Method for improving microsphere matrix spatial transcriptome capture area and application thereof

Abstract

The present application relates to a method for improving the area of microsphere matrix spatial transcriptome capture and its application. The method comprises: carrying out tissue permeation treatment, using a microsphere matrix capture chip for capture, and then carrying out reverse transcription and cDNA single strand synthesis, cDNA double strand synthesis, partitioning the microsphere matrix capture chip, denaturing each region, and cDNA amplification. The present application designs a method for improving the area of microsphere matrix spatial transcriptome capture, which uses a broken partition or a hybridization gasket to set a partition for detection, is simple and easy to operate, does not need to directly improve the type of barcode, reduces the number of synthesized microsphere primers and the type of decoding probes when the microsphere pool is expanded, effectively saves costs, and also does not need more decoding rounds to realize the decoding of the microsphere chip, can reduce the damage of the decoding process to the capture chip, and effectively improves the efficiency of large-size spatial transcriptome chip capture.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and relates to a method for improving the capture area of a microsphere matrix spatial transcriptome and application thereof. BACKGROUND

[0002] Spatial transcriptome combines imaging and sequencing technologies, and can map the location of specific transcripts on a tissue, thereby indicating the expression of specific genes at specific sites. The emergence of spatial transcriptome technology provides a powerful tool for humans to analyze the nature of life in higher dimensions. Spatial transcriptome provides new insights in developmental biology, tumorigenesis, neuroscience, etc.

[0003] Currently, spatial transcriptome mainly faces two problems. One is how to improve the resolution of capture to meet the analysis of single-cell level spatial transcriptome. On the solid-phase capture chip, the number of transcripts captured needs to be further improved to realize more accurate single-cell segmentation analysis. On the other hand, how to effectively use large-size capture chips to meet the analysis of panoramic view of large tissues.

[0004] The mainstream platform 10x Genomics platform launched a commercial Visium spatial transcriptome chip in 2019, with a capture area of 6.5 mm x 6.5 mm. This size of capture area can only be limited to the analysis of small tissues at the millimeter level. The Visium CytAssist platform launched in 2023 only supports a capture area of 11 mm x 11 mm. Moreover, the CytAssist sample is based on the principle of probe capture to obtain gene expression information in cells, which is different from the conventional single-cell and spatial transcriptome which capture mRNA through 3'-polyA, and is not conducive to the joint analysis in the later stage. Another mainstream platform is the Stereo-Seq platform of BGI, which provides a centimeter-level large-field section spatial transcriptome capture chip from 5 mm x 5 mm to 130 mm x 130 mm. However, the silicon-based chip cannot perform high-definition HE image scanning, and the empty conversion chip based on the sequencing chip is extremely expensive.

[0005] HDST (High-definition spatial transcriptomics) is a high-resolution spatial transcriptomics technology that captures RNA on tissue sections using a dense matrix of microspheres, enabling high-resolution gene expression analysis in situ in tissues. HDST uses three-step microspheres for synthesis, with approximately 2,893,865 different microspheres, which makes the microsphere library only suitable for small-area capture analysis. When the capture area increases, the repeatability of the microspheres increases, and the resolution decreases due to the waste of data caused by the splitting of barcodes. Moreover, this method uses a 65x211x211 microsphere synthesis method, and 14 rounds of decoding are used for decoding. If the number of microsphere libraries is increased, the decoding rounds of the chip are increased, and more decoding probes need to be synthesized to combine the decoding probe pool. The S1000 chip is a 6.8 mm x 6.8 mm matrix, containing about 2.2 million micro-pits. When using a 384x384x384 microsphere pool, the total number of microspheres is 56623104, and the repetition rate is about 4%. When the number of micro-pits is increased to 4.1 million, the repetition rate is increased to 7%. When the number of micro-pits is increased to 10 million, the repetition rate is about 17%. If the number of microsphere pools is increased, the 768x768x768 microsphere pool has 2.2 million micro-pits, and the repetition rate is about 0.5%. When the number of micro-pits is increased to 4.1 million, the repetition rate is 1%. When the number of micro-pits is increased to 10 million, the repetition rate is 2%. However, the cost of microsphere primer synthesis needs to be doubled, and the synthesis and decoding rounds of decoding probes also need to be increased to a certain extent, which undoubtedly limits the update of the product. Using a 384x384x384 microsphere pool, if the capture area is increased to 13.6 mm x 13.6 mm, and the number of micro-pits is 16.4 million, the repetition rate is about 25%. If the capture area is further increased, the number of micro-pits will exceed the number of microsphere types, so a larger microsphere pool is needed for chip assembly, and the decoding probe and decoding process of the microsphere also need to be updated accordingly, which greatly increases the cost.

[0006] In summary, how to improve the capture area of the microsphere matrix spatial transcriptome is still one of the problems to be solved in the field of spatial transcriptome. SUMMARY

[0007] In view of the deficiencies of the prior art and actual needs, the present application provides a method for improving the capture area of the microsphere matrix spatial transcriptome and its application, in order to realize capture and library construction using large-size microsphere matrix capture chips.

