Method for improving performance of spatial transcriptome capture and application thereof

Abstract

The application discloses a method for improving the performance of spatial transcriptome capture and application thereof. The method comprises the following steps: tissue permeation of a sample, mRNA release and chip capture, mRNA reverse transcription and cDNA single strand synthesis, cDNA double strand synthesis, cDNA denaturation and PCR amplification, repeated cDNA double strand synthesis, cDNA denaturation and PCR amplification, and purification of the two amplification products, respectively. The double strand synthesis buffer used in the cDNA double strand synthesis and the repeated cDNA double strand synthesis comprises any one or a combination of at least two of Tween-20, glycerol, bovine serum albumin, trehalose or betaine. The method can effectively reduce the loss of mRNA captured by the chip in the subsequent cDNA synthesis and library construction process, improve the number of effective molecules in the library, improve the gene detection amount, and thus improve the performance of spatial transcriptome capture.

Description

Technical Field

[0001] This application belongs to the field of biotechnology and relates to a method for improving the performance of spatial transcriptome capture and its application. Background Technology

[0002] Spatial transcriptomics is an innovative high-resolution technology that combines tissue imaging with sequencing to visualize and quantify gene expression in the spatial context of intact tissues and cells.

[0003] BGI Genomics' Stereo-seq uses a self-developed high-density DNA nanosphere (DNB) chip array to achieve a subcellular resolution of 0.5 μm. It also provides a spatial transcriptome capture chip for centimeter-level large-field-of-view tissue sections. This platform captures mRNA in situ using probes on the DNB on the chip surface, reverse transcribes it into cDNA, then washes off the single-stranded cDNA, and then performs cDNA double-strand synthesis. However, during this process, single-stranded nucleic acids are easily degraded, and genes with high GC content are prone to interruption during elongation, which greatly reduces the efficiency of cDNA double-strand synthesis. This affects the efficiency of converting the mRNA captured in situ by the probes on the DNB into the effective number of genes obtained from subsequent sequencing, resulting in a significant gap in the effective gene detection rate compared to conventional transcriptome sequencing. The BMKM ANU S series from Baichuang Intelligent Manufacturing utilizes a fixed microsphere array chip. The newly released S3000 has a spot diameter of 0.2 μm and a center-to-center distance of 3.5 μm between spots, containing approximately 4.14 million spots within a capture area of ​​6.8 mm × 6.8 mm. This platform captures mRNA in situ using oligo probes on microspheres within the chip's microwells, synthesizes one-stranded cDNA via reverse transcription, and then performs two-stranded cDNA synthesis after unwinding. However, this process also faces challenges such as the easy degradation of single-stranded nucleic acids and the tendency for genes with high GC content to be interrupted during elongation. Furthermore, since cDNA two-strand synthesis occurs on a solid-phase chip, the steric hindrance of the oligo probes on the microspheres hinders the polymerase from covering the full-length single-stranded cDNA template. This makes it difficult to synthesize complete complementary two-stranded strands from many long transcripts, resulting in the retention of numerous incomplete single-stranded cDNA fragments. Additionally, the insufficient efficiency of two-strand synthesis in a solid-phase chip environment makes single-stranded cDNA molecules prone to degradation during subsequent cDNA amplification and purification. To address the issues of unstable single-stranded cDNA and low double-strand synthesis efficiency, an additional cDNA double-strand synthesis reaction is added to convert more single-stranded cDNA into double-stranded cDNA. Simultaneously, the double-strand synthesis system is optimized to increase polymerase reaction efficiency and reduce the loss of unstable single-stranded cDNA during the double-strand synthesis process. This results in an increased number of effective molecules in the library, improved detection of effective genes, and enhanced sequencing data quality. Ultimately, this improves the efficiency of spatial transcriptome mRNA capture and enhances the capture performance of the spatial transcriptome.

[0004] In summary, although spatial transcriptomics can preserve the spatial information of all transcript distributions and has significantly improved upon traditional batch RNA sequencing methods, there is still much room for improvement in the technical performance of mainstream spatial transcriptomics products currently on the market. One of the urgent problems to be solved is how to improve the capture performance of spatial transcriptomics. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this application provides a method for improving the capture performance of spatial transcriptomes and its application. With low-cost investment, it captures more cDNA molecules and significantly improves the capture performance of spatial transcriptomes.

[0006] To achieve the purpose of this application, the following technical solution is adopted:

[0007] In a first aspect, this application provides a method for improving the performance of spatial transcriptome capture, the method comprising: tissue patching, fixation and staining of a sample; tissue permeation; release of mRNA and capture of mRNA by a microarray; reverse transcription of mRNA and one-stranded cDNA synthesis; two-stranded cDNA synthesis; cDNA unwinding and PCR amplification; repeating the two-stranded cDNA synthesis, cDNA unwinding and PCR amplification once; and purifying the amplification products of the two amplifications respectively; wherein the two-stranded cDNA synthesis and the two-stranded cDNA synthesis buffer used for the two-stranded cDNA synthesis and the repeating of the two-stranded cDNA synthesis include any one or a combination of at least two of Tween-20, glycerol, bovine serum albumin, trehalose or betaine.

[0008] This application achieves the synthesis of more cDNA from reverse-transcribed mRNA by adding an extra cDNA double-strand synthesis process and optimizing the double-strand synthesis buffer system, thereby increasing the effective number of molecules in the final library.

[0009] In some embodiments, the Tween-20 mass percentage in the double-strand synthesis buffer is 0.5% to 5% (e.g., 0.8%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0%), the glycerol mass percentage is 1% to 10% (e.g., 1.5%, 2.0%, 4.0%, 6.0%, 8.0%, 8.5%, 9.0%, 9.5%, or 10%), the final concentration of bovine serum albumin is 0.1 to 1 mg / mL (e.g., 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1.0 mg / mL), and the final concentration of trehalose is 0.5 to 2 M (e.g., 0.8 M, 1.0 M, 1.2 M, 1.5 M, 1.8 M). The final concentration of betaine is 0.5 to 1.5 M (e.g., 0.8 M, 1.0 M, 1.2 M, or 1.5 M).

