A high-throughput sequencing method for spatial transcriptomics based on a laser microdissection system and DNA barcode labeling and its application

Abstract

The present invention provides a high-throughput sequencing method for spatial transcriptome based on a laser microdissection system and DNA barcode labeling and its application, which specifically belongs to the technical field of gene sequencing. The high-throughput sequencing method for spatial transcriptome of the present invention comprises the following steps: synthesizing DNA spatial barcodes by a two-step method; mixing the DNA spatial barcodes and cell lysate with single cells, lysing, capturing mRNA, reverse transcribing, and performing PCR amplification to obtain cDNA; constructing a cDNA library, sequencing, processing the sequencing results, and performing spatial transcriptome data analysis. The high-throughput sequencing method for spatial transcriptome of the present invention has a 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 particularly relates to a high-throughput sequencing method for spatial transcriptome based on a laser microdissection system and DNA barcode labeling and its application. Background Art

[0002] Spatial transcriptome technology can retain the spatial information of gene transcriptional expression, revolutionizing the way people understand tissues and organs. It is considered a revolutionary technology for revealing tissue heterogeneity, elucidating disease mechanisms, and promoting the development of new drugs. The research and development of its instruments have become a high ground for technological competition in the field of life and health. Limited by the deficiencies of traditional labeling methods, there is currently no instrument that can achieve high-throughput whole-transcriptome sequencing at the single-cell spatial resolution, and a breakthrough is urgently needed.

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

[0004] Spatial omics technologies are rapidly becoming key tools for in-depth understanding of complex biological systems. By revealing the spatial heterogeneity of cells and tissues in complex environments, they have greatly enriched our understanding of life sciences. With the increasing attention of the scientific community to the spatial differences between cells, the demand for high-precision and unbiased spatial omics technologies is also growing urgently. Laser capture microdissection (LCM) technology stands out with its minimal contamination, highly flexible tissue capture ability, and wide application range. It is particularly suitable for processing different tissue or patient samples and shows great potential in clinical research. However, the problem of its low cell throughput still needs to be solved. Summary of the Invention

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

[0006] The present invention provides a high-throughput sequencing method for spatial transcriptomics based on a laser microdissection system and DNA barcode labeling, comprising the following steps:

[0007] Mix carboxyl magnetic beads with EDC and carry out an activation reaction to obtain activated carboxyl magnetic beads;

[0008] Respectively mix the activated carboxyl magnetic beads with the first spatial barcode primer and carry out a dehydration condensation reaction to obtain the first magnetic beads;

[0009] Mix the first magnetic beads with the second spatial barcode primer, the third spatial barcode primer, and Phanta Super-Fidelity DNA Polymerase, and carry out a PCR reaction connection in a well plate to obtain a DNA spatial barcode;

[0010] Freeze-section an animal tissue, use an electrostatic removal device to remove static electricity from the surface of the section to obtain a static-free section; use a laser capture microdissection instrument to perform microdissection on the static-free section, and use a well plate to collect single cells;

[0011] Add the DNA spatial barcode and cell lysate to the well plate containing single cells, use the DNA spatial barcode to lyse the cells in the well plate, and capture mRNA; the raw materials for preparing the cell lysate include an RNase inhibitor;

[0012] Reverse transcribe the captured mRNA, wash it, and obtain the washed magnetic beads; resuspend the washed magnetic beads with the Pre-Amp PCR system, perform the first PCR amplification, place it on a magnetic stand, take the supernatant to obtain the first PCR product; use VAHTS DNAClean Beads to purify the first PCR product, mix the purified product with 2× Kapa HiFi HotStart Readymix and TSO-PCR primers, perform the second PCR amplification to obtain the second PCR amplification product; use 0.7 × VAHTS DNA Clean Beads to purify the second PCR amplification product again to obtain cDNA;

[0013] Use the TruePrep Flexible DNA Library Prep Kit for Illumina free library construction kit to construct a cDNA library; use a Salus Pro gene sequencer to perform PE150 paired-end sequencing;

[0014] Process the sequencing results and perform spatial transcriptome data analysis.

[0015] Preferably, the first spatial barcode primer includes A1 to A4, and the nucleotide sequences are respectively as shown in SEQ ID NO.1 to 4; the second spatial barcode primer includes B1 to B12, and the nucleotide sequences are respectively as shown in SEQ ID NO.5 to 16; the third spatial barcode primer includes C1 to C8, and the nucleotide sequences are respectively as shown in SEQ ID NO.17 to 24;

[0016] The TSO-PCR primer includes TSO LNA and TSO-PCR; the nucleotide sequence of TSO LNA is as shown in SEQ ID NO.27; the nucleotide sequence of TSO-PCR is as shown in SEQ ID NO.28.

[0017] Preferably, the well plate includes a 384-well plate; the reaction system of the PCR reaction per well includes 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.

[0018] Preferably, the reaction program of the PCR reaction is: 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.

[0019] Preferably, the section thickness obtained by the frozen section is 10 μm; the time for the static elimination treatment is 3 min; the conditions for the microdissection are as follows: using a laser capture microdissection instrument to automatically identify the cutting area, with the parameter settings of objective lens 10×, Final Pulse mode, power = 17, aperture = 1, speed = 20, bridging size = 5, and final pulse = 16.

[0020] Preferably, the cell lysate includes Tris-HCl, LiCl, SDS, EDTA, DTT solution, RNase inhibitor, and nuclease-free water; in every 1200 μL of the cell lysate, it 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, 12 μL of 40U / μL RNase inhibitor, and the balance of nuclease-free water.

[0021] Preferably, the length of the cDNA is above 650 bp.

[0022] Preferably, the processing of the sequencing results includes: according to the differences in the spatial barcode sequences, using a custom Python script to split the sequencing data into independent files, and following the Drop-seq core analysis process for processing, and then using STAR v2.7.8a for gene alignment; the quality control and preliminary processing are completed by the FastQC tool.

[0023] Preferably, the spatial transcriptome data analysis includes: using the calcNormFactors function of the edgeR package v3.40.2 to perform normalization processing on the generated digital expression matrix; using the likelihood ratio test method to evaluate the differential expression of each gene in different groups; the likelihood ratio test judges the significance of the effect by comparing the goodness of fit of the full model containing specific effects with the reduced model without these effects; P performing multiple testing correction on the P p values, and using the False Discovery Rate (FDR) method to control the false positive rate; finally, setting the threshold ( <0.05, |log2 fold change| > 1) to determine the genes with significantly differential expression; using the Metascape online platform to perform GO and KEGG pathway enrichment analysis on the differentially expressed genes; after the enrichment analysis is completed, the enrichment result data provided by Metascape is exported and visualized using the ggplot2 package of the R language.

[0024] The present invention also provides an application of the spatial transcriptome high-throughput sequencing method described in the above technical solution in screening disease treatment drugs, studying disease pathogenesis, studying developmental biology or studying regenerative medicine; the research is for the purpose of non-disease treatment.

[0025] The present invention provides a spatial transcriptome high-throughput sequencing method based on a laser microdissection system and DNA barcode labeling. Compared with the current spatial transcriptome sequencing technologies on the market, the sequencing method of the present invention can achieve single-cell resolution, perform custom acquisition and analysis of regions of interest, and has a high cell throughput. The test results show that the spatial transcriptome high-throughput sequencing method of the present invention simplifies the DNA spatial barcode synthesis method, has a high synthesis efficiency, saves reagents, and saves synthesis time; moreover, it improves the quality of RNA; and it enhances the Spots capture rate; and increases the number of detected genes. The spatial transcriptome high-throughput sequencing method of the present invention can be more widely and efficiently applied to the research of disease pathogenesis, the screening of disease treatment drugs, the research of developmental biology, and the research of regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0027] Figure 1 It is a graph showing the 24-hour pharmacodynamic evaluation results of PNS and PGS on MCAO mice provided by the present invention; wherein, A: Schematic diagram of the overall administration plan; B: Graph showing the effects of PNS and PGS on the neurological behavior scores of MCAO mice; C: Representative graph of TTC staining of mouse brain tissue; D: Graph showing the effects of PNS and PGS on the infarct volume of mouse brain tissue; the data are all expressed as mean ± SEM, n = 6; compared with the model group in the administration group, ** P <0.01, *** P <0.001;

[0028] Figure 2 It is a representative graph of H&E staining of the cerebral cortex region of mouse brain tissue provided by the present invention; wherein n = 3, and the scale bars in the figure are 2000 μm and 100 μm;

[0029] Figure 3Spatial-seq 2.0 spatial transcriptome data quality control chart provided by the present invention; wherein, A: Quality inspection report chart of 384 different barcode capture beads; B: Size and array distribution diagram of 384 spatial spots; C: Quality inspection report chart of the sequencing library; D: Quality control report chart of the sequencing results;