[0008] To achieve this purpose, the present application adopts the following technical solutions:

[0009] In a first aspect, the present application provides a method for improving the capture area of the microsphere matrix spatial transcriptome, comprising:

[0010] The microsphere matrix capture chip is broken into multiple partitions by a chip breaking partition method or a hybrid gasket partition method.

[0011] In view of the problems of a large number of barcodes, microsphere pools, complicated operation and high cost in the use of a large-size microsphere matrix capture chip at present, a novel idea is adopted in the application, that is, after the transcripts are captured by using the large-size microsphere matrix capture chip, the second-strand cDNA is uniformly produced, then the chip substrate is broken into partitions or is partitioned by using a hybrid gasket, and the generated second-strand cDNA is dissociated from the partitions, after sequencing, the sequencing results can be traced back and integrated according to the partition mode, and finally a large-field spatial transcriptome data is generated. Compared with the traditional method, the number of barcodes is not increased, the number of synthesized microsphere primers and the number of decoding probes are reduced when the microsphere pool is expanded, the cost is effectively saved, and the decoding of the microsphere chip can be realized without more decoding rounds, the damage to the capture chip in the decoding process is reduced, and the capture efficiency of the large-size microsphere matrix capture chip is effectively improved.

[0012] The microsphere matrix capture chip in the application refers to a chip for spatial transcriptome capture or spatial multi-omics analysis, and it can be understood that functionally similar capture chips in the art are suitable for the application, such as a capture chip with a microsphere matrix obtained by synthesizing three-section microspheres in HDST technology.

[0013] In the application, the hybrid gasket refers to a silica gel gasket with an adhesive, which can be combined with the capture chip within a few seconds, and then different partition regions are formed.

[0014] Preferably, the number of regions arranged is 2-225, for example, 2, 3, 4, 5, 6, 10 or 20, and the like, which are not listed one by one.

[0015] Preferably, the area of the microsphere matrix capture chip is 0.25 cm 2 ~225 cm 2 .

[0016] In the application, the shape of the partition can be designed as a grid, such as a sun-shaped grid, a field-shaped grid, and the like, and the area of different partitions can be 0.125 cm 2 ~10 cm 2 .

[0017] Preferably, the tissue permeabilization solution for the tissue permeabilization treatment contains at least one of pepsin, Triton X-100 or HC1; the mass percentage of pepsin in the tissue permeabilization solution is 0.01% to 1% (for example, it can be 0.02%, 0.03%, 0.05%, 0.1%, 0.12%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% or 0.9%, etc.); the concentration of Triton X-100 in the tissue permeabilization solution is 0.1 to 2 mM (for example, it can be 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8 or 1.9 mM, etc.); the concentration of HC1 in the tissue permeabilization solution is 0.1 to 1 M (for example, it can be 0.2, 0.3, 0.5, 0.6, 0.7, 0.8 or 0.9 M, etc.).

[0018] Preferably, the reagents for reverse transcription and cDNA single strand synthesis include reverse transcription buffer, reverse transcriptase, dNTPs, DTT and strand displacement primer; wherein the reverse transcription buffer contains 100 to 500 mM Tris-HCl, 200 to 500 mM KCl, 10 to 30 mM MgCl2 and 10 to 100 mM DTT.

[0019] The above numerical ranges represent optional ranges within the listed ranges, such as 100 to 500 mM can be selected as 101, 102, 110, 120, 150, 200, 220, 240, 260, 300, 320, 350, 380, 400, 450, 460, 470, 480 or 490 mM, etc., 200 to 500 mM can be 201, 202, 205, 210, 220, 240, 280, 300, 320, 350, 380, 400, 450, 460, 470, 480 or 490 mM, etc., 10 to 30 mM can be 11, 12, 13, 14, 15, 20, 22, 26, 28 or 29 mM, etc., 10 to 100 mM can be 11, 12, 15, 18, 20, 25, 28, 30, 35, 40, 45, 50, 60, 70, 80, 90, 92, 94, 96 or 98 mM, etc.

[0020] In the present application, common reverse transcriptases on the market can be used, such as one or more of SuperScript™ II, SuperScript™ III, SuperScript™ IV of Thermofisher Company, HiScript II Reverse Transcriptase, HiScript III Reverse Transcriptase, HiScript Reverse Transcriptase of Vazyme Company, etc.

[0021] dNTPs: 1~20 mM dNTPs; DTT: 1~200 mM DTT; SSP: 10~100 μM strand displacement primer.

[0022] Preferably, the reagents for cDNA second strand synthesis include second strand synthesis buffer, dNTPs, DNA polymerase and second strand reaction primer; wherein the second strand synthesis buffer contains 100~300 mM Tris-HCl, 50~200 mM (NH4)2SO4, 100~1000 mM KCl, 10~50 mM MgSO4and 0.5%~5% Tween-20, pH 7~9.