[0010] In some embodiments, the tissue permeation solution used for tissue permeation is an acidic buffer solution containing pepsin, wherein the mass percentage of pepsin is 0.01% to 2% (e.g., 0.02%, 0.5%, 1.0%, 1.2%, 1.5%, 1.8%), and the pepsin is dissolved in HCl.

[0011] In some embodiments, the reverse transcription reaction system includes: reverse transcription buffer, reverse transcriptase, dNTPs, dithiothreitol, and strand displacement primers.

[0012] In some embodiments, the reverse transcription buffer comprises Tris-HCl (e.g., 105 mM, 110 mM, 120 mM, 150 mM, 200 mM, 300 mM, 400 mM or 500 mM) at a final concentration of 100-500 mM, KCl (e.g., 205 mM, 210 mM, 220 mM, 250 mM, 300 mM, 400 mM or 500 mM) at a final concentration of 100-500 mM, MgCl2 (e.g., 12 mM, 15 mM, 18 mM, 20 mM, 25 mM, 28 mM or 30 mM) at a final concentration of 100-500 mM, dithiothreitol (e.g., 12 mM, 15 mM, 18 mM, 20 mM, 50 mM, 80 mM or 100 mM) at a final concentration of 100-500 mM, KCl (e.g., 205 mM, 210 mM, 220 mM, 25 mM, 30 mM, 25 mM or 30 mM) at a final concentration of 100-500 mM, KCl (e.g., 105 mM, 110 mM, 120 mM, 120 mM, 15 mM, 18 mM, 20 mM, 50 mM, 80 mM or 100 mM) at a final concentration of 100-500 mM, KCl (e.g., 105 mM, 110 mM, 120 mM, 120 mM, 15 mM, 18 mM,

[0013] The aforementioned reverse transcriptase can be one or more of the following: SuperScript™ III or SuperScript™ IV from Thermofisher; HiScript II Reverse Transcriptase or HiScript III Reverse Transcriptase from Vazyme; or ProtoScript II Reverse Transcriptase from NEB.

[0014] In some embodiments, the final concentration of dNTPs in the reaction system is 1–20 mM (e.g., 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 18 mM, or 20 mM), the final concentration of dithiothreitol is 100–200 mM (e.g., 102 mM, 105 mM, 108 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM), and the final concentration of the chain substitution primer is 10–100 mM (e.g., 12 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM). mM, 80mM, 85mM, 90mM, 95mM or 100mM).

[0015] In some embodiments, the reaction system for cDNA double-strand synthesis and repeated cDNA double-strand synthesis includes a double-strand synthesis buffer, dNTPs, cDNA double-strand synthase, and double-strand reaction primers.

[0016] In some embodiments, the double-chain synthesis buffer further includes Tris-HCl (e.g., 105 mM, 110 mM, 120 mM, 150 mM, 180 mM, 200 mM, 220 mM, 250 mM, 280 mM, or 300 mM) at a final concentration of 100-300 mM, (e.g., 55 mM, 60 mM, 65 mM, 70 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, 190 mM, or 2 ...1000 mM, (e.g., 110 mM, 120 mM, 150 mM, 180 mM, 200 mM, 400 mM, 600 mM, 800 mM, 850 mM, or 900 mM) at a final concentration of 100-1000 mM, (e.g., 110 mM, 120 mM, 150 mM, 180 mM, 200 mM, 400 mM, 600 mM, 800 mM, 850 mM, or 900 mM) at a final concentration of 100-300 mM, (e.g., 105 mM, 110 mM, 110 MgSO4 in the range of 10-50 mM (e.g., 12 mM, 15 mM, 18 mM, 20 mM, 25 mM, 28 mM, 30 mM, 35 mM, 38 mM, 40 mM, 45 mM, 48 mM or 50 mM).

[0017] The aforementioned cDNA two-strand synthases can be one or more of the polymerases required for DNA synthesis, such as RNase H and E. coli DNA Polymerase I from Thermo Fisher Scientific, RNase H and E. coli DNA Pol I from Vazyme Scientific, or Hieff RNase H and E. coli DNA Pol I from Yeasen Scientific.

[0018] As a preferred technical solution, this application provides a method for improving the performance of spatial transcriptome capture, the method comprising the following steps:

[0019] (1) The tissue was attached to the chip area containing the mRNA capture probe, fixed with methanol and then stained;

[0020] (2) Add tissue permeation solution to the chip reaction area and permeate at 35~38℃ (e.g., 35℃, 36℃, 37℃ or 38℃) for 5~30 min (e.g., 5 min, 10 min, 20 min or 30 min). Remove the tissue permeation solution and add cleaning solution to the chip reaction area for incubation and cleaning.

[0021] (3) Remove the cleaning solution, add reverse transcription and cDNA one-strand synthesis reagents to the chip reaction area, and incubate at 37~55℃ (e.g., 38℃, 40℃, 45℃ or 55℃) for 60~120 min (e.g., 60 min, 100 min or 120 min).

[0022] (4) Remove the reverse transcription and cDNA first-strand synthesis reagents, add alkaline solution to the chip reaction area for de-stranding, wash with washing buffer after de-stranding, add cDNA second-strand synthesis reagent to the chip reaction area, and incubate at 45~65℃ (e.g., 45℃, 55℃ or 65℃) for 30~60 min (e.g., 30 min, 40 min or 60 min).

