Method for increasing capture area of spatial transcriptome of microsphere matrix and application of method

Abstract

The invention relates to a method for increasing the capture area of a microsphere matrix space transcriptome and application of the method. The method comprises the following steps: carrying out tissue permeabilization treatment, capturing by using a microsphere matrix capturing chip, then carrying out reverse transcription and cDNA one-chain synthesis, carrying out cDNA two-chain synthesis, partitioning the microsphere matrix capturing chip, carrying out melting on each region, carrying out cDNA amplification, and purifying an amplification product. According to the method for increasing the capture area of the space transcriptome of the microsphere matrix, the chip is broken and partitioned or partitioned by using a hybridization gasket for detection, the operation is simple and easy to implement, the variety of bar codes does not need to be directly increased, the number of synthesized microsphere primers and the variety of decoding probes are reduced when a microsphere pool is expanded, and the detection efficiency is improved. According to the method, the cost is effectively saved, in addition, decoding of the microsphere chip can be achieved without more decoding rounds, damage to the capture chip in the decoding process can be reduced, and the capture efficiency of the large-size space transcriptome chip is effectively improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for increasing the spatial transcriptome capture area of ​​a microsphere matrix and an application thereof. Background Art

[0002] Spatial transcriptomics combines imaging and sequencing technologies to map the location of specific transcripts on tissues, thereby indicating the specific site expression of specific genes. The emergence of spatial transcriptomics technology provides humans with a powerful tool to analyze the essence of life in a higher dimension. Spatial transcriptomics provides new insights in developmental biology, tumorigenesis, neuroscience, and other fields.

[0003] There are two main problems facing spatial transcriptomics. One is how to improve the capture resolution to meet the needs of spatial transcriptome analysis at the single-cell level. On the solid-phase capture chip, the number of transcripts captured needs to be further increased to achieve more accurate single-cell segmentation analysis. On the other hand, how to effectively use large-size capture chips to meet the needs of panoramic view analysis of large tissues.

[0004] The mainstream platform 10×Genomics platform launched the commercial Visium spatial transcriptome chip in 2019, with a capture area of ​​6.5 mm×6.5 mm. This size of capture area can only be limited to small tissue analysis at the millimeter level. The Visium CytAssist platform launched in 2023 only supports a maximum capture area of ​​11 mm×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 conventional single cells and spatial transcriptomes that capture mRNA through 3'-polyA, which is not conducive to subsequent joint analysis. Another mainstream platform is BGI's Stereo-Seq platform, which provides centimeter-level large-field-of-view sliced ​​spatial transcriptome capture chips ranging from 5mm×5 mm-130 mm×130 mm, but silicon-based chips cannot perform high-definition HE image scanning, and the idle chips made based on sequencing chips are extremely expensive.

[0005] HDST (High-definition spatial transcriptomics) is a high-resolution spatial transcriptomics technology that uses a dense microsphere matrix on tissue sections to capture RNA, thereby achieving high-resolution gene expression analysis in situ in tissues. HDST uses three-segment 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 barcode splitting cannot be guaranteed, resulting in data waste and reduced resolution. This method uses a 65×211×211 microsphere synthesis method, and 14 rounds of decoding are used for decoding. If the number of microsphere libraries is increased, the chip decoding rounds will increase, and more decoding probes need to be synthesized to combine the decoding probe pool. The S1000 chip is a 6.8 mm × 6.8 mm matrix containing about 2.2 million micro-pits. When a 384 × 384 × 384 micro-ball pool is used, the total number of micro-ball types is 56,623,104, 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 types of micro-ball pools is increased, a 768 × 768 × 768 micro-ball pool, 2.2 million micro-pits, and a repetition rate of 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%, but the cost of micro-ball primer synthesis needs to be doubled, and the synthesis of decoding probes and decoding rounds must also be increased to a certain extent, which undoubtedly limits the update of the product. Using a 384×384×384 microsphere pool, if the capture area is increased to 13.6 mm×13.6 mm, the number of micropits is 16.4 million, and the repetition rate is about 25%. If the capture area is further increased, the number of micropits will exceed the number of microsphere types, and a larger microsphere pool will be needed for chip assembly. At the same time, the decoding probes and decoding processes of the microspheres must also be updated accordingly, which is very costly.