[0030] Figure 4 Analysis diagram of the correlation between key spatial regions and the ischemic penumbra of stroke provided by the present invention; wherein, A: Result diagram of the number of differential genes at each spatial position in the Sham group and the Model group; B: Spatial positions of the top 25% sorted by the number of differential genes; C: Spatial distribution of the ischemic penumbra (red) and the infarct core area (blue) shown by the H&E staining diagram;

[0031] Figure 5 Pathway enrichment analysis result diagram provided by the present invention; wherein, A: Classical pathway diagram involved in key genes of PNS callback; B: Classical pathway diagram involved in key genes of PGS callback;

[0032] Figure 6 GO pathway enrichment result diagram of marker genes in each brain region of mice provided by the present invention;

[0033] Figure 7 Comparison result diagram of the quality of RNA with different cell lysates provided by the present invention; wherein A is Spatial-seq and B is Spatial-seq 2.0. Detailed implementation mode

[0034] The present invention provides a high-throughput sequencing method for spatial transcriptome based on a laser microdissection system and DNA barcode labeling, comprising the following steps:

[0035] Mix carboxyl beads with EDC and carry out an activation reaction to obtain activated carboxyl beads;

[0036] Respectively mix the activated carboxyl beads with the first spatial barcode primer and carry out a dehydration condensation reaction to obtain the first beads;

[0037] Mix the first beads with the second spatial barcode primer, the third spatial barcode primer and Phanta Super-Fidelity DNA Polymerase, and carry out a PCR reaction connection in a well plate to obtain a DNA spatial barcode;

[0038] Freeze-section the animal tissue, use an electrostatic removal device to remove static electricity from the surface of the section to obtain a static-free section; use a laser capture microdissection instrument to microdissect the static-free section, and use a well plate to collect single cells;

[0039] Add DNA spatial barcodes and cell lysate to the well plate containing single cells, use the DNA spatial barcodes to lyse the cells in the well plate, and capture mRNA; the raw materials for preparing the cell lysate include RNase inhibitors;

[0040] Reverse transcribe the captured mRNA, wash, and obtain the washed magnetic beads; resuspend the washed magnetic beads with the Pre-Amp PCR system, perform the first PCR amplification, place on a magnetic stand, take the supernatant to obtain the first PCR product; use VAHTS DNAClean Beads to purify the first PCR product, mix the purified product with 2× Kapa HiFi HotStart Readymix and TSO-PCR primers, perform the second PCR amplification to obtain the second PCR amplification product; use 0.7 × VAHTS DNA Clean Beads to purify the second PCR amplification product again to obtain cDNA;

[0041] Use the TruePrep Flexible DNA Library Prep Kit for Illumina free library construction kit to construct a cDNA library; use a Salus Pro gene sequencer to perform PE150 paired-end sequencing;

[0042] Process the sequencing results and perform spatial transcriptome data analysis.

[0043] In the present invention, carboxyl magnetic beads are mixed with EDC for an activation reaction to obtain 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 washes 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 carried out in MES buffer. In a specific embodiment, the temperature of the activation reaction is 15 to 30 °C and the time is 20 min.

[0044] The activated carboxyl magnetic beads are respectively mixed with the first spatial barcode primers for dehydration condensation reaction to obtain the first magnetic beads. In a specific embodiment, the first spatial barcode primers include A1 to A4, and the nucleotide sequences are respectively shown in SEQ ID NO.1 to 4. In a specific embodiment, after 400 μL of the activated carboxyl magnetic beads are mixed with 80 μL of 0.2 M MES buffer and 80 μL of the first spatial barcode primers, the dehydration condensation reaction is carried out. In a specific embodiment, the concentration of the first spatial barcode primers is 400 μM. In a specific embodiment, the rotation speed of the dehydration condensation reaction (low-speed rotation) is 10 to 20 rpm. In the present invention, the time of the dehydration condensation reaction can be 20 min. After low-speed rotation, 32.36 μL (0.45 mg) of EDC solution is added in the present invention, and low-speed rotation is repeated, including one 20-min low-speed rotation and one 80-min low-speed rotation, and the temperature is 15 to 30 °C. Repeated addition of EDC can improve the cross-linking efficiency, avoid premature inactivation of reaction intermediates, and ensure the quality and yield of the final product. After the rotation is completed, it is placed on a magnetic rack, the supernatant is aspirated, washed and resuspended to obtain the first magnetic beads. In a specific embodiment, the washing and resuspension are sequentially resuspended and washed with 100 μL of 0.1 M PBS containing 0.02% Tween-20, enzyme-free water, and TE with pH = 8.0 in sequence.

[0045] After obtaining the first magnetic beads, the present invention mixes the first magnetic beads with the second spatial barcode primer, the third spatial barcode primer, and Phanta Super-Fidelity DNA Polymerase, and performs a PCR reaction for ligation to obtain a DNA spatial barcode. In a specific embodiment, the second spatial barcode primer includes B1 to B12, and the nucleotide sequences are respectively as shown in SEQ ID NO.5 to 16; the third spatial barcode primer includes C1 to C8, and the nucleotide sequences are respectively as shown in SEQ ID NO.17 to 24. In a specific embodiment, 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 the first magnetic beads, 1 μL of the second spatial barcode primer (50 μM), and 1.5 μL of the third spatial barcode primer (50 μM) per well. In a specific embodiment, the concentrations of the second and third spatial barcode primers are both 50 μM. In a specific embodiment, the reaction program of the PCR reaction is: 94 °C for 5 min; 95 °C for 15 s, 48.8 °C for 4 min, 72 °C for 4 min, for 5 cycles; 94 °C for 5 min, 48.8 °C for 20 min, 72 °C for 20 min. After the PCR reaction, the present invention also includes post-treatment to ensure the accuracy and usability of the barcode. In a specific embodiment, the post-treatment 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 to remove the residual PCR reaction mixture. Then incubate with ExoI exonuclease Mix to digest the unamplified single-stranded DNA. To ensure the full progress of the reaction, the magnetic beads need to be suspended from time to time, about once every 6 min, to promote the full contact between the enzyme and the DNA. After that, washing and resuspension are carried out, including continuously resuspending the magnetic beads with 10 μL of TE-SDS, TE-TW, and 20 μL of enzyme-free water to thoroughly remove the residual substances that may affect the next experiment. Then, cover the sealing film, place the well plate in a metal bath at 95 °C for 6 min, then quickly take out the plate and place it on a magnetic stand and aspirate the supernatant, and add 20 μL of enzyme-free water to each well again, and this step is repeated 2 times. Finally, add 15 μL of TE-TW to each well to resuspend the magnetic beads and store at 4 °C to obtain a Beads storage plate (magnetic bead storage plate). These delicate post-treatment steps not only ensure the quality of the DNA barcode but also provide a stable and reliable basis for subsequent applications. In the traditional scheme, the second spatial barcode primer is first ligated to the magnetic beads and then ligated to the third spatial barcode primer, which has cumbersome steps and high time costs; moreover, the efficiency of the ligation reaction is low, especially under the pairing conditions of two different barcode primers.The present invention adopts a one-step method, adding the second and third spatial barcode primers into the PCR reaction system together. Through the high amplification ability of Phanta enzyme, the ligation and amplification of these two steps are simultaneously completed in the same reaction system, which can improve the efficiency (by synchronizing primer amplification and ligation, reducing experimental steps and time, and improving the overall reaction efficiency), simplify the operation (reducing multiple reaction steps, reagent addition, and cleaning processes, thus reducing the operation complexity and the probability of operation errors), and improve the reaction consistency (because it is carried out synchronously 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, by selecting Phanta Super-Fidelity DNA Polymerase and performing one-step PCR reaction in a new reaction system, the present invention can improve the accuracy of barcode ligation and the reliability of amplification; significantly simplify the experimental steps, reduce the consumption of time and reagents; ensure high fidelity, reduce error introduction, and ensure the accuracy of barcodes. Therefore, compared with the traditional two-step reaction, the new system setup of the present invention can generate the target DNA spatial barcode more efficiently and accurately, providing a more stable basis for subsequent experiments.

[0046] After synthesizing the DNA spatial barcode, the present invention also includes quality control of the DNA spatial barcode. In a specific embodiment, the storage well plate of the DNA spatial barcode is taken out, placed on a magnetic rack, the supernatant is aspirated, nuclease-free water is added, and the magnetic beads are diluted with nuclease-free water to obtain a diluted magnetic bead liquid. Then, the diluted magnetic bead liquid is mixed with the qPCR reaction solution, universal primer, and tail-specific primer for qPCR reaction. After the reaction, it is resuspended with TE-EW. 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 each of the universal primer and tail-specific primer, and 7 μL of nuclease-free water per 20 μL. In the present invention, the reaction procedure of the qPCR is: 95°C for 1 min; 95°C for 5 s, 55°C for 15 s, for 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 ligated through qPCR, and ensure the correct pairing and amplification of the primers in the target region. qPCR (quantitative polymerase chain reaction) can monitor the product accumulation during the PCR amplification process in real time, and judge the success or failure of amplification according to the Ct value (Cycle threshold).