[0023] (5) Remove the cDNA double-strand synthesis reagent, incubate with washing buffer, add alkaline solution to the chip reaction area to denature, incubate at 20~30℃ (e.g., 20℃, 25℃ or 30℃) for 10~20 min (e.g., 10 min, 15 min or 20 min), aspirate the incubated solution, add reagent to neutralize the alkaline solution, add cDNA amplification reagent for PCR amplification;

[0024] (6) Add washing buffer to the chip reaction area for incubation and washing. After washing, add cDNA double strand synthesis reagent again and incubate at 45~65℃ (e.g., 45℃, 55℃ or 65℃) for 30~60 min (e.g., 30 min, 40 min or 60 min).

[0025] (7) Remove the cDNA double-strand synthesis reagent, incubate and clean with washing buffer, add alkaline solution to the chip reaction area to de-strand, incubate at 20~30℃ (e.g., 20℃, 25℃ or 30℃) for 10~20 min (e.g., 10 min, 15 min or 20 min), aspirate the de-stranded solution, add reagent to neutralize alkaline solution, add cDNA amplification reagent for PCR amplification;

[0026] (8) The PCR amplification products from steps (5) and (7) are purified by magnetic beads, and the products on the magnetic beads are washed off using washing buffer.

[0027] In some embodiments, the cleaning solution in step (2) includes an SSC solution.

[0028] In some embodiments, the alkaline solution in step (4) includes a KOH solution, and the washing buffer includes an EB buffer.

[0029] In some embodiments, the washing buffer in step (5) includes EB buffer, the alkaline solution includes KOH solution, and the reagent for neutralizing the alkaline solution includes 0.5~1 M Tris, for example, 0.6 M, 0.7 M, 0.8 M, 0.9 M or 1.0 M.

[0030] In some embodiments, the PCR amplification program in step (5) is as follows: 95~98℃ (e.g., 95℃, 96℃, or 98℃), 20~40 s (e.g., 20 s, 30 s, or 40 s); 95~98℃ (e.g., 95℃, 96℃, or 98℃), 15~30 s (e.g., 15 s, 20 s, or 30 s), 62~68℃ (e.g., 62℃, 65℃, or 68℃), 15~30 s (e.g., 15 s, 20 s, or 30 s), 60~70℃ (e.g., 60℃, 65℃, or 70℃), 4~8 min (e.g., 4 min, 6 min, or 8 min), 10~20 cycles (e.g., 10 cycles, 15 cycles, or 20 cycles); 60~70℃ (e.g., 60℃, 65℃, or 70℃), 4~6 min (e.g., 4 min, 5 ... (min or 6 min).

[0031] In some embodiments, the cleaning buffer in step (6) includes EB buffer.

[0032] In some embodiments, the washing buffer in step (7) includes EB buffer, the alkaline solution includes KOH solution, and the reagent for neutralizing the alkaline solution includes 0.5~1 M Tris, for example, 0.5 M, 0.8 M or 1 M.

[0033] In some embodiments, the PCR amplification program in step (7) is as follows: 95~98℃ (e.g., 95℃, 96℃, or 98℃), 20~40 s (e.g., 20 s, 30 s, or 40 s); 95~98℃ (e.g., 95℃, 96℃, or 98℃), 15~30 s (e.g., 15 s, 20 s, or 30 s), 62~68℃ (e.g., 62℃, 65℃, or 68℃), 15~30 s (e.g., 15 s, 20 s, or 30 s), 60~70℃ (e.g., 60℃, 65℃, or 70℃), 4~8 min (e.g., 4 min, 6 min, or 8 min), 10~20 cycles (e.g., 10 cycles, 15 cycles, or 20 cycles); 60~70℃ (e.g., 60℃, 65℃, or 70℃), 4~6 min (e.g., 4 min, 5 ... (min or 6 min).

[0034] In some embodiments, the cleaning buffer in step (8) includes EB buffer.

[0035] In this application, the final concentration of the alkaline KOH solution is 0.05~0.2 M, for example, it can be 0.06 M, 0.07 M, 0.08 M, 0.09 M, 0.1 M, 0.12 M, 0.15 M, 0.18 M or 0.2 M.

[0036] Secondly, this application provides the application of the method for improving spatial transcriptome capture performance described in the first aspect in the construction of spatial transcriptome sequencing libraries.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] (1) By adding a step of cDNA double-strand synthesis, this application greatly improves the efficiency of synthesizing complete cDNA after mRNA reverse transcription, increases the effective number of molecules in the spatial transcriptome library, enriches the number of detectable effective genes, and thus improves the capture efficiency of the spatial transcriptome.

[0039] (2) In this application, trehalose, betaine, glycerol, bovine serum albumin and Tween-20 are added alone or in combination to the cDNA double-strand synthesis buffer, which improves the stability of DNA polymerase, increases the efficiency of cDNA, and greatly improves the integrity of long fragments of cDNA amplification.

[0040] (3) The method for improving spatial transcriptome capture performance in this application is simple to operate and highly operable. Attached Figure Description

[0041] Figure 1 This is a flowchart illustrating the method for improving spatial transcriptome capture performance in this application. Detailed Implementation

[0042] To further illustrate the technical means and effects adopted in this application, the following description, in conjunction with embodiments and accompanying drawings, will provide further details. It is understood that the specific embodiments described herein are merely for explaining this application and not for limiting it.

[0043] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0044] Spatial transcriptomics is susceptible to factors such as the physicochemical properties of the sample itself, sample quality, permeation conditions, low tissue reverse transcription efficiency, and stepwise molecular loss during cDNA two-strand synthesis and amplification, resulting in low capture performance of current mainstream spatial transcriptomics platforms. To address these issues, this application proposes a novel method to improve spatial transcriptomics capture performance. The combination is attached to a capture chip, fixed and stained, then placed in a reaction clip for tissue permeation. After removing the permeation solution, reverse transcription and cDNA one-strand synthesis reagents are added. Following this, the chips are unwound and washed, cDNA two-strand synthesis reagents are added, and the cDNA is unwound and amplified again. After washing the chip reaction area, freshly prepared cDNA two-strand synthesis reagents are added again, followed by another round of unwound and amplification. The cDNA amplification products from both processes are purified and then used for library construction and sequencing.

