Construction method and sequencing method of plant tissue space transcriptome sequencing library

Abstract

The invention provides a construction method of a plant tissue space transcriptome sequencing library, which comprises the following steps: fixing and embedding plant tissues to obtain embedded blocks; performing autofluorescence detection and tissue permeabilization treatment on part of the embedded blocks, and determining an autofluorescence threshold value and target tissue permeabilization time; slicing, pasting and fixing the remaining embedding blocks, and performing microscope fluorescence scanning detection and tissue permeabilization treatment on the tissue-containing chip according to an autofluorescence threshold value and target tissue permeabilization time to obtain a permeabilized tissue chip; and carrying out reverse transcription, tissue removal, cDNA release, recovery and amplification on the permeabilized tissue chip to obtain a cDNA amplification product. The plant tissue transcript constructed by the construction method is not easy to diffuse and high in capture rate, high-quality in-situ capture time-space group data can be obtained, the accuracy and the credibility are high, the sequencing accuracy is ensured, and the application value is high.

Description

Technical Field

[0001] This invention relates to the field of biology, and more specifically, to a method for constructing and sequencing a spatial transcriptome sequencing library of plant tissues. Background Technology

[0002] Spatial transcriptomics is an important high-throughput sequencing technology that can simultaneously obtain spatial location information of cells and gene expression data. This has advanced the study of true gene expression in in situ cells within tissues, providing crucial research tools for multiple fields such as tissue cell function, microenvironment interactions, developmental lineage tracing, and disease pathology. Stereo-seq is a technique that utilizes nanosphere DNB sequencing to achieve spatiotemporal transcriptome sequencing at subcellular resolution. However, when using this technology for spatiotemporal transcriptome sequencing, it was found that some plant tissues exhibit problems such as easy transcript diffusion and low capture rates.

[0003] Therefore, the construction and sequencing methods for spatial transcriptome sequencing libraries of plant tissues still need further research. Summary of the Invention

[0004] This invention aims to at least partially address the technical problems existing in the prior art. To this end, this invention proposes a method for constructing plant tissue spatial transcriptome sequencing libraries, a method for plant tissue spatial transcriptome sequencing, and a kit for using plant tissue spatial transcriptome sequencing libraries. The plant tissue transcripts constructed using this invention are not easily diffused, have a high capture rate, and can obtain high-quality in-situ captured spatiotemporal data with high accuracy and reliability, ensuring sequencing accuracy and high application value.

[0005] In one aspect of the present invention, a method for constructing a spatial transcriptome sequencing library of plant tissue is proposed. According to an embodiment of the present invention, the method includes: (1) fixing and embedding plant tissue to obtain embedded blocks; (2) performing autofluorescence detection and tissue permeation treatment on a portion of the embedded blocks to determine the autofluorescence threshold and the target tissue permeation time; (3) slicing, mounting and fixing the remaining portion of the embedded blocks, and performing microscopic fluorescence scanning detection and tissue permeation treatment on the chip containing the tissue according to the autofluorescence threshold and the target tissue permeation time to obtain a permeated tissue chip; (4) performing reverse transcription, tissue removal, cDNA release, recovery and amplification on the permeated tissue chip to obtain a cDNA amplification product; wherein, the fixation treatment includes: immersing the plant tissue in a paraformaldehyde solution.

[0006] In the method according to an embodiment of the present invention, step (1) involves fixing and embedding the plant tissue to preserve it. Existing plant tissue fixation methods typically involve paraformaldehyde (PFA) fixation, which requires fixing the tissue for 16 hours, followed by overnight dehydration in 10%, 20%, and 30% sucrose solutions. This cumbersome process easily leads to RNA degradation in the tissue. Therefore, the inventors have simplified the fixation method by soaking the plant tissue in paraformaldehyde solution for only a few minutes, achieving the fixation purpose while reducing the risk of RNA degradation and saving experimental time.

[0007] When performing microscopic fluorescence scanning on tissue sections, it is necessary to set the autofluorescence threshold for detection. Plant cell tissues are highly specific, and due to the presence of cell walls and various secondary metabolites, their autofluorescence will generate a lot of interference. Therefore, before performing step (3), it is necessary to pre-determine the fluorescence threshold of the plant tissue to be tested. Similarly, different plant tissue sections usually have applicable tissue permeation times. Before performing step (3), it is also necessary to pre-determine the target tissue permeation time of the plant tissue to be tested.

[0008] According to embodiments of the present invention, the method for constructing the above-described plant tissue spatial transcriptome sequencing library may further include the following additional technical features:

[0009] According to an embodiment of the present invention, the concentration of the paraformaldehyde solution is 0.1 to 2% by volume; the fixation time is 2 to 3 minutes.

[0010] According to an embodiment of the present invention, the tissue permeation treatment includes: adding the prepermeation reagent to a tissue chip after microscopic fluorescence scanning detection, incubating, rinsing the incubated tissue chip with a cleaning reagent, adding the permeation reagent to the tissue chip again, incubating, and then absorbing the liquid on the tissue chip and cleaning the tissue chip.

[0011] According to an embodiment of the present invention, the pre-permeabilization reagent includes PVP, BSA, RNase inhibitor and EXO I buffer; the permeabilization reagent includes PVP, HCl and PR Enzyme; the cleaning reagent includes PVP and HCl.

[0012] According to an embodiment of the present invention, based on the volume of the pre-permeabilization reagent, the concentration of PVP in the pre-permeabilization reagent is 1-5% by volume, the concentration of BSA is 0.01-0.1% by volume, and the concentration of RNase inhibitor is 1-5 U / μl.

[0013] According to an embodiment of the present invention, based on the volume of the permeation reagent, the concentration of PVP in the permeation reagent is 1 to 5% by volume, and the concentration of HCl is 0.001 to 0.1N.

[0014] According to an embodiment of the present invention, based on the volume of the cleaning reagent, the concentration of PVP in the cleaning reagent is 1 to 5% by volume, and the concentration of HCl is 0.001 to 0.1N.