[0006] In summary, how to increase the spatial transcriptome capture area of ​​microsphere matrix chips is still one of the urgent problems to be solved in the field of spatial transcriptome. Summary of the invention

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

[0008] To achieve this object, the present invention adopts the following technical solutions:

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

[0010] The tissue is permeabilized and captured using a microsphere matrix capture chip, followed by reverse transcription and cDNA single-strand synthesis and cDNA double-strand synthesis. The microsphere matrix capture chip is partitioned, each area is denatured and cDNA is amplified, and the amplified product is purified. The partitioning method includes a chip fragmentation partitioning method or a hybridization gasket partitioning method. The chip fragmentation partitioning method includes fragmenting the microsphere matrix capture chip into multiple partitions. The hybridization gasket partitioning method includes using a hybridization gasket to partition the microsphere matrix capture chip.

[0011] In view of the current large-size microsphere matrix capture chip, a large number of barcodes and microsphere pools are required, and the operation is cumbersome and the cost is high. The present invention adopts a new design idea. After the large-size microsphere matrix capture chip is used to capture the transcript, the two-strand cDNA is first produced by unified operation, and then the chip substrate is broken and partitioned or the hybridization gasket partition is used to unwind the generated two-stranded regions. After the sequencing is completed, the sequencing results can be retrospectively integrated according to the partition method, and finally a large-field spatial transcriptome data is generated. Compared with the traditional method, it does not need to increase the type of barcode, and reduces the number of synthetic microsphere primers and the type of decoding probes when expanding the microsphere pool, which effectively saves costs. In addition, it does not require more decoding rounds to achieve the decoding of the microsphere chip, which can reduce the damage to the capture chip during the decoding process and effectively improve the capture efficiency of the large-size microsphere matrix capture chip.

[0012] The microsphere matrix capture chip described in the present invention refers to a chip used for spatial transcriptome capture or spatial multi-omics analysis. It can be understood that capture chips with similar functions in the art are suitable for the present invention, such as a capture chip with a microsphere matrix obtained by synthesizing three-segment microspheres in HDST technology.

[0013] In the present invention, the hybridization pad refers to a silicone pad with an adhesive, which can be combined with the capture chip within a few seconds and then form different segmentation areas.

[0014] Preferably, the number of areas in the partition setting is 2 to 225, for example, 2, 3, 4, 5, 6, 10 or 20, etc., 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 present invention, the shape of the partition can be designed as a grid, such as a Japanese grid, a Tian grid, etc., and the area between different partitions can be 0.125 cm 2 ~10 cm 2 .

[0017] Preferably, the tissue permeabilization solution for tissue permeabilization treatment contains at least one of pepsin, TritonX-100 or HCl; the mass percentage of pepsin in the tissue permeabilization solution is 0.01%~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 TritonX-100 in the tissue permeabilization solution is 0.1~2 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 HCl in the tissue permeabilization solution is 0.1~1 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 primers; wherein the reverse transcription buffer contains 100-500 mM Tris-HCl, 200-500 mM KCl, 10-30 mM MgCl 2 and 10–100 mM DTT.