[0023] The above numerical ranges represent options within the listed ranges, such as 100~300 mM can be selected as 101, 102, 110, 120, 150, 200, 220, 240, 260, 270, 280 or 290 mM, etc., 50~200 mM can be 51, 52, 55, 60, 80, 90, 100, 120, 150, 180 or 190 mM, etc., 100~1000 mM can be 101, 102, 103, 140, 180, 200, 220, 280, 300, 350, 380, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 920, 940, 960, 970, 980 or 990 mM, etc., 10~50 mM can be 11, 12, 15, 18, 20, 25, 28, 30, 35, 40, 45, 46 or 48 mM, etc., 0.5%~5% can be 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 4.6%, 4.7%, 4.8% or 4.9%.

[0024] dNTPs: 1-20 mM dNTPs; Second Strand Enzyme: one or more of DNA polymerase I large fragment (Klenow), DNA polymerase I (E. coli), and other polymerases with DNA synthesis function; Second Strand Primer: 1-100 μM second strand primer.

[0025] In a second aspect, the application provides an application of the method for increasing the capture area of a microsphere matrix spatial transcriptome in constructing a spatial transcriptome sequencing library.

[0026] The application provides an operating method of a large field spatial transcriptome chip, which recovers second strand products by physical fragmentation or hybridization pad partitioning.

[0027] In a third aspect, the application provides a method for constructing a spatial transcriptome sequencing library, which uses the method for increasing the capture area of a microsphere matrix spatial transcriptome in the first aspect for capture, and specifically includes the following steps: (1) tissue patching, fixing, and staining treatment; (2) tissue permeation treatment; (3) reverse transcription and cDNA first strand synthesis; (4) cDNA second strand synthesis; (5) cDNA partitioning melting and PCR amplification; and (6) cDNA purification.

[0028] Preferably, step (1) includes: fixing and staining treatment by pasting a tissue section on a microsphere matrix capture chip, wherein the microsphere matrix capture chip is provided with mRNA capture probes.

[0029] Preferably, step (2) includes: placing the microsphere matrix capture chip obtained in step (1) into the tissue permeation solution and incubating at 30-40℃ for 2-30 min, removing the tissue permeation solution, and adding SSC solution (Saline Sodium Citrate, concentration of 0.1×-5×) into the well of the microsphere matrix capture chip.

[0030] Preferably, step (3) includes: removing the SSC solution in the well of the microsphere matrix capture chip, adding the reagents for reverse transcription and cDNA first strand synthesis into the well, and incubating at 37-65℃ (for example, 38, 39, 40, 41, 42, 43, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64℃, etc.) for 0.5-2 h (preferably, 42℃ for 1.5 h).

[0031] Preferably, step (4) comprises: removing the reagents for reverse transcription and cDNA first strand synthesis in the microparticle matrix capture chip wells, adding KOH (concentration of 0.05-0.5 M) solution for denaturation, then washing with EB buffer, and then adding the reagents for cDNA second strand synthesis, incubating at 45-70°C (for example, it can be 46, 47, 48, 49, 50, 55, 60, 61, 62, 65, 66, 67, 68 or 69°C, etc.) for 10-40 min (preferably, 65°C for 30 min).

[0032] Preferably, the EB buffer is 10 mM Tris-HCl, pH 7.5.

[0033] Preferably, step (5) comprises: removing the reagents for cDNA second strand synthesis in the microparticle matrix capture chip wells, washing with EB buffer, adding KOH after partitioning the microparticle matrix capture chip, incubating at 20-30°C for 2-10 min, and then neutralizing the KOH with 0.1-1 M Tris (pH=6-8), and then adding cDNA amplification reagents to each region for PCR amplification.

[0034] Preferably, the PCR amplification program is: 95-98°C, 20-40 s; 95-98°C, 15-30 s, 62-68°C, 15-30 s, 60-70°C, 4-8 min, 10-20 cycles; 60-70°C, 4-6 min.

[0035] Further preferably, the PCR amplification program is: 95°C, 30 s; 95°C, 15 s, 62°C, 15 s, 65°C, 8 min, 15 cycles; 65°C, 6 min.

[0036] Preferably, step (6) comprises: purifying the amplification product of step (5) with magnetic beads, then washing the magnetic beads with ethanol, and finally eluting the amplification product from the magnetic beads with EB buffer.

[0037] Preferably, the method for constructing a spatial transcriptome sequencing library further comprises a step of cDNA quality control.

[0038] Preferably, the cDNA quality control comprises determining the concentration of the purified cDNA product and determining the peak type of the cDNA product.

[0039] In a fourth aspect, the present application provides a spatial transcriptome sequencing method, which utilizes the method for constructing a spatial transcriptome sequencing library of the third aspect to construct a library, sequences each partition, backtracks and integrates the sequencing results in a partitioned manner, and obtains spatial transcriptome sequencing results.

[0040] Preferably, the spatial transcriptome sequencing method specifically comprises: (1) constructing a library using the method for constructing a spatial transcriptome sequencing library of the third aspect; (2) sequencing each partition, respectively identifying barcode and umi (unique molecular marker) of data of each partition, aligning the genome and determining the barcode position; (3) combining the expression information corresponding to the barcode of different partitions; (4) combining the barcode position information of different partitions, and determining the target barcode information according to the barcode and umi results identified by different partitions for the spot (spot) near the edge of the partition; (5) filtering the repeated barcodes in different partitions; (6) obtaining the above results to generate a spatial expression matrix file, performing data analysis, and obtaining complete spatial transcriptome data.