[0015] According to an embodiment of the present invention, step (2) includes: slicing, mounting, fixing, and autofluorescence detection of a portion of the embedded block to determine the autofluorescence threshold; performing tissue permeation treatment on the tissue chip after autofluorescence detection in the previous step under different tissue permeation time conditions to obtain different permeated tissue chips; performing reverse transcription, tissue removal, and microscopic fluorescence scanning detection on the different permeated tissue chips respectively. In the microscopic fluorescence scanning detection, the signal intensity and diffusion degree detected by each permeated tissue chip are scored. The signal intensity score is recorded as A, and the higher the score, the stronger the signal; the diffusion degree score is recorded as B, and the higher the score, the weaker the diffusion degree; the tissue permeation time corresponding to the permeated tissue chip with the lowest A and B is selected as the target tissue permeation time.

[0016] According to an embodiment of the present invention, the slicing, patching and fixation steps in step (2) are the same as the slicing, patching and fixation steps in step (3); the tissue permeation treatment conditions in step (2) are the same as the tissue permeation treatment conditions in step (3) except for time.

[0017] According to an embodiment of the present invention, the method for determining the autofluorescence threshold includes: setting the fluorescence microscope to epifluorescence, adjusting the light intensity and exposure gain, switching to the DAPI channel, and adjusting the exposure until tissue autofluorescence can no longer be seen. The exposure value at this point is the autofluorescence threshold.

[0018] In another aspect of the invention, a method for spatial transcriptome sequencing of plant tissues is provided. According to an embodiment of the invention, the method includes: constructing a sequencing library using the aforementioned method for constructing a spatial transcriptome sequencing library of plant tissues; and sequencing the sequencing library.

[0019] In another aspect, the present invention provides a kit for spatial transcriptome sequencing libraries of plant tissues. According to an embodiment of the present invention, the kit comprises the following reagents: a fixation reagent comprising paraformaldehyde solution; and a pre-permeabilization reagent comprising PVP, BSA, an RNase inhibitor, and EXO I buffer.

[0020] Permeation reagents, including PVP, HCl, and PR Enzyme.

[0021] According to an embodiment of the present invention, based on the volume of the pre-permeabilization reagent, the concentration of PVP in the pre-permeabilization reagent is 1-5% by volume, the concentration of BSA is 0.01-0.1% by volume, and the concentration of RNase inhibitor is 1-5 U / μl.

[0022] According to an embodiment of the present invention, based on the volume of the permeation reagent, the concentration of PVP in the permeation reagent is 1 to 5% by volume, and the concentration of HCl is 0.001 to 0.1N.

[0023] According to embodiments of the present invention, the kit further comprises at least one of the following: reverse transcription reagent, OCT embedding agent, staining agent, cDNA release agent, tissue removal agent, and amplification reagent.

[0024] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0026] Figure 1 A schematic flowchart of a method for constructing a plant tissue spatial transcriptome sequencing library according to an embodiment of the present invention is shown;

[0027] Figure 2 A schematic flowchart of a method for constructing a plant tissue spatial transcriptome sequencing library according to another embodiment of the present invention is shown;

[0028] Figure 3 Electron micrographs of tissues at different permeation times according to an embodiment of the present invention are shown;

[0029] Figure 4 A PCR amplification procedure diagram according to an embodiment of the present invention is shown;

[0030] Figure 5 A visual heatmap of the present invention, according to Embodiment 1, is shown. Detailed Implementation

[0031] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0032] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0033] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0034] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0035] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0036] This invention proposes a method for constructing a plant tissue spatial transcriptome sequencing library, a method for sequencing a plant tissue spatial transcriptome, and a kit for sequencing a plant tissue spatial transcriptome library, which will be described in detail below.

[0037] Methods for constructing spatial transcriptome sequencing libraries from plant tissues

[0038] In one aspect of the invention, a method for constructing a spatial transcriptome sequencing library of plant tissues is provided. See embodiments of the invention for details. Figure 1 The method includes: S100 fixation and embedding; S200 determination of autofluorescence threshold and target tissue permeation time; S300 sectioning, mounting, fixation, microscopic fluorescence scanning detection, and tissue permeation; S400 reverse transcription, tissue removal, cDNA release, recovery, and amplification. Each step will be described in detail below.

[0039] S100 Fixing and Embedding

[0040] In this step, the plant tissue is fixed and embedded to obtain embedded blocks.

[0041] Plant tissue fixation aims to maintain its morphology, structure, RNA, and protein stability, preventing degradation and structural loss during experiments. Existing plant tissue fixation methods typically involve paraformaldehyde (PFA) fixation, a cumbersome and time-consuming process that easily leads to RNA degradation. To address this, the inventors have simplified the fixation method by simply immersing the plant tissue in a paraformaldehyde solution. This achieves the fixation goal while reducing the risk of RNA degradation and saving experimental time.

[0042] According to embodiments of the present invention, the fixation treatment includes immersing the plant tissue in a paraformaldehyde solution. In some embodiments, the concentration of the paraformaldehyde solution is 0.1–2% by volume; the fixation time is 2–3 minutes. Under the above fixation conditions, paraformaldehyde can penetrate into the tissue to coagulate intracellular proteins, minimizing or terminating the reactions of exogenous and endogenous enzymes; preventing cell autolysis, thus preventing antigen diffusion into the interstitial tissue, and maintaining the inherent morphology and structure of the tissue. Simultaneously, it can maintain the antigenicity of the tissue or cells, avoiding antigen inactivation and diffusion, thereby preventing excessively dark background during staining that could affect the identification of positive analytes. In some embodiments, the paraformaldehyde solution can be water or a buffer solution, such as phosphate buffer, and the pH of the paraformaldehyde solution is near neutral; exemplarily, the pH can be 7.0–7.4.

[0043] Before fixation, the detached fresh plant tissue can be trimmed to the required size, such as less than 0.9cm × 0.9cm × 0.4cm.

[0044] Embedding fixed tissues preserves their shape and structure, preventing damage during processing and sectioning. This facilitates observation and study of cellular structure, tissue morphology, and various cell-molecule interactions. Common embedding media include paraffin or OCT, yielding paraffin sections and frozen sections respectively. Compared to paraffin sections, frozen sections better preserve the tissue's antigenic immunoreactivity.

[0045] S200 determines the autofluorescence threshold and target tissue permeation time.