[0019] The above numerical ranges indicate that any value can be selected within the listed range, such as 100-500 mM can be 101, 102, 110, 120, 150, 200, 220, 240, 260, 300, 320, 350, 380, 400, 450, 460, 470, 480 or 490 mM, etc. 200-500 mM can be 201, 202, 205, 210, 220, 240, 280, 300, 320, 350, 380, 400, 450, 460, 470, 480 or 490 mM, etc. 10-30 mM can be 11, 12, 13, 14, 15, 20, 22, 26, 28 or 29 mM, etc. 10-100 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 invention, one or more reverse transcriptases commonly available on the market can be used, such as SuperScript™ II, SuperScript™ III, SuperScript™ IV from Thermofisher; HiScript II ReverseTranscriptase, HiScript III Reverse Transcriptase, HiScript ReverseTranscriptase from Vazyme, 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 a second-strand synthesis buffer, dNTPs, DNA polymerase and a second-strand reaction primer; wherein the second-strand synthesis buffer contains 100-300 mM Tris-HCl, 50-200 mM (NH 4 ) 2 SO 4 , 100~1000 mM KCl, 10~50 mM MgSO 4 and 0.5%~5% Tween-20, pH 7~9.

[0023] The above numerical ranges indicate that any value can be selected within the listed range, such as 100-300 mM can be 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 polymerases with DNA synthesis function such as DNA polymerase I large fragment (Klenow), DNA polymerase I (E. coli), etc.; Second Strand Primer: 1-100 μM Second Strand Primer.

[0025] In a second aspect, the present invention provides application of the method for increasing the spatial transcriptome capture area of ​​a microsphere matrix as described in the first aspect in constructing a spatial transcriptome sequencing library.

[0026] The present invention provides an operation method for a large-field spatial transcriptome chip, which performs partition recovery of double-strand products by physical fragmentation or hybridization gasket partitioning.

[0027] In a third aspect, the present invention provides a method for constructing a spatial transcriptome sequencing library, wherein the method uses the method for increasing the capture area of ​​the spatial transcriptome of the microsphere matrix described in the first aspect for capture, and specifically comprises the following steps: (1) tissue patching, fixation and staining; (2) tissue permeabilization; (3) reverse transcription and cDNA single-strand synthesis; (4) cDNA second-strand synthesis; (5) cDNA partitioning and denaturing and PCR amplification; and (6) cDNA purification.

[0028] Preferably, step (1) comprises: attaching the tissue section to a microsphere matrix capture chip for fixation and staining, wherein the microsphere matrix capture chip is provided with an mRNA capture probe.

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

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

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

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

[0033] Preferably, step (5) comprises: removing the reagent for cDNA second-strand synthesis in the microsphere array capture chip holes, adding EB buffer for washing, partitioning the microsphere array capture chip and then adding KOH, 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 reagent to each area 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) using 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 includes determining the concentration of the purified cDNA product and determining the peak shape of the cDNA product.

[0039] In a fourth aspect, the present invention provides a spatial transcriptome sequencing method, which utilizes the method for constructing a spatial transcriptome sequencing library described in the third aspect to construct a library, sequences each partition, and retrospectively integrates the sequencing results in a partitioned manner to obtain a spatial transcriptome sequencing result.

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

[0041] In the present invention, a spatial transcriptome sequencing method using a large-area capture chip is developed to be convenient and efficient, 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 mechanisms).

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

[0043] The present invention designs a method for increasing the spatial transcriptome capture area of ​​a microsphere matrix. The method adopts a new idea to break up and partition the chip or use a hybridization gasket to set up partitions for detection. The operation is simple and easy. Compared with the traditional method, while using a large-area capture chip, it is not necessary to directly increase the type of barcode (Barcode), and the number of synthesized microsphere primers and the type of decoding probes are reduced when expanding the microsphere pool, which effectively saves costs. In addition, the decoding of the microsphere chip can be achieved without more decoding rounds, which can reduce the damage to the capture chip during the decoding process, and effectively improve the capture efficiency of the large-size spatial transcriptome chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the method of partitioning and unzipping by the fragmentation method.