[0045] The following examples use the Baichuang S3000 spatial transcriptome capture chip from Qingdao Baichuang Intelligent Manufacturing Technology Co., Ltd. for experiments.

[0046] Example 1

[0047] This embodiment uses fresh animal tissue as an example to provide a method for increasing the effective number of cDNA molecules by adding one cDNA double-strand synthesis and subsequent melting and amplification reaction, thereby improving the capture performance of the spatial transcriptome, as detailed below:

[0048] 1. Tissue mounting, fixation and staining imaging: Fresh frozen tissue (mouse brain, pig bladder) sections were mounted on a permeabilized chip for fixation and staining, wherein the permeabilized chip was equipped with an mRNA capture probe.

[0049] 1.1 Organizational stability

[0050] 1.1.1 Prepare 40 mL of methanol in a 50 mL centrifuge tube and pre-cool it at -20℃.

[0051] 1.1.2 Set the PCR temperature to 37°C, the hot lid temperature to 50°C, and the time to hold. Place the adapter on the PCR instrument to equilibrate to 37°C.

[0052] 1.1.3 Place the chip with the tissue attached in the cryostat (transported on dry ice), with the tissue-attached side facing up, on a 37°C adapter and bake for 1 minute (do not cover with a heat cap).

[0053] 1.1.4 The chip was fixed in pre-cooled methanol at -20°C. Animal samples were fixed for 30 min and plant samples for 1 h.

[0054] 1.1.5 During this period, the reagents required for staining can be prepared.

[0055] 1.2 Tissue staining and imaging

[0056] 1.2.1 Cell segmentation and fluorescent staining.

[0057] 1.2.1.1 Prepare the cell segmentation fluorescent reagent according to Table 1:

[0058] Table 1

[0059]

[0060] 1.2.1.2 Remove the fixed chip, and after the methanol on the chip has completely evaporated, add the above reagent that has been vortexed and mixed, and incubate at room temperature in the dark for 5 min.

[0061] 1.2.1.3 Prepare 40 mL of 0.1×SSC according to Table 2:

[0062] Table 2

[0063]

[0064] 1.2.1.4 After the fluorescent staining reagent incubation is complete, pour off the reagent on the chip and gently rinse it about 15 times in the 0.1×SSC solution that has been vortexed and mixed.

[0065] 1.2.1.5 Wipe away excess liquid outside the tissue area with lint-free paper, place horizontally on a 37°C metal bath, and bake away from light to dry excess liquid on the tissue for about 2 minutes.

[0066] 1.2.2 Cell segmentation and fluorescence staining imaging (using a Baichuang BH1000 scanner)

[0067] 1.2.2.1 Place the chip on the stage and create a new scan.

[0068] 1.2.2.2 Double-click the chip area, click the real-time image, and adjust the focal plane in the FITC channel until the fluorescence is clear.

[0069] 1.2.2.3 Select the area with the chip patch, add a focal point, select the bright field and FITC channels, name the image, and click Start Scan.

[0070] 1.2.3 HE staining

[0071] 1.2.3.1 Add isopropanol to the tissue area of ​​the chip after scanning the fluorescence image above, so that the isopropanol completely covers the tissue, and incubate at room temperature for 1 min.

[0072] 1.2.3.2 Discard the isopropyl alcohol and gently shake the chip to allow the isopropyl alcohol to evaporate completely.

[0073] 1.2.3.3 Add hematoxylin to the tissue area dropwise, so that the hematoxylin completely covers the tissue, and incubate at room temperature for 3 min.

[0074] 1.2.3.4 Discard the hematoxylin and eosin, place the chip in enzyme-free water and gently rinse it about 15 times, then wipe off the excess liquid with a lint-free paper towel.

[0075] 1.2.3.5 Add blue dye to the tissue area until the blue dye completely covers the tissue, and incubate at room temperature for 1 min.

[0076] 1.2.3.6 Discard the blue residue, place the chip in enzyme-free water and gently rinse it about 15 times, then wipe off the excess liquid with a lint-free paper towel.

[0077] 1.2.3.7 Add eosin to the tissue area dropwise until the eosin completely covers the tissue, and incubate at room temperature for 1 min.

[0078] 1.2.3.8 Discard the eosin, place the chip in enzyme-free water and gently rinse it about 15 times, then wipe off the excess liquid with a lint-free paper towel.

[0079] 1.2.3.9 Place the chip in a 50 mL centrifuge tube, balance and centrifuge at 250 g for 1 min.

[0080] 1.2.4 HE Imaging

[0081] 1.2.4.1 Place the chip on the stage and create a new scan.

[0082] 1.2.4.2 Double-click the chip area, click the real-time image, and adjust the focal plane in the bright field channel until the image is clear.

[0083] 1.2.4.3 Select the area with the chip patch, add a focal point, select the bright field channel, name the image, and click Start Scan.

[0084] 2. Tissue permeability

[0085] 2.1 Place 100 μL of tissue permeation solution in a 37℃ metal bath for 10 min to preheat.

[0086] 2.2 Install the chip into the reaction card holder, and add 100 μL of preheated tissue permeation solution to the reaction area so that the tissue permeation solution evenly covers the entire inverse matrix.

[0087] 2.3 Seal the container with sealing film, place the reaction card holder into the PCR instrument set in 1.1.2, and use a timer to start the process.

[0088] 2.4 Dilute 20×SSC buffer to 0.1×SSC buffer, vortex to mix, and then briefly centrifuge.

[0089] 2.5 After the permeation time is complete, remove the cartridge, place it on ice, tear open the sealing film, aspirate the tissue permeation solution along the edge of the matrix, add the prepared 0.1×SSC buffer into the well and evenly cover the reaction matrix, and seal it again.