[0046] In this step, a portion of the embedded block is subjected to autofluorescence detection and tissue permeation treatment to determine the autofluorescence threshold and the target tissue permeation time.

[0047] When performing microscopic fluorescence scanning on stained tissue sections, it is necessary to set the autofluorescence threshold for detection. Plant cell tissues are highly specific, and due to the presence of cell walls and various secondary metabolites, their autofluorescence will generate a lot of interference. Therefore, before performing S300, it is necessary to pre-determine the fluorescence threshold of the plant tissue to be tested.

[0048] The inventors of this invention have discovered that the appropriate tissue permeation time may vary for different plant tissues, and the tissue permeation time significantly affects the detection and analysis results. If the tissue permeation time is insufficient, mRNA cannot be fully released; if the tissue permeation time is too long, mRNA may diffuse into nearby areas, affecting the accuracy of the analysis results. Therefore, it is necessary to pre-determine the optimal tissue permeation time for plant tissues. For example, 6 minutes is optimal for testing SAM samples at the R1 stage of soybean flowering; 8 minutes is optimal for testing Arabidopsis cotyledon embryos.

[0049] According to an embodiment of the present invention, step S200 includes: sectioning, mounting, fixing, and autofluorescence detection of a portion of the embedded block to determine the autofluorescence threshold; performing tissue permeation treatment on the tissue chips after the previous autofluorescence detection under different tissue permeation time conditions to obtain different permeated tissue chips; performing reverse transcription, tissue removal, and microscopic fluorescence scanning detection on the different permeated tissue chips, wherein the signal intensity and diffusion degree of each permeated tissue chip are scored in the microscopic fluorescence scanning detection, the signal intensity score is denoted as A, the higher the score, the stronger the signal; the diffusion degree score is denoted as B, the higher the score, the weaker the diffusion degree; the tissue permeation time corresponding to the permeated tissue chip with the lowest sum of A and B is the target (optimal) tissue permeation time. Therefore, the above method can be used to screen out target tissue permeation times based on different types of plant tissues.

[0050] According to an embodiment of the present invention, the slicing, patching, and fixation steps in step S200 are consistent with the slicing, patching, and fixation steps in step S300; the tissue permeation treatment conditions in step S200 are consistent with the tissue permeation treatment conditions in step S300 except for the time. This ensures that the target tissue permeation time determined in step S200 is suitable for the tissue permeation treatment in step S300.

[0051] According to an embodiment of the present invention, the method for determining the autofluorescence threshold includes: setting the fluorescence microscope to epifluorescence, adjusting the light intensity and exposure gain, switching to the DAPI channel, and adjusting the exposure until tissue autofluorescence can no longer be seen. The exposure value at this point is the autofluorescence threshold.

[0052] S300 sectioning, mounting, fixation, microscopic fluorescence scanning detection, and tissue permeation.

[0053] In this step, the remaining embedded blocks are sliced, mounted, and fixed. The stained tissue chip is then subjected to microscopic fluorescence scanning and tissue permeation according to the autofluorescence threshold and the target tissue permeation time to obtain the permeated tissue chip.

[0054] According to an embodiment of the present invention, the tissue permeation treatment includes: adding the prepermeation reagent to a tissue chip after microscopic fluorescence scanning detection, incubating, washing the incubated tissue chip with a cleaning reagent, adding the permeation reagent to the tissue chip again, incubating, removing the liquid on the tissue chip, and then washing and drying the tissue chip.

[0055] Currently, conventional tissue permeabilization involves directly permeating the tissue microarray. The inventors of this invention have discovered that the low gene capture rate in plant spatiotemporal sequencing is primarily due to the presence of numerous secondary metabolites in plants, which can affect reverse transcriptase activity. Furthermore, while PFA fixation reduces transcript diffusion, it also decreases gene capture to some extent. By employing a pre-permeabilization reagent, the capture rate can be improved, ensuring that the simplified PFA fixation results meet the requirements of subsequent analysis.

[0056] According to embodiments of the present invention, the pre-permeabilization reagent includes PVP (PVP10-500G, Sigma), BSA (bovine serum albumin), RNase inhibitor, and EXO I buffer (BO293S, NEB); the permeabilization reagent includes PVP (PVP10-500G, Sigma), HCl, and PR Enzyme (BGI, 1000028500); the washing reagent includes PVP and hydrochloric acid solution. Therefore, using the above-mentioned pre-permeabilization reagent can further improve the capture efficiency.

[0057] According to an embodiment of the present invention, based on the volume of the pre-permeabilizing reagent, the concentration of PVP in the pre-permeabilizing reagent is 1-5% by volume, the concentration of BSA is 0.01-0.1% by volume, and the concentration of RNase inhibitor is 1-5 U / μl. Therefore, by using the above-mentioned pre-permeabilizing reagent, the capture efficiency can be further improved.

[0058] According to an embodiment of the present invention, based on the volume of the permeation reagent, the concentration of PVP in the permeation reagent is 1-5% by volume, the concentration of HCl is 0.001-0.1N, and the concentration of RNase inhibitor is 1-5 U / μl. Therefore, by using the above-mentioned permeation reagent, the capture efficiency can be further improved.

[0059] According to an embodiment of the present invention, based on the volume of the cleaning reagent, the concentration of PVP in the cleaning reagent is 1-5% by volume, and the concentration of HCl is 0.001-0.1N. This avoids the possibility of residues affecting subsequent operations.

[0060] S400 reverse transcription, tissue removal, cDNA release, recovery, and amplification.

[0061] In this step, the permeabilized tissue microarray undergoes reverse transcription, tissue removal, cDNA release, recovery, and amplification to obtain a sequencing library. After reverse transcription, the mRNA in the tissue is transcribed into cDNA. However, the tissue itself may contain other impurities, such as cell debris and proteins. These impurities may interfere with subsequent experimental steps, affecting sample purity, such as probe capture and sequencing, thus hindering the reliability of experimental results. Therefore, tissue removal is necessary after reverse transcription. Since the tissue microarray is pre-set with probes that can capture mRNA from the tissue, these probes act as primers during reverse transcription. Therefore, the reverse-transcribed cDNA sequence can be directly spliced ​​onto the probes, avoiding removal during tissue removal and microarray washing.