[0041] In the present application, a spatial transcriptome sequencing method using a large-area capture chip is developed, which can effectively analyze large-area tissues and can be effectively applied to disease diagnosis or non-disease diagnosis fields (such as basic research on gene expression mechanism) and the like.

[0042] Compared with the prior art, the present application has the following beneficial effects:

[0043] The method for improving the microsphere matrix spatial transcriptome capture area of the present application adopts a new idea to detect by breaking and partitioning the chip or using a hybrid gasket to set partitions, which is simple and easy to operate. Compared with the traditional method, while using a large-area capture chip, the present application does not need to directly increase the types of barcodes, reduces the number of synthesized microsphere primers and the types of decoding probes when expanding the microsphere pool, effectively saves costs, and also does not need more decoding rounds to realize decoding of the microsphere chip, which can reduce damage to the capture chip in the decoding process and effectively improve the efficiency of large-size spatial transcriptome chip capture. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Schematic diagram of the method for breaking and partitioning the melting.

[0045] Figure 2 Schematic diagram of the method for hybrid gasket partitioning and melting. DETAILED DESCRIPTION

[0046] The technical solutions of the present application will be further described below in combination with the drawings and through specific embodiments. However, the following examples are only simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0047] Unless otherwise indicated, the techniques or conditions of the embodiments described and techniques or conditions described in the literature are used. Unless otherwise indicated, the reagents or instruments used are conventional products that can be purchased from a regular channel.

[0048] At present, to improve the spatial transcriptome capture area of microsphere matrix chip, the number of micropits will exceed the number of microsphere types, so a larger microsphere pool is needed for chip assembly, and the decoding probe and decoding process of the microsphere also need to be updated accordingly. The cost of this improvement is very large. In view of the above problems, based on a new idea, a method for improving the spatial transcriptome capture area of microsphere matrix is ingeniously designed, which directly uses a large-area microsphere matrix capture chip. After fixing and staining the tissue section on the capture chip, it is placed in a clamp, and the prepared permeabilization enzyme is used for tissue permeabilization, then the tissue permeabilization solution is removed, and a one-chain cDNA synthesis mixed solution is poured into the chip, and reverse transcription is carried out. After completion, the RNA is denatured and then the synthesis of cDNA double strand is carried out. The chip is broken into partitions or partitioned using a hybridization gasket (the method for partitioning and denaturing the chip by breaking is schematically shown in Figure 1 , and the method for partitioning and denaturing the chip by using a hybridization gasket is schematically shown in Figure 2 ), then the cDNA in each region is denatured, and then the cDNA sample is amplified to obtain a sample that can be used to construct a second-generation library, and then sequencing analysis can be carried out. While using a large-area capture chip, it is not necessary to directly increase the number of barcodes.

[0049] Example 1

[0050] The present embodiment provides a method for improving the spatial transcriptome capture area of microsphere matrix based on partitioning by breaking, and a method for interpreting the results by using high-sensitivity Agilent 2100 and its application. By using this method, RNA capture, cDNA one-chain synthesis, cDNA two-chain synthesis, chip breaking partition denaturation, and cDNA amplification can be carried out on the tissue sample to obtain a cDNA sample with spatial information of the tissue sample. Downstream sequencing can be carried out. Compared with the traditional method, the method has the advantages of simple and convenient operation, stable performance, and large capture area.

[0051] Take a 13.6 mm x 13.6 mm capture chip (preliminary microetching on the back of the chip using a laser to facilitate later fragmentation) as an example, the capture chip is a S series spatial transcriptome capture chip purchased from Qingdao Baichuang Intelligent Manufacturing Technology Co., Ltd., and the method for breaking into four regions for spatial transcriptome gene expression includes the following steps:

[0052] 1. Tissue patch, fixation and staining: cut the fresh frozen tissue (mouse whole brain) into sections and fix and stain them on the permeabilization chip, wherein the permeabilization chip has mRNA capture probes.

[0053] Tissue staining and imaging:

[0054] 1.1 Tissue fixation

[0055] 40 mL of methanol was pre-cooled to -20°C in a 50 mL centrifuge tube.

[0056] PCR machine was set to 37°C, hot lid temperature was set to 37°C or 50°C, and the adapter was equilibrated to the temperature.

[0057] The chip with tissue was taken out from -80°C (dry ice transport) and the tissue side was placed on the 37°C adapter for 1 min (without hot lid); if the tissue was just mounted on the chip in the cryostat, this step was also performed.

[0058] The chip was placed in the pre-cooled methanol and incubated at -20°C for 30 min.

[0059] During this time, the reagents for tissue staining were prepared.

[0060] 1.2 Tissue staining

[0061] 1.2.1 Three 1 L beakers were prepared in advance, each containing no less than 800 mL of ultrapure water.

[0062] 1.2.2 The eosin mix was prepared according to Table 1 below.

[0063] Table 1

[0064]

[0065] The hematoxylin stain solution needed to be filtered using a 0.2 pm filter membrane before use.