[0047] The animal tissue is cryosectioned, and the surface of the section is de-electrostatically treated using an electrostatic removal device to obtain a de-electrostatic section; the de-electrostatic section is microdissected using a laser capture microdissection instrument, and single cells are collected using a well plate. Before cryosectioning the animal tissue, rapid freezing is also included and it is stored at -80°C. After taking out the frozen animal tissue and embedding it, cryosectioning is performed. In a specific embodiment, the section thickness obtained by the cryosectioning is 10 μm. In a specific embodiment, the time for the de-electrostatic treatment is 3 min. In a specific embodiment, the electrostatic removal device includes a bench-top ion blower LA-211. The static electricity on the LCM instrument and the section is very serious, affecting the capture rate. By adding an electrostatic removal device in the present invention, an air flow of positive and negative charges is blown out, and charge neutralization can increase the capture rate of Spots from 50% to 95%. In a specific embodiment, the conditions for the microdissection are: using a laser capture microdissection instrument to automatically identify the cutting area, with the parameter settings being objective lens 10×, Final Pulse mode, power = 17, aperture = 1, speed = 20, bridging size = 5, and final pulse = 16. After cutting, it is collected using a well plate and immediately stored at -80°C after collection.

[0048] Add DNA spatial barcodes and cell lysate to the well plate containing single cells, use the DNA spatial barcodes to lyse the cells in the well plate, and capture mRNA; the raw materials for preparing the cell lysate include RNase inhibitors. In a specific embodiment, before adding the DNA spatial barcodes, the operations also include aspirating the supernatant (TE-TW), as well as washing and resuspending with enzyme-free water, and the whole process is carried out on ice. In a specific embodiment, the cell lysate includes Tris-HCl, LiCl, SDS, EDTA, DTT solution, RNase inhibitor and nuclease-free water; in every 1200 μL of cell lysate, it includes 120 μL of Tris-HCl with a pH of 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 40 U / μL RNase inhibitor and the remaining nuclease-free water. The cell lysate of the present invention can inhibit the influence of exogenous RNA enzymes on RNA degradation and improve the quality of RNA. In the present invention, the volume ratio of DNA spatial barcodes to cell lysate is 1:19. The present invention preferably first mixes the cell lysate with the DNA spatial barcodes, and then adds the cell lysate containing the DNA spatial barcodes to the well plate collecting single cells. In a specific embodiment, the lysis conditions include: lysing at room temperature for 5 min and incubating on ice for 12 min, and resuspending the magnetic beads every 2 min during this period to promote the effective capture of mRNA. In the present invention, the room temperature refers to lysing at 15-30°C. After lysis, the present invention washes the magnetic beads. In a specific embodiment, 6 × SSC is used to wash the magnetic beads. Then centrifuge and discard the supernatant.

[0049] Reverse transcribe the captured mRNA, wash, and obtain the washed magnetic beads; resuspend the washed magnetic beads with the Pre-Amp PCR system, perform the first PCR amplification, place on a magnetic stand, take the supernatant, and obtain the first PCR product. In a specific embodiment, it is preferred to resuspend the magnetic beads with 50 mM Tris pH 8.0, and then immediately discard the supernatant and add 20 μL of reverse transcription premix RT Mix prepared on ice to perform reverse transcription of mRNA. In the present invention, the conditions for reverse transcription are 42°C for 90 min. In a specific embodiment, resuspend once every 15 min. After the reaction, centrifuge, place on a magnetic stand, and aspirate 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, resuspend the magnetic beads with an exonuclease system, incubate at 37°C for 60 min, and resuspend once every 10 min. After the reaction, place on a magnetic stand, discard the supernatant, and then wash the magnetic beads using TE-SDS, TE-TW, and 10 mM Tris-HCl (pH = 8.0) respectively. Subsequently, resuspend the magnetic beads with the Pre-Amp PCR system to perform the first PCR amplification. In the present invention, the reaction program 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.

[0050] The first PCR product was purified using VAHTS DNA Clean Beads. The purified product was mixed with 2× KapaHiFi HotStart Readymix and TSO-PCR primers, and the second PCR amplification was carried out to obtain the second PCR amplification product. In a specific embodiment, 0.8× VAHTS DNA Clean Beads were used to purify the PCR product. In a specific embodiment, the following steps were preferably included: 0.8× VAHTS DNA Clean Beads were mixed with the first PCR product, placed at room temperature for 15 min, the supernatant was aspirated and discarded, washed twice with 80% aqueous ethanol solution, incubated at room temperature for 30 s, the supernatant was removed, and the cDNA was eluted with nuclease-free water and placed at room temperature for 10 min to obtain the purified product. The purified product was mixed with 2× Kapa HiFi HotStart Readymix and TSO-PCR primers, and the second PCR amplification was carried out. In the present invention, the reaction program of the second PCR amplification was: 98°C for 3 min; 98°C for 20 s, 72°C for 6 min, for 10 cycles; 72°C for 10 min. In the present invention, the purification conditions were preferably the same as above. In a specific embodiment, the TSO-PCR primers included TSO LNA and TSO-PCR; the nucleotide sequence of TSO LNA was as shown in SEQ ID NO.27, and there were two trans-modified guanines / rG / rG and one LNA (locked nucleic acid)-modified guanine / iXNA_G at the end of TSO LNA to enhance its binding stability to the template and reduce non-specific binding; the nucleotide sequence of TSO-PCR was as shown in SEQ ID NO.28.

[0051] After obtaining the second PCR amplification product, the present invention used 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 was more than 650 bp.

[0052] The present invention used the TruePrep Flexible DNA Library Prep Kit for Illumina to construct a cDNA library. The PE150 paired-end sequencing was carried out using a Salus Pro gene sequencer.

[0053] The present invention processes sequencing results. In a specific embodiment, processing the sequencing results includes: according to different spatial barcode sequences, using a custom Python script to split the sequencing data into independent files and processing it following the Drop-seq core analysis pipeline (specifically, performing quality control and preliminary processing using Drop-seq_tools v2.5.1 according to Drop-seq Core Computational Protocol v2.0.0), and then performing gene alignment using STAR v2.7.8a; the quality control and preliminary processing are completed by the FastQC tool.

[0054] The present invention conducts 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 differential expression of each gene in different groups; the likelihood ratio test determines the significance of the effect by comparing the goodness of fit of the full model containing specific effects with the reduced model without these effects; performing multiple testing correction on the P p values, and using the FDR method to control the false positive rate; finally, setting a threshold ( P <0.05, |log2 fold change| > 1) to determine significantly differentially expressed genes; using the Metascape online platform to perform GO and KEGG pathway enrichment analysis on the differentially expressed genes; after the enrichment analysis is completed, the enrichment result data provided by Metascape is exported and visualized using the ggplot2 package of the R language.

[0055] The present invention also provides the application of the above-mentioned spatial transcriptome high-throughput sequencing method in screening disease treatment drugs, studying disease pathogenesis, studying developmental biology, or studying regenerative medicine; the research is for the purpose of non-disease treatment.

[0056] To further illustrate the present invention, the following describes in detail a spatial transcriptome high-throughput sequencing method and its application based on a laser microdissection system and DNA barcode labeling provided by the present invention with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0057] Example 1

[0058] 1. Experimental materials and reagents

[0059] 20 μm carboxyl magnetic beads, Suzhou Beaver Biomedical Engineering Co., Ltd., product number 40200;

[0060] EDC: Thermo Fisher Scientific, USA, product number PG82079;

[0061] MES: Sigma-Aldrich Co., LLC, Catalog No. M3671;

[0062] 0.1M PBS: Solarbio Science & Technology Co., Ltd., Catalog No. P1022;

[0063] Tween-20: Sigma-Aldrich Co., LLC, Catalog No. P7949;

[0064] TE buffer: Invitrogen, Catalog No. AM9849;

[0065] Phanta Super-Fidelity DNA Polymerase: Novoprotein Scientific Inc., Catalog No. P501;

[0066] Exonuclease I ( E. coli ): New England Biolabs, Catalog No. M0293L;

[0067] 1M Tris-HCl buffer (pH = 7.5): Solarbio Science & Technology Co., Ltd., Catalog No. T1140;

[0068] LiCl precipitation solution (7.5 M): Invitrogen, Catalog No. AM9480;

[0069] 10% SDS solution (RNase-free): Invitrogen, Catalog No. AM9823;

[0070] EDTA: Beyotime Biotechnology, Catalog No. ST066;