[0062] To avoid impurities in the recovered cDNA product affecting subsequent amplification, the product is purified beforehand. After amplification, the resulting amplification product is further processed into nanospheres (DNB).

[0063] Methods for spatial transcriptome sequencing of plant tissues

[0064] In another aspect, the present invention provides a method for spatial transcriptome sequencing of plant tissues. According to an embodiment of the present invention, the method includes: constructing a sequencing library using the aforementioned method for constructing a plant tissue spatial transcriptome sequencing library; and sequencing the sequencing library. Therefore, the sequencing results obtained using the method of the present invention are highly accurate.

[0065] It should be noted that the features and advantages described above for the construction method of plant tissue spatial transcriptome sequencing libraries also apply to this plant tissue spatial transcriptome sequencing method, and will not be repeated here.

[0066] Kits for Plant Tissue Spatial Transcriptome Sequencing Libraries

[0067] In another aspect, the present invention provides a kit for spatial transcriptome sequencing libraries of plant tissues. According to embodiments of the present invention, the kit comprises the following reagents: a fixation reagent comprising paraformaldehyde solution; a pre-permeabilization reagent comprising PVP, BSA, an RNase inhibitor, and EXO I buffer; and a permeabilization reagent comprising PVP, HCl, and PR Enzyme. When constructing spatial transcriptome sequencing libraries of plant tissues using the kit of the present invention, plant tissue transcripts are less prone to diffusion, resulting in a high capture rate and the acquisition of high-quality in-situ captured spatiotemporal data with high accuracy and reliability, ensuring sequencing accuracy and demonstrating high application value.

[0068] According to an embodiment of the present invention, based on the volume of the pre-permeabilizing reagent, the concentration of PVP in the pre-permeabilizing reagent is 1-5% by volume, the concentration of BSA is 0.01-0.1% by volume, and the concentration of RNase inhibitor is 1-5 U / μl. Therefore, by using the above-mentioned pre-permeabilizing reagent, the capture efficiency can be further improved.

[0069] According to an embodiment of the present invention, based on the volume of the permeation reagent, the concentration of PVP in the permeation reagent is 1-5% by volume, and the concentration of HCl is 0.001-0.1N. Therefore, by using the above-mentioned permeation reagent, the capture efficiency can be further improved.

[0070] According to embodiments of the present invention, the kit further comprises at least one of the following: a reverse transcription reagent, an OCT embedding agent, a staining agent, a cDNA release agent, a tissue removal agent, and an amplification reagent. The reverse transcription reagent is a reagent used to perform reverse transcription and may include reverse transcriptase, dNTPs, buffers, and other reagents commonly used in the art for reverse transcription. The staining agent is a reagent used to stain tissue sections and may include Fluorescent Brightener 28 tissue fluorescence staining solution, etc. The tissue removal agent is a reagent used to remove tissue and may include SSC buffer and Tris-HCl buffer, etc. The cDNA release reagent is used to release cDNA from the tissue chip. The amplification reagent is used to perform cDNA amplification and may include buffers, DNA polymerase, dNTPs, etc.

[0071] It should be noted that the features and advantages described above for the construction method of plant tissue spatial transcriptome sequencing libraries also apply to this kit for plant tissue spatial transcriptome sequencing libraries, and will not be repeated here.

[0072] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0073] Example 1

[0074] See Figure 2 Spatial transcriptome sequencing libraries of soybean SAM tissue samples at the R1 stage of initial flowering were constructed using the following methods:

[0075] 1. Plant tissue treatment and embedding

[0076] 1.1. Sample Processing

[0077] Under laboratory conditions, after fresh samples are removed from the body, plant tissues are sampled, trimmed, and rapidly fixed and embedded using sterilized instruments to minimize RNA degradation within the tissue. Tissue blocks are smaller than 0.9cm × 0.9cm × 0.4cm.

[0078] 1.2. Simplified PFA fixing

[0079] (1) Thaw the 4% PFA solution at room temperature in advance and prepare 1% PFA Mix (Table 1) in a 1.5ml centrifuge tube.

[0080] Table 1 Composition of 1% PFA Mix

[0081]

[0082] (2) Immerse the plant tissue in 1% PFA Mix and incubate on ice for 2.5 min.

[0083] (3) Wash the plant tissue with 1×PBS 3 times.

[0084] (4) Use lint-free paper to absorb the surface moisture of the plant tissue and place it on ice to await embedding.

[0085] 1.3. Sample embedding

[0086] Prepare an embedding cassette slightly larger than the size of the tissue block. Place it on dry ice and slowly add an appropriate amount of pre-cooled OCT at 4°C, about 2-4 mm thick, ensuring the surface is flat and free of air bubbles. Use tweezers to transfer the plant tissue into the OCT-covered embedding cassette and add more OCT to cover the tissue surface. If there are air bubbles, use a pipette tip to remove them and quickly transfer the tissue to dry ice. After the OCT solidifies and turns white, mark the tissue embedding location and pre-section orientation on the embedding cassette.

[0087] 1.4. Sample Preservation and Transportation

[0088] Wrap the embedded tissue in aluminum foil, label it, and place it in a sealed bag. Store it in a refrigerator at -80°C. For long-distance transportation, it can be mailed via dry ice.

[0089] 2. Autofluorescence assay and permeation test of plant tissues

[0090] 2.1 Experimental Preparation and Chip Preprocessing

[0091] (1) Pre-cool the cryostat chamber (-20℃) and sample head (-10℃~-15℃); at the same time, place the brush, blade and tweezers in the cryostat for pre-cooling; adjust the temperature of the curing machine to 37℃.

[0092] (2) Take the embedding cassette containing the tissue sample to be tested out of the -80℃ freezer and transfer it to the cryostat for equilibration (about 30 min); pre-cool methanol at -20℃, add it to the 24-well plate (1 mL / well), cover it, and place it in the cryostat or the -20℃ freezer for later use.