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

[0046] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0047] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0048] At present, if the spatial transcriptome capture area of ​​the microsphere matrix chip is increased, the number of micropits will exceed the number of microsphere types, and a larger microsphere pool will be needed for chip assembly. At the same time, the decoding probes and decoding processes of the microspheres must also be updated accordingly. The cost of this increase is extremely high. In response to the above problems, the present application is based on a new idea and cleverly designs a method to increase the spatial transcriptome capture area of ​​the microsphere matrix. A large-area microsphere matrix capture chip is directly used. The tissue slices are attached to the capture chip for fixation and staining, and then placed in a clamp. The tissue is permeabilized using a configured permeabilization enzyme, and then the tissue permeabilization solution is removed. A first-chain cDNA synthesis mixture is added for reverse transcription. After completion, the RNA is denatured and then the second-chain cDNA is synthesized. The chip is broken and partitioned or partitioned using hybridization gaskets (a schematic diagram of the partition denaturation method of the fragmentation method is shown in FIG. 1 ). Figure 1 As shown, the schematic diagram of the hybridization gasket partitioning and melting method is as follows Figure 2 As shown in the figure, the cDNA is denatured and melted in different regions, and then the cDNA samples are amplified to obtain samples that can be used to construct the second-generation library, and then sequencing analysis can be performed. When using a large-area capture chip, it is not necessary to directly increase the type of barcode.

[0049] Example 1

[0050] This embodiment provides a method for improving the capture area of ​​the microsphere matrix spatial transcriptome based on the fragmentation method, and a method and application thereof for interpreting the results using the highly sensitive Agilent2100. This method can be used to capture RNA from tissue samples, synthesize one strand of cDNA, synthesize two strands of cDNA, perform fragmentation chip partitioning and denaturation, and amplify cDNA to obtain a final cDNA sample with spatial information of the tissue sample, which can be connected to the second and third generation sequencing downstream. Compared with the traditional method, this method has the advantages of simple and convenient operation, stable performance, and a large capture area.

[0051] Taking a 13.6 mm × 13.6 mm capture chip (micro-etching is performed on the back of the chip using a laser in advance to facilitate later splitting), the capture chip is an S series spatial transcriptome capture chip purchased from Qingdao Baichuang Intelligent Manufacturing Technology Co., Ltd., and is broken into four regions for spatial transcriptome gene expression as an example, the following steps are included:

[0052] 1. Tissue patching, fixation and staining: Fresh frozen tissue (whole mouse brain) slices are placed on a permeabilized chip for fixation and staining, wherein the permeabilized chip is equipped with an mRNA capture probe.

[0053] Tissue staining and imaging:

[0054] 1.1 Tissue fixation

[0055] Place 40 mL of methanol in a 50 mL centrifuge tube and precool to -20°C.

[0056] Set the thermal cycler to 37 °C, the heated lid to 37 °C or 50 °C, and place the adapter to equilibrate the temperature.

[0057] Take out the chip with attached tissue (transported on dry ice) from -80℃, place it on the 37℃ adapter with the tissue facing upward and incubate for 1 minute (do not cover with hot cover); if the tissue has just been attached in the freezing microtome, do this as well.

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

[0059] During this time, reagents required for tissue staining can be prepared.

[0060] 1.2 Tissue staining

[0061] 1.2.1 Prepare three 1 L beakers in advance and add no less than 800 mL of ultrapure water to each beaker.

[0062] 1.2.2 Prepare the eosin mixture according to Table 1 below.

[0063] Table 1

[0064]

[0065] The hematoxylin stain solution must be filtered through a 0.2 μm filter membrane before use.

[0066] 1.2.3 Wipe off excess methanol from the fixed chip and place it on a clean surface of the platform.

[0067] 1.2.4 Add isopropanol to the tissue surface and cover it evenly. Incubate at room temperature for 1 min.

[0068] 1.2.5 Tilt the chip to remove isopropanol and wipe off excess liquid. Gently shake the chip to evaporate excess isopropanol on the tissue. This process should not exceed 5 minutes to avoid over-drying.

[0069] 1.2.6 Add filtered hematoxylin stain to the tissue surface and cover evenly, incubate at room temperature for 1-7 min (depending on the tissue).

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

[0071] 1.2.8 Add indigo dye solution to the tissue surface and cover it evenly. Incubate at room temperature for 30 s~2 min (depending on the tissue).