[0071] SSC (20×): Invitrogen, Catalog No. AM9770;

[0072] Betaine solution: Sigma-Aldrich Co., LLC, Catalog No. B0300;

[0073] 0.5 M DTT (DNase, RNase & Protease free): Beyotime Biotechnology, Catalog No. ST041;

[0074] MgCl2: Invitrogen, Catalog No. AM9530G;

[0075] PrimeScript™ II Reverse Transcriptase: Takara Bio Inc., Catalog No. 2690A;

[0076] Recombinant RNasin® Ribonuclease Inhibitor: Promega Corporation, USA, catalog number N2515;

[0077] SUPERase In RNase inhibitor: ABI, USA, catalog number AM2694;

[0078] KAPA HiFi HotStart ReadyMix DNA Polymerase: Kapa Biosystems, USA, catalog number KK2602;

[0079] Tris-HCl (pH 8.0): Shanghai Beyotime Biotechnology Co., Ltd., catalog number ST780;

[0080] Tris-HCl (pH 7.5): Beijing Solarbio Science & Technology Co., Ltd., catalog number T1140;

[0081] Fast Fluorescent Quantitative PCR Kit (SYBR Green): Biosharp, China, catalog number BL705A;

[0082] Qubit® dsDNA HS Assay Kits: Thermo, USA, catalog number Q32851;

[0083] Leica Cryo-Gel: Leica, Germany, catalog number 14020108926;

[0084] Nissl Stain: Wuhan Sevier Biotechnology Co., Ltd., catalog number G1036-100ML;

[0085] VAHTS DNA Clean Beads: Nanjing Novoprotein Scientific Inc., catalog number N411-01;

[0086] TruePrep Flexible DNA Library Prep Kit for Illumina: Nanjing Novoprotein Scientific Inc., catalog number TD504;

[0087] PCR primers: As shown in Table 1, all synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.

[0088] Table 1 Primer List

[0089]

[0090] 2. Preparation of experimental reagents

[0091] 0.1M MES: Dissolve 491 mg of MES in sufficient deionized water, adjust to pH = 5.0 using NaOH solution, and make up to a total volume of 25 mL;

[0092] 0.2M MES: Dissolve 982 mg of MES in sufficient deionized water, adjust to pH = 5.0 using NaOH solution, and make up to a total volume of 25 mL;

[0093] TE-TW solution: Add 5 μL of 10% Tween-20 solution to sufficient TE buffer with a pH value of 8.0, and make up to a total volume of 50 mL;

[0094] TE-SDS solution: Add 500 μL of 10% SDS solution to sufficient TE buffer with a pH value of 8.0, and make up to a total volume of 10 mL;

[0095] EDC solution: Dissolve 300 mg of EDC in sufficient 0.1M MES buffer, and make up to a total volume of 5 mL;

[0096] Cell lysis solution: 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 (40 U / μL) to sufficient nuclease-free water to prepare a specific cell lysis solution;

[0097] RT mix: 16.16 μL of nuclease-free water, 8 μL of 5× RT buffer, 8 μL of betaine solution, 4 μL of dNTP (10 mM), 0.2 μL of DTT, 0.24 μL of MgCl2, 0.4 μL of TSO primer, 2 μL of PrimeScriptII reverse transcriptase (200 U / μL), 1 μL of RNase inhibitor, for a total of 40 μL.

[0098] 3. Experimental instruments

[0099] Gradient PCR amplifier: Thermo Fisher Scientific, USA, model Applied biosystem ProFlexBase;

[0100] Real-time fluorescence quantitative PCR system: Bio-Rad, USA, model CFX96 Touch;

[0101] Laser capture microdissection system: Leica Company, Germany, including an upright microscope (model DM68), a laser cutter (model LMD6), and a single-cell capture and recovery device (model LMT350);

[0102] Cryostat: Thermo Company, USA, model NX50;

[0103] Agilent nucleic acid microfluidic electrophoresis analysis system: Agilent Company, USA, model 4200 TapeStation;

[0104] Gene sequencer: Salus Medical Company, China, model Salus Pro;

[0105] Static elimination equipment: desktop ion blower LA-211, LAOGE (Leger) Shanghai Company.

[0106] 4. Operation steps

[0107] Step 1: Synthesis of DNA spatial barcodes

[0108] In the first step of synthesizing the first segment of the spatial barcode for the positioning magnetic beads, first take four 1.5 mL centrifuge tubes, and add about 1.67 × 10 6 carboxyl magnetic beads (about 232 μL) to each tube. After that, aspirate the supernatant and wash 1 - 2 times with 0.1M MES buffer. After washing, add about 358 μL of 0.1M MES buffer to each tube, and add 2.06 mg (34.4 μL) of EDC to make the final volume about 424 μL. After mixing, take out 400 μL of the mixed solution from each centrifuge tube. Subsequently, add 80 μL of 0.2M MES and 80 μL of the corresponding first spatial barcode primer (primers A1 - A4) to each tube and mix well. Fix the centrifuge tubes on a rotator and rotate at low speed at room temperature for 20 min. Then add 32.36 μL (0.45 mg) of EDC solution to each tube and repeat the low-speed rotation step, including one 20 - minute and one 80 - minute low-speed rotation. After rotation, place the centrifuge tubes on a magnetic rack, aspirate the supernatant, and then resuspend and wash the magnetic beads (Beads) successively with 100 μL of 0.1M PBS containing 0.02% Tween-20, enzyme-free water, and TE with pH = 8.0. Finally, resuspend the magnetic beads in 280 μL of enzyme-free water to obtain the first magnetic beads.

[0109] Next, in step 2, the synthesis is to ligate the second and third spatial barcodes to the positioning beads. First, the second spatial barcode primer (primer B, B1 - B12), the third spatial barcode primer (primer C, C1 - C8), and Phanta Super-Fidelity DNA Polymerase need to be prepared. Take them out from 4 °C in advance, mix well and then centrifuge quickly to prepare a concentration of 50 μM.

[0110] In the PCR ligation stage, in a 384-well PCR plate, the reactants in each well include 13.5 μL of Phanta Super-Fidelity DNA Polymerase, 4 μL of the first beads, 1 μL of the second spatial barcode primer (50 μM), and 1.5 μL of the third spatial barcode primer (50 μM), as shown in Table 2.

[0111] Table 2 Reactants and volumes per well

[0112]

[0113] After mixing and quickly centrifuging the 384-well plate, carry out the subsequent PCR amplification, and the amplification program is shown in Table 3.

[0114] Table 3 Reaction program settings for PCR ligation of the second and third sequences

[0115]

[0116] 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 usability of the barcodes. First, place the 384-well plate on a magnetic rack and operate on ice to aspirate the supernatant. Subsequently, wash each well once with enzyme-free water to remove the residual PCR reaction mixture. Then, add 10 μL of ExoI exonuclease Mix to each well and incubate at 37 °C for 15 min to digest the unamplified single-stranded DNA. To ensure the full progress of the reaction, the magnetic beads need to be suspended from time to time, about once every 6 min, to promote the full contact between the enzyme and DNA. After that, perform a series of washing and resuspension steps, including continuously resuspending the magnetic beads with 10 μL of TE-SDS, TE-TW, and 20 μL of enzyme-free water to thoroughly remove the residual substances that may affect the next experiment. Then, cover the plate with a sealing film, place the 384-well plate in a metal bath at 95 °C for about 6 min, then quickly remove the plate and place it on the magnetic rack to aspirate the supernatant. Add 20 μL of enzyme-free water to each well again, and repeat this step 2 times. Finally, add 15 μL of TE-TW to each well to resuspend the magnetic beads, label them, and store them at 4 °C to obtain the Beads storage plate (magnetic bead storage plate). These delicate post-processing steps not only ensure the quality of the DNA barcodes but also provide a stable and reliable basis for subsequent applications.

[0117] Step 2: Quality control of DNA spatial barcodes

[0118] Take out the Beads storage plate, place it on a magnetic rack, aspirate and discard the supernatant, add 20 μL of enzyme-free water to each well, and take 1 μL of the magnetic bead-enzyme-free water from each well and dilute it to 100 μL. Take a 384-well plate, add 9 μL of qPCR Mix and 1 μL of the diluted magnetic bead suspension to each well to prepare a complete qPCR system, as shown in Table 4.

[0119] Table 4 qPCR reactants and volumes per well

[0120]

[0121] After mixing and quickly centrifuging the 384-well plate, perform the subsequent qPCR reaction, and the reaction program is shown in Table 5. After the experiment, the magnetic beads in the 384-well plate are resuspended with 15 μL of TE-TW and stored at 4 °C.