[0093] (3) Transfer the fluorescent chip to a new 24-well plate, paying attention to the front and back of the chip, and do not scratch the front of the chip. Record the chip number. Rinse twice with 400 μL 0.1×SSC, and aspirate the liquid. Cover the chip surface with 100 μL Nuclease-Free Water (containing RNase Inhibitor, 5%) and let it stand for 3 min. Aspirate the liquid on the chip surface, and rinse twice with 400 μL Nuclease-Free Water, and aspirate the liquid. Wipe the back and surrounding area of ​​the chip with lint-free paper, and bake the chip at 37°C in a chip oven until there is no liquid residue on the chip surface. Place the chip in a petri dish and wait for mounting.

[0094] 2.2 Cryosectioning, mounting, and fixation of embedded blocks

[0095] (1) Take out the embedding block from the embedding cassette and fix it to the sample holder using OCT according to the slide orientation; after successful adhesion, trim the embedding block, cut off the excess OCT, and install it to the sample head to prepare for slide preparation.

[0096] (2) When starting to slice, the slice thickness can be adjusted to 50μm. After the tissue is exposed, adjust the slice thickness to 10μm (adjustments can be made according to cell size and tissue slice condition); after successful slicing, use the sample stage to quickly pre-cool the brush head and carefully flatten the slice, trying to avoid touching the tissue area. Carefully flip the slice so that the back side is facing up to complete the shaping and prepare for mounting.

[0097] (3) Cold bonding: Place the chip face up in the slicer and pre-cool for 3 minutes. Carefully cover the center of the chip with the frozen slice, gently adjust the position, and use your fingertips to warm the back of the chip to make the slice adhere.

[0098] (4) After successful patching, place the patch at 37°C for 3 minutes with the front side facing up. Do not place it at room temperature. After baking, immediately place the patch in a pre-cooled methanol solution at -20°C for 30 to 40 minutes to fix it.

[0099] (5) To ensure sample quality and reduce experimental risks, it is recommended to cut 10-20 pieces of tissue with a thickness of 10μm and store them in 1.5mL EP tubes pre-cooled at -20℃ for subsequent extraction and quality testing of Total RNA (RIN≥7 is considered a qualified sample, and plant tissue is only used as a reference).

[0100] 2.3 Autofluorescence determination

[0101] (1) Remove the fixed tissue chip from the methanol, blot the back and surrounding methanol with lint-free paper, and transfer it to a culture dish with a sealing film. After the methanol evaporates and the chip surface dries, clean the chip once with 0.1×SSC (100μL / chip; containing RNase Inhibitor, 5%) to keep the chip surface moist.

[0102] (2) Carefully transfer the chip onto the glass slide, add 15 μL of Image Reagent (containing RNase Inhibitor, 5%) to cover the entire chip, ensuring that there is no obvious liquid bulge on the chip surface, and slowly transfer the glass slide containing the chip to the microscope scanning stage.

[0103] (3) Open the microscope control system, select epifluorescence, light intensity modulation 30, exposure gain 100 (tissues with weak autofluorescence can be adjusted according to the actual situation); first rotate to the FITC channel, adjust the exposure under 10x magnification until the tissue autofluorescence can no longer be seen, and record the exposure value at this time; then rotate to the DAPI channel, keep the magnification unchanged, adjust the exposure, again until the tissue autofluorescence can no longer be seen, and record the exposure value. The exposure value at this time is the autofluorescence threshold.

[0104] 2.4 Permeability Test

[0105] (1) Prepare the pre-permeability reagent (2% PVP, 1×EXO I Buffer, 0.02% BSA, 1×RNaseInhibitor 2U / μl, 100μl / chip, prepared and used on the same day); transfer the chip to a culture dish with a sealing film, slowly add the pre-permeability reagent to cover the entire chip, transfer the culture dish containing the chip to a 37℃ constant temperature incubator, and incubate for 10 min.

[0106] (2) Prepare 0.01N HCl containing 2% PVP (pH value is 2, prepare and use on the same day); slowly add 0.01N HCl containing 2% PVP to rinse the tissue on the chip (100μL / chip).

[0107] (3) Dissolve the dry powdered PR Enzyme in 1 mL of 0.01 N HCl to prepare a 10× stock solution (mix by pipetting, do not vortex), dispense as needed and freeze to avoid repeated freeze-thaw cycles; prepare permeation reagent in advance (2% PVP, 0.01 N HCl, 1× PR Enzyme), transfer the permeation reagent to a 37°C constant temperature incubator to equilibrate for ≥3 min, and add the permeation reagent dropwise (100 μL / chip, add dropwise in the constant temperature incubator to avoid diffusion caused by movement); the permeation time gradient control can be set according to the actual situation, and gradient tests can be performed at 3 min intervals (e.g., 1 min, 4 min, 7 min).

[0108] (4) After the reaction is complete, remove the liquid on the chip surface, clean the chip once with 0.1×SSC solution (100μL / chip, containing RNase inhibitor, 5%), dry the back and surrounding area of ​​the chip with lint-free paper, and transfer the chip to a new culture dish with a sealing film, and immediately carry out the reverse transcription reaction.

[0109] 2.5 Reverse transcription reaction

[0110] (1) Set the temperature of the constant temperature chamber to 42℃ in advance; thaw the RT QC Reagent, RT Additive and RT QC Enzyme on ice in advance; prepare the RT QC Mix (Table 2) and equilibrate to room temperature (away from light).

[0111] Table 2 RT QC Mix System Preparation

[0112]

[0113] (2) Gently add RT QC Mix (90 μl / well) to the surface of the chip, making sure the liquid level is submerged in the tissue; seal the culture dish containing the chip with sealing film and completely wrap it with aluminum foil (to protect from light), and slowly transfer it to a 42°C constant temperature incubator for 10 h.

[0114] 2.6 Tissue Removal and Microscopic Fluorescence Scanning

[0115] (1) Set the temperature of the incubator to 55℃ in advance; take the culture dish containing the chip out of the 42℃ incubator, gently remove the sealing film, and aspirate the liquid on the surface of the chip; carefully transfer the chip to a 24-well plate, and clean the chip with 0.1×SSC (400μL / well) (pipette tip up and down 5 times on the outside of the chip) twice; prepare the tissue removal mix according to Table 3, transfer the chip to a new well, add the tissue removal mix (400μL / well, TR Buffer should be thawed at room temperature in advance, if white precipitate is formed, it can be dissolved at 55℃), and place in a 55℃ incubator for 10 min.