[0072] 1.2.9 Tilt the chip to remove the indigo dye, place it in the second cup of ultrapure water and gently rinse it about 5 times, wiping off the excess liquid.

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

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

[0075] 1.2.12 Place the chip in a clean 50 mL centrifuge tube and centrifuge at 250 × g for 30 s.

[0076] 1.2.13 Take out the chip and incubate it on a 37℃ adapter for 1 min.

[0077] 1.3 Brightfield tissue imaging

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

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

[0080] 1.3.3 Adjust the focus value to ±50, select a folder to save the image and name it.

[0081] 1.3.4 Frame the tissue location and click Scan.

[0082] 2. Tissue Permeabilization

[0083] 2.1 Preheat the tissue permeabilization enzyme (0.1% pepsin) in a 37°C metal bath for 10 min.

[0084] 2.2 Place the adapter in a PCR instrument and set the PCR instrument to the program shown in Table 2.

[0085] Table 2

[0086]

[0087] 2.3 Install the chip into the chip holder and add 300 μL of preheated tissue permeabilization enzyme along the side of the well to ensure that the enzyme evenly covers the entire matrix (do not introduce bubbles).

[0088] 2.4 Seal with sealing film, place on the adapter in the PCR instrument, cover with the hot cover, incubate at 37℃ for the determined time, and use a timer to count.

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

[0090] 2.6 The permeabilization time varies according to different tissues and is determined through tissue permeabilization experiments. At the end, remove the card clip, tear open the seal, aspirate the tissue permeabilization enzyme along the edge of the matrix, add 300 μL of the above-prepared 0.1×SSC buffer into the well and evenly cover the matrix (avoid introducing bubbles), and seal again.

[0091] 3. cDNA Single-Strand Synthesis

[0092] 3.1 Prepare the cDNA single-strand synthesis mixture (mix) as shown in Table 3 on ice.

[0093] Table 3

[0094]

[0095] The sequence of the strand displacement primer is: 5′-TTTCTGTTGGTGCTGATATTGCTrGrGrG-3′ (SEQ ID NO. 1), where rG represents the G base in RNA form.

[0096] Vortex to mix and centrifuge briefly.

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

[0098] Table 4

[0099]

[0100] 3.3 Tear open the seal, aspirate the buffer along the side of the well, add the first-strand synthesis mixture (evenly cover and avoid bubbles), seal again, place the card holder on the adapter, cover with the hot cover, skip the preheating step, and start the first-strand synthesis.

[0101] 4. cDNA Second Strand Synthesis and Denaturation

[0102] 4.1 Remove the cartridge from the adapter, tear open the seal, and aspirate one strand of the synthesis mixture along the side of the hole.

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

[0104] 4.3 Pipette slowly and evenly along the side of the well for 5 times (do not pipette violently) and discard the 0.08 M KOH.

[0105] 4.4 Add 300 μL EB buffer to the wells and incubate at room temperature for 2 min.

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

[0107] Table 5

[0108]

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

[0110] Vortex to mix and centrifuge briefly.

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

[0112] Table 6

[0113]

[0114] 4.7 Pipette slowly and evenly along the side of the well for 5 times (do not pipette violently) and discard the EB buffer.

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

[0116] 4.9 After the cDNA second-strand synthesis is completed, remove the second-strand synthesis mixture from the well.

[0117] 4.10 Add 300 μL EB buffer into the wells and incubate at room temperature for 2 min.

[0118] 4.11 Pipette slowly and evenly along the side of the well for 5 times (do not pipette violently) and discard the EB buffer.

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

[0120] 4.13 Pipette out 42 μL 0.08M KOH and 6 μL Tris (1M, pH 7.0) and mix them in a 0.2 mL low-absorption centrifuge tube. Centrifuge briefly and place on ice.

[0121] 5. cDNA Amplification

[0122] 5.1 qPCR cycle number determination

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

[0124] Table 7

[0125]

[0126] 1×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] Vortex to mix and centrifuge briefly.