[0122] Table 5 qPCR reaction program settings

[0123]

[0124] Step 3: Animal tissue sampling and sectioning

[0125] Prepare enzyme-free PBS and pre-cooled RNA later in advance. After anesthetizing the animal with 1.5% sodium pentobarbital, perfuse it with 20 mL of enzyme-free PBS and 5 mL of RNA later. Then quickly remove the animal tissue, 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 - 1 min, and then store it in a -80 °C refrigerator.

[0126] After taking out the animal tissue embedded at -80 °C, use a cryostat to cut frozen sections of the brain tissue, with a section thickness of 10 μm.

[0127] Step 4: Microdissection and barcode capture labeling

[0128] Gently blow the surface of the frozen sections of the animal tissue with an anti-static device (desktop ion blower LA-211) for 3 min, and then place it on a laser microdissection instrument (LCM) for microscopic high-definition scanning. Each section can be divided into multiple Spot regions according to user needs. The radius of each Spot can be customized, with a minimum of about 10 μm, reaching the single-cell level. Use the LCM instrument to automatically identify and cut the region, with the parameter settings: objective lens 10 ×, Final Pulse mode, power = 17, aperture = 1, speed = 20, bridging size = 5, final pulse = 16. Collect with an enzyme-free well plate and immediately store at -80 °C after collection to obtain a collection plate.

[0129] Centrifuge the magnetic bead storage plate synthesized in Step 1 and stored at 4 °C quickly to remove residual liquid. Then place the plate on a magnetic rack to suck out the supernatant (TE-TW). Next, wash the magnetic beads with enzyme-free water 1 - 2 times, adding 15 - 20 μL of enzyme-free water each time, and then quickly centrifuge and resuspend the magnetic beads to remove the supernatant. After washing, resuspend the magnetic beads in each well with 20 μL of enzyme-free aqueous solution, and the whole operation is carried out on ice.

[0130] Subsequently, take a deep-well 384-well plate as a container plate, add 19 μL of Lysis Buffer to each well, and keep it on ice during the operation. Take 1 μL of the magnetic bead suspension from each well of the original magnetic bead storage plate and transfer it to the corresponding well of the container plate. This step is also completed on ice to obtain a cell lysate containing magnetic beads. Take out the stored collection plate from -80 °C and perform instantaneous centrifugation to remove possible ice crystals. Add 20 μL of the cell lysate containing magnetic beads to each well of the collection plate, incubate at room temperature for 5 min, and then incubate on ice for 12 min. Resuspend the magnetic beads every 2 min during this period to promote the effective capture of mRNA. After completing the incubation, collect all the magnetic beads into a 1.5 mL centrifuge tube.

[0131] Next, wash the magnetic beads 2 - 3 times with 500 μL of 6 × SSC. After the last wash, centrifuge quickly for a few seconds and discard the supernatant. Then add 300 μL of 50 mM Tris pH 8.0 to resuspend the magnetic beads, and immediately discard the supernatant and add 20 μL of reverse transcription premix RT Mix prepared on ice to perform reverse transcription of mRNA.

[0132] Step 5: Reverse Transcription and cDNA Amplification

[0133] The reverse transcription reaction conditions are as follows: react in a metal bath at 42°C for 90 min, and resuspend once every 15 min. After the reaction, centrifuge quickly for a few seconds, place the centrifuge tube on a magnetic stand, and carefully aspirate and discard the supernatant. Wash the magnetic beads with 200 μL of TE - SDS, TE - TW, and 10 mM Tris - HCl (pH = 8.0) respectively. Resuspend the magnetic beads with 200 μL of exonuclease system and incubate in an incubator at 37°C for 60 min, resuspend once every 10 min. After the reaction, place it on a magnetic stand and discard the supernatant. Wash the magnetic beads with 200 μL of TE - SDS, TE - TW, and 10 mM Tris - HCl (pH = 8.0) respectively.

[0134] Subsequently, resuspend the magnetic beads with the Pre - Amp PCR system and then perform PCR amplification. The PCR program is shown in Table 6:

[0135] Table 6 PCR Amplification Reaction Program Settings

[0136]

[0137] Take out VAHTS DNA Clean Beads 30 min in advance and equilibrate to room temperature, then vortex to mix evenly. Place the PCR product on a magnetic stand and transfer the supernatant to a new centrifuge tube. Purify the PCR product with 0.8× VAHTS DNA Clean Beads. The steps are as follows: add VAHTS DNA Clean Beads to the centrifuge tube, mix well and let it stand at room temperature for 15 min, aspirate and discard the supernatant, wash twice with 200 μL of freshly prepared 80% ethanol, incubate at room temperature for 30 s, remove the supernatant, take the centrifuge tube off the magnetic stand, elute the cDNA with 13 μL of nuclease - free water, let it stand at room temperature for 10 min, take 12 μL of the supernatant and place it in a new centrifuge tube, add 12.5 μL of 2×Kapa HiFi HotStart Readymix and 0.5 μL of 10 μM TSO - PCR primer. Perform the second PCR amplification, and the program is shown in Table 7.

[0138] Table 7 qPCR Amplification Reaction Program Settings

[0139]

[0140] Purify the amplified product with 0.7 × VAHTS DNA Clean Beads again, following the same steps as above. Measure the cDNA concentration using a Qubit 4.0 fluorometer and detect the cDNA bands by Agilent 4200 nucleic acid microfluidic electrophoresis.

[0141] Step 6: cDNA library construction and sequencing on the machine

[0142] Construct the cDNA library according to the operation guide of the TruePrep Flexible DNA Library Prep Kit for Illumina. Perform paired-end 150 (PE150) sequencing using the Salus Pro genetic sequencer from Salus Medical Co., Ltd., China.

[0143] Step 7: Processing of sequencing results

[0144] The amount of data output from Spatial-seq high-throughput multiplexed sequencing is huge. To achieve effective gene alignment, first, according to the differences in the spatial barcode sequences (Barcode 1 - 384), use a custom Python script to separate the sequencing data into individual files, and perform quality control and preliminary processing 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 meet the requirements of subsequent analysis. After the above processing, use the ultra-fast RNA sequence alignment tool STAR v2.7.8a version, combined with the genomic sequence file (FASTA format) and gene annotation file (GTF format) of the reference genome GRCm39 (Release-109 version) provided by the Ensembl database for precise sequence alignment. The preliminary quality control of the sequencing data is completed by the FastQC tool, which is developed based on Java and performs a rapid quality assessment of the sequencing files in the "fq" format to ensure that the data quality meets the requirements of subsequent analysis. Use a custom Python script to carefully split and organize the original sequencing data "fq" files according to the differences in Barcode 1 - 384 to prepare for further molecular identification.

[0145] Step 9: Analysis of Spatial-seq 2.0 spatial transcriptome data

[0146] The generated digital expression matrix was normalized using the calcNormFactors function of the edgeR package v3.40.2 to eliminate the influence of differences in sequencing depth and RNA composition on the estimation of expression levels. The likelihood ratio test (LRT) method was used to evaluate the differential expression of each gene in the Sham group, Model group, PNS group, and PGS group. LRT determines the significance of an effect by comparing the goodness of fit of a full model that includes a specific effect with a reduced model that does not include these effects. For P values, multiple testing correction was performed, and the FDR method was used to control the false positive rate. Finally, the set threshold ( P <0.05, |log2 fold change| > 1) was used to determine significantly differentially expressed genes. Using the Metascape online platform, GO and KEGG pathway enrichment analyses were performed 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 in R language.

[0147] Example 2

[0148] Comparative study on the anti-acute stroke effects of notoginsenosides and ginsenosides based on the spatial transcriptome high-throughput sequencing method (abbreviated as Spatial-seq 2.0) provided in Example 1

[0149] Experimental animals

[0150] C57BL / 6 mice: SPF grade, male, 8 - 10 weeks old, 22 - 25 g, purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Animal Quality Certificate Number: 20220004002200). The mice were housed in the SPF-grade animal room of the Experimental Animal Center of Zhejiang University, placed in a constant temperature room (25 ± 1 °C), and had free access to food and water in an environment with 12 h of light and 12 h of darkness.

[0151] Experimental drugs and reagents

[0152] Notoginsenosides: Shanghai Yuanye Bio-Technology Co., Ltd., product number S27243;

[0153] Ginsenosides: Shanghai Yuanye Bio-Technology Co., Ltd., product number S25997;

[0154] Edaravone: Nanjing Simcere Pharmaceutical Co., Ltd., specification 5 mL: 10 mg, batch number 80 - 151203;

[0155] Normal saline: Shandong Qidu Pharmaceutical Co., Ltd., product number 1250850;

[0156] Silicone-coated suture: Guangzhou Jialing Biotechnology Co., Ltd., model L1800, product number 1800AAA;

[0157] 2,3,5-Triphenyltetrazolium chloride (TTC): Sigma, USA, purity ≥ 95%, batch number BCBL1305V;

[0158] 4% Paraformaldehyde: Solarbio Science & Technology Co., Ltd., Beijing, product number P1110;

[0159] High-glucose DMEM medium: Gibco, USA, product number C11995500BT;

[0160] RNeasy Mini Kit (250): Qiagen, Germany, product number BIO-000002;

[0161] HiFiScript cDNA Synthesis Kit: CWBIO Co., Ltd., Jiangsu, product number CW2569M;

[0162] Hieff UNICON® Power qPCR SYBR Green Master Mix (antibody method, No Rox): Yeasen Biotechnology (Shanghai) Co., Ltd., China, product number 11195ES08.