[0116] Table 3. Formulation of the tissue removal mix system

[0117]

[0118] (2) Discard the tissue removal solution and rinse the chip twice with 0.1×SSC (400μL / well) (for tissues that are difficult to remove, rinse repeatedly); after the tissue is removed, add an appropriate amount of 0.1×SSC to soak the chip to prevent it from drying out.

[0119] (3) Aspirate 0.1×SSC, add Nuclease-free water (400μL / well), and rinse the chip surface with a suction and agitation to remove salt. Wipe the chip's periphery and back with lint-free paper, place it on a glass slide, add Image Reagent (15μl / chip), and transfer it to the microscope stage. Turn on the microscope control system, select epifluorescence, rotate to the TRITC channel, and scan the entire image with a 10x microscope. The exposure time for the same group of experiments must be consistent. Score the signal intensity and diffusion degree of each permeable tissue chip. The signal intensity score is recorded as A, where a higher score indicates a stronger signal. The diffusion degree score is recorded as B, where a higher score indicates a weaker diffusion degree. Select the chip with higher signal intensity and weaker diffusion, i.e., the lowest sum of A and B. The corresponding time is the optimal permeation time for this tissue. The optimal permeation time was finally determined to be 4 min. Figure 3 ).

[0120] 3. Simplified standard operating procedure for plant spatiotemporal transcriptomics using PFA+PVP pre-permeabilization method

[0121] 3.1 Experimental Preparation and Chip Preprocessing

[0122] (1) Pre-cool the cryostat chamber (-20℃) and sample head (-10℃~-15℃); at the same time, place the brush, blade and tweezers in the cryostat for pre-cooling; adjust the temperature of the curing machine to 37℃.

[0123] (2) Take the embedding cassette corresponding to the tissue sample determined by the test out of the -80℃ freezer and transfer it to the cryostat for equilibration (about 30 min); add methanol (1 mL / well) to the 24-well plate, cover it, and place it in the cryostat or -20℃ freezer for pre-cooling (not exceeding 30 min).

[0124] (3) Transfer the spatiotemporal chip to a new 24-well plate, paying attention to the front and back of the chip, and do not scratch the front of the chip. Record the chip number. Rinse the chip twice with 400 μL 0.01×SSC, and then rinse twice with 400 μL Nuclease-Free Water. Discard the liquid. Prepare ssDNA fluorescent staining solution according to Table 4 below. Add ssDNA fluorescent staining solution (100 μL / chip) to cover the tissue on the chip. Stain at room temperature in the dark for 5 min. Slowly discard the liquid on the chip surface. Rinse twice with 400 μL Nuclease-Free Water. Discard the liquid. Use lint-free paper to dry the liquid remaining on the back and around the chip. Place the chip on a chip oven and bake at 37°C for 1 min. Store in the dark until ready for use.

[0125] Table 4. Preparation of ssDNA fluorescent staining solution

[0126]

[0127] 3.2 Cryosectioning, mounting, and fixation of embedded blocks

[0128] (1) Take out the embedding block from the embedding cassette and fix it to the sample holder using OCT according to the slide orientation; after successful adhesion, trim the embedding block, cut off the excess OCT, and install it to the sample head to prepare for slide preparation.

[0129] (2) When starting to slice, the slice thickness can be adjusted to 50μm. After the tissue is exposed, adjust the slice thickness to 10μm (adjustments can be made according to cell size and tissue slice condition); after successful slicing, use the sample stage to quickly cool the brush head and carefully flatten the slice, try to avoid touching the tissue area, carefully flip the slice so that the back is facing up to complete the shaping and prepare for mounting.

[0130] (3) Cold bonding: Place the chip face up in the slicer and pre-cool for 3 minutes. Carefully cover the center of the chip with the frozen slice, gently adjust the position, and use your fingertips to warm the back of the chip to make the slice adhere.

[0131] (4) After successful patching, place the patch at 37°C for 3 minutes with the front side facing up. Do not place it at room temperature. After baking, immediately place the patch in a pre-cooled methanol solution at -20°C for 30 to 40 minutes to fix it.

[0132] 3.3 FB28 staining and microscopic fluorescence scanning

[0133] (1) Remove the fixed tissue chip from the methanol, blot the methanol on the back and around the chip with lint-free paper, and transfer it to a culture dish with a sealing film. After the methanol evaporates and the chip surface dries, perform staining in sequence.

[0134] (2) Prepare Fluorescent Brightener 28 (FB28) tissue fluorescence staining solution according to Table 5 below. Add FB28 fluorescence staining solution (100 μL / chip; working final concentration 0.01%) to cover the tissue on the chip. Stain at room temperature in the dark for 3 min. Slowly aspirate the liquid on the chip surface. Then gently wash the chip once with 0.1×SSC (100 μL / chip; containing RNase Inhibitor, 5%) to keep the chip surface moist and prepare for microscope scanning.

[0135] Table 5. Preparation of FB28 Fluorescent Staining Solution

[0136]

[0137] (3) Remove the liquid from the chip surface, carefully transfer it onto a glass slide, add 15 μL Image Reagent (containing RNase Inhibitor, 5%) to cover the entire chip, ensuring that there is no obvious liquid protrusion on the chip surface, and slowly transfer the glass slide containing the chip to the microscope scanning stage.

[0138] (4) Open the microscope control system and select epifluorescence; first, rotate to the FITC channel, create a new slide file, create a map with 4x magnification, manually scan with 10x magnification, quickly add model points (4-5 evenly distributed, focus based on clear track lines), adjust the exposure (within the tissue autofluorescence test threshold under the FITC channel, based on tissue brightness, with clear and visible boundaries, no high-brightness blur, and avoid overexposure) and scan the entire chip; then rotate to the DAPI channel (keeping the chip position and magnification unchanged), create a new slide file 1 again, skip map creation (retain the ssDNA map creation result), adjust the exposure (within the tissue autofluorescence test threshold under the DAPI channel) 2, switch to manual scanning, clear all points, re-add model points (4-5 evenly distributed, focus based on clear tissue) 3, and scan the entire chip.