[0090] 3. Reverse transcription and cDNA first-strand synthesis

[0091] 3.1 Prepare the reverse transcription and cDNA one-strand synthesis reagents on ice according to Table 3:

[0092] Table 3

[0093]

[0094] Strand substitution primer sequence: 5'-TTTCTGTTGGTGCTGATATTGCTrGrGrG-3' (SEQ ID NO.1), where rG represents the G base in RNA form.

[0095] 3.2 Set the PCR instrument program as shown in Table 4:

[0096] Table 4

[0097]

[0098] 3.3 Tear open the seal of the reaction card holder, aspirate 0.1×SSC buffer along the edge of the reaction well, add the reverse transcription and cDNA one-strand synthesis reagents obtained by vortexing and momentarily dissociating in step 3.1, place the reaction card holder on the PCR instrument, close the heat cap, skip the preheating step, and start the one-strand synthesis reaction.

[0099] 4 cDNA double strand synthesis

[0100] 4.1 Remove the reaction cartridge from the PCR instrument, tear open the seal, and aspirate the first-strand synthesis reagent along the edge of the reaction well.

[0101] 4.2 Add 200 μL of 0.08 M KOH to the reaction well and incubate at room temperature for 5 min.

[0102] 4.3 Use a pipette to remove 0.08 M KOH along the edge of the reaction well, add 200 μL of EB buffer to the reaction well, and incubate at room temperature for 2 min.

[0103] 4.4 Prepare the cDNA double-strand synthesis system on ice according to Table 5:

[0104] Table 5

[0105]

[0106] The sequence of the primer for the two-strand reaction is: 5'-TTTCTGTTGGTGCTGATATTGC-3' (SEQ ID NO.2).

[0107] 4.5 Set the PCR instrument program as shown in Table 6:

[0108] Table 6

[0109]

[0110] 4.6 Tear open the seal of the reaction card, aspirate the EB buffer along the edge of the reaction well, add the cDNA two-strand synthesis reagent that was vortexed and briefly separated in step 4.4, place the reaction card on the PCR instrument, close the heat cap, skip the preheating step, and start the two-strand synthesis reaction.

[0111] 5. cDNA unwinding and PCR amplification

[0112] 5.1 After the cDNA double-strand synthesis is completed, remove the double-strand synthesis reagent from the edge of the reaction well.

[0113] 5.2 Add 200 μL of EB buffer to the reaction well and incubate at room temperature for 2 min.

[0114] 5.3 Remove EB buffer from the edge of the reaction well, add 45 μL of 0.08M KOH, and incubate at room temperature for 10 min.

[0115] 5.4 Aspirate 43 μL of 0.08M KOH into a 0.2 mL low-adsorption centrifuge tube, add 6 μL of Tris (1M, pH 7.0) and mix by pipetting and aspiration. After a brief centrifugation, place the tube on ice.

[0116] 5.5 Prepare the PCR amplification reaction system on ice according to Table 7:

[0117] Table 7

[0118]

[0119] The primer sequences for the 15×cDNA primers are as follows:

[0120] Primer F: 5'-CTACACGACGCTCTTCCGATCT-3' (SEQ ID NO.3).

[0121] Primer R: 5'-TTTCTGTTGGTGCTGATATTGC-3' (SEQ ID NO.4).

[0122] 5.6 After vortexing and momentarily centrifuging the above amplification mixture, perform amplification on a PCR instrument according to the procedure in Table 8:

[0123] Table 8

[0124]

[0125] 6. cDNA purification

[0126] 6.1 Vortex the SPRIselect magnetic beads to mix them thoroughly. Add 8 μL of SPRIselect magnetic beads (0.8×) to the above cDNA amplification product (total 100 μL), and mix by pipetting. Note that it is necessary to mix completely and avoid air bubbles.

[0127] 6.2 Incubate at room temperature for 4 min.

[0128] 6.3 Perform instant centrifugation, place on a high position on a magnetic rack, and let stand for 2 minutes. Note that the supernatant must be completely clear.

[0129] 6.4 Remove and discard the supernatant, being careful not to pick up the magnetic beads to avoid damage.

[0130] 6.5 Add 200 μL of 80% ethanol, being careful to avoid rinsing the magnetic beads directly. After 30 seconds at room temperature, remove and discard the ethanol. Be careful not to pick up the magnetic beads, as this may result in loss.

[0131] 6.6 Repeat the previous step.

[0132] 6.7 Remove the PCR tubes from the magnetic rack, centrifuge briefly, and place them at the bottom of the magnetic rack.

[0133] 6.8 Carefully aspirate any excess liquid using a 10 μL pipette.

[0134] 6.9 Open the tube cap and let it stand for about 2 minutes until the excess ethanol evaporates. Be careful to avoid the magnetic beads drying out too much and cracking.

[0135] 6.10 Remove the PCR tube from the magnetic rack, add 40 μL of EB into the tube, and mix by pipetting and aspirating. Make sure to mix completely, about 20 times.

[0136] 6.11 Let stand at room temperature for 4 minutes.

[0137] 6.12 Perform instant centrifugation, place on a low position on a magnetic rack, and let stand for 2 minutes. Note that the supernatant must be completely clear.

[0138] 6.13 Carefully aspirate 40 μL of supernatant into a clean 1.5 mL centrifuge tube, avoiding aspirating the magnetic beads.

[0139] 6.14 The cDNA amplification product was purified, and the cDNA library of the tissue sample was obtained.

[0140] 6.15 Take 1 μL of cDNA product for qubit concentration determination.

[0141] 6.16 Take another 1 μL based on the concentration for quality inspection.

[0142] 7. Secondary Synthesis of cDNA Second Strand

[0143] 7.1 Add 200 μL of EB buffer to the reaction wells to which 0.08 M KOH was aspirated in step 5.4, and incubate at room temperature for 2 min.