[0066] 1.2.3 The fixed chip was wiped off excess methanol liquid and placed on a clean benchtop.

[0067] 1.2.4 Isopropyl alcohol was added to the tissue surface and evenly covered, and incubated at room temperature for 1 min.

[0068] 1.2.5 The chip was tilted to remove the isopropyl alcohol, and excess liquid was wiped off. The chip could be gently shaken to evaporate the excess isopropyl alcohol on the tissue. This process should not exceed 5 min to avoid excessive drying.

[0069] 1.2.6 The filtered hematoxylin stain solution was added to the tissue surface and evenly covered, and incubated at room temperature for 1-7 min (depending on the tissue).

[0070] 1.2.7 Tilt the chip to remove the hematoxylin stain and rinse the chip in the first cup of ultrapure water for about 15 times, then rinse the chip in the second cup of ultrapure water for about 15 times, and wipe off the excess liquid.

[0071] 1.2.8 Add the indigo stain to the tissue side and cover evenly, incubate at room temperature for 30 s~2 min (vary depending on the tissue).

[0072] 1.2.9 Tilt the chip to remove the indigo stain and rinse the chip in the second cup of ultrapure water for about 5 times, and wipe off the excess liquid.

[0073] 1.2.10 Add the prepared eosin mixture to the tissue side and cover evenly, incubate at room temperature for 30 s~1 min (vary depending on the tissue).

[0074] 1.2.11 Tilt the chip to remove the eosin mixture and rinse the chip in the third cup of ultrapure water for about 20 times, and wipe off the excess liquid.

[0075] 1.2.12 Place the chip in a clean 50 mL centrifuge tube and balance the centrifuge, 250 x g, 30 s.

[0076] 1.2.13 Take out the chip and incubate on the 37°C adapter for 1 min.

[0077] 1.3 Brightfield tissue imaging

[0078] 1.3.1 Place the chip on the stage and load into the instrument.

[0079] 1.3.2 After the system pre-acquires the image, place the cursor point on the tissue image for automatic focusing.

[0080] 1.3.3 Set the focus value to ±50, select the folder for image saving and name it.

[0081] 1.3.4 Frame the tissue position and click to scan.

[0082] 2. Tissue permeation

[0083] 2.1 Place the tissue permeation enzyme (Permeabilization Enzyme, i.e. 0.1% pepsin) in a 37°C metal bath and preheat for 10 min.

[0084] 2.2 Place the adapter in the PCR instrument, and set the PCR instrument as shown in Table 2.

[0085] Table 2

[0086]

[0087] 2.3 Mount the chip into the chip card holder, add 300 μL of pre-warmed tissue permeation enzyme to the side of the well, and make sure the enzyme covers the entire matrix (do not introduce air bubbles).

[0088] 2.4 Seal with sealing film, place on the adapter in the PCR machine, cover with the heat cover, and incubate at 37°C for a determined time, using the timer.

[0089] 2.5 Dilute the 20x SSC buffer to 0.1x SSC buffer, vortex, and centrifuge briefly.

[0090] 2.6 The permeation time varies according to the tissue and is determined by the tissue permeation experiment. At the end, remove the card holder, tear the seal, and aspirate the tissue permeation enzyme along the edge of the matrix. Add 300 μL of the prepared 0.1x SSC buffer to the well and cover the matrix evenly (avoid introducing air bubbles), and reseal.

[0091] 3. cDNA first strand synthesis

[0092] 3.1 Prepare the cDNA first strand synthesis mix as shown in Table 3 on ice.

[0093] Table 3

[0094]

[0095] Chain displacement primer sequence: 5'-TTTCTGTTGGTGCTGATATTGCTrGrGrG-3' (SEQ ID NO. 1), rG represents a G base in RNA form.

[0096] Vortex, and centrifuge briefly.

[0097] 3.2 Place the adapter in the PCR machine, and set the PCR machine to the program in Table 4.

[0098] Table 4

[0099]

[0100] 3.3 Tear the seal, aspirate the buffer along the side of the well, add the first strand synthesis mix (evenly cover, avoiding air bubbles), reseal, place the card holder on the adapter, cover with the heat cover, skip the preheating step, and start the first strand synthesis.

[0101] 4. cDNA second strand synthesis and denaturation

[0102] 4.1 Remove the card holder from the adapter, tear the seal, and aspirate the first strand synthesis mix along the side of the well.

[0103] 4.2 Add 300 μΐ of 0.08 M KOH to the well, and incubate at room temperature for 5 min.

[0104] 4.3 Discard the 0.08 M KOH after pipetting along the side of the well at a slow and steady pace for 5 times (do not be too vigorous).

[0105] 4.4 Add 300 μΐ of EB buffer to the well, and incubate at room temperature for 2 min.

[0106] 4.5 Prepare the cDNA second strand synthesis mix as shown in Table 5 on ice.

[0107] Table 5

[0108]

[0109] The sequence of the second strand reaction primer is: 5'-TTTCTGTTGGTGCTGATATTGC-3' (SEQ ID NO. 2).

[0110] Vortex and centrifuge briefly.

[0111] 4.6 Place the adapter in the PCR machine, and set the PCR machine to the program shown in Table 6.