[0139] (5) Aspirate the liquid on the chip surface, slowly add 0.1×SSC (100μL / chip, containing RNase Inhibitor, 5%) to wash away the residual Image Reagent, blot dry the liquid on the back and around the chip with lint-free paper (carefully move the chip with tweezers and place it flat on lint-free paper), and immediately perform tissue permeation.

[0140] 3.4 Tissue permeability

[0141] (1) Prepare the pre-permeability reagent (2% PVP, 1×EXO I Buffer, 0.02% BSA, 1×RNaseInhibitor 2U / μl, 100μl / chip, prepared and used on the same day); transfer the chip to a culture dish with a sealing film, slowly add the pre-permeability reagent to cover the entire chip, transfer the culture dish containing the chip to a 37℃ constant temperature incubator, and incubate for 10 min.

[0142] (2) Prepare 0.01N HCl containing 2% PVP (pH value is 2, prepare and use on the same day); slowly add 0.01N HCl containing 2% PVP to rinse the tissue on the chip (100μL / chip).

[0143] (3) Dissolve the dry powdered PR Enzyme in 1 mL of 0.01 N HCl to prepare a 10× stock solution (mix by pipetting, do not vortex), dispense as needed and freeze to avoid repeated freeze-thaw cycles; prepare the permeation reagent in advance (2% PVP, 0.01 N HCl, 1× PR Enzyme), transfer the permeation reagent to a 37°C constant temperature incubator to equilibrate for ≥3 min, add the permeation reagent (100 μL / chip, add in the constant temperature incubator to avoid diffusion caused by movement); permeate for 4 min.

[0144] (4) After the reaction is complete, remove the liquid on the chip surface, clean the chip once with 0.1×SSC solution (100μL / chip, containing RNase inhibitor, 5%), dry the back and surrounding area of ​​the chip with lint-free paper, and transfer the chip to a new culture dish with a sealing film, and immediately carry out the reverse transcription reaction.

[0145] 3.5 Reverse transcription reaction

[0146] (1) Set the temperature of the constant temperature chamber to 42℃ in advance; briefly separate the centrifuge tube containing RT Oligo powder, add 79μL TEbuffer, cover the tube, and vortex at maximum speed for 15 seconds; briefly separate the tube again, take the corresponding volume of RT Oligo to prepare RT Mix (Table 6), and if there is any left, store the RT Oligo at -20℃ immediately.

[0147] Table 6 RT Mix System Preparation

[0148]

[0149] (2) Slowly add RT Mix (90μL-100μL / chip) from one corner of the chip, making sure the liquid covers the tissue evenly; carefully seal the culture dish with sealing film and slowly transfer it to a 42℃ incubator for reaction for 3 hours or more.

[0150] 3.6 Organization Removal

[0151] (1) Set the temperature of the constant temperature chamber to 55℃ in advance; remove the reaction solution on the surface of the chip, transfer the chip to a 24-well plate, and clean the chip once with 0.1×SSC (400μL / well); add TR Buffer (400μL / well, thaw at room temperature in advance, if white precipitate is formed, it can be dissolved at 55℃), and place it in a 55℃ incubator for 10 min.

[0152] (2) Discard the TR Buffer and rinse the chip twice with 0.1×SSC (400μL / well). (For tissues that are difficult to remove, rinse repeatedly, but be careful not to poke the chip with the pipette tip.) Transfer the chip with the tissue removed to a new reaction well for the next reaction.

[0153] 3.7 cDNA Release and Recovery

[0154] (1) Prepare cDNA Release Mix according to Table 7. Add cDNA Release Mix (400 μL / well) to the chip reaction wells in the previous step, seal the reaction wells containing the chip with sealing film, cover the plate with the cap and seal it again with sealing film, and wrap it with aluminum foil to prevent evaporation. Transfer it to a 55°C constant temperature incubator and react overnight.

[0155] Table 7. Preparation of cDNA Release Mix System

[0156]

[0157]

[0158] Thaw the cDNA Release Buffer at room temperature beforehand. If a white precipitate forms, dissolve it at 55°C.

[0159] (2) Collect the liquid in the reaction well into a 1.5 mL centrifuge tube, and add 55°C preheated Nuclease-Free Water (350 μL / well) to clean the reaction well and chip. The cleaning can be done in two steps. The first cleaning produces more foam, so you need to transfer it patiently and collect it into the same corresponding centrifuge tube. After merging, proceed to the next step.

[0160] 3.8 cDNA Purification and Amplification

[0161] (1) Take out the magnetic beads 30 minutes in advance, vortex to mix thoroughly, and equilibrate to room temperature; mix the above-mentioned recovered liquid with the magnetic beads at a ratio of 1:0.8, vortex to mix, and incubate at room temperature for 30 minutes; centrifuge briefly, place the centrifuge tube on a magnetic rack and let it stand for 3 minutes, and carefully remove the supernatant after the liquid becomes clear; add 1 mL of freshly prepared 80% ethanol equilibrated to room temperature to rinse, operate the pipette tip away from the tube wall of the magnetic beads, do not aspirate or stir the magnetic beads, discard the supernatant, and keep the centrifuge tube on the magnetic rack at all times; repeat the rinsing once, discard the supernatant, and air dry at room temperature for 5-8 minutes until the surface of the magnetic beads is no longer reflective and no cracks; add 42 μL of TE buffer preheated at 55℃ to reconstitute, vortex to mix, and let it stand at room temperature for 10 minutes; centrifuge briefly, and then place the centrifuge tube on a magnetic rack and let it stand for 3-5 minutes, and recover the supernatant after the liquid becomes clear.

[0162] (2) Prepare the PCR Mix according to Table 8. Add 42 μL of the recovered sample. If the amount is less than 42 μL, make up the difference with TE buffer. Add 8 μL of cDNA Primer and 50 μL of cDNA Amplification Mix, for a total of 100 μL. After short-term centrifugation, proceed with the PCR reaction program (100 μL system). Figure 4 ), and PCR products were obtained.

[0163] Table 8. Preparation of PCR Mix

[0164]

[0165] (3) Prepare QubitdsDNA Mix according to Table 9, vortex and mix well, then measure and record the concentration of PCR product (the concentration is usually higher than 5 ng / μL).