[0130] 5.1.2 In the qPCR well plate, add 9 μL of qPCR mixture to each well (including 2 sample replicates, 1 negative control, and a positive control can also be set up. The qPCR mixture system can be increased proportionally).

[0131] 5.1.3 Pipette the samples in step 4.13 and add 1 μL to each sample duplicate well. Add 1 μL of water to the negative control. If a positive control is added, add 1 μL of quality-controlled cDNA sample to the well.

[0132] 5.1.4 Perform qPCR reaction according to the procedure in Table 8.

[0133] Table 8

[0134]

[0135] 5.1.5 Record the obtained Cq value.

[0136] 5.2 cDNA amplification

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

[0138] Table 9

[0139]

[0140] 15× cDNA primers The 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 and centrifuge briefly.

[0144] 5.2.2 Add 51 μL of cDNA amplification mixture to each of the four partitions in the sample in 4.13, mix well by pipetting, and centrifuge briefly to avoid bubbles.

[0145] 5.2.3 Set the following program in Table 10 on the PCR instrument to amplify cDNA.

[0146] Table 10

[0147]

[0148] 5.2.4 Quality control node: Qubit can be used to measure the concentration of post-PCR products.

[0149] 6. cDNA Purification

[0150] 6.1 Vortex the SPRIselect beads to mix thoroughly. Add 60 μL of SPRIselect beads (0.6×) to each sample tube (100 μL) and mix by pipetting 15 times.

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

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

[0153] 6.4 Remove the supernatant.

[0154] 6.5 Add 200 μL of 80% ethanol to the tube and wait for 30 s.

[0155] 6.6 Remove ethanol.

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

[0157] 6.8 Centrifuge briefly and transfer to a magnetic rack.

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

[0159] 6.10 Remove the test tube from the magnetic rack, add 40.5 μL EB buffer, and mix by pipetting 15 times.

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

[0161] 6.12 Place the test tube on a magnetic rack until the solution becomes clear.

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

[0163] 6.14 The sample can be stored at 4℃ for 72 h, or at -20℃ for 4 weeks, or directly proceed to the next step.

[0164] 7. cDNA Quantification and Quality Control

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

[0166] 7.2 Use the High sensitivity Agilent Technologies 2100 Bioanalyzerd to measure the peak shape of the cDNA product.

[0167] 8. Next-generation sequencing and analysis

[0168] Sequence each partition, and retrospectively integrate the sequencing results according to the partition method, and finally generate a large-field spatial transcriptome data, including:

[0169] 8.1 Perform barcode and umi identification on the data of each partition, compare the genome and determine the barcode position.

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

[0171] 8.3 Merge the barcode position information of different partitions, and determine the appropriate barcode information for the spots near the edge of the partition according to the barcode and UMI results identified 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 and perform data analysis to obtain a complete spatial transcriptome data.

[0174] The test without splitting was used as the control group (the rest of the operations refer to the above operations), and sequencing analysis was performed as a whole.

[0175] The specific test results are shown in Table 11. The control group did not split the chips and operated directly, resulting in a high microsphere repetition rate and low data utilization rate. The present invention adopts a new idea to break the chip into partitions for detection. While using a large-area capture chip, it does not need to directly increase the type of barcode, which can effectively reduce the microsphere repetition rate and improve data utilization rate.

[0176] Table 11

[0177]

[0178] Example 2

[0179] This embodiment provides a method for partitioning a hybrid gasket to increase the capture area of ​​the microsphere matrix spatial transcriptome, and a method for interpreting the results using the highly sensitive Agilent 2100 and its application. This method can be used to capture RNA from tissue samples, synthesize one strand of cDNA, synthesize two strands of cDNA, perform partitioning of hybrid gaskets, and perform cDNA amplification to obtain a final cDNA sample with spatial information of the tissue sample, which can be connected to second and third generation sequencing downstream. Compared with the traditional method, this method has the advantages of simple and convenient operation, stable performance, and a large capture area.