[0112] Table 6

[0113]

[0114] 4.7 Discard the EB buffer after pipetting along the side of the well at a slow and steady pace for 5 times (do not be too vigorous).

[0115] 4.8 Add the cDNA second strand synthesis mix to the well (avoid air bubbles), place the adapter on the well, skip the pre-heat, and start the cDNA second strand synthesis.

[0116] 4.9 After the cDNA second strand synthesis is complete, remove the second strand synthesis mix from the well.

[0117] 4.10 Add 300 μΐ of EB buffer to the well, and incubate at room temperature for 2 min.

[0118] 4.11 Discard the EB buffer after pipetting along the side of the well at a slow and steady pace for 5 times (do not be too vigorous).

[0119] 4.12 Split the capture area from the back of the chip along the laser-etched line, and then add 45 μΐ of 0.08 M KOH to each partition, making sure that it covers the entire partition matrix, and incubate at room temperature for 10 min, during which time use the pipette to pipette twice every two minutes.

[0120] 4.13 Pipette 42 μL of 0.08M KOH and 6 μL of Tris (1M, pH 7.0) into a 0.2 mL low-adsorption centrifuge tube, mix and centrifuge briefly then place on ice.

[0121] 5. cDNA amplification

[0122] 5.1 Determination of number of qPCR cycles

[0123] 5.1.1 Prepare qPCR mix as shown in Table 7 on ice.

[0124] Table 7

[0125]

[0126] 1x cDNA primer Primer sequence is:

[0127] Primer F: 5'-CTACACGACGCTCTTCCGATCT-3' (SEQ ID NO. 3).

[0128] Primer R: 5'-TTTCTGTTGGTGCTGATATTGC-3' (SEQ ID NO. 4).

[0129] Mix by vortexing and centrifuge briefly.

[0130] 5.1.2 Add 9 μL of qPCR mix to each well of a qPCR plate (2 sample replicates per well, 1 negative control, and optionally a positive control, scale up qPCR mix as necessary).

[0131] 5.1.3 Pipette 1 μL of sample from step 4.13 into each sample replicate well and 1 μL of water into the negative control well, and 1 μL of a quality controlled cDNA sample into the positive control well if present.

[0132] 5.1.4 Perform qPCR according to the program shown in Table 8.

[0133] Table 8

[0134]

[0135] 5.1.5 Record the resulting Cq values.

[0136] 5.2 cDNA amplification

[0137] 5.2.1 Prepare cDNA amplification mix as shown in Table 9 on ice.

[0138] Table 9

[0139]

[0140] 15x cDNA primer Primer sequences are as follows:

[0141] Primer F: 5'-CTACACGACGCTCTTCCGATCT-3' (SEQ ID NO. 5).

[0142] Primer R: 5'-TTTCTGTTGGTGCTGATATTGC-3' (SEQ ID NO. 6).

[0143] Vortex to mix, spin down briefly.

[0144] 5.2.2 To 4.13 samples, add 51 μΐ of cDNA amplification mix to each of the 4 partitions, vortex to mix, spin down briefly, avoiding air bubbles.

[0145] 5.2.3 Set up the following Table 10 program on the PCR machine for cDNA amplification.

[0146] Table 10

[0147]

[0148] 5.2.4 Quality control node, use Qubit to determine the concentration of the post-PCR product.

[0149] 6. cDNA purification

[0150] 6.1 Vortex SPRIselect magnetic beads to mix well, add 60 μΐ of SPRIselect magnetic beads (0.6x) to each sample tube (100 μΐ), pipette mix 15 times.

[0151] 6.2 Incubate at room temperature for 5 min.

[0152] 65.3 Place on a magnetic stand until the solution is clear.

[0153] 6.4 Remove the supernatant.

[0154] 6.5 Add 200 μΐ of 80% ethanol to the tube, stand for 30 s.

[0155] 6.6 Remove the ethanol.

[0156] 6.7 Repeat steps 6.5 and 6.6, a total of 2 washes.

[0157] 6.8 Spin down briefly and transfer to a magnetic stand.

[0158] 6.9 Remove residual alcohol, dry at room temperature for 2 min.

[0159] 6.10 The tube is removed from the magnetic stand and 40.5 μL EB buffer is added and mixed by pipetting 15 times.

[0160] 6.11 Incubate at room temperature for 2 min.

[0161] 6.12 Place the tube on the magnetic stand until the solution is clear.

[0162] 6.13 Pipette 40 μL of the liquid into a new tube.

[0163] 6.14 Store at 4°C for 72 h, or at -20°C for 4 weeks, or directly proceed to the next step.

[0164] 7. cDNA quantification and quality control

[0165] 7.1 Use Qubit to determine the concentration of the purified cDNA product and dilute to 2 ng / μL.

[0166] 7.2 Use High sensitivity Agilent Technologies 2100 Bioanalyzer to determine the peak type of the cDNA product.

[0167] 8. Next-generation sequencing and analysis

[0168] Sequence each partition, and integrate the sequencing results according to the partition, and finally generate a large field of view of the spatial transcriptome data, which specifically includes:

[0169] 8.1 Perform barcode and umi recognition, genome alignment, and barcode position determination for the data of each partition.