[0163] Preparation of experimental reagents

[0164] Solutions of total saponins of Panax notoginseng and total saponins of Panax ginseng for animal experiments: Prepared freshly before use. Dissolve the drugs with 0.5% sodium carboxymethylcellulose solution before the experiment, and prepare total saponin solutions with low concentration (10 mg / mL) and high concentration (20 mg / mL) respectively. In this example, the administration methods of total saponins of Panax notoginseng and total saponins of Panax ginseng are intragastric (i.g.), and the doses are 100 mg / kg and 200 mg / kg respectively.

[0165] Solutions of total saponins of Panax notoginseng and total saponins of Panax ginseng for cell experiments: Stock solution of total saponin solution with a concentration of 1600 μg / mL prepared with DMEM culture medium. After ultrasonic dissolution, dilute the concentration to 800 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL and 12.5 μg / mL.

[0166] Edaravone solution: Referring to 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 dose of the positive drug. The original concentration of the edaravone injection was 2 mg / mL, and it was diluted with normal saline to a solution with a concentration of 0.4 mg / mL. The administration volume for mice was 0.1 mL / 10 g.

[0167] 0.5% Carboxymethylcellulose sodium (0.5% CMC-Na) solution: Accurately measure 1000 mL of deionized water and boil it. Then, slowly add 5 g of carboxymethylcellulose sodium powder in one direction, keep stirring until it becomes transparent. Ultrasonic acceleration can be used for dissolution. Let it cool and store it in a refrigerator at 4 °C for later use.

[0168] 1.0% Pentobarbital sodium: Weigh an appropriate amount of pentobarbital sodium powder and dissolve it in normal saline to prepare a solution with a concentration of 10 mg / mL. Prepare it freshly before use.

[0169] 0.25% TTC solution: Weigh an appropriate amount of TTC powder and dissolve it in normal saline. Prepare a solution with a concentration of 2.5 mg / mL under light protection. Prepare it freshly before use.

[0170] Experimental instruments

[0171] Electronic balance: Mettler Toledo, Switzerland, model AL104;

[0172] Centrifuge: Eppendorf, USA, models 5810R and 5424R;

[0173] Milli-Q ultrapure water treatment system: Millipore, USA, model Milli-Q (18.2i);

[0174] Fluorescence microscope: OLYMPUS, Japan, model BX63;

[0175] Constant temperature water bath: Joan Lab, China, model BHS-1;

[0176] Establishment of the mouse middle cerebral artery occlusion (MCAO) model

[0177] After male C57BL / 6 mice were adaptively fed for one week, a permanent middle cerebral artery occlusion (pMCAO) model was established in mice using the suture method. The specific operation is as follows:

[0178] Before the operation, mice were anesthetized with 1.0% sodium pentobarbital. After exposing the skull of the anesthetized mice, the optical fiber probe of the laser Doppler flowmeter was fixed on the surface of the skull (central area: 2 mm posterior to the bregma, 6 mm to the right of the midline suture; peripheral area: 2 mm posterior to the bregma, 3 mm to the right of the midline 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, a permanent focal cerebral ischemia model was made by occluding the right middle cerebral artery of the mice using the suture occlusion method, referring to the experimental methods 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 injury [D]. Zhejiang University, 2022) and (Liu Mengru. Protective effect and antioxidant mechanism of TIGAR via the non-oxidative pentose phosphate pathway in ischemic brain injury [D]. Zhejiang University, 2022).

[0179] Animal grouping and drug administration

[0180] Using the random number table method, all mice were divided into the following 7 groups: (1) Sham operation group (Sham): Mice underwent MCAO surgery but without inserting the suture; (2) Model group (Model): Mice successfully established the MCAO model; (3) Low-dose ginsenoside group (PGS-L): After mice established the MCAO model, they were given 100 mg / kg ginsenoside suspension by gavage; (4) High-dose ginsenoside group (PGS-H): After mice established the MCAO model, they were given 200 mg / kg ginsenoside suspension by gavage; (5) Low-dose notoginsenoside group (PNS-L): After mice established the MCAO model, they were given 100 mg / kg notoginsenoside suspension by gavage; (6) High-dose notoginsenoside group (PNS-H): After mice established the MCAO model, they were given 200 mg / kg notoginsenoside suspension by gavage; (7) Positive drug edaravone group (Edaravone): After mice established the MCAO model, they were intraperitoneally injected with 4 mg / kg edaravone solution, and this dose was the approximate equivalent dose for mice converted from the clinical administration dose of edaravone injection (30 mg / time, twice a day). The Sham operation group and the Model group were given an equal volume of carboxymethyl cellulose sodium solution by gavage. After 24 h of modeling, neurological scoring evaluation and sample collection were performed.

[0181] Neurological scoring of mice

[0182] The neurological scoring of mice was performed 24 h after ischemia. The Longa neurological scoring was carried out, and the specific evaluation criteria were as follows: ① Asymptomatic: 0 points; ② The paralyzed forepaw could not be fully extended: 1 point; ③ Circling towards the paralyzed side when walking: 2 points; ④ Falling towards the paralyzed side when walking: 3 points; ⑤ Unable to walk automatically and having a loss of consciousness: 4 points.

[0183] TTC staining of mouse brain tissue

[0184] Twenty-four hours after mouse model establishment, the mice were quickly decapitated and sacrificed. The brain tissue was carefully removed, and then the mouse brain was cut into 5 brain slices about 2 mm thick along the coronal plane. The brain slices were placed in 0.25% TTC solution and stained in the dark at 37 °C for 20 min. During this period, the brain slices were turned over with forceps in a timely manner to make the staining uniform. After staining, the infarct area was white, the normal tissue was red, and the infarct and normal transition area was pink. Finally, it was fixed with 4% paraformaldehyde for 4 h, washed with normal saline, and then photographed. The relative cerebral infarct volume of each mouse was calculated using Image J software.

[0185] Sampling and sectioning of mouse brain tissue

[0186] Prepare enzyme-free PBS and pre-cooled RNA later in advance. After anesthetizing four groups of mice (3 mice in each group, 12 mice in total) of Sham, Model, PNS-H, and PGS-H with 1.5% sodium pentobarbital, they were perfused with 20 mL of enzyme-free PBS and 5 mL of RNA later. Subsequently, the brain tissue was quickly removed and rinsed with pre-cooled PBS solution, and then the liquid was blotted dry with clean absorbent paper. Finally, the brain tissue was quickly frozen in liquid nitrogen for 30 s to 1 min and then stored in a -80 °C refrigerator.

[0187] After taking out the brain tissue embedded at -80 °C, the brain tissue was cut into frozen sections using a cryostat. The section thickness was 10 μm, and a total of 10 consecutive sections were retained for subsequent experiments such as no-load experiment and H&E staining.

[0188] Hematoxylin-eosin (H&E) staining of mouse brain tissue

[0189] After embedding the brain tissue in the Spatial-seq protocol, serial sections were made. One of the sections was placed in 4% paraformaldehyde solution for fixation for about 10 - 15 min. Subsequently, the sections were rinsed with PBS to remove the fixative, usually rinsed 2 - 3 times, 5 min each time. After washing, the sections were placed in hematoxylin stain for 5 min. The sections were rinsed with water to remove the excess dye. Then the sections were placed in eosin stain for 3 min. After staining, the sections were successively placed in alcohol with different concentration gradients (70%, 85%, 95%, and 100% ethanol) for dehydration, 1 min for each level of dehydration. Subsequently, the sections were placed in xylene for 2 min for transparency treatment. Finally, the sections were sealed with neutral gum. The stained sections were observed under a microscope, and the required images were collected through an image acquisition system for analysis.

[0190] Spatial Transcriptome Sequencing and Data Analysis of Mouse Brain Tissue Using Spatial-seq 2.0 Technology

[0191] Brains were taken from 12 mice in each of the Sham, Model, PNS-H, and PGS-H groups. First, the first frozen section was scanned microscopically in high definition. The right side area of each section was divided into 32 Spot regions in an array. The radius of each Spot was approximately 125 μm, and the area was approximately 50,000 μm 2 , and there were approximately 600 cells. A total of 384 Spot regions were cut from all the mice. The LCM instrument was used to automatically identify the cutting regions. The parameter settings were objective lens 10 ×, Final Pulse mode, power = 17, aperture = 1, speed = 20, bridging size = 5, final pulse = 16. Collection was done using an enzyme-free well plate and immediately stored at -80°C after collection. For the specific protocol after collection, refer to Example 1.