[0180] Taking a 13.6 mm × 13.6 mm capture chip as an example, a method for dividing the capture area after two-strand synthesis into four areas using a hybridization gasket for spatial transcriptome gene expression includes the following steps:

[0181] 1. Tissue patching, fixation and staining: Fresh frozen tissue (whole mouse brain) slices are placed on a permeabilized chip for fixation and staining, wherein the permeabilized chip is equipped with an mRNA capture probe.

[0182] Tissue staining and imaging:

[0183] 1.1 Tissue fixation

[0184] Refer to step 1.1 in Example 1.

[0185] 1.2 Tissue staining

[0186] Refer to step 1.2 in Example 1.

[0187] 1.3 Brightfield tissue imaging

[0188] Refer to step 1.3 in Example 1.

[0189] 2. Tissue Permeabilization

[0190] Refer to step 2 in Example 1.

[0191] 3. cDNA Single-Strand Synthesis

[0192] Refer to step 3 in Example 1.

[0193] 4. cDNA Second Strand Synthesis and Denaturation

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

[0195] 4.12 Use the hybridization pad to divide the capture area into four areas, add 45 μL 0.08M KOH to each area, making sure to cover the entire partition matrix, and incubate at room temperature for 10 minutes. During this period, use a pipette to pipette twice every two minutes, and do not contaminate different areas.

[0196] 4.13 Aspirate 42 μL 0.08M KOH from the four regions and mix with 6 μL Tris (1 M, pH 7.0) in a 0.2 mL low-absorption centrifuge tube. Centrifuge briefly and place on ice. Then add 45 μL 0.08M KOH to melt the uneluted region of the second chain caused by the width of the hybridization pad. Aspirate 42 μL 0.08M KOH from this region and mix with 6 μL Tris (1M, pH 7.0) in a 0.2 mL low-absorption centrifuge tube.

[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 51 μL of cDNA amplification mixture to each of the four partitions and one gasket-covered area in the sample in 4.13, and mix thoroughly by pipetting. Centrifuge briefly to avoid bubbles.

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

[0204] 5.2.4 Quality control node: Qubit can be used to measure the concentration of post-PCR products.

[0205] 6. cDNA Purification

[0206] Refer to step 6 in Example 1.

[0207] 7. cDNA Quantification and Quality Control

[0208] Refer to step 7 in Example 1.

[0209] 8. Next-generation sequencing and analysis

[0210] Each partition is sequenced, and the sequencing results are retrospectively integrated according to the partition method, and finally a large-field spatial transcriptome data is generated.

[0211] The test without partitioning was used as the control group, and sequencing analysis was performed as a whole.

[0212] The specific test results are shown in Table 12. The control group did not split the slices and operated directly, resulting in a high microsphere repetition rate and low data utilization. The present invention adopts a new idea and uses hybridization pads to set partitions for detection. While using a large-area capture chip, it does not need to directly increase the type of barcode, which can effectively reduce the microsphere repetition rate and improve data utilization.

[0213] Table 12

[0214]

[0215] In summary, the present invention designs a method for increasing the spatial transcriptome capture area of ​​a microsphere matrix, adopts a new idea, breaks the chip into partitions or uses hybridization gaskets to set partitions for detection, and the operation is simple and easy. Compared with the traditional method, while using a large-area capture chip, it is not necessary to directly increase the type of barcode (Barcode), and the number of synthesized microsphere primers and the type of decoding probes are reduced when expanding the microsphere pool, which effectively saves costs. In addition, the decoding of the microsphere chip can be achieved without more decoding rounds, which can reduce the damage to the capture chip during the decoding process, and effectively improve the capture efficiency of the large-size spatial transcriptome chip.

[0216] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for increasing the spatial transcriptome capture area of ​​a microsphere matrix, characterized in that: The method comprises: The tissue is permeabilized and captured using a microsphere array capture chip, followed by reverse transcription and cDNA first-strand synthesis and cDNA second-strand synthesis. The microsphere array capture chip is partitioned, each area is melted and cDNA is amplified, and the amplified product is purified; The partitioning method includes a chip crushing partitioning method or a hybridization pad partitioning method; The chip fragmentation and partitioning method comprises fragmenting a microsphere matrix capture chip into a plurality of partitions; The hybridization pad partitioning method includes partitioning a microsphere matrix capture chip using a hybridization pad.

