A spatial whole-transcription sequencing method for tobacco petiole tissue and its application

Abstract

This invention proposes a spatial whole-transcriptional sequencing method and application suitable for tobacco petiole tissue. The method employs tissue embedding and frozen sectioning techniques suitable for tobacco petioles, effectively ensuring uniform and flat sections and maintaining structural integrity. A pretreatment solution suitable for plant tissue permeation effectively enzymatically dissolves plant cell walls and increases cell membrane permeability, facilitating better RNA release. An in-situ polyadenylation treatment method suitable for tobacco petiole tissue effectively polyadenylates non-coding RNA in the sectioned samples, improving non-coding RNA capture efficiency. A method suitable for spatial whole-transcriptional sequencing workflows to remove rRNA from cDNA libraries effectively removes rRNA from the library through wet experimental ends, avoiding reduced sequencing depth for other RNA types due to excessive ribosomal RNA capture, thus greatly improving data utilization. This invention has broad application prospects.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedical technology, and relates to a spatial whole transcriptome sequencing method suitable for tobacco leaf petiole tissue and application thereof. BACKGROUND

[0002] Spatial transcriptomics is a technique for studying gene expression in different cell types and regions within a tissue. In recent years, the rise of spatial transcriptomics has revealed the spatial context of many mammalian gene expressions, such as by identifying different cell populations and spatial structures in tumors, revealing their relationship with tumor development and treatment response. In addition to this, it has also been applied in botany, such as exploring the gene expression patterns in different regions of leaves during photosynthesis, but the range of application on plants is still not comparable to that on mammals, the main reasons being: 1. Plant tissues usually have complex, multi-level differentiated structures, so it is difficult to prepare samples with higher resolution and accuracy. 2. The presence of plant cell walls has certain influence on RNA diffusion and sample fixation steps, and special treatment methods different from animal tissues need to be taken. 3. The genomic data of many plant species is missing or incomplete, which also limits the range and depth of application of spatial transcriptomics in plant tissue research to some extent.

[0003] The current mature spatial transcriptomics method is only limited to determining transcripts with poly-A tails, and is not sensitive to many non-A tail RNAs. It is known that 85% of the regions of plant genomes are transcribed into RNA, including mRNA, miRNA, long ncRNA, snoRNA and other types of RNA molecules, of which mRNA only accounts for 3% of the whole genome, and most other types of RNA molecules are non-coding RNAs. These RNA molecules also have very important regulatory effects on cell function and state. Expanding the scope of plant spatial transcriptomics to whole transcriptome can understand the expression patterns of all genes in plants under different conditions, which will be able to observe the spatial distribution of regulatory RNAs and their targets, link non-host RNAs to host transcription processes, and reveal the interaction relationships and multi-level regulatory networks between genes at different parts and levels, which will help to deeply understand the mechanisms of plant growth and development, environmental adaptation and stress resistance, etc. It may also excavate many unknown functional genes or regulatory factors, thereby expanding the understanding and application fields of plant gene functions. These new genes may affect plant metabolism, signal transduction, protein synthesis and other aspects, and have important biological and agricultural values.

[0004] The application of spatial transcriptome on animal tissues is very mature at present, but there is a big gap between animal and plant tissues. Due to the characteristics of plant cells, such as cell wall, large vacuole and high water content in tissues, it is difficult to slice and permeate the tissues. The same method as animal tissues cannot obtain ideal results. Based on the defects of the spatial transcriptome method, the spatial transcriptome has not been well developed and applied in plants. In summary, it is urgent to develop a spatial whole transcriptome operation method and process suitable for plant tissues.

[0005] At present, the existing 10XVisium spatial transcriptome sequencing technology is widely used in the research of mammalian tissues. This technology can combine RNA sequencing and tissue imaging to realize high-resolution visualization and quantitative analysis of gene expression in space in tissues. However, this technology is developed for mammalian tissues, and the permeation agent mainly composed of pepsin is not suitable for plant tissues with cell walls. Moreover, this method is limited to detecting polyadenylated transcripts, and cannot explore some non-coding RNAs which play an important role in gene expression regulation, signal transmission, metabolic regulation and environmental response. SUMMARY

[0