[0144] 7.2 Configure the two-chain synthesis system according to Table 5 in 4.4.

[0145] 7.3 Set up the PCR reaction program according to Table 6 in 4.5.

[0146] 7.4 Tear open the seal of the reaction card holder, aspirate the EB buffer along the edge of the reaction well, add the cDNA two-strand synthesis reagent that was vortexed and briefly separated in step 7.2, place the reaction card holder on the PCR instrument, close the heat cap, skip the preheating step, and start the two-strand synthesis reaction.

[0147] 8. cDNA unwinding and PCR amplification

[0148] 8.1 After the cDNA double-strand synthesis is completed, remove the double-strand synthesis reagent from the edge of the reaction well.

[0149] 8.2 Add 200 μL of EB buffer to the reaction well and incubate at room temperature for 2 min.

[0150] 8.3 Remove EB buffer from the edge of the reaction well, add 45 μL of 0.08M KOH, and incubate at room temperature for 10 min.

[0151] 8.4 Aspirate 43 μL of 0.08 M KOH into a 0.2 mL low-adsorption centrifuge tube, add 6 μL of Tris (1 M, pH 7.0) and mix by pipetting and aspiration. After a brief centrifugation, place the tube on ice.

[0152] 8.5 Prepare the PCR amplification reaction system on ice according to Table 7 in 5.5.

[0153] 8.6 After vortexing and briefly centrifuging the above amplification mixture, perform amplification on a PCR instrument according to the procedure in Table 6.

[0154] 9. cDNA purification

[0155] 9.1 Vortex the SPRIselect magnetic beads to mix them thoroughly. Add 8 μL of SPRIselect magnetic beads (0.8×) to the above cDNA amplification product (total 100 μL), and mix by pipetting. Note that it is necessary to mix completely and avoid air bubbles.

[0156] 9.2 Incubate at room temperature for 4 min.

[0157] 9.3 Instant centrifugation: Place the sample on a high magnetic rack and let it stand for 2 minutes. Note that the supernatant must be completely clear.

[0158] 9.4 Remove and discard the supernatant, being careful not to pick up the magnetic beads to avoid damage.

[0159] 9.5 Add 200 μL of 80% ethanol, being careful to avoid rinsing the magnetic beads directly. After 30 seconds at room temperature, remove and discard the ethanol. Be careful not to pick up the magnetic beads, as this may result in loss.

[0160] 9.6 Repeat the previous step.

[0161] 9.7 Remove the PCR tubes from the magnetic rack, centrifuge briefly, and place them at the bottom of the magnetic rack.

[0162] 9.8 Carefully aspirate any excess liquid using a 10 μL pipette.

[0163] 9.9 Open the tube cap and let it stand for about 2 minutes until the excess ethanol evaporates. Be careful to avoid the magnetic beads drying out too much and cracking.

[0164] 9.10 Remove the PCR tube from the magnetic rack, add 40 μL of EB into the tube, and mix by pipetting and aspirating. Make sure to mix completely, about 20 times.

[0165] 9.11 Let stand at room temperature for 4 minutes.

[0166] 9.12 Instant centrifuge, place on a low magnetic rack, and let stand for 2 minutes, ensuring the supernatant is completely clear.

[0167] 9.13 Carefully aspirate 40 μL of supernatant into a clean 1.5 mL centrifuge tube, avoiding aspirating the magnetic beads.

[0168] 9.14 The cDNA amplification product was purified, and a secondary double-stranded cDNA library of the tissue sample was obtained.

[0169] 9.15 Take 1 μL of cDNA product for qubit concentration determination.

[0170] 9.16 Take another 1 μL based on the concentration for quality inspection.

[0171] 10. Next-generation sequencing and analysis

[0172] The two tubes of cDNA obtained above were mixed with the same molar mass to construct a library for sequencing, and then analyzed according to the spatial transcriptome data analysis process of the Baichuang S3000.

[0173] Example 2

[0174] This embodiment uses fresh plant tissue as an example to provide a method for increasing the effective number of cDNA molecules by adding one cDNA double-strand synthesis and subsequent melting and amplification reaction, thereby improving the capture performance of the spatial transcriptome, as detailed below:

[0175] 1. Tissue mounting, fixation and staining imaging: Fresh frozen tissue (honeysuckle) sections are mounted on a permeabilized chip for fixation and staining, wherein the permeabilized chip is equipped with an mRNA capture probe.

[0176] 1.1 Organizational stability

[0177] Refer to step 1.1 in Example 1.

[0178] 1.2 Tissue staining and imaging

[0179] Refer to step 1.2 in Example 1.

[0180] 2. Tissue permeability

[0181] Refer to step 2 in Example 1.

[0182] 3. Reverse transcription and cDNA first-strand synthesis

[0183] Refer to step 3 in Example 1.

[0184] 4 cDNA double strand synthesis

[0185] Referring to step 4 in Example 1, the cDNA double-strand synthesis system was prepared according to Table 9.

[0186] Table 9

[0187]

[0188] 5. cDNA unwinding and PCR amplification

[0189] Refer to step 5 in Example 1.

[0190] 6. cDNA purification

[0191] Refer to step 6 in Example 1.

[0192] 7. Secondary Synthesis of cDNA Second Strand

[0193] Referring to step 7 in Example 1, the cDNA double-strand synthesis system was prepared according to Table 8.

[0194] 8. cDNA unwinding and PCR amplification

[0195] Refer to step 8 in Example 1.

[0196] 9. cDNA purification

[0197] Refer to step 9 in Example 1.

[0198] Example 3

[0199] The only difference between this embodiment and Example 1 is that the cDNA double-strand synthesis system in step 4.4 does not contain trehalose, and its mass fraction is allocated to betaine and bovine serum albumin in proportion.

[0200] Example 4

[0201] The only difference between this embodiment and Example 1 is that the cDNA double-strand synthesis system in step 4.4 does not contain betaine, and its mass fraction is allocated to trehalose and bovine serum albumin in proportion.

