the microfluidic PDMS chip 1 and the microfluidic PDMS chip 2 are bonded together, the transverse microchannels intersect with the longitudinal microchannels 15 times to form 15 areas with position coordinates. S2, attach a microfluidic PDMS chip 1 on the positively charged substrate, and introduce B0 into the microchannel; Among them, B0 is fixed to the substrate with positive charge on the surface through the mutual attraction between positive and negative charges. At the same time, B0 contains a sequence that is complementary to the base pair of barcode-A. After incubation, 48 kinds of barcode-A are introduced into the microchannel; S3, replacing the microfluidic PDMS chip 1 on the positively charged substrate with the microfluidic PDMS chip 2, and then introducing 48 barcode-B and T4 DNA ligase ligation systems into the microchannel, and incubating after the reaction solution is introduced into the microfluidic system; S4, replacing the microfluidic PDMS chip 2 on the positively charged substrate with the bottomless microwell PDMS chip 3, and then adding the T4 DNA ligase ligation system containing 15 barcode-C sequences into different microwells for incubation; S5. Peel off the bottomless microporous PDMS chip from the positively charged substrate. Paste the tissue slice onto the substrate loaded with the spatially encoded array, and then permeabilize the cell membrane to release the mRNA in the cell. The released mRNA is captured by the spatially encoded poly(T) on the surface of the substrate. S6, mRNA synthesizes cDNA on the substrate surface under the catalysis of reverse transcriptase; S7, digesting the tissue slices, and destroying the base complementary pairing between B0 and barcode-A by adding an alkaline solution, releasing and recovering the RNA / DNA hybrid chain product from the substrate surface; S8, synthesizing double-stranded cDNA and amplifying double-stranded cDNA products; S9, library construction and sequencing; S10. Perform data analysis.

2. The high-throughput spatial transcriptome sequencing method based on microfluidics technology according to claim 1, characterized in that: In the step S1, each microchannel on the microfluidic PDMS chip 1 and the microfluidic PDMS chip 2 respectively comprises 48 parallel microchannels, and the width of each microchannel is 30 μm.

3. The high-throughput spatial transcriptome sequencing method based on microfluidics technology according to claim 1, characterized in that: The incubation operation in step S2 is performed at room temperature for 30 minutes.

4. The high-throughput spatial transcriptome sequencing method based on microfluidics technology according to claim 1, characterized in that: In step S2, barcode-A includes a complementary sequence of B0, an index sequence and a complementary sequence of ligation linker 1, and Barcode-A is combined with B0 through complementary base pairing.

5. The high-throughput spatial transcriptome sequencing method based on microfluidics technology according to claim 1, characterized in that: In step S3, barcode-B includes the complementary sequence of ligation linker1, UMI, Index sequence and the complementary sequence of ligation linker2.

6. The high-throughput spatial transcriptome sequencing method based on microfluidics technology according to claim 5, characterized in that: During the incubation operation in step S3, the mixture is placed in a humidified box and maintained at 37° C. for 30 minutes to connect barcode-A and barcode-B under the action of T4 DNA ligase.

7. The high-throughput spatial transcriptome sequencing method based on microfluidics technology according to claim 1, characterized in that: During the incubation operation in step S4, the cells were placed in a humidified box and maintained at 37° C. for 30 minutes.

8. The high-throughput spatial transcriptome sequencing method based on microfluidics technology according to claim 4, characterized in that: In step S4, the Barcode-C sequence contains the complementary sequence of ligation linker 2, the Index sequence and the poly (T) structure for RNA capture and labeling. After this reaction, barcode-B and barcode-C are connected under the action of T4 DNA ligase to form a triple combination code.

9. The high-throughput spatial transcriptome sequencing method based on microfluidics technology according to claim 1, characterized in that: The substrate is a glass slide. The manufacturing method of the positively charged substrate is to physically or chemically modify the surface of the substrate so that it has a positive charge and generates a charge characteristic for electrostatic adsorption with oligonucleotides; wherein the modification includes poly-lysine modification.

10. The high-throughput spatial transcriptome sequencing method based on microfluidics technology according to claim 6, characterized in that: The Index sequence generates at most 4 8 The complementary sequence to B0, the complementary sequence to ligation linker 1, the complementary sequence to ligation linker 2, and the poly(T) structure are all constant sequences.