Space transcriptome high-throughput sequencing method based on laser microdissection system and DNA bar code marking and application of space transcriptome high-throughput sequencing method

Abstract

The invention provides a space transcriptome high-throughput sequencing method based on a laser microdissection system and DNA bar code marking and application of the space transcriptome high-throughput sequencing method, and particularly belongs to the technical field of gene sequencing. The high-throughput sequencing method for the space transcriptome comprises the following steps: synthesizing a DNA space bar code by a two-step method; mixing the DNA space bar code and a cell lysis solution with a single cell, and carrying out lysis, mRNA capture, reverse transcription and PCR amplification to obtain cDNA; cDNA library construction and sequencing are carried out, sequencing results are processed, and spatial transcriptome data analysis is carried out. The space transcriptome high-throughput sequencing method disclosed by the invention has relatively high cell throughput and can obtain single cell spatial resolution.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene sequencing, and in particular relates to a spatial transcriptome high-throughput sequencing method based on a laser microdissection system and DNA barcode labeling and an application thereof. Background Art

[0002] Spatial transcriptome technology can preserve the spatial information of gene transcription expression, which has completely changed the way people understand tissues and organs. It is considered to be a revolutionary technology for revealing tissue heterogeneity, clarifying disease mechanisms, and promoting the creation of new drugs. Its instrument research and development has become a high ground for technological competition in the field of life and health. Limited by the shortcomings of traditional labeling methods, there is currently no instrument that can achieve high-throughput whole transcriptome sequencing at the spatial resolution of a single cell, and a breakthrough is urgently needed.

[0003] The traditional bulk sequencing analysis method obtains the average value of all cells, which will cause the loss of spatial information of cells, fail to reflect the heterogeneity of functions at different positions in space, and thus fail to accurately reveal the mechanism of action of traditional Chinese medicine at the molecular level. The spatial location information of gene expression is crucial for understanding the functions and pathological changes of complex tissues such as the brain. In order to obtain this key spatial location information, a variety of spatial transcriptomics technologies have emerged. At present, they are mainly divided into three categories: multiple RNA sequencing technology based on spatial barcodes, in situ targeted methods based on imaging, and RNA sequencing methods based on laser capture microdissection (LCM). The first category, spatial barcode-based technologies such as Stereo-seq, Slide-seq, 10x Visium and Spatial Transcriptomics, accurately divide tissues through regular array segmentation. Although it improves the throughput of sample processing, the single-cell analysis capability is limited. The second category is based on imaging-targeted methods, including MERFISH, seqFISH, STARmap, and ISS, which can detect specific gene expression at the single-cell level, but the number of target genes is limited, making it difficult to fully detect gene variation. The third category is RNA sequencing technology combined with LCM, such as TSCS, LCM-seq, GEO-seq, and Spatial-seq. However, the cell throughput of LCM technology is extremely low.

[0004] Spatial omics technology is rapidly becoming a key tool for in-depth understanding of complex biological systems. It has greatly enriched people's understanding of life sciences by revealing the spatial heterogeneity of cells and tissues in complex environments. As the scientific research community continues to pay more and more attention to spatial differences between cells, the demand for high-precision, unbiased spatial resolution omics technology is becoming increasingly urgent. LCM technology stands out for its minimal contamination, highly flexible tissue capture capabilities and a wide range of applications. It is particularly suitable for processing different tissues or patient samples and has shown great potential in clinical research, but its low cell throughput problem remains to be solved. Summary of the invention

[0005] The object of the present invention is to provide a spatial transcriptome high-throughput sequencing method based on a laser microdissection system and DNA barcode labeling and its application. The spatial transcriptome high-throughput sequencing method of the present invention has a high cell throughput, can obtain single-cell spatial resolution, and can customize the selection of regions or cells of interest.

[0006] The present invention provides a spatial transcriptome high-throughput sequencing method based on a laser microdissection system and DNA barcode labeling, comprising the following steps: The carboxyl magnetic beads are mixed with EDC to perform an activation reaction to obtain activated carboxyl magnetic beads; The activated carboxyl magnetic beads are mixed with the first spatial barcode primers respectively to carry out a dehydration condensation reaction to obtain first magnetic beads; The first magnetic beads are mixed with the second spatial barcode primer, the third spatial barcode primer and Phanta Super-Fidelity DNA Polymerase, and PCR reaction is performed in a well plate to obtain a DNA spatial barcode; The animal tissue is frozen and sectioned, and the surface of the section is treated for static electricity removal using a static electricity removal device to obtain static electricity-free sections; the static electricity-free sections are microcut using a laser capture microdissection instrument, and single cells are collected using a well plate; Adding DNA spatial barcodes and cell lysis solution to a well plate containing single cells, using the DNA spatial barcodes to lyse the cells in the well plate to capture mRNA; the raw materials used to prepare the cell lysis solution include RNase inhibitors; The captured mRNA was reverse transcribed and washed to obtain washed magnetic beads; the washed magnetic beads were resuspended with the Pre-Amp PCR system, the first PCR amplification was performed, and the beads were placed on a magnetic stand, and the supernatant was taken to obtain the first PCR product; the first PCR product was purified using VAHTS DNAClean Beads, and the purified product was mixed with 2× Kapa HiFi HotStart Readymix and TSO-PCR primers for a second PCR amplification to obtain a second PCR amplification product; the second PCR amplification product was purified again using 0.7× VAHTSDNA Clean Beads to obtain cDNA; The TruePrep Flexible DNA Library Prep Kit for Illumina was used to construct the cDNA library. The Salus Pro gene sequencer was used for PE150 double-end sequencing. The sequencing results were processed and spatial transcriptome data analysis was performed.

[0007] Preferably, the first spatial barcode primers include A1 to A4, and the nucleotide sequences are shown in SEQ ID NOs. 1 to 4, respectively; the second spatial barcode primers include B1 to B12, and the nucleotide sequences are shown in SEQ ID NOs. 5 to 16, respectively; the third spatial barcode primers include C1 to C8, and the nucleotide sequences are shown in SEQ ID NOs. 17 to 24, respectively; The TSO-PCR primers include TSO LNA and TSO-PCR; the nucleotide sequence of the TSO LNA is shown in SEQ ID NO.27; the nucleotide sequence of the TSO-PCR is shown in SEQ ID NO.28.

[0008] Preferably, the well plate comprises a 384-well plate; and the reaction system of the PCR reaction comprises 13.5 μL of Phanta Super-Fidelity DNA Polymerase, 4 μL of the first magnetic beads, 1 μL of the second spatial barcode primer and 1.5 μL of the third spatial barcode primer in each well.

[0009] Preferably, the reaction program of the PCR reaction is: 94°C 5 min; 95°C 15s, 48.8°C 4 min, 72°C 4 min, 5 cycles; 94°C 5 min, 48.8°C 20 min, 72°C 20 min.

[0010] Preferably, the slice thickness obtained by the frozen section is 10 μm; the time of the static electricity removal treatment is 3 minutes; the conditions of the microdissection are: using a laser capture microdissection instrument to automatically identify the cutting area, the parameters are set to objective lens 10×, Final Pulse mode, power = 17, aperture = 1, speed = 20, bridge size = 5, and final pulse = 16.

[0011] Preferably, the cell lysis solution comprises Tris-HCl, LiCl, SDS, EDTA, DTT solution, RNase inhibitor and nuclease-free water; each 1200 μL of cell lysis solution comprises 120 μL of Tris-HCl at pH 7.5, 80 μL of LiCl, 120 μL of 10% SDS solution, 16 μL of EDTA, 12 μL of 500 mM DTT solution, 12 μL of 40U / μL RNase inhibitor and the remainder of nuclease-free water.

[0012] Preferably, the length of the cDNA is greater than 650 bp.

[0013] Preferably, processing the sequencing results includes: splitting the sequencing data into independent files according to the different spatial barcode sequences using a custom Python script, and processing them according to the Drop-seq core analysis process, and then using STAR v2.7.8a for gene alignment; the quality control and preliminary processing are completed by the FastQC tool.

[0014] Preferably, the spatial transcriptome data analysis includes: using the calcNormFactors function of the edgeR package v3.40.2 to standardize the generated digital expression matrix; using the likelihood ratio test method to evaluate the difference in expression of each gene in different groups; the likelihood ratio test determines the significance of the effect by comparing the fit of the full model containing specific effects with the simplified model without these effects; P The value was corrected for multiple testing, and the false discovery rate (FDR) method was used to control the false positive rate; the final threshold ( P <0.05, |log2 fold difference|>1) to determine the genes with significant differential expression; the Metascape online platform was used to perform GO and KEGG pathway enrichment analysis on the differentially expressed genes; after the enrichment analysis was completed, the enrichment result data provided by Metascape was exported and visualized using the ggplot2 package of the R language.

[0015] The present invention also provides the application of the spatial transcriptome high-throughput sequencing method described in the above technical scheme in screening disease treatment drugs, disease pathogenesis research, developmental biology research or regenerative medicine research; the research is aimed at non-disease treatment.