[0192] Results and Discussion

[0193] Pharmacodynamic Evaluation of PNS and PGS on MCAO Mice

[0194] According to the technical scheme ( Figure 1 A in it), 24 hours after MCAO modeling, neurobehavioral scoring and statistical analysis were performed on the mice in each group. The results are as shown in Figure 1 B in it. In the sham operation group, no mice showed signs of nerve function damage, and the scores were all 0 points. Compared with the sham operation group, the model group showed obvious nerve function defects, such as circling and falling to the contralateral side, and the scores increased significantly. Compared with the model group, the nerve function damage in each drug administration group was improved, and the scores decreased significantly, indicating that 200 mg / kg of PGS and PNS significantly improved the nerve function of pMCAO mice ( P <0.01), and was superior to the positive drug edaravone. The results of TTC staining of mouse brain tissue are as shown in Figure 1 C in it and Figure 1 D in it. Figure 1 In C in it, the red area represents normal tissue, the white area is the infarcted part, and the middle pink area is the ischemic transition zone. The results showed that no infarct lesions were seen in the brain tissue of mice in the sham operation group, while large white infarcted areas appeared in the brain tissue of mice in the model group, and the average infarct area reached 30.73%. Compared with the model group, the infarct areas of the brain tissue of mice in each drug administration group showed a downward trend. After the action of PNS-H, the average infarct area of the brain tissue of mice was 11.70%, and the infarct area decreased significantly by 61.9% compared with the model group, and was approximately the same as the infarct area of the group of mice treated with the positive drug edaravone. The above results indicate that both PNS-H and PGS-H have the effect of anti-acute ischemic stroke, but the pharmacodynamic effect of PNS-H is better than that of PGS-H.

[0195] Figure 1 Pharmacodynamic evaluation results of PNS and PGS on MCAO mice at 24 h; among them, A: Schematic diagram of the overall administration plan; B: Influence diagram of PNS and PGS on the neurological behavior score of MCAO mice; C: Representative diagram of TTC staining of mouse brain tissue; D: Influence result diagram 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, ** P <0.01, *** P <0.001.

[0196] Effects of PNS and PGS on the pathological morphological changes of the brain tissue of MCAO mice

[0197] Observe the effects of PNS and PGS on the pathological morphological changes of the brain tissue of MCAO mice. H&E staining was used, and the results are as Figure 2 shown. In the sham operation group, the coloring of the left and right cerebral hemispheres of the mice was consistent, the cell morphology was intact, there was no edema or vacuoles, the cell nuclei were plump, and the nucleoli were clear; in the model group, the overall coloring of the infarcted area of the mouse brain tissue was lighter than that of the normal tissue, severe edema occurred, a large number of vacuoles were scattered, and the nucleoli were shrunk and deeply stained; compared with the model group, after administration of PNS and PGS, the brain tissue edema and nucleolus shrinkage in MCAO mice were significantly alleviated. The above results indicate that both of them have obvious improvement effects on the pathological changes of the brain tissue of MCAO mice.

[0198] Figure 2 It is a representative diagram of H&E staining of the cerebral cortex area of mouse brain tissue; among them, n = 3, and the scale bar in the figure is 2000 μm and 100 μm.

[0199] Acquisition and quality control of Spatial-seq 2.0 spatial transcriptome data

[0200] The experimental process of Spatial-seq 2.0 mainly includes the synthesis of different spatial barcodes, the sampling and freezing section of mouse brain tissue, the selection of spatial Spots regions of tissue sections on LCM and laser microdissection, and high-throughput pooled sequencing. Among them, 3 parts require precise quality control, namely the quality control of magnetic bead synthesis efficiency, the quality control of cDNA of sequencing library, and the quality control of sequencing data analysis. First, it is necessary to evaluate the number of oligoT sequences fixed on the surface of the synthesized magnetic beads, and this parameter is crucial for the subsequent efficiency of capturing mRNA. As Figure 3 shown in A of Figure 3As shown in B of [Figure 0], the right brain region of the frozen brain sections of each mouse was captured in an array, with a total of 4 groups, 3 replicates in each group, and a total of 384 Spots were captured. Subsequently, 384 synthesized magnetic beads were used for mRNA capture, reverse transcription, multiple rounds of amplification, and library construction in the spatial region of the mouse brain tissue. To evaluate the quality of the sequencing library, the length distribution was detected using the Agilent 4200 system. Observation Figure 3 The results in C show that the library length is mainly distributed between 350 and 1000 bp, meeting the quality standards for on-machine sequencing. Finally, the quality of the cell samples was jointly judged by analyzing the number of genes detected in each Spot in each group (nFeature) and the total transcript count in each Spot in each group (nCount). By Figure 3 It was found in D of [Figure 0] that all 384 Spots could be detected, ensuring the accuracy of subsequent analysis. Among them, it was found that Spots with more damaged or dead cells may express fewer genes, so they have a lower nFeature value and nCount value, which is in line with the complexity or biological state of stroke disease. Through the overall expression analysis of the 4 groups of samples, it was found that the median values of nFeature and nCount in the PNS group were higher than those in the PGS group, indicating that PNS showed better therapeutic effects in MCAO mice.

[0201] Figure 3 It is the quality control chart of Spatial-seq 2.0 spatial transcriptome data; among them, A: the quality inspection report chart of 384 different barcode capture magnetic beads; B: the size and array distribution chart of 384 spatial Spots; C: the quality inspection report chart of the sequencing library; D: the quality control report chart of the sequencing results.

[0202] Analysis of the correlation between the key spatial regions and the ischemic penumbra of stroke based on Spatial-seq 2.0 of the present invention

[0203] The penumbra region is a specific spatial region that may have therapeutic potential during the recovery period after a stroke, and is a region expected to promote recovery through drugs or other treatment means. Spatial-seq 2.0 can analyze the gene expression changes in each spatial region. 192 times of edgR differential gene analysis were performed on 192 Spots of the model group and the sham operation group respectively, as Figure 4 , and sorted according to the magnitude of the differential change. As Figure 4 in A of [Figure 0] and Figure 4 in B of [Figure 0], the spatial regions with the top 25% (8 / 32) of the magnitude of the differential change were used as the key spatial regions affected by stroke disease. As Figure 4 shown in C of [Figure 0], combined with Figure 2The H&E staining results showed that the range of the 8 Spots circled in the middle was almost equal to the range of the ischemic penumbra. This result indicates that specific spatial regions with therapeutic potential after stroke can be located by the gene group with the most significant spatial expression changes in the brain tissue; there is a spatial co-localization relationship between this region and the penumbra region.

[0204] Figure 4 It is an analysis diagram of the correlation between the key spatial region and the ischemic penumbra of stroke; among them, A: The result diagram of the number of differentially expressed genes at each spatial position in the Sham group and the Model group; B: The spatial positions ranked in the top 25% according to the number of differentially expressed genes; C: The spatial distribution of the ischemic penumbra (red) and the infarct core area (blue) shown in the H&E staining diagram.

[0205] Pathway enrichment analysis of differentially expressed genes of PNS and PGS in the penumbra based on the Spatial-seq 2.0 of the present invention

[0206] It is known that the "analysis of the correlation between the key spatial region and the ischemic penumbra of stroke based on the Spatial-seq 2.0 of the present invention" found that the 8 spots regions circled can correspond to the penumbra part of the stroke. Next, continue to analyze the gene differential callback situation in the penumbra region obtained by the Spatial-seq 2.0 technology. As Figure 5 shown in A of Figure 5 and B of Figure 5 shown, PNS and PGS have some identical regulatory effects on acute stroke in the penumbra region. It was found that under stroke conditions, PNS highly callbacks two biological processes: regulation of actin filament-based process and regulation of actin cytoskeleton. The PNS pathway regulates the actin-related pathway and can perform functions such as regulation of blood flow, blood vessel expansion, and blood vessel repair. These effects are consistent with the "activating blood circulation" effect of Panax notoginseng, that is, improving blood circulation and promoting the recovery of damaged tissues (Figure 5 In part B) of this. These all indicate that total ginsenosides can regulate the energy conversion efficiency, increase the 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 "Qi-tonifying" efficacy of ginseng, that is, enhancing physical strength and vitality.

[0207] In summary, PNS and PGS jointly regulate the penumbra region of acute stroke by affecting multiple biological processes and signaling pathways, but their respective characteristics and mechanisms of action are different, respectively reflecting the concepts of "activating blood circulation" of Panax notoginseng and "Qi-tonifying" of ginseng in traditional Chinese medicine theory. The above analysis reveals the potential value of these two traditional Chinese medicinal materials in modern medical research and provides a scientific basis for further research and application.