[0170] 8.2 Merge the expression information corresponding to the barcodes of different partitions.

[0171] 8.3 Merge the barcode position information of different partitions, and determine the appropriate barcode information for the spot near the edge of the partition according to the barcode and umi results recognized by different partitions.

[0172] 8.4 Filter the repeated barcodes in different partitions.

[0173] 8.5 After obtaining the above results, generate a spatial expression matrix file, perform data analysis, and obtain a complete spatial transcriptome data.

[0174] Perform overall sequencing analysis without splitting the test as the control group (the remaining operations refer to the above operations).

[0175] The specific detection results are shown in Table 11. The control group is not split, direct operation, the microsphere repetition rate is high, and the data utilization rate is low. The application adopts a new idea to detect the chip by breaking and partitioning. While using a large area of capture chip, it does not need to directly increase the type of barcode (Barcode), which can effectively reduce the microsphere repetition rate and improve the data utilization rate.

[0176] Table 11

[0177]

[0178] Example 2

[0179] The embodiment provides a method for partitioning hybridization pads to further improve the space transcriptome capture area of microsphere matrix, a method for interpreting results by using high-sensitivity Agilent 2100 and applications thereof. The method can be used for RNA capture of tissue samples, single-strand synthesis of cDNA, double-strand synthesis of cDNA, partitioning and melting of hybridization pads, and cDNA amplification to obtain a final cDNA sample with spatial information of tissue samples, and downstream second-generation or third-generation sequencing. Compared with the traditional method, the method has the advantages of simple and convenient operation, stable performance, and large capture area.

[0180] Taking a method for spatial transcriptome gene expression by using a hybridization pad to divide the capture area after double-strand synthesis into four areas on a 13.6 mm*13.6 mm capture chip as an example, the method comprises the following steps.

[0181] 1. Tissue patching, fixing and staining: fresh frozen tissue (mouse whole brain) is cut into slices and attached to a permeabilization chip for fixing and staining, wherein the permeabilization chip is provided with mRNA capture probes.

[0182] Tissue staining and imaging:

[0183] 1.1 Tissue fixing

[0184] Refer to step 1.1 in embodiment 1.

[0185] 1.2 Tissue staining

[0186] Refer to step 1.2 in embodiment 1.

[0187] 1.3 Bright field tissue imaging

[0188] Refer to step 1.3 in embodiment 1.

[0189] 2. Tissue permeabilization

[0190] Refer to step 2 in embodiment 1.

[0191] 3. Single-strand synthesis of cDNA

[0192] Refer to Step 3 in Example 1.

[0193] 4. cDNA second strand synthesis and denaturation

[0194] Steps 4.1-4.11 refer to Steps 4.1-4.11 in Example 1.

[0195] 4.12 Divide the capture area into four areas using hybridization spacers, and add 45 μL of 0.08M KOH to each area, making sure to cover the entire partition matrix. Incubate at room temperature for 10 minutes, blowing and sucking with a pipette every two minutes during the incubation, and making sure that the different areas do not contaminate each other.

[0196] 4.13 Mix 42 μL of 0.08M KOH and 6 μL of Tris (1 M, pH 7.0) in a 0.2 mL low-adsorption centrifuge tube for each of the four areas, and place it on ice after centrifugation. Then add 45 μL of 0.08M KOH to the area where the second strand was not eluted due to the width of the hybridization spacer, and mix 42 μL of 0.08M KOH and 6 μL of Tris (1 M, pH 7.0) in a 0.2 mL low-adsorption centrifuge tube for this area.

[0197] 5. cDNA amplification

[0198] 5.1 qPCR cycle number determination

[0199] Refer to Step 5.1 in Example 1.

[0200] 5.2 cDNA amplification

[0201] 5.2.1 Prepare the following cDNA amplification mixture on ice, referring to Step 5.2.1 in Example 1.

[0202] 5.2.2 Add a spacer to cover the area in the 4.13 sample, and add a total of 51 μL of cDNA amplification mixture to each of the 5 tubes, mix well by blowing, and centrifuge immediately to avoid air bubbles.

[0203] 5.2.3 Set the following program on the PCR instrument for cDNA amplification, referring to Example 1.

[0204] 5.2.4 Quality control node, use Qubit to determine the concentration of the PCR product.

[0205] 6. cDNA purification

[0206] Refer to Step 6 in Example 1.

[0207] 7. cDNA quantification and quality control

[0208] Reference to step 7 in Example 1.

[0209] 8. Second-generation sequencing and analysis

[0210] Each partition is sequenced, and the sequencing results are back-integrated in a partitioned manner, and finally a large field of view of spatial transcriptome data is generated.

[0211] The test without partitioning is used as a control group, and the whole is sequenced and analyzed.

[0212] The specific detection results are shown in Table 12. The control group is not split, directly operated, the microsphere repetition rate is high, and the data utilization rate is low. The application uses a new idea, uses a hybrid gasket to set partitions for detection, while using a large area capture chip, without directly increasing the number of barcodes (Barcode), it can effectively reduce the microsphere repetition rate and improve the data utilization rate.