[0202] Example 5

[0203] The only difference between this embodiment and Example 1 is that the cDNA double-strand synthesis system in step 4.4 does not contain bovine serum albumin, and its mass fraction is allocated to trehalose and betaine in proportion.

[0204] Example 6

[0205] The only difference between this embodiment and Example 1 is that the cDNA double-strand synthesis system in step 4.4 does not contain betaine and trehalose, and their mass fractions are allocated to bovine serum albumin in proportion.

[0206] Example 7

[0207] The only difference between this embodiment and Example 1 is that the cDNA double-strand synthesis system in step 4.4 does not contain betaine and bovine serum albumin, and their mass fractions are allocated to trehalose in proportion.

[0208] Example 8

[0209] The only difference between this embodiment and Example 1 is that the cDNA double-strand synthesis system in step 4.4 does not contain trehalose and bovine serum albumin, and their mass fractions are allocated to betaine in proportion.

[0210] Example 9

[0211] The only difference between this embodiment and Example 1 is that in step 4.4, trehalose in the cDNA double-strand synthesis system is replaced with sucrose of the same concentration.

[0212] Example 10

[0213] The only difference between this embodiment and Example 1 is that in step 4.4, betaine in the cDNA double-strand synthesis system is replaced with betaine phosphate of equal concentration.

[0214] Example 11

[0215] The only difference between this embodiment and Example 2 is that the cDNA double-strand synthesis system in step 4 does not contain trehalose, and its mass fraction is proportionally allocated to glycerol and Tween-20.

[0216] Example 12

[0217] The only difference between this embodiment and Example 2 is that the cDNA double-strand synthesis system in step 4 does not contain glycerol, and its mass fraction is proportionally allocated to trehalose and Tween-20.

[0218] Example 13

[0219] The only difference between this embodiment and Example 2 is that the cDNA double-strand synthesis system in step 4 does not contain Tween-20, and its mass fraction is allocated to trehalose and glycerol in proportion.

[0220] Example 14

[0221] The only difference between this embodiment and Example 2 is that the cDNA double-strand synthesis system in step 4 does not contain trehalose and glycerol, and their mass fractions are allocated to Tween-20 in proportion.

[0222] Example 15

[0223] The only difference between this embodiment and Example 2 is that the cDNA double-strand synthesis system in step 4 does not contain trehalose and Tween-20, and their mass fractions are allocated to glycerol in proportion.

[0224] Example 16

[0225] The only difference between this embodiment and Example 2 is that the cDNA double-strand synthesis system in step 4 does not contain glycerol and Tween-20, and their mass fractions are allocated to trehalose in proportion.

[0226] Example 17

[0227] The only difference between this embodiment and Embodiment 2 is that in step 4, trehalose in the cDNA two-strand synthesis system is replaced with sucrose of the same concentration.

[0228] Example 18

[0229] The only difference between this embodiment and Example 2 is that in step 4, glycerol in the cDNA double-strand synthesis system is replaced with an equal concentration of 1,2-propanediol.

[0230] Example 19

[0231] The only difference between this embodiment and Example 2 is that in step 4, Tween-20 in the cDNA double-strand synthesis system is replaced with an equal concentration of Tween-80.

[0232] Comparative Example 1

[0233] The only difference between this comparative example and Example 1 is that the cDNA double-strand synthesis reagent system in step 4.4 does not contain trehalose, betaine, and bovine serum albumin.

[0234] Comparative Example 2

[0235] The only difference between this comparative example and Example 1 is that steps (7)-(9) were not performed, and only one tube of cDNA was used for library construction and sequencing.

[0236] Comparative Example 3

[0237] The only difference between this comparative example and Example 2 is that the cDNA double-strand synthesis reagent system in step 4.4 does not contain trehalose, glycerol, and Tween-20.

[0238] Comparative Example 4

[0239] The only difference between this comparative example and Example 2 is that steps (7)-(9) were not performed, and only one tube of cDNA was used for library construction and sequencing.

[0240] Test case

[0241] For fresh frozen animal tissues (mouse brain, pig bladder), the same amount of data was extracted and analyzed for each example and comparative example. The cDNA yield of the two strands was compared between each group, and the specific results are shown in Table 10. The cDNA yield of Example 1 was significantly improved, and the median gene number was also significantly increased. This application adopts a novel approach, adding one cDNA two-strand synthesis and adding trehalose, betaine, and bovine serum albumin to the two-strand synthesis buffer. These substances work synergistically to effectively improve the efficiency of converting captured mRNA into cDNA, increase the effective number of molecules in the library, and thus improve the capture efficiency of spatial transcriptomics.

[0242] Table 10

[0243]

[0244]

[0245]

[0246] For fresh plant tissues, the same amount of data was extracted for analysis in each example and comparative example, and the cDNA yield of the two double strands was compared between each group. The specific results are shown in Table 11. The cDNA yield of Example 2 was significantly improved, and the number of median genes was also significantly increased. This application adopts a novel approach, adding one cDNA double strand synthesis and adding trehalose, glycerol, and Tween-20 to the double strand synthesis buffer. These synergistic effects effectively improve the efficiency of converting captured mRNA into cDNA, increase the effective number of molecules in the library, and thus improve the capture efficiency of spatial transcriptomics.

[0247] Table 11

[0248]

[0249]

[0250] In summary, the method for improving spatial transcriptome capture performance in this application adopts a novel approach by adding a cDNA double-strand synthesis step. Trehalose, betaine, glycerol, bovine serum albumin, and Tween-20 are added individually or in combination to the double-strand synthesis buffer, which increases both the yield of cDNA and the number of effective molecules in the library, thereby improving the gene detection rate and achieving the goal of improving capture efficiency. In addition, the method of this application is simple to operate and improves spatial transcriptome capture performance while minimizing the increase in cost.