2. The method for increasing the spatial transcriptome capture area of ​​a microsphere matrix according to claim 1, characterized in that: The area of ​​the microsphere matrix capture chip is 0.25 cm 2 ~225 cm 2 ; The number of areas in the partition setting is 2 to 225.

3. The method for increasing the spatial transcriptome capture area of ​​a microsphere matrix according to claim 1, characterized in that: The tissue permeabilization solution of the tissue permeabilization treatment contains at least one of pepsin, TritonX-100 or HCl; the mass percentage of pepsin in the tissue permeabilization solution is 0.01%~1%; the concentration of TritonX-100 in the tissue permeabilization solution is 0.1~2 mM; the concentration of HCl in the tissue permeabilization 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 primers; 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 primers; 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.

4. Use of the method for increasing the spatial transcriptome capture area of ​​a microsphere matrix according to any one of claims 1 to 3 in constructing a spatial transcriptome sequencing library.

5. A method for constructing a spatial transcriptome sequencing library, characterized in that: The method uses the method for increasing the spatial transcriptome capture area of ​​the microsphere matrix according to any one of claims 1 to 3 for capture, and specifically comprises the following steps: (1) Tissue mounting, fixation and staining; (2) Tissue permeabilization; (3) Reverse transcription and single-strand cDNA synthesis; (4) cDNA second-strand synthesis; (5) cDNA partitioning and PCR amplification; (6) cDNA purification.

6. The method for constructing a spatial transcriptome sequencing library according to claim 5, characterized in that: Step (1) comprises: attaching the tissue section to a microsphere matrix capture chip for fixation and staining, wherein the microsphere matrix capture chip is provided with an mRNA capture probe.

7. The method for constructing a spatial transcriptome sequencing library according to claim 5, characterized in that: Step (2) comprises: placing the chip obtained in step (1) in the tissue permeabilization solution and incubating at 30-40° C. for 2-30 min, removing the tissue permeabilization solution and adding 0.1×-5× SSC solution into the wells of the microsphere matrix capture chip.

8. The method for constructing a spatial transcriptome sequencing library according to claim 5, characterized in that: Step (3) includes: removing the SSC solution in the microsphere array capture chip wells, adding reagents for reverse transcription and cDNA single-strand synthesis into the wells, and incubating at 37-65° C. for 0.5-2 h; Step (4) includes: removing the reagents for reverse transcription and cDNA first-strand synthesis in the microsphere array capture chip wells, adding KOH solution for denaturation, washing with EB buffer, and then adding the reagent for cDNA second-strand synthesis, and incubating at 45-70°C for 10-40 min.

9. The method for constructing a spatial transcriptome sequencing library according to claim 5, characterized in that: Step (5) includes: removing the reagent for cDNA second-strand synthesis in the microsphere array capture chip hole, adding EB buffer for washing, partitioning the microsphere array capture chip and then adding KOH, incubating at 20-30°C for 2-10 min, neutralizing KOH with 0.1-1 M Tris, and adding cDNA amplification reagent to each area for PCR amplification; The procedure of the PCR amplification is: 95~98℃、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℃, 4~6 min; Step (6) comprises: purifying the amplification product of step (5) using magnetic beads, then washing the magnetic beads with ethanol, and then eluting the amplification product from the magnetic beads with EB buffer; The method for constructing a spatial transcriptome sequencing library also includes a cDNA quality control step.

10. A spatial transcriptome sequencing method, characterized in that: The spatial transcriptome sequencing method uses the method for constructing a spatial transcriptome sequencing library according to any one of claims 5 to 9 to construct a library, sequence each partition, and retrospectively integrate the sequencing results according to the partition method to obtain the spatial transcriptome sequencing results.

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