[0213] Table 12

[0214]

[0215] In summary, the application designs a method for improving the microsphere matrix spatial transcriptome capture area, which uses a new idea to break and partition the chip or use a hybrid gasket to set partitions for detection. The operation is simple and easy to operate. Compared with the traditional method, while using a large area capture chip, without directly increasing the number of barcodes (Barcode), reducing the number of synthesized microsphere primers and the number of decoding probes when expanding the microsphere pool, effectively saving costs, and without more decoding rounds to realize the decoding of the microsphere chip, it can reduce the damage to the capture chip in the decoding process, and effectively improve the efficiency of the large size spatial transcriptome chip capture.

[0216] The applicant declares that the above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the application can be easily thought of by any person skilled in the art, and all fall within the protection scope and disclosure scope of the application.

Claims

1. A spatial transcriptome sequencing method, characterized by, The spatial transcriptome sequencing method utilizes a method for constructing a spatial transcriptome sequencing library to construct the library, sequences each partition, respectively identifies the barcode and umi of the data of each partition, aligns the genome and determines the barcode position, merges the expression information corresponding to the barcodes of different partitions, and merges the barcode position information of different partitions. The target barcode information is determined according to the barcode and umi results identified by different partitions for the points near the edge of the partition; The barcodes that appear repeatedly in different partitions are filtered; after obtaining the above results, a spatial expression matrix file is generated, data analysis is performed, and complete spatial transcriptome data is obtained; The method for constructing a spatial transcriptome sequencing library comprises the following steps: (1) tissue slice, fixation and staining treatment; (2) tissue permeation treatment; (3) reverse transcription and cDNA single strand synthesis; (4) cDNA double strand synthesis; (5) cDNA partition melting and PCR amplification; (6) cDNA purification; Step (1) comprises: attaching a tissue slice to a microsphere matrix capture chip for fixation and staining treatment, wherein the microsphere matrix capture chip is provided with mRNA capture probes; Step (2) comprises: placing the chip obtained in step (1) into the tissue permeation solution and incubating at 30-40℃ for 2-30 min, removing the tissue permeation solution and adding 0.1×-5×SSC solution into the hole of the microsphere matrix capture chip; Step (3) comprises: removing the SSC solution in the hole of the microsphere matrix capture chip, adding reverse transcription and cDNA single strand synthesis reagents into the hole, and incubating at 37-65℃ for 0.5-2 h; Step (4) comprises: removing the reverse transcription and cDNA single strand synthesis reagents in the hole of the microsphere matrix capture chip, adding KOH solution for melting denaturation, then washing with EB buffer, and then adding cDNA double strand synthesis reagents, and incubating at 45-70℃ for 10-40 min; Step (5) comprises: removing the cDNA double strand synthesis reagents in the hole of the microsphere matrix capture chip, adding EB buffer for washing, dividing the microsphere matrix capture chip into partitions, then adding KOH, incubating at 20-30℃ for 2-10 min, neutralizing the KOH with 0.1-1M Tris, and adding cDNA amplification reagents to each region for PCR amplification; The PCR amplification program is as follows: 95 ～ 98℃、20～40s； 95-98℃, 15-30s, 62-68℃, 15-30s, 60-70℃, 4-8min, 10-20 cycles; 60-70℃, 4-6min; Step (6) comprises: purifying the amplification product of step (5) with magnetic beads, then washing the magnetic beads with ethanol, and then eluting the amplification product from the magnetic beads with EB buffer; The partition method comprises a chip breaking partition method or a hybridization gasket partition method; The chip breaking partition method comprises breaking the microsphere matrix capture chip into multiple partitions; The hybridization gasket partition method comprises partitioning the microsphere matrix capture chip using a hybridization gasket; The microsphere matrix capture chip has an area of 0.25 cm 2 ~ 225 cm 2 ; The number of regions set by the partitioning is 2-225; The tissue permeation solution for the tissue permeation treatment contains at least one of pepsin, Triton X-100 or HCl; the mass percentage of pepsin in the tissue permeation solution is 0.01%-1%; the concentration of Triton X-100 in the tissue permeation solution is 0.1-2 mM; and the concentration of HCl in the tissue permeation solution is 0.1-1 M; The reagents for reverse transcription and cDNA single strand synthesis include reverse transcription buffer, reverse transcriptase, dNTPs, DTT and strand displacement primer; wherein the reverse transcription buffer contains 100-500 mM Tris-HCl, 200-500 mM KCl, 10-30 mM MgCl2 and 10-100 mM DTT; The reagents for cDNA double strand synthesis include double strand synthesis buffer, dNTPs, DNA polymerase and double strand reaction primer; wherein the double strand synthesis buffer contains 100-300 mM Tris-HCl, 50-200 mM (NH4)2SO4, 100-1000 mM KCl, 10-50 mM MgSO4 and 0.5%-5% Tween-20, and the pH is 7-9.

2. The method of spatial transcriptome sequencing according to claim 1, wherein, The method for constructing a spatial transcriptome sequencing library further comprises a cDNA quality control step.

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