[0208] Figure 5 This is the result map of pathway enrichment analysis; among them, A: The classical pathway map related to the key genes callback by PNS; B: The classical pathway map related to the key genes callback by PGS.

[0209] Based on the Spatial-seq 2.0 of the present invention to study the effects of PNS and PGS on different brain regions of mice

[0210] Similarly, in order to discover the efficacy of PNS and PGS on different brain regions of mice, GO and KEGG pathway enrichment analysis was performed on the brain region gene data obtained by the gene Spatial-seq 2.0 technology, and the six brain regions of the key hypothalamus, thalamus, hippocampus, cerebral cortex, striatum region, and amygdala and the pathways most relevant to cerebral ischemia diseases were selected ( Figure 6 ). In summary, it can be found through the analysis of the Spatial-seq 2.0 technology that in acute ischemic stroke, PNS and PGS show different action differences 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 reflects that PNS regulates more widely than PGS in acute stroke ( Figure 6 ). This may be because the multiple bioactive compounds of PNS affect multiple aspects of stroke pathology such as blood flow restoration, inflammation reduction, and enhanced cell metabolism.

[0211] Figure 6 This is the result map of GO pathway enrichment of the marker genes in each brain region of mice.

[0212] Conclusion

[0213] The present invention uses the Spatial-seq 2.0 technology. Starting from the full perspective of space, it breaks through the technical problem that "existing Bulk transcriptome sequencing technologies 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 both on each Spot at different positions 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 region of stroke by the Spatial-seq 2.0 technology, it is found that PNS has a more extensive impact on the pathways related to blood circulation, inflammation, and cell metabolism, reflecting the "blood-activating" characteristics of Panax notoginseng. In contrast, PGS may be more specifically targeted at energy metabolism, stress response, and neuron protection pathways, which is consistent with the "qi-tonifying" effect of ginseng. In the comparative analysis of brain regions, it is found that PNS and PGS exhibit their potential mechanisms for treating acute ischemic stroke through different molecular pathways in six different brain regions. At the same time, PNS covers more extensive pathways and genes than PGS, which also reflects that the regulatory effect of PNS on anti-acute stroke is more extensive than that of PGS.

[0214] Comparative Example 1

[0215] The Spatial-seq protocol (a spatial transcriptome sequencing technology published by scholars such as Liao Jie 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 registration of mouse brain tissues; ③ Microdissection and barcode capture and labeling; ④ Reverse transcription, amplification, and cDNA library preparation; ⑤ High-throughput pooled sequencing and data analysis.

[0216] Spatial-seq 2.0 optimizes the Spatial-seq protocol, mainly involving: on the basis of Spatial-seq: ① Improving the barcode synthesis efficiency (corresponding to step 1 of Example 1); ② Improving the RNA quality (corresponding to steps 3 and 4 of Example 1); ③ Increasing the capture rate of Spots (corresponding to step 4 of Example 1); ④ Optimizing the reagents to improve the efficiency of reverse transcription, amplification, and library preparation (corresponding to steps 5 and 6 of Example 1).

[0217] There are technical differences and effect differences between the technologies of Spatial-seq and Spatial-seq 2.0:

[0218] 1. Spatial-seq is a 96-well plate system; Spatial-seq 2.0 is a 384-well plate system. The advantages of Spatial-seq 2.0 compared to Spatial-seq are: reagent saving and efficiency increase. The volume is reduced, making it more suitable for small-volume and high-throughput reaction operations. It can greatly increase the number of barcoded synthesis samples that can be processed simultaneously under limited experimental space and reagent usage, effectively improving the overall synthesis efficiency.

[0219] 2. For Spatial-seq, the addition amount of the first spatial barcode primer per well is 320 μL, and the addition amount of the key synthesis reagent EDC is 120 μL. For Spatial-seq 2.0, the addition amount of the first spatial barcode primer per well is 80 μL, and the addition amount of the key synthesis reagent EDC is 32.36 μL. The advantages of Spatial-seq 2.0 compared to Spatial-seq are: reagent saving. The 384-well plate can process more samples at one time and does not need to repeat the reaction as frequently as the 96-well plate, thus further significantly saving the overall reagent usage.

[0220] 3. For 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 perform a ligation reaction on the B-series primers based on the first step; the third step is to synthesize the C-series primers on the preliminary barcode structure formed in the second step). For 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; in the second step, the B-series primers and C-series primers are put into the reaction together, and through combination with reagents, the ligation of the B-series primers and C-series primers of the barcode can be completed simultaneously in one step). The advantages of Spatial-seq 2.0 compared to Spatial-seq: The 3-step synthesis method of Spatial-seq (the whole process is relatively cumbersome, each step requires specific reaction conditions and time control, and intermediate product detection and processing may be required between steps to ensure the smooth progress of the reaction) is optimized to the 2-step synthesis method of Spatial-seq 2.0 (directly obtaining relatively stable and recognizable barcode products), simplifying the synthesis process and shortening the synthesis time. For the synthesis of 384 barcodes, Spatial-seq requires 28 h, while Spatial-seq 2.0 requires 5 h. For the total synthesis time, Spatial-seq requires 3 days, and Spatial-seq 2.0 requires 1 day.

[0221] 4. Spatial-seq: Cell lysate: 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 lysate; Spatial-seq 2.0: Cell lysate (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 nuclease-free water to prepare a specific cell lysate; Compared with Spatial-seq, Spatial-seq 2.0 improves the RNA quality by more than 1 time, as shown in Figure 7 , the comparison result graph of the RNA quality of different cell lysates, where A is Spatial-seq and B is Spatial-seq 2.0;

[0222] 5. After obtaining the frozen sections in step 4 of Spatial-seq, directly perform LCM cutting. After obtaining the frozen sections in step 4 of Spatial-seq 2.0, first use an electrostatic removal device and then perform LCM cutting; The static electricity on the LCM instrument and the sections is very serious, affecting the capture rate. Compared with Spatial-seq, Spatial-seq 2.0 adds an electrostatic removal device (desktop ion blower LA-211) to blow out the airflow of positive and negative charges, neutralize the charges, and increase the capture rate of Spots from 50% to 93.75%. The results are shown in Table 8, the comparison result table of the capture rate records;

[0223] 6. The reverse transcription reagent for Spatial-seq is: Nanjing Novoprotein Scientific Co., Ltd., product number P511 (2×Phanta Master Mix), and the library construction reagent is: Novoprotein Scientific Co., Ltd. TD501 quantitative library construction (TruePrep DNA Library Prep Kit V2 for Illumina). The sequencing scheme is: second-generation sequencer: Illumina Xten, USA; The reverse transcription reagent for Spatial-seq 2.0 is: P501 mixed enzyme (Phanta Super-Fidelity DNA Polymerase), and the library construction reagent is: TD504 free library construction (TruePrep Flexible DNA Library Prep Kit for Illumina). The sequencing scheme is: Salus Medical Co., Ltd. in China, model Salus Pro; The advantage of Spatial-seq 2.0 compared with Spatial-seq is that the number of genes detected is twice that of the original Spatial-seq scheme (12464 / 6392≈2). The specific results are shown in Table 9.

[0224] Table 8 Comparison results of the capture rate of Spots

[0225]

[0226] Table 9 Comparison results of the quality of sequencing data

[0227]

[0228] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative efforts based on this embodiment, 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 and 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 first spatial barcode primer includes A1 to A4, and the nucleotide sequences are shown in SEQ ID NO.1 to 4 respectively; the second spatial barcode primer includes B1 to B12, and the nucleotide sequences are shown in SEQ ID NO.5 to 16 respectively; the third spatial barcode primer includes C1 to C8, and the nucleotide sequences are shown in SEQ ID NO.17 to 24 respectively; 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, lysing the cells in the well plate, and capturing 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 a Pre-Amp PCR system, a first PCR amplification was performed, the beads were placed on a magnetic stand, and the supernatant was taken to obtain a 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 to perform 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 nucleotide sequence of the TSO-PCR is shown in SEQ ID NO.28; 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 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.

3. 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.

4. 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.

5. 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.

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

7. 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, performing quality control and preliminary processing using Drop-seq_tools v2.5.1 according to the Drop-seq Core Computational Protocol v2.0.0, and then performing gene alignment using STAR v2.7.8a; the quality control and preliminary processing are completed using the FastQC tool.

8. 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 value was corrected for multiple testing, and the false positive rate was controlled by the false discovery rate method; the threshold value was finally set 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.

9. Application of the spatial transcriptome high-throughput sequencing method according to any one of claims 1 to 8 in developmental biology research or regenerative medicine research; the research is aimed at non-disease treatment.

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