[0251] The applicant declares that this application illustrates the detailed method of this application through the above embodiments, but this application is not limited to the above detailed method, that is, it does not mean that this application must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent substitutions of the raw materials of this application's product, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this application.

Claims

1. A method for improving the performance of spatial transcriptome capture, characterized in that, The method includes: tissue patching, fixation and staining of the sample; tissue permeation; release of mRNA and capture of mRNA by a microarray; mRNA reverse transcription and cDNA one-strand synthesis; cDNA two-strand synthesis; cDNA unwinding and PCR amplification; repeating cDNA two-strand synthesis, cDNA unwinding and PCR amplification once; and purifying the amplification products from both amplification processes. The two-strand synthesis buffer used for the cDNA two-strand synthesis and the repeated cDNA two-strand synthesis includes any one or a combination of at least two of Tween-20, glycerol, bovine serum albumin, trehalose or betaine.

2. The method for improving spatial transcriptome capture performance according to claim 1, characterized in that, The two-chain synthesis buffer contains 0.5% to 5% Tween-20 by mass, 1% to 10% glycerol by mass, 0.1 to 1 mg / mL bovine serum albumin, 0.5 to 2 M trehalose, and 0.5 to 1.5 M betaine.

3. The method for improving spatial transcriptome capture performance according to claim 1, characterized in that, The tissue permeation solution used for the tissue permeation is an acidic buffer solution containing pepsin, wherein the pepsin mass percentage is 0.01% to 2%, and the pepsin is dissolved in HCl.

4. The method for improving spatial transcriptome capture performance according to claim 1, characterized in that, The reverse transcription reaction system includes: reverse transcription buffer, reverse transcriptase, dNTPs, dithiothreitol and strand displacement primers; the reverse transcription buffer includes Tris-HCl with a final concentration of 100-500 mM, KCl of 200-500 mM, MgCl2 of 10-30 mM and dithiothreitol of 10-100 mM.

5. The method for improving spatial transcriptome capture performance according to claim 4, characterized in that, The final concentration of dNTPs in the reaction system is 1-20 mM, the final concentration of dithiothreitol is 100-200 mM, and the final concentration of the chain substitution primer is 10-100 mM.

6. The method for improving spatial transcriptome capture performance according to claim 1, characterized in that, The reaction system for the second-stranded synthesis of cDNA and the repeated second-stranded synthesis of cDNA includes a second-stranded synthesis buffer, dNTPs, cDNA second-stranded synthase, and second-stranded reaction primers.

7. The method for improving spatial transcriptome capture performance according to claim 1, characterized in that, The double-chain synthesis buffer also includes Tris-HCl at a final concentration of 100-300 mM, (NH4)2SO4 at 50-200 mM, KCl at 100-1000 mM, and MgSO4 at 10-50 mM.

8. The method for improving spatial transcriptome capture performance according to claim 1, characterized in that, The method includes the following steps: (1) The tissue was attached to the chip area containing the mRNA capture probe, fixed with methanol and then stained; (2) Add tissue permeation solution to the chip reaction area, permeate at 35~38℃ for 5~30 min, remove tissue permeation solution, and add cleaning solution to the chip reaction area for incubation and cleaning; (3) Remove the cleaning solution, add reverse transcription and cDNA one-strand synthesis reagents to the chip reaction area, and incubate at 37~55℃ for 60~120 min; (4) Remove the reverse transcription and cDNA first-strand synthesis reagents, add alkaline solution to the chip reaction area for de-stranding, wash with washing buffer after de-stranding, add cDNA second-strand synthesis reagent to the chip reaction area, and incubate at 45~65℃ for 30~60min; (5) Remove the cDNA double-strand synthesis reagent, incubate with washing buffer to clean, add alkaline solution to the chip reaction area to melt, incubate at 20~30°C for 10~20 min, aspirate the incubated solution, add reagent to neutralize alkaline solution, add cDNA amplification reagent to perform PCR amplification; (6) Add washing buffer to the chip reaction area for incubation and washing. After washing, add cDNA double strand synthesis reagent again and incubate at 45~65℃ for 30~60 min. (7) Remove the cDNA double-strand synthesis reagent, incubate and clean with washing buffer, add alkaline solution to the chip reaction area to de-strand, incubate at 20~30°C for 10~20 min, aspirate the de-stranded solution, add reagent to neutralize alkaline solution, add cDNA amplification reagent for PCR amplification; (8) The PCR amplification products from steps (5) and (7) are purified by magnetic beads, and the products on the magnetic beads are washed off using washing buffer.

9. The method for improving spatial transcriptome capture performance according to claim 8, characterized in that, The cleaning solution mentioned in step (2) includes SSC solution; The alkaline solution mentioned in step (4) includes KOH solution, and the washing buffer includes EB buffer. The washing buffer in step (5) includes EB buffer, the alkaline solution includes KOH solution, and the reagent for neutralizing the alkaline solution includes 0.5~1 M Tris; The PCR amplification program described in step (5) is as follows: 95~98℃, 20~40 s; 95~98℃, 15~30 s, 62~68℃, 15~30 s, 60~70℃, 4~8 min, 10~20 cycles; 60~70℃, 4~6 min; The cleaning buffer described in step (6) includes EB buffer; The washing buffer in step (7) includes EB buffer, the alkaline solution includes KOH solution, and the reagent for neutralizing the alkaline solution includes 0.5~1 M Tris; The PCR amplification program described in step (7) is as follows: 95~98℃, 20~40 s; 95~98℃, 15~30 s, 62~68℃, 15~30 s, 60~70℃, 4~8 min, 10~20 cycles; 60~70℃, 4~6 min; The cleaning buffer described in step (8) includes EB buffer.

10. The application of the method for improving spatial transcriptome capture performance according to any one of claims 1-9 in the construction of spatial transcriptome sequencing libraries.

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