[0016] The present invention provides a spatial transcriptome high-throughput sequencing method based on a laser microdissection system and DNA barcode labeling. Compared with the spatial transcriptome sequencing technology currently on the market, the sequencing method of the present invention can achieve single-cell resolution, perform customized acquisition and analysis of regions of interest, and has high cell throughput. The experimental results show that the spatial transcriptome high-throughput sequencing method of the present invention simplifies the DNA spatial barcode synthesis method, has high synthesis efficiency, saves reagents, and saves synthesis time; moreover, it improves the quality of RNA; and improves the Spots capture rate; and increases the number of genes detected. The spatial transcriptome high-throughput sequencing method of the present invention can be more widely and efficiently applied to the study of disease pathogenesis, the screening of disease treatment drugs, the study of developmental biology, and the study of regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 The results of the 24-hour efficacy evaluation of PNS and PGS on MCAO mice provided by the present invention are shown in Figure 1; wherein, A: a schematic diagram of the overall dosing regimen; B: a diagram showing the effects of PNS and PGS on the neurobehavioral scores of MCAO mice; C: a representative diagram of TTC staining of mouse brain tissue; D: a diagram showing the effects of PNS and PGS on the infarct volume of mouse brain tissue; all data are expressed as mean ± SEM, n = 6; compared with the model group, ** P <0.01, *** P <0.001; Figure 2 The representative images of H&E staining of the cortical area of ​​mouse brain tissue provided by the present invention; wherein n = 3, the scale bars in the figure are 2000 μm and 100 μm; Figure 3The Spatial-seq 2.0 spatial transcriptome data quality control diagram provided by the present invention; wherein, A: 384 different barcode capture beads quality inspection report diagram; B: 384 spatial Spots size and array distribution diagram; C: sequencing library quality inspection report diagram; D: sequencing result quality control report diagram; Figure 4 The correlation analysis diagram between the key spatial regions provided by the present invention and the ischemic penumbra of stroke; wherein, A: the result diagram of the number of differential genes at each spatial position of the Sham group and the Model group; B: the spatial positions of the top 25% according to the number of differential genes; C: the spatial distribution of the ischemic penumbra (red) and the infarct core area (blue) shown in the H&E staining diagram; Figure 5 The diagram of the pathway enrichment analysis results provided by the present invention; wherein, A: the classical pathway diagram involving the key genes in PNS callback; B: the classical pathway diagram involving the key genes in PGS callback; Figure 6 This is a graph showing the enrichment results of GO pathways for marker genes in various brain regions of mice provided by the present invention; Figure 7 This is a comparison result diagram of RNA quality of different cell lysates provided by the present invention; wherein A is Spatial-seq and B is Spatial-seq 2.0. DETAILED DESCRIPTION

[0019] The present invention provides a spatial transcriptome high-throughput sequencing method based on a laser microdissection system and DNA barcode labeling, comprising the following steps: The carboxyl magnetic beads are mixed with EDC to perform an activation reaction to obtain activated carboxyl magnetic beads; The activated carboxyl magnetic beads are mixed with the first spatial barcode primers respectively to carry out a dehydration condensation reaction to obtain first magnetic beads; The first magnetic beads are mixed with the second spatial barcode primer, the third spatial barcode primer and Phanta Super-Fidelity DNA Polymerase, and PCR reaction is performed in a well plate to obtain a DNA spatial barcode; The animal tissue is frozen and sectioned, and the surface of the section is treated for static electricity removal using a static electricity removal device to obtain static electricity-free sections; the static electricity-free sections are microcut using a laser capture microdissection instrument, and single cells are collected using a well plate; Adding DNA spatial barcodes and cell lysis solution to a well plate containing single cells, using the DNA spatial barcodes to lyse the cells in the well plate to capture mRNA; the raw materials used to prepare the cell lysis solution include RNase inhibitors; The captured mRNA was reverse transcribed and washed to obtain washed magnetic beads; the washed magnetic beads were resuspended with the Pre-Amp PCR system, the first PCR amplification was performed, and the beads were placed on a magnetic stand, and the supernatant was taken to obtain the first PCR product; the first PCR product was purified using VAHTS DNAClean Beads, and the purified product was mixed with 2× Kapa HiFi HotStart Readymix and TSO-PCR primers for a second PCR amplification to obtain a second PCR amplification product; the second PCR amplification product was purified again using 0.7× VAHTSDNA Clean Beads to obtain cDNA; The TruePrep Flexible DNA Library Prep Kit for Illumina was used to construct the cDNA library. The Salus Pro gene sequencer was used for PE150 double-end sequencing. The sequencing results were processed and spatial transcriptome data analysis was performed.

[0020] The present invention mixes carboxyl magnetic beads with EDC, performs an activation reaction, and obtains activated carboxyl magnetic beads. Before the carboxyl magnetic beads of the present invention are mixed with EDC, they are washed with MES buffer. In the present invention, the number of washings can be 1 to 2 times. In a specific embodiment, the present invention uses 0.1M MES buffer for washing. In a specific embodiment, the activation reaction is performed in MES buffer. In a specific embodiment, the temperature of the activation reaction is 15 to 30°C and the time is 20 minutes.

[0021] The activated carboxyl magnetic beads are mixed with the first space barcode primers, respectively, and a dehydration condensation reaction is performed to obtain the first magnetic beads. In a specific embodiment, the first space barcode primers include A1~A4, and the nucleotide sequences are shown in SEQ ID NO.1~4, respectively. In a specific embodiment, after each 400 μL of activated carboxyl magnetic beads are mixed with 80 μL of 0.2M MES buffer and 80 μL of the first space barcode primer, the dehydration condensation reaction is performed. In a specific embodiment, the concentration of the first space barcode primer is 400 μM. In a specific embodiment, the rotation speed of the dehydration condensation reaction (slow speed rotation) is 10~20rpm. In the present invention, the time of the dehydration condensation reaction can be 20min. After low-speed rotation, the present invention adds 32.36 μL (0.45 mg) of EDC solution and repeats low-speed rotation, including one 20min and one 80min low-speed rotation, and the temperature is 15~30°C. Repeated addition of EDC can improve the efficiency of cross-linking, avoid premature inactivation of reaction intermediates, and ensure the quality and yield of the final product. After the rotation is completed, place it on a magnetic stand, aspirate the supernatant, wash and resuspend to obtain the first magnetic beads. In a specific embodiment, the washing and resuspension is sequentially resuspended and washed with 100 μL 0.1M PBS containing 0.02% Tween-20, enzyme-free water, and TE with pH = 8.0.

[0022] After obtaining the first magnetic beads, the present invention mixes the first magnetic beads with the second spatial barcode primers, the third spatial barcode primers and Phanta Super-Fidelity DNA Polymerase, performs PCR reaction connection, and obtains DNA spatial barcodes. In a specific embodiment, the second spatial barcode primers include B1~B12, and the nucleotide sequences are respectively as shown in SEQ ID NO.5~16; the third spatial barcode primers include C1~C8, and the nucleotide sequences are respectively as shown in SEQ ID NO.17~24. In a specific embodiment, the well plate includes a 384-well plate; the reaction system of the PCR reaction includes Phanta Super-Fidelity DNA Polymerase 13.5μL, the first magnetic beads 4μL, the second spatial barcode primer (50μM) 1μL and the third spatial barcode primer (50μM) 1.5μL per well. In a specific embodiment, the concentrations of the second and third spatial barcode primers are 50μM, respectively. In a specific embodiment, the reaction procedure of the PCR reaction is: 94°C 5min; 95°C 15s, 48.8°C 4min, 72°C 4min, 5 cycles; 94°C 5min, 48.8°C 20min, 72°C 20min. After the PCR reaction, the present invention also includes post-processing to ensure the accuracy and availability of the barcode. In a specific embodiment, the post-processing includes: first placing the well plate for the PCR reaction on a magnetic stand, operating on ice, aspirating the supernatant, washing with enzyme-free water, and removing the residual PCR reaction mixture. Then incubate with ExoI exonuclease Mix to digest the single-stranded DNA that has not been amplified. In order to ensure that the reaction is fully carried out, the magnetic beads need to be suspended from time to time, about every 6 min, to promote sufficient contact between the enzyme and DNA. Afterwards, washing and resuspension are performed, including continuous resuspension of the magnetic beads with 10 μL of TE-SDS, TE-TW and 20 μL of enzyme-free water to thoroughly remove residual substances that may affect the next step of the experiment. Then, cover the plate with a sealing film, place the plate in a 95 °C metal bath for 6 min, then quickly take out the plate and place it on a magnetic stand and remove the supernatant, add 20 μL of enzyme-free water to each well again, and repeat this step twice. Finally, add 15 μL of TE-TW to each well to resuspend the magnetic beads and store them at 4 °C to obtain the Beads preservation plate (magnetic bead preservation plate). These delicate post-processing steps not only ensure the quality of the DNA barcode, but also provide a stable and reliable foundation for subsequent applications. The traditional scheme first connects the second space barcode primer to the magnetic beads, and then connects it with the third space barcode primer. The steps are cumbersome and time-consuming; and the efficiency of the connection reaction is low, especially under the pairing conditions of two different barcode primers.The present invention adopts a one-step method to add the second and third spatial barcode primers to the PCR reaction system together. Through the efficient amplification ability of the Phanta enzyme, the connection and amplification of these two steps are completed simultaneously in the same reaction system, which can improve efficiency (through synchronous primer amplification and connection, the experimental steps and time are reduced, and the overall reaction efficiency is improved), simplify operation (reducing multiple reaction steps, reagent addition and cleaning processes, thereby reducing the complexity of operation and the probability of operation errors), and improve reaction consistency (because it is synchronized in the same reaction system, it can ensure that the second and third barcode primers react under the same conditions, reducing the variation caused by differences in different reaction systems). Specifically, the present invention can improve the accuracy of barcode connection and the reliability of amplification by selecting Phanta Super-Fidelity DNA Polymerase and performing a one-step PCR reaction in a new reaction system; significantly simplify the experimental steps, reduce the consumption of time and reagents; ensure high fidelity, reduce error introduction, and ensure the accuracy of the barcode. Therefore, compared with the traditional two-step reaction, the new system setting of the present invention can generate the target DNA spatial barcode more efficiently and accurately, providing a more stable foundation for subsequent experiments.