[0166] Table 9 QubitdsDNA Mix

[0167]

[0168] (4) Mix the PCR product with the magnetic beads equilibrated at room temperature at a ratio of 1:0.6, vortex to mix, and incubate at room temperature for 10 min; centrifuge briefly, place the centrifuge tube on a magnetic rack and let it stand for 3 min, and carefully remove the supernatant after the liquid becomes clear; add 200 μL of freshly prepared 80% ethanol equilibrated to room temperature to rinse, and operate the pipette tip away from the tube wall of the magnetic beads without agitating or stirring the magnetic beads, discard the supernatant, and keep the centrifuge tube on the magnetic rack at all times; repeat the rinsing once, discard the supernatant, and air dry at room temperature for 5-8 min until the surface of the magnetic beads is no longer reflective and no longer cracked; add 42 μL of TE buffer preheated at 55℃ to reconstitute, vortex to mix, and let it stand at room temperature for 10 min; centrifuge briefly, and then place the centrifuge tube on a magnetic rack and let it stand for 3-5 min, and recover the supernatant after the liquid becomes clear.

[0169] (5) Take 1 μL of cDNA sample and use the QubitdsDNA HS Kit to detect the concentration (the purified yield is usually greater than 20 ng). After labeling the concentration, store the sample at -20℃ or send it to the warehouse using an ice box.

[0170] Test case

[0171] The sequencing libraries constructed in Example 1 and Comparative Example 1 were sequenced, and the obtained sequencing data were analyzed to obtain corresponding visualization heatmaps.

[0172] Depend on Figure 5 It can be seen that the transcripts obtained by the method in Example 1 did not show obvious extra-tissue diffusion, and the tissue outline was clear.

[0173] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a plant tissue spatial transcriptome sequencing library, characterized in that, The method comprises the following steps: (1) fixing and embedding the plant tissue to obtain an embedding block; (2) detecting the autofluorescence of part of the embedding block and performing tissue permeation treatment to determine the autofluorescence threshold and the target tissue permeation time; (3) slicing, patching and fixing the remaining part of the embedding block, and performing microscope fluorescence scanning detection and tissue permeation treatment on the chip containing the tissue according to the autofluorescence threshold and the target tissue permeation time to obtain a permeated tissue chip; (4) performing reverse transcription, tissue removal, cDNA release, recovery and amplification on the permeated tissue chip to obtain a cDNA amplification product. The fixing treatment comprises immersing the plant tissue in a paraformaldehyde solution.

2. The method of claim 1, wherein, The concentration of the paraformaldehyde solution is 0.1-2% by volume. Optionally, the fixing treatment time is 2-3 min.

3. The method of claim 1, wherein, The tissue permeation treatment comprises: adding a pre-permeation reagent to the tissue chip after microscope fluorescence scanning detection, incubating, rinsing the incubated tissue chip with a washing reagent, adding the permeation reagent to the tissue chip, incubating, and then washing the tissue chip after the liquid on the tissue chip is absorbed.

4. The method of claim 3, wherein, The pre-permeation reagent comprises PVP, BSA, RNase inhibitor and EXO I buffer. The permeation reagent comprises PVP, HCl and PR Enzyme. The washing reagent comprises PVP and HCl.

5. The method of claim 4, wherein, Based on the volume of the pre-permeation reagent, the concentration of PVP in the pre-permeation reagent is 1-5% by volume, the concentration of BSA is 0.01-0.1% by volume, and the concentration of RNase inhibitor is 1-5 U / μl. Optionally, based on the volume of the permeation reagent, the concentration of PVP in the permeation reagent is 1-5% by volume, and the concentration of HCl is 0.001-0.1 N. Optionally, based on the volume of the washing reagent, the concentration of PVP in the washing reagent is 1-5% by volume, and the concentration of HCl is 0.001-0.1 N.

6. The method of claim 1, wherein, Step (2) comprises: slicing, patching, fixing and autofluorescence detection of part of the embedding block to determine the autofluorescence threshold; performing tissue permeation treatment on the tissue chip after autofluorescence detection under different tissue permeation time conditions to obtain different permeated tissue chips; respectively performing reverse transcription, tissue removal and microscope fluorescence scanning detection on the different permeated tissue chips, and respectively scoring the signal intensity and diffusion degree of each permeated tissue chip in the microscope fluorescence scanning detection, the score of the signal intensity is A, and the higher the score, the stronger the signal; the score of the diffusion degree is B, and the higher the score, the weaker the diffusion degree; the tissue permeation time corresponding to the permeated tissue chip with the lowest A and B is selected as the target tissue permeation time; Optionally, the slicing, patching and fixing steps in step (2) are consistent with the slicing, patching and fixing steps in step (3). Optionally, the tissue permeation treatment conditions in step (2) are consistent with the tissue permeation treatment conditions in step (3) except for the time.

7. The method of claim 6, wherein, The method for determining the autofluorescence threshold comprises: Set up the fluorescent microscope for epifluorescence, adjust the light intensity and exposure gain, and set to the DAPI channel. Adjust the exposure until no tissue autofluorescence is visible. The exposure value at this point is the autofluorescence threshold.

8. A method for spatial transcriptome sequencing of plant tissues, characterized in that, The method comprises the following steps: constructing a sequencing library by using the method for constructing a plant tissue spatial transcriptome sequencing library according to any one of claims 1-7; sequencing the sequencing library.

9. A kit for plant tissue spatial transcriptome sequencing library, characterized in that, The kit comprises the following reagents: a fixing reagent comprising a paraformaldehyde solution; a pre-permeation reagent comprising PVP, BSA, an RNase inhibitor, and an EXO I buffer; a permeation reagent comprising PVP, HCl, and a PR Enzyme.

10. The kit of claim 9, wherein Based on the volume of the pre-permeation reagent, the concentration of PVP in the pre-permeation reagent is 1-5% by volume, the concentration of BSA is 0.01-0.1% by volume, and the concentration of the RNase inhibitor is 1-5 U / μl; Optionally, based on the volume of the permeation reagent, the concentration of PVP in the permeation reagent is 1-5% by volume, and the concentration of HCl is 0.001-0.1 N; Optionally, the kit further comprises at least one of the following: a reverse transcription reagent, an OCT embedding agent, a staining agent, a cDNA release agent, a tissue removal agent, and an amplification reagent.

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