[0023] After synthesizing the DNA spatial barcode, the present invention also includes quality control of the DNA spatial barcode. In a specific embodiment, the preservation well plate of the DNA spatial barcode is taken out, placed on a magnetic stand, the supernatant is aspirated and discarded, enzyme-free water is added, and the magnetic beads are diluted with enzyme-free water to obtain a diluted magnetic bead liquid, and then the diluted magnetic bead liquid is mixed with the qPCR reaction solution and the primer universal primer and tail-specific primer to perform a qPCR reaction. After the reaction, TE-EW is used to resuspend. In the present invention, the system of the qPCR reaction includes 10 μL of 2×SYBR Green premix, 1 μL of DNA spatial barcode, 1 μL of universal primer, 1 μL of tail-specific primer and 7 μL of enzyme-free water per 20 μL. In the present invention, the reaction procedure of the qPCR is: 95°C 1min; 95°C 5s, 55°C 15s, 35 cycles. The core principle of the quality control of the present invention is to detect whether the specific sequence of the DNA spatial barcode is successfully connected by qPCR, and to ensure the correct pairing and amplification of the primers in the target area. qPCR (quantitative polymerase chain reaction) can monitor the product accumulation during PCR amplification in real time and determine the success of amplification based on the Ct value (Cycle threshold).

[0024] The animal tissue is frozen and sectioned, and the surface of the section is treated with a static electricity removal device to remove static electricity to obtain a static electricity-free section; the static electricity-free section is microcut using a laser capture microdissection instrument, and a single cell is collected using a well plate. Before the animal tissue is frozen and sectioned, it is also included in rapid freezing and stored at -80°C. After the frozen animal tissue is taken out and embedded, it is frozen and sectioned. In a specific embodiment, the thickness of the section obtained by the frozen section is 10μm. In a specific embodiment, the static electricity removal time is 3min. In a specific embodiment, the static electricity removal device includes a desktop ion blower LA-211. The static electricity on the LCM instrument and the section is very serious, which affects the capture rate. The present invention increases the capture rate of Spots from 50% to 95% by adding a static electricity removal device to blow out an airflow of positive and negative charges and neutralize the charge. In a specific embodiment, the microdissection conditions are as follows: using a laser capture microdissection instrument to automatically identify the cutting area, the parameters are set to objective lens 10×, Final Pulse mode, power = 17, aperture = 1, speed = 20, bridge size = 5, and final pulse = 16. After cutting, the samples are collected using a well plate and stored at -80°C immediately after collection.

[0025] DNA spatial barcodes and cell lysis solution are added to a well plate containing single cells, and the cells in the well plate are lysed using the DNA spatial barcode to capture mRNA; the raw materials for preparing the cell lysis solution include RNase inhibitors. In a specific embodiment, before the DNA spatial barcode is added, the operation of aspirating the supernatant (TE-TW) and washing and resuspending with enzyme-free water are also included, and the whole process is performed on ice. In a specific embodiment, the cell lysis solution includes Tris-HCl, LiCl, SDS, EDTA, DTT solution, RNase inhibitors and nuclease-free water; each 1200 μL cell lysis solution includes 120 μL of Tris-HCl at pH 7.5, 80 μL of LiCl, 120 μL of 10% SDS solution, 16 μL of EDTA, 12 μL of 500 mM DTT solution, 12 μL of 40U / μL of RNase inhibitor and the remainder of nuclease-free water. The cell lysate of the present invention can inhibit the effect of exogenous RNase on RNA degradation and improve RNA quality. In the present invention, the volume ratio of DNA spatial barcode to cell lysate is 1:19. The present invention preferably mixes the cell lysate with the DNA spatial barcode first, and then adds the cell lysate containing the DNA spatial barcode to the well plate where single cells are collected. In a specific embodiment, the lysis conditions include: lysis at room temperature for 5 minutes, incubation on ice for 12 minutes, and resuspending the magnetic beads every 2 minutes during the period to promote the effective capture of mRNA. In the present invention, the room temperature refers to 15~30°C. After lysis, the present invention rinses the magnetic beads. In a specific embodiment, 6 × SSC is used to rinse the magnetic beads. Centrifuge afterwards and discard the supernatant.

[0026] The captured mRNA is reverse transcribed and washed to obtain washed magnetic beads; the washed magnetic beads are resuspended with the Pre-Amp PCR system, the first PCR amplification is performed, the beads are placed on a magnetic stand, and the supernatant is taken to obtain the first PCR product. In a specific embodiment, it is preferred to use 50 mM Tris pH 8.0 to resuspend the magnetic beads, then discard the supernatant and immediately add 20 μL of the reverse transcription premix RT Mix prepared on ice to reverse transcribe the mRNA. In the present invention, the reverse transcription condition is 42°C for 90 min. In a specific embodiment, the beads are suspended once every 15 min. After the reaction is completed, centrifuge, place on a magnetic stand, and discard the supernatant. In a specific embodiment, the washing can be performed using TE-SDS, TE-TW, and 10 mM Tris-HCl (pH = 8.0), respectively. After washing, the magnetic beads are resuspended using an exonuclease system, incubated at 37°C for 60 min, and suspended once every 10 min. After the reaction, the beads were placed on a magnetic stand, the supernatant was discarded, and then the magnetic beads were washed with TE-SDS, TE-TW, and 10 mM Tris-HCl (pH = 8.0), respectively. Subsequently, the magnetic beads were resuspended with the Pre-AmpPCR system for the first PCR amplification. In the present invention, the reaction procedure for the first PCR amplification is: 98°C for 3 min; 98°C for 20 s, 65°C for 45 s, 72°C for 6 min, 6 cycles; 72°C for 10 min.

[0027] The first PCR product is purified using VAHTS DNA Clean Beads, and the purified product is mixed with 2× KapaHiFi HotStart Readymix and TSO-PCR primers for a second PCR amplification to obtain a second PCR amplification product. In a specific embodiment, 0.8× VAHTS DNA Clean Beads are used to purify the PCR product. In a specific embodiment, the following steps are preferably included: 0.8× VAHTS DNA Clean Beads are mixed with the first PCR product, placed at room temperature for 15 minutes, the supernatant is discarded, washed twice with 80% ethanol aqueous solution, incubated at room temperature for 30 seconds, the supernatant is removed, the cDNA is eluted with nuclease-free water, and placed at room temperature for 10 minutes to obtain a purified product. The purified product is mixed with 2× Kapa HiFi HotStart Readymix and TSO-PCR primers for a second PCR amplification. In the present invention, the reaction procedure for the second PCR amplification is: 98°C 3min; 98°C 20s, 72°C 6min, 10 cycles; 72°C 10min. In the present invention, the purification conditions are preferably the same as above. In a specific embodiment, the TSO-PCR primer includes TSO LNA and TSO-PCR; the nucleotide sequence of the TSO LNA is shown in SEQ ID NO.27, and the end of TSO LNA has two trans-modified guanine / rG / rG and one LNA (locked nucleic acid) modified guanine / iXNA_G to enhance its stability of binding to the template and reduce non-specific binding; the nucleotide sequence of the TSO-PCR is shown in SEQ ID NO.28.

[0028] After obtaining the second PCR amplification product, the present invention uses 0.7×VAHTS DNA Clean Beads to purify the second PCR amplification product again to obtain cDNA. In a specific embodiment, the length of the cDNA is more than 650 bp.

[0029] The present invention uses the TruePrep Flexible DNA Library Prep Kit for Illumina free library construction kit to construct the cDNA library and uses the Salus Pro gene sequencer to perform PE150 double-end sequencing.

[0030] The present invention processes the sequencing results. In a specific embodiment, the processing of the sequencing results includes: according to the difference of the spatial barcode sequence, the sequencing data is split into independent files using a custom Python script, and the processing is carried out according to the Drop-seq core analysis process (specifically, according to the Drop-seq Core Computational Protocol v2.0.0, Drop-seq_tools v2.5.1 is used for quality control and preliminary processing), and then STAR v2.7.8a is used for gene alignment; the quality control and preliminary processing are completed by the FastQC tool.

[0031] The present invention performs spatial transcriptome data analysis. In a specific embodiment, the spatial transcriptome data analysis includes: using the calcNormFactors function of the edgeR package v3.40.2 to standardize the generated digital expression matrix; using the likelihood ratio test method to evaluate the difference in expression of each gene in different groups; the likelihood ratio test determines the significance of the effect by comparing the fit of the full model containing specific effects with the simplified model that does not contain these effects; P The value was corrected for multiple testing, and the FDR method was used to control the false positive rate; the final threshold ( P <0.05, |log2 fold difference|>1) to determine the genes with significant differential expression; the Metascape online platform was used to perform GO and KEGG pathway enrichment analysis on the differentially expressed genes; after the enrichment analysis was completed, the enrichment result data provided by Metascape was exported and visualized using the ggplot2 package of the R language.

[0032] The present invention also provides the application of the spatial transcriptome high-throughput sequencing method described in the above technical scheme to screening disease therapeutic drugs, disease pathogenesis research, developmental biology research or regenerative medicine research; the research is aimed at the treatment of non-diseases.

[0033] To further illustrate the present invention, a spatial transcriptome high-throughput sequencing method based on a laser microdissection system and DNA barcode labeling and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 1. Experimental Materials and Reagents 20 μm carboxyl magnetic beads, Suzhou Beaver Biomedical Engineering Co., Ltd., catalog number 40200; EDC: Thermo Fisher Scientific, USA, catalog number PG82079; MES: Sigma, USA, catalog number M3671; 0.1M PBS: Beijing Solebow Technology Co., Ltd., catalog number P1022; Tween-20: Sigma, USA, catalog number P7949; TE buffer: Invitrogen, USA, catalog number AM9849; Phanta Super-Fidelity DNA Polymerase: Nanjing Novozyme Biotechnology Co., Ltd., catalog number P501; Exonuclease I ( E. coli ): American NEB Company, catalog number M0293L; 1M Tris-HCl buffer (PH=7.5): Beijing Solebow Technology Co., Ltd., catalog number T1140; LiCl precipitation solution (7.5 M): Invitrogen, USA, catalog number AM9480; 10% SDS solution (RNase-free): Invitrogen, USA, catalog number AM9823; EDTA: Shanghai Bio-Tech Biotechnology Co., Ltd., catalog number ST066; SSC (20×): Invitrogen, USA, catalog number AM9770; Betaine solution: Sigma, USA, product number B0300; 0.5 M DTT (DNase, RNase & Protease free): Shanghai Biotime Biotechnology Co., Ltd., catalog number ST041; MgCl 2 : Invitrogen, USA, catalog number AM9530G; PrimeScript™ II Reverse Transcriptase: Takara Co., Ltd., Japan, product number 2690A; Recombinant RNasin® Ribonuclease Inhibitor: Promega, USA, catalog number N2515; SUPERase In RNase inhibitor: ABI, USA, catalog number AM2694; KAPA HiFi High-Fidelity Hot Start DNA Polymerase Master Mix: Kapa Biosystems, USA, catalog number KK2602; Tris-HCl (pH 8.0): Shanghai Bio-Tech Biotechnology Co., Ltd., catalog number ST780; Tris-HCl (pH 7.5): Beijing Solebow Technology Co., Ltd., catalog number T1140; Rapid fluorescence quantitative PCR kit (SYBR Green): Biosharp, China, catalog number BL705A; Qubit® dsDNA HS Assay Kits: Thermo Fisher Scientific, USA, catalog number Q32851; Leica Cryo-Gel cryosection embedding medium: Leica, Germany, catalog number 14020108926; Nissl stain: Wuhan Saiweier Biotechnology Co., Ltd., catalog number G1036-100ML; VAHTS DNA Clean Beads: Nanjing Novozyme Biotechnology Co., Ltd., catalog number N411-01; TruePrep Flexible DNA Library Prep Kit for Illumina: Nanjing Novogene Biotech Co., Ltd., catalog number TD504; PCR primers: As shown in Table 1, all were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

[0035] Table 1 Primer list

[0036] 2. Preparation of experimental reagents 0.1M MES: Dissolve 491 mg of MES in sufficient deionized water, adjust to pH 5.0 with NaOH solution, and add to a total volume of 25 mL; 0.2M MES: Dissolve 982 mg of MES in sufficient deionized water, adjust to pH 5.0 with NaOH solution, and add to a total volume of 25 mL; TE-TW solution: Add 5 μL of 10% Tween-20 solution to sufficient TE buffer, pH 8.0, to a total volume of 50 mL; TE-SDS solution: Add 500 μL of 10% SDS solution to sufficient TE buffer, pH 8.0, to a total volume of 10 mL; EDC solution: Dissolve 300 mg of EDC in enough 0.1 M MES buffer to make a total volume of 5 mL; Cell lysis buffer: Add 120 μL of Tris-HCl (pH 7.5), 80 μL of LiCl, 120 μL of 10% SDS solution, 16 μL of EDTA, 12 μL of 500 mM DTT solution and 12 μL of RNase inhibitor (40U / μL) to sufficient amount of nuclease-free water to prepare a specific cell lysis buffer; RT mix: 16.16 μL nuclease-free water, 8 μL 5× RT buffer, 8 μL betaine solution, 4 μL dNTP (10 mM), 0.2 μL DTT, 0.24 μL MgCl 2 , 0.4 μL TSO primer, 2 μL PrimeScriptII reverse transcriptase (200U / μL), 1 μL RNase inhibitor, a total of 40 μL.

[0037] 3. Experimental Instruments Gradient PCR amplifier: Thermo Fisher, USA, model Applied biosystem ProFlexBase; Real-time fluorescence quantitative PCR system: Bio-Rad, USA, model CFX96 Touch; Laser capture microdissection system: Leica, Germany, including upright microscope (model DM68), laser cutter (model LMD6) and single cell capture and recovery device (model LMT350); Cryostat: Thermo Corporation, USA, model NX50; Agilent nucleic acid microfluidics electrophoresis analysis system: Agilent, USA, model 4200 TapeStation; Gene sequencer: Salus Medical Company, China, model Salus Pro; Static electricity removal equipment: desktop ion blower LA-211, LAOGE Shanghai Company.

[0038] 4. Operation steps Step 1: Synthesis of DNA spatial barcodes Step 1 Synthesis To locate the magnetic beads and connect the first segment of spatial barcode, first take four 1.5 mL centrifuge tubes and add about 1.67 × 10 6Carboxyl magnetic beads (approximately 232 μL). After that, the supernatant was aspirated and washed 1 to 2 times with 0.1M MES buffer. After washing, approximately 358 μL of 0.1M MES buffer was added to each tube, and 2.06 mg (34.4 μL) of EDC was added to make the final volume approximately 424 μL. After mixing, 400 μL of the mixture was taken out from each centrifuge tube. Subsequently, 80 μL of 0.2M MES and 80 μL of the corresponding first space barcode primer (primers A1~A4) were added to each tube and mixed thoroughly. The centrifuge tube was fixed on the rotator and rotated at low speed for 20 min at room temperature. Then 32.36 μL (0.45 mg) of EDC solution was added to each tube, and the low-speed rotation step was repeated, including one low-speed rotation for 20 min and one low-speed rotation for 80 min. After the rotation is completed, place the centrifuge tube on the magnetic stand, aspirate the supernatant, and then use 100 μL of 0.1M PBS containing 0.02% Tween-20, enzyme-free water, and TE with pH = 8.0 to resuspend and wash the magnetic beads. Finally, resuspend the magnetic beads in 280 μL of enzyme-free water to obtain the first magnetic beads.

[0039] Next, the second step is to synthesize the second and third spatial barcodes for the positioning magnetic beads. First, you need to prepare the second spatial barcode primer (primer B, B1~B12), the third spatial barcode primer (primer C, C1~C8) and Phanta Super-Fidelity DNA Polymerase, take them out from 4 ℃ in advance, mix them and quickly centrifuge them to a concentration of 50μM.

[0040] In the PCR connection stage, in a 384-well PCR plate, the reaction materials in each well included 13.5 μL of Phanta Super-Fidelity DNA Polymerase, 4 μL of the first magnetic beads, 1 μL of the second space barcode primer (50 μM), and 1.5 μL of the third space barcode primer (50 μM), see Table 2.

[0041] Table 2 Reaction materials and volumes per well

[0042] After the 384-well plate was mixed and quickly centrifuged, subsequent PCR amplification was performed. The amplification program is shown in Table 3.

[0043] Table 3 PCR reaction program settings for connecting the second and third segments

[0044] After completing the PCR reaction for DNA spatial barcode synthesis, the next step is to post-process the 384-well plate to ensure the accuracy and availability of the barcode. First, place the 384-well plate on a magnetic stand and operate on ice to remove the supernatant, then wash each well once with enzyme-free water to remove the residual PCR reaction mixture. Next, add 10 μL of ExoI exonuclease Mix to each well and incubate at 37 °C for 15 min to digest the single-stranded DNA that has not been amplified. To ensure that the reaction is fully carried out, the magnetic beads need to be suspended from time to time, approximately every 6 min, to promote sufficient contact between the enzyme and DNA. After that, a series of washing and resuspension steps are performed, including continuous resuspension of the magnetic beads with 10 μL of TE-SDS, TE-TW, and 20 μL of enzyme-free water to thoroughly remove residual substances that may affect the next experiment. Then, cover the plate with a sealing film and place the 384-well plate in a 95 °C metal bath for about 6 min. Then quickly take the plate out and place it on a magnetic stand and remove the supernatant. Add 20 μL of enzyme-free water to each well again, and repeat this step twice. Finally, add 15 μL of TE-TW to each well to resuspend the magnetic beads, store them at 4 °C after labeling, and obtain the Beads storage plate (magnetic bead storage plate). These meticulous post-processing steps not only ensure the quality of the DNA barcode, but also provide a stable and reliable foundation for subsequent applications.

[0045] Step 2: Quality control of DNA spatial barcoding Take out the Beads storage plate, place it on the magnetic stand, discard the supernatant, add 20 μL of enzyme-free water to each well, take out 1 μL of magnetic beads and dilute to 100 μL with enzyme-free water. Take a 384-well plate, add 9 μL of qPCR Mix and 1 μL of diluted magnetic bead suspension to each well to prepare a complete qPCR system, see Table 4.

[0046] Table 4 qPCR reaction materials and volumes per well

[0047] After the 384-well plate was mixed and quickly centrifuged, the subsequent qPCR reaction was performed. The reaction procedure is shown in Table 5. After the experiment, the magnetic beads in the 384-well plate were resuspended with 15 μL TE-TW and stored at 4 °C.

[0048] Table 5 qPCR reaction program settings

[0049] Step 3: Animal tissue collection and sectioning Prepare enzyme-free PBS and RNA later in advance, anesthetize the animal with 1.5% sodium pentobarbital, and perfuse with 20 mL enzyme-free PBS and 5 mL RNA later. Then quickly take the animal tissue and rinse it with pre-cooled PBS solution, and then dry the liquid with clean absorbent paper. Finally, quickly freeze the animal tissue in liquid nitrogen for 30 s~1min, and store it in a -80 ℃ refrigerator.

[0050] After the animal tissues were taken out from -80 °C and embedded, the brain tissues were cut into frozen sections using a freezing microtome with a section thickness of 10 μm.

[0051] Step 4: Microdissection and barcode capture labeling The frozen sections of animal tissues were blown gently on the surface for 3 minutes using a static removal device (desktop ion blower LA-211), and then placed in a laser cutting instrument (LCM) for microscopic high-definition scanning. Each section can be divided into multiple Spots according to user needs. The radius of each Spot can be customized, with a minimum of about 10 μm, reaching the single-cell level. The LCM instrument was used to automatically identify the cutting area, and the parameters were set to objective lens 10 ×, Final Pulse mode, power = 17, aperture = 1, speed = 20, bridge size = 5, final pulse = 16, and collected using an enzyme-free well plate. After collection, it was immediately stored at -80°C to obtain a collection plate.

[0052] The magnetic bead storage plate synthesized in step 1 and stored at 4 °C was quickly centrifuged to remove the residual liquid. Then, the plate was placed on a magnetic stand to aspirate the supernatant (TE-TW). Next, the magnetic beads were washed 1 to 2 times with enzyme-free water, adding 15~20 μL of enzyme-free water each time, and then quickly centrifuged and resuspended to remove the supernatant. After washing, the magnetic beads in each well were resuspended with 20 μL of enzyme-free water solution, and the entire operation was performed on ice.

[0053] Subsequently, a deep-well 384-well plate was used as a container plate, and 19 μL of Lysis Buffer was added to each well, which was kept on ice during the operation. 1 μL of magnetic bead suspension was taken from each well of the original magnetic bead storage plate and transferred to the corresponding well of the container plate. This step was also completed on ice to obtain a cell lysate containing magnetic beads. The collection plate stored at -80 °C was taken out and centrifuged instantly to remove possible ice crystals. 20 μL of cell lysate containing magnetic beads was added to each well of the collection plate. After lysis at room temperature for 5 minutes, it was incubated on ice for 12 minutes. During this period, the magnetic beads were resuspended every 2 minutes to promote the effective capture of mRNA. After the incubation is completed, all magnetic beads were collected in a 1.5 mL centrifuge tube.

[0054] Next, wash the beads 2-3 times with 500 μL of 6 × SSC, centrifuge for a few seconds for the last time and discard the supernatant. Then add 300 μL of 50 mM Tris pH 8.0 to resuspend the beads, discard the supernatant and immediately add 20 uL of RT Mix prepared on ice to reverse transcribe the mRNA.

[0055] Step 5: Reverse transcription and cDNA amplification The reverse transcription reaction conditions are 90 min in a 42°C metal bath, and the tubes should be suspended every 15 min. After the reaction is completed, centrifuge quickly for a few seconds, place the centrifuge tube on a magnetic rack, and carefully discard the supernatant. Wash the magnetic beads with 200 μL TE-SDS, TE-TW, and 10 mM Tris-HCl (pH = 8.0), respectively. Resuspend the magnetic beads with 200 μL exonuclease system, incubate in a 37°C incubator for 60 min, and resuspend once every 10 min. After the reaction, place on a magnetic rack and discard the supernatant. Wash the magnetic beads with 200 μL TE-SDS, TE-TW, and 10 mM Tris-HCl (pH = 8.0), respectively.

[0056] Subsequently, the magnetic beads were resuspended using the Pre-Amp PCR system and PCR amplification was performed. The PCR program is shown in Table 6: Table 6 PCR amplification reaction program settings

[0057] VAHTS DNA Clean Beads were taken out 30 minutes in advance and equilibrated to room temperature, and vortexed to mix. The PCR product was placed on a magnetic rack and the supernatant was transferred to a new centrifuge tube. The PCR product was purified using 0.8× VAHTS DNA Clean Beads. The steps are as follows: VAHTS DNA Clean Beads were added to the centrifuge tube, mixed and placed at room temperature for 15 minutes, the supernatant was discarded, and the tube was washed twice with 200 μL of freshly prepared 80% ethanol, incubated at room temperature for 30 seconds, the supernatant was removed, the centrifuge tube was removed from the magnetic rack, and cDNA was eluted with 13 μL of nuclease-free water, placed at room temperature for 10 minutes, 12 μL of supernatant was placed in a new centrifuge tube, and 12.5 μL of 2×Kapa HiFi HotStart Readymix and 0.5 μL of 10 μM TSO-PCR primer were added. The second PCR amplification was performed, and the procedure is shown in Table 7.

[0058] Table 7 qPCR amplification reaction program settings

[0059] The amplified product was purified again using 0.7 × VAHTS DNA Clean Beads, and the steps were the same as above. The cDNA concentration was determined using a Qubit 4.0 fluorometer, and the cDNA bands were detected using an Agilent 4200 nucleic acid microfluidics electrophoresis.

[0060] Step 6: cDNA library construction and sequencing The cDNA library was constructed according to the TruePrep Flexible DNA Library Prep Kit for Illumina free library construction kit operating instructions. PE150 double-end sequencing was performed by the Salus Pro gene sequencer of Salus Medical Company in China.

[0061] Step 7: Sequencing result processing The amount of data generated by high-throughput mixed-sample sequencing in Spatial-seq is huge. To achieve effective gene alignment, the sequencing data was first separated into separate files using a custom Python script according to the different spatial barcode sequences (Barcode 1 - 384), and quality control and preliminary processing were performed using Drop-seq_tools v2.5.1 according to the Drop-seq Core Computational Protocol v2.0.0 to ensure that the data quality and format met the requirements of subsequent analysis. After completing the above processing, the ultra-fast RNA sequence alignment tool STAR v2.7.8a was used to perform accurate sequence alignment in combination with the genome sequence file (FASTA format) and gene annotation file (GTF format) of the reference genome GRCm39 (Release-109 version) provided by the Ensembl database. The initial quality control of the sequencing data was completed using the FastQC tool, which was developed based on Java and performed rapid quality assessment on sequencing files in the "fq" format to ensure that the data quality met the requirements of subsequent analysis. Using a customized Python script, the raw sequencing data "fq" files were carefully split and organized according to the differences in Barcode 1-384, providing preparation for further molecular identification.

[0062] Step 9: Spatial-seq 2.0 spatial transcriptome data analysis The generated digital expression matrix was standardized using the calcNormFactors function of the edgeR package v3.40.2 to eliminate the effects of differences in sequencing depth and RNA composition on expression estimates. The likelihood ratio test (LRT) method was used to evaluate the differences in expression of each gene in the Sham group, Model group, PNS group, and PGS group. LRT judges the significance of an effect by comparing the fit of a full model that includes specific effects with a simplified model that does not include these effects. P The value was corrected for multiple testing and the FDR method was used to control the false positive rate. The final threshold ( P <0.05, |log2 fold difference|>1) to determine the genes with significant differential expression. The Metascape online platform was used to perform GO and KEGG pathway enrichment analysis on the differentially expressed genes. After the enrichment analysis was completed, the enrichment result data provided by Metascape was exported and visualized using the ggplot2 package of the R language.

[0063] Example 2 Comparative study on the anti-acute stroke effects of Panax notoginseng total saponins and ginseng total saponins based on the spatial transcriptome high-throughput sequencing method (referred to as Spatial-seq 2.0) provided in Example 1 Experimental animals C57BL / 6 mice: SPF grade, male, 8-10 weeks, 22-25 g, purchased from Shanghai Slake Experimental Animal Co., Ltd. (Animal Quality Certificate No.: 20220004002200). Mice were housed in the SPF animal room of the Experimental Animal Center of Zhejiang University, placed in a constant temperature room (25 ± 1 ℃), and had free access to food and water in an environment with 12 h of light and 12 h of darkness.

[0064] Experimental drugs and reagents Total saponins of Panax notoginseng: Shanghai Yuanye Biotechnology Co., Ltd., product number S27243; Ginseng total saponins: Shanghai Yuanye Biotechnology Co., Ltd., product number S25997; Edaravone: Nanjing Simcere Pharmaceutical Co., Ltd., specification 5 mL: 10 mg, batch number 80-151203; Physiological saline: Shandong Qidu Pharmaceutical Co., Ltd., catalog number 1250850; Silicone-coated suture: Guangzhou Jialing Biotechnology Co., Ltd., model L1800, catalog number 1800AAA; 2,3,5-Triphenyltetrazolium chloride (TTC): Sigma, USA, purity ≥ 95%, batch number BCBL1305V; 4% paraformaldehyde: Beijing Solebow Technology Co., Ltd., catalog number P1110; DMEM high-glucose medium: Gibco, USA, product number C11995500BT; RNeasy Mini Kit (250): Qiagen, Germany, catalog number BIO-000002; HiFiScript cDNA Synthesis Kit: Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number CW2569M; Hieff UNICON® Power qPCR SYBR Green Master Mix (antibody method, No Rox): China Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 11195ES08.

[0065] Experimental reagent preparation Solution for animal experiment of total saponins of Panax notoginseng and total saponins of ginseng: Prepare and use immediately. Dissolve the drug with 0.5% sodium carboxymethyl cellulose solution before the experiment to prepare low concentration (10 mg / mL) and high concentration (20 mg / mL) total saponins solutions. In this example, the administration method of total saponins of Panax notoginseng and total saponins of ginseng is intragastric administration (ig), and the dosage is 100 mg / kg and 200 mg / kg, respectively.

[0066] Solution for cell experiments with Panax notoginseng total saponins and ginseng total saponins: The stock solution of total saponins with a concentration of 1600 μg / mL was prepared with DMEM culture medium. The dilution concentrations after ultrasonic dissolution were 800 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL and 12.5 μg / mL.

[0067] Edaravone solution: Based on the dosage of edaravone injection in the literature (Zhong Fangfang, Wu Chenglong, Sun Xinfang, et al. Effects of Buyang Huanwu Decoction combined with edaravone on cerebral ischemia-reperfusion injury in mice [J]. Chinese Journal of Gerontology, 2017, 37 (21): 5251-5253), 4 mg / kg was selected as the positive drug dose. The concentration of edaravone injection stock solution was 2 mg / mL, which was diluted to 0.4 mg / mL solution with normal saline, and the dosage volume for mice was 0.1 mL / 10 g.

[0068] 0.5% sodium carboxymethyl cellulose (0.5% CMC-Na) solution: Accurately measure 1000 mL of deionized water and boil it, then slowly add 5 g of sodium carboxymethyl cellulose powder in one direction, stirring continuously until it becomes transparent. Ultrasound can be used to accelerate the dissolution. Cool it and store it in a refrigerator at 4 ℃ for later use.

[0069] 1.0% sodium pentobarbital: Weigh an appropriate amount of sodium pentobarbital powder and dissolve it in normal saline to prepare a solution with a concentration of 10 mg / mL. Prepare it for immediate use. 0.25% TTC solution: Weigh an appropriate amount of TTC powder and dissolve it in physiological saline. Shield from light and prepare a solution with a concentration of 2.5 mg / mL. Prepare and use immediately. Experimental instruments Electronic balance: Mettler Toledo, Switzerland, model AL104; Centrifuge: Eppendorf, USA, models 5810R and 5424R; Milli-Q ultrapure water treatment system: Millipore, USA, model Milli-Q (18.2i); Fluorescence microscope: Japan OLYMPUS, model BX63; Constant temperature water bath: Joan Lab, China, model BHS-1; Establishment of the mouse middle cerebral artery occlusion (MCAO) model After one week of adaptive feeding of male C57BL / 6 mice, the permanent middle cerebral artery occlusion (pMCAO) model was established in mice using the suture method. The specific operation is as follows: Before surgery, mice were anesthetized with 1.0% sodium pentobarbital, and the skulls of the anesthetized mice were exposed. The fiber optic probe of the laser Doppler blood flowmeter was fixed on the skull surface (central area: 2mm posterior to the bregma and 6mm to the right of the middle suture; peripheral area: 2mm posterior to the bregma and 3mm to the right of the middle suture) to monitor the changes in cortical cerebral blood flow corresponding to the middle cerebral artery in real time. After the optical fiber was fixed, the right middle cerebral artery of the mouse was occluded by suture to produce a permanent focal cerebral ischemia model, referring to the experimental method of middle cerebral artery occlusion (MCAO) in mice described in previous studies (Yao Jianbiao. Analysis of organic acid components in Ginkgo biloba extract and its protective effect against cerebral ischemia [D]. Zhejiang University, 2022) and (Liu Mengru. Study on the protective effect and antioxidant mechanism of TIGAR in ischemic brain injury via the non-pentose phosphate pathway [D]. Zhejiang University, 2022) Animal grouping and drug administration All mice were divided into the following 7 groups using the random number table method: (1) sham group (Sham): mice underwent MCAO surgery but no suture was inserted; (2) model group (Model): mice successfully established MCAO model; (3) low-dose ginsenosides group (PGS-L): mice were given 100 mg / kg ginsenosides suspension by gavage after MCAO model was established; (4) high-dose ginsenosides group (PGS-H): mice were given 200 mg / kg ginsenosides suspension by gavage after MCAO model was established; (5) low-dose notoginseng saponins group (PNS-L): mice were given 100 mg / kg notoginseng saponins suspension by gavage after MCAO model was established; (6) high-dose notoginseng saponins group (PNS-H): mice were given 200 mg / kg notoginseng saponins suspension by gavage after MCAO model was established; (7) positive drug edaravone group (Edaravone): mice were given 4 The sham operation group and the model group were given an equal volume of sodium carboxymethylcellulose solution by gavage. Neurological scoring and sampling were performed 24 hours after modeling.

[0070] Mouse neurobehavioral scoring The neurobehavioral scoring of mice was performed 24 hours after ischemia, and the Longa neurobehavioral scoring was performed. The specific evaluation criteria are as follows: ① No symptoms: 0 points; ② The front paw on the paralyzed side cannot be fully extended: 1 point; ③ Turning in circles toward the paralyzed side when walking: 2 points; ④ Leaning toward the paralyzed side when walking: 3 points; ⑤ Unable to walk automatically and loss of consciousness: 4 points.

[0071] TTC staining of mouse brain tissue 24 hours after the modeling, the mice were quickly killed by dislocating the neck, and the brain tissue was carefully removed. Then, the mouse brain was cut into 5 slices of about 2 mm along the coronal plane. The brain slices were placed in 0.25% TTC solution and stained in a 37 ℃ constant temperature box in the dark for 20 minutes. During this period, the brain slices were turned over with tweezers to make them evenly stained. After staining, the infarct area was white, the normal tissue was red, and the transition area between infarction and normal was pink. Finally, the mice were fixed with 4% paraformaldehyde for 4 hours, washed with physiological saline, and images were taken. The relative brain infarction volume of each mouse was calculated using Image J software.

[0072] Mouse brain tissue collection and sectioning Prepare enzyme-free PBS and RNA later in advance, and anesthetize the four groups of mice (3 mice in each group, a total of 12 mice) of Sham, Model, PNS-H and PGS-H with 1.5% sodium pentobarbital, and perfuse with 20 mL enzyme-free PBS and 5 mL RNA later. Then quickly take the brain tissue and rinse it with pre-cooled PBS solution, and then dry the liquid with clean absorbent paper. Finally, the brain tissue was quickly frozen in liquid nitrogen for 30 s~1 min and stored in a -80 ℃ refrigerator.

[0073] After the brain tissue was taken out from -80 °C and embedded, it was cut into frozen sections using a freezing microtome. The section thickness was 10 μm, and a total of 10 consecutive sections were retained for subsequent idling experiments, H&E staining, and other experiments.

[0074] Hematoxylin-eosin (H&E) staining of mouse brain tissue After embedding the brain tissue in the Spatial-seq protocol, it was sliced ​​continuously, and one of the slices was fixed in 4% paraformaldehyde solution for about 10-15 min. Subsequently, the slices were rinsed with PBS to remove the fixative, usually 2-3 times, 5 min each time. After washing, the slices were placed in hematoxylin staining solution for 5 min. The slices were rinsed with water to remove excess dye. The slices were then placed in eosin staining solution for 3 min. After staining, the slices were placed in different concentrations of gradient alcohol (70%, 85%, 95% and 100% ethanol) for dehydration, with each level of dehydration for 1 min. Subsequently, the slices were placed in xylene for 2 min to achieve transparency. Finally, the slices were sealed with neutral gum. The stained slices were observed under a microscope, and the required images were collected for analysis through the image acquisition system.

[0075] Spatial transcriptome sequencing and data analysis of mouse brain tissue using Spatial-seq 2.0 technology The brains of 12 mice in the four groups of Sham, Model, PNS-H and PGS-H were taken out respectively. The first frozen section was scanned by microscopy. The right area of ​​each section was divided into 32 spots in an array. The radius of each spot was about 125 μm and the area was about 50,000 μm. 2 , there are about 600 cells. A total of 384 Spots were cut from all mice. The LCM instrument was used to automatically identify the cutting area, and the parameters were set to objective lens 10×, Final Pulse mode, power = 17, aperture = 1, speed = 20, bridge size = 5, final pulse = 16, and collected using an enzyme-free well plate, and immediately stored at -80°C after collection. The specific scheme after collection is referenced to Example 1.

[0076] Results and Discussion Pharmacodynamic evaluation of PNS and PGS on MCAO mice According to the technical solution ( Figure 1 (A) 24 h after MCAO modeling, the neurobehavioral scores and statistical analysis were performed on the mice in each group. Figure 1 As shown in Figure B, the mice in the sham operation group showed no neurological impairment, and the scores were all 0 points. Compared with the sham operation group, the model group showed obvious neurological defects, such as circling and falling to the contralateral side, and the scores were significantly increased. Compared with the model group, the neurological impairment in each drug-treated group was improved, and the scores were significantly decreased, indicating that 200 mg / kg of PGS and PNS significantly improved the neurological function of pMCAO mice ( P <0.01), and was superior to the positive drug edaravone. The TTC staining results of mouse brain tissue are shown in Figure 1 C and Figure 1 As shown in D, Figure 1 The red area in C represents normal tissue, the white area is the infarcted part, and the pink area in the middle is the ischemic transition zone. The results showed that no infarcts were found in the brain tissue of the mice in the sham operation group, while the mice in the model group had larger white infarct areas, with an average infarct area of ​​30.73%; compared with the model group, the infarct area of ​​the brain tissue of the mice in each drug-treated group showed a downward trend. After the action of PNS-H, the average infarct area of ​​the mouse brain tissue was 11.70%, which was significantly reduced by 61.9% compared with the model group, and was similar to the infarct area of ​​the mice in the positive drug edaravone group. The above results show that both PNS-H and PGS-H have the effect of resisting acute ischemic stroke, but the efficacy of PNS-H is better than that of PGS-H.

[0077] Figure 1 Results of the 24h efficacy evaluation of PNS and PGS on MCAO mice; A: Schematic diagram of the overall dosing regimen; B: Effects of PNS and PGS on the neurobehavioral scores of MCAO mice; C: Representative diagram of TTC staining of mouse brain tissue; D: Results of the effects of PNS and PGS on the infarct volume of mouse brain tissue. Data are expressed as mean ± SEM, n = 6; Compared with the model group, the drug group ** P <0.01, *** P <0.001.

[0078] Effects of PNS and PGS on pathological changes of brain tissue in MCAO mice The effects of PNS and PGS on the pathological morphological changes of brain tissue in MCAO mice were observed. H&E staining was used, and the results were as follows Figure 2As shown in the figure, the left and right hemispheres of the sham-operated mice were uniformly colored, with intact cell morphology, no edema or vacuoles, full nuclei, and clear nucleoli; the infarcted area of ​​the brain tissue of the model group mice was lighter than the normal tissue, with severe edema, scattered vacuoles, and shrunken and darkly stained nucleoli; and compared with the model group, the administration of PNS and PGS could significantly reduce the edema and shrunken nucleoli of the brain tissue of MCAO mice. The above results show that both have a significant improvement effect on the pathological changes of brain tissue in MCAO mice.

[0079] Figure 2 Representative images of H&E staining of the cortical area of ​​mouse brain tissue; n = 3, and the scale bars in the figures are 2000 μm and 100 μm.

[0080] Spatial-seq 2.0 spatial transcriptome data acquisition and quality control The Spatial-seq 2.0 experimental process is mainly divided into the synthesis of different spatial barcodes, the collection and frozen sectioning of mouse brain tissue, the selection of spatial spots on tissue sections on LCM and laser microdissection, and high-throughput mixed sample sequencing. Three of these parts require precise quality control, namely magnetic bead synthesis efficiency quality control, sequencing library cDNA quality control, and sequencing data analysis quality control. First, the number of oligoT sequences fixed on the surface of synthetic magnetic beads must be evaluated. This parameter is crucial to the efficiency of subsequent mRNA capture. Figure 3 As shown in Figure A, the CT values ​​of qRT-PCR are mainly distributed between 5 and 15, and are accompanied by a normal amplification curve, which indicates that the number of oligoT sequences on the surface of the magnetic beads is sufficient, the synthesis quality is stable and uniform, and meets the experimental requirements. Figure 3 As shown in B, the right brain region of each mouse brain tissue frozen section was arrayed and captured, with a total of 4 groups, 3 mice in each group, and a total of 384 spots captured. Subsequently, 384 synthetic magnetic beads were used for mRNA capture, reverse transcription, multiple rounds of amplification and library construction in the spatial region of mouse brain tissue. In order to evaluate the quality of the sequencing library, the length distribution was detected using the Agilent4200 system. Observation Figure 3 The C results in Figure 2 show that the library length is mainly distributed between 350 and 1000 bp, which meets the quality standards for sequencing. Finally, the quality of the cell samples was evaluated by analyzing the number of genes detected by each Spot in each group (nFeature) and the total transcript counts of each Spot in each group (nCount). Figure 3The D in the results showed that all 384 Spots can be detected, ensuring the accuracy of subsequent analysis. Among them, Spots with more damaged or dead cells may express fewer genes, and therefore have a lower nFeature value and nCount value, which is consistent with the complexity or biological state of stroke. The overall expression analysis of the four groups of samples found that the median values ​​of nFeature and nCount in the PNS group were higher than those in the PGS group, reflecting that PNS showed better therapeutic effects on MCAO mice.

[0081] Figure 3 These are the quality control graphs of Spatial-seq 2.0 spatial transcriptome data; A: 384 different barcode capture beads quality inspection report graph; B: 384 spatial Spots size and array distribution graph; C: sequencing library quality inspection report graph; D: sequencing result quality control report graph.

[0082] Correlation analysis between key spatial regions and ischemic penumbra of stroke based on Spatial-seq 2.0 of the present invention The penumbra region is a specific spatial region that may have therapeutic potential during the recovery period after stroke, and is expected to promote recovery through drugs or other treatments. Spatial-seq 2.0 can analyze the changes in gene expression in various regions in space. 192 spots in the model group and the sham operation group were analyzed for 192 edgR differential gene analyses, such as Figure 4 , sorted by the magnitude of the difference. Figure 4 A and Figure 4 Figure B takes the spatial regions with the top 25% (8 / 32) of the difference change as the key spatial regions affected by stroke. Figure 4 As shown in C, combined with Figure 2 The H&E staining results showed that the range of the 8 spots in the middle was almost equal to the range of the penumbra of stroke. This result shows that the gene group with the most significant spatial expression changes in brain tissue can be used to locate a specific spatial area with therapeutic potential after stroke; this area has a spatial co-localization relationship with the penumbra area.

[0083] Figure 4 The correlation analysis diagram between the key spatial area and the ischemic penumbra of stroke; A: the result diagram of the differential gene number at each spatial position between the Sham group and the Model group; B: the top 25% spatial positions ranked by the differential gene number; C: H&E staining image showing the spatial distribution of the ischemic penumbra (red) and the infarct core area (blue).

[0084] Pathway enrichment analysis of differentially expressed genes in PNS and PGS in the penumbra based on Spatial-seq 2.0 of the present invention It is known that the "Correlation analysis of key spatial regions and ischemic penumbra of stroke based on Spatial-seq 2.0 of the present invention" found that the 8 circled spots can correspond to the penumbra part of stroke. Next, we will continue to analyze the genetic difference callback of the penumbra region obtained by Spatial-seq 2.0 technology. Figure 5 A and Figure 5 As shown in B, PNS and PGS have some of the same regulatory effects on acute stroke in the penumbra area. It was found that under stroke conditions, PNS highly adjusted the two biological processes of regulation of actin filament-based process and regulation of actin cytoskeleton. PNS pathways regulate actin-related pathways and can perform related functions such as blood flow regulation, vascular expansion and vascular repair. These effects are consistent with the efficacy of Panax notoginseng in "activating blood circulation", that is, improving blood circulation and promoting the recovery of damaged tissues ( Figure 5 A in the figure). PGS has a significant impact on biological pathways such as the TCA cycle and NADH dehydrogenase complex assembly. The TCA cycle is one of the key pathways for cells to produce energy and can promote the generation of ATP. PGS can increase energy production by promoting the TCA cycle; NADH dehydrogenase is the first complex in the mitochondrial electron transport chain and is essential for maintaining energy conversion efficiency within cells. PGS can enhance the energy metabolism efficiency of cells by promoting the normal assembly and function of the NADH dehydrogenase complex, further supporting the body's energy supply ( Figure 5 B in the above). These all show that ginsenosides can regulate the efficiency of energy conversion and increase energy supply to improve the vitality and endurance of the body, and support the survival and function of brain cells after stroke. These effects are consistent with the effect of ginseng in "replenishing qi", that is, improving physical strength and enhancing vitality.

[0085] In summary, PNS and PGS jointly regulate the penumbra area of ​​acute stroke by affecting multiple biological processes and signaling pathways, but their respective characteristics and mechanisms of action are different, reflecting the concepts of Panax notoginseng "activating blood circulation" and ginseng "replenishing qi" in TCM theory. The above analysis reveals the potential value of these two traditional Chinese medicines in modern medical research and provides a scientific basis for further research and application.

[0086] Figure 5 These are the results of pathway enrichment analysis; A: the classic pathway diagram involving key genes in PNS callback; B: the classic pathway diagram involving key genes in PGS callback.

[0087] Study on the effects of PNS and PGS on different brain regions of mice based on Spatial-seq 2.0 of the present invention In order to discover the effects of PNS and PGS on different brain regions of mice, the brain region gene data obtained by gene Spatial-seq 2.0 technology were analyzed for GO and KEGG pathway enrichment, and the six key brain regions of hypothalamus, thalamus, hippocampus, cerebral cortex, striatum, and amygdala were selected to be the most relevant pathways for cerebral ischemic diseases ( Figure 6 ). In summary, Spatial-seq 2.0 technology analysis revealed that in acute ischemic stroke, PNS and PGS showed different effects in different brain regions through different molecular pathways. At the same time, PNS covers a wider range of pathways and genes than PGS, which also shows that PNS regulates more extensively than PGS in acute stroke ( Figure 6 This may be due to the fact that multiple bioactive compounds of the PNS affect multiple aspects of stroke pathology, from restoration of blood flow to reduced inflammation and enhanced cellular metabolism.

[0088] Figure 6 This is the GO pathway enrichment result diagram of marker genes in each brain region of mice.

[0089] in conclusion The present invention uses Spatial-seq 2.0 technology, starting from the full perspective of space, to break through the technical difficulty that "existing bulk transcriptome sequencing technology cannot retain spatial location information", and conducts a systematic comparative study on the effects of PNS and PGS in anti-acute stroke, obtains the effects of the two on each spot at different locations in the brain, and analyzes the similarities and differences between PNS and PGS in the treatment of stroke diseases. Through the analysis of the ischemic penumbra area of ​​stroke using Spatial-seq 2.0 technology, it was found that PNS had a wider impact on pathways related to blood circulation, inflammation and cell metabolism, reflecting the "blood circulation" characteristics of Panax notoginseng. In contrast, PGS may be more specifically targeted at energy metabolism, stress response and neuronal protection pathways, which is consistent with the "qi-tonifying" effect of ginseng. In the comparative analysis of brain regions, it was found that PNS and PGS demonstrated their potential mechanism for treating acute ischemic stroke in six different brain regions through different molecular pathways. At the same time, PNS covers a wider range of pathways and genes than PGS, which also reflects that PNS has a broader regulatory effect on anti-acute stroke than PGS.

[0090] Comparative Example 1 The Spatial-seq protocol (a spatial transcriptome sequencing technology published by Liao Jie et al. in Nature Communications (Nat Commun, 2022, 13(1):6498)) includes the following steps: ① Synthesis and quality control of DNA spatial barcodes; ② Sampling, sectioning, staining and spatial alignment of mouse brain tissue; ③ Microdissection and barcode capture labeling; ④ Reverse transcription, amplification and cDNA library preparation; ⑤ High-throughput mixed sample sequencing and data analysis.

[0091] Spatial-seq 2.0 optimizes the Spatial-seq scheme, mainly involving: on the basis of Spatial-seq: ① improving the efficiency of barcode synthesis (corresponding to step 1 in Example 1); ② improving the quality of RNA (corresponding to steps 3 and 4 in Example 1); ③ increasing the capture rate of Spots (corresponding to step 4 in Example 1); ④ optimizing reagents to improve the efficiency of reverse transcription, amplification and library preparation (corresponding to steps 5 and 6 in Example 1).

[0092] There are differences in technology and effects between Spatial-seq and Spatial-seq 2.0: 1. Spatial-seq is a 96-well plate system; Spatial-seq 2.0 is a 384-well plate system; the advantages of Spatial-seq2.0 over Spatial-seq are: saving reagents and increasing efficiency: the volume is reduced, which is more suitable for small-volume, high-throughput reaction operations. In the case of limited experimental space and reagent usage, it can greatly increase the number of barcode synthesis samples that can be processed simultaneously, effectively improving the overall synthesis efficiency; 2. The amount of the first spatial barcode primer added to each well of Spatial-seq is 320μL, and the amount of the key synthesis reagent EDC added is 120μL; the amount of the first spatial barcode primer added to each well of Spatial-seq 2.0 is 80μL, and the amount of the key synthesis reagent EDC added is 32.36μL; the advantages of Spatial-seq 2.0 over Spatial-seq are: Saving reagents: A 384-well plate can process a large number of samples at a time, and does not need to repeat the reaction as frequently as a 96-well plate, thereby further significantly saving the use of reagents overall; 3. Spatial-seq: The synthesis of DNA spatial barcodes is divided into three steps (the first step is to synthesize magnetic beads and A series primers; the second step is to connect B series primers based on the first step; the third step is to synthesize C series primers on the preliminary barcode structure formed in the second step); Spatial-seq 2.0: The synthesis of DNA spatial barcodes requires two steps (the first step is to synthesize magnetic beads and A series primers; the second step is to put B series primers and C series primers into the reaction together, and by matching with reagents, the connection of barcode B series primers and C series primers can be completed in one step); Advantages of Spatial-seq 2.0 over Spatial-seq: The three-step synthesis method of Spatial-seq (the whole process is relatively cumbersome, each step requires specific reaction conditions and time control, and some intermediate product detection and processing may be required between steps to ensure the smooth progress of the reaction) is optimized to Spatial-seq The two-step synthesis method of 2.0 (directly obtains relatively stable and identifiable barcode products) simplifies the synthesis process and shortens the synthesis time. It takes 28 hours for Spatial-seq to synthesize 384 barcodes, while Spatial-seq 2.0 takes 5 hours. The total synthesis time is 3 days for Spatial-seq and 1 day for Spatial-seq2.0. 4. Spatial-seq: Cell lysis buffer: Add 120 μL of Tris-HCl (pH 7.5), 80 μL of LiCl, 120 μL of 10% SDS solution, 16 μL of EDTA, and 12 μL of 500 mM DTT solution to 852 μL of nuclease-free water to prepare a specific cell lysis buffer; Spatial-seq 2.0: Cell lysis buffer (1200 μL system): Add 120 μL of Tris-HCl (pH 7.5), 80 μL of LiCl, 120 μL of 10% SDS solution, 16 μL of EDTA, 12 μL of 500 mM DTT solution, and 8 μL of RNase Inhibitor (40 U / μL) to the remaining amount of nuclease-free water to prepare a specific cell lysis buffer; Spatial-seq 2.0 improves RNA quality by more than 1 times compared to Spatial-seq, see Figure 7 , Comparison of RNA quality results of different cell lysates, where A is Spatial-seq and B is Spatial-seq2.0; 5. After obtaining frozen sections in step 4 of Spatial-seq, LCM cutting is performed directly. After obtaining frozen sections in step 4 of Spatial-seq 2.0, static electricity removal equipment is used first before LCM cutting. The static electricity on the LCM instrument and sections is very serious, which affects the capture rate. Compared with Spatial-seq, Spatial-seq 2.0 adds a static electricity removal equipment (desktop ion blower LA-211) to blow out positive and negative charged airflow to neutralize the charge, so that the capture rate of Spots is increased from 50% to 93.75%. The results are shown in Table 8, which is a comparison result table of capture rate records; 6. Spatial-seq reverse transcription reagent: Nanjing Novezan Biotech Co., Ltd., catalog number P511 (2×Phanta Master Mix), library construction reagent: Novezan Biotech Co., Ltd. TD501 quantitative library construction (TruePrep DNA Library Prep Kit V2 for Illumina), sequencing scheme: second-generation sequencer: Illumina Xten, USA; Spatial-seq 2.0 reverse transcription reagent: P501 mixed enzyme (Phanta Super-FidelityDNA Polymerase), library construction reagent: TD504 free library construction (TruePrep Flexible DNA Library PrepKit for Illumina), sequencing scheme: Salus Medical Co., Ltd., China, model Salus Pro; The advantage of Spatial-seq2.0 over Spatial-seq is that the number of genes detected is twice that of the original scheme Spatial-seq (12464 / 6392≈2), and the specific results are shown in Table 9.

[0093] Table 8 Comparison results of Spots capture rate

[0094] Table 9 Sequencing data quality comparison results

[0095] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A spatial transcriptome high-throughput sequencing method based on a laser microdissection system and DNA barcode labeling, characterized in that: The following steps are involved: The carboxyl magnetic beads are mixed with EDC to perform an activation reaction to obtain activated carboxyl magnetic beads; The activated carboxyl magnetic beads are mixed with the first spatial barcode primers respectively to carry out a dehydration condensation reaction to obtain first magnetic beads; The first magnetic beads are mixed with the second spatial barcode primer, the third spatial barcode primer and Phanta Super-Fidelity DNA Polymerase, and PCR reaction is performed in a well plate to obtain a DNA spatial barcode; The animal tissue is frozen and sliced, and the surface of the slice is treated to remove static electricity using a static electricity removal device to obtain a static electricity-free slice; Electrostatically deionized sections were microdissected using a laser capture microdissection instrument, and single cells were collected using a well plate; Adding DNA spatial barcodes and cell lysis solution to a well plate containing single cells, using the DNA spatial barcodes to lyse the cells in the well plate to capture mRNA; the raw materials used to prepare the cell lysis solution include RNase inhibitors; The captured mRNA was reverse transcribed and washed to obtain washed magnetic beads; the washed magnetic beads were resuspended with the Pre-Amp PCR system, the first PCR amplification was performed, and the beads were placed on a magnetic stand, and the supernatant was taken to obtain the first PCR product; the first PCR product was purified using VAHTS DNA CleanBeads, and the purified product was mixed with 2× Kapa HiFi HotStart Readymix and TSO-PCR primers for a second PCR amplification to obtain a second PCR amplification product; the second PCR amplification product was purified again using 0.7× VAHTS DNAClean Beads to obtain cDNA; The TruePrep Flexible DNA Library Prep Kit for Illumina was used to construct the cDNA library. The Salus Pro gene sequencer was used for PE150 double-end sequencing. The sequencing results were processed and spatial transcriptome data analysis was performed.

2. The spatial transcriptome high-throughput sequencing method according to claim 1, characterized in that: The first spatial barcode primers include A1 to A4, and the nucleotide sequences are shown in SEQ ID NOs. 1 to 4, respectively; the second spatial barcode primers include B1 to B12, and the nucleotide sequences are shown in SEQ ID NOs. 5 to 16, respectively; the third spatial barcode primers include C1 to C8, and the nucleotide sequences are shown in SEQ ID NOs. 17 to 24, respectively; The TSO-PCR primers include TSO LNA and TSO-PCR; the nucleotide sequence of the TSO LNA is shown in SEQ ID NO.27; the nucleotide sequence of the TSO-PCR is shown in SEQ ID NO.

28.

3. The spatial transcriptome high-throughput sequencing method according to claim 1, characterized in that: The well plate includes a 384-well plate; the reaction system of the PCR reaction includes 13.5 μL of Phanta Super-Fidelity DNA Polymerase, 4 μL of first magnetic beads, 1 μL of second space barcode primers and 1.5 μL of third space barcode primers in each well.

4. The spatial transcriptome high-throughput sequencing method according to claim 1, characterized in that: The reaction program of the PCR reaction was: 94°C for 5 min; 95°C for 15 s, 48.8°C for 4 min, 72°C for 4 min, 5 cycles; 94°C for 5 min, 48.8°C for 20 min, 72°C for 20 min.

5. The spatial transcriptome high-throughput sequencing method according to claim 1, characterized in that: The slice thickness obtained by the frozen section is 10 μm; the time of the antistatic treatment is 3 minutes; the conditions of the microdissection are: using a laser capture microdissection instrument to automatically identify the cutting area, the parameters are set to objective lens 10×, Final Pulse mode, power = 17, aperture = 1, speed = 20, bridge size = 5, and final pulse = 16.

6. The spatial transcriptome high-throughput sequencing method according to claim 1, characterized in that: The cell lysis solution includes Tris-HCl, LiCl, SDS, EDTA, DTT solution, RNase inhibitor and nuclease-free water; each 1200 μL of cell lysis solution includes 120 μL of Tris-HCl with pH 7.5, 80 μL of LiCl, 120 μL of 10% SDS solution, 16 μL of EDTA, 12 μL of 500 mM DTT solution, 8 μL of 40U / μL RNase inhibitor and the remainder of nuclease-free water.

7. The spatial transcriptome high-throughput sequencing method according to claim 1, characterized in that: The length of the cDNA is more than 650 bp.

8. The spatial transcriptome high-throughput sequencing method according to claim 1, characterized in that: The processing of sequencing results includes: splitting the sequencing data into independent files according to the different spatial barcode sequences using a custom Python script, and processing them according to the Drop-seq core analysis process, and then using STAR v2.7.8a for gene alignment; the quality control and preliminary processing are completed by the FastQC tool.

9. The spatial transcriptome high-throughput sequencing method according to claim 1, characterized in that: The spatial transcriptome data analysis includes: using the calcNormFactors function of the edgeR package v3.40.2 to standardize the generated digital expression matrix; using the likelihood ratio test method to evaluate the difference in expression of each gene in different groups; the likelihood ratio test determines the significance of the effect by comparing the fit of the full model containing specific effects with the simplified model without these effects; P The values ​​were corrected for multiple testing, and the false positive rate was controlled using the false discovery rate method; the final threshold ( P <0.05, |log2 fold difference| > 1) to determine the genes with significant differential expression; the Metascape online platform was used to perform GO and KEGG pathway enrichment analysis on the differentially expressed genes; after the enrichment analysis was completed, the enrichment result data provided by Metascape was exported and visualized using the ggplot2 package of the R language.

10. Application of the spatial transcriptome high-throughput sequencing method according to any one of claims 1 to 9 in screening disease therapeutic drugs, disease pathogenesis research, developmental biology research or regenerative medicine research; the research is for the purpose of non-disease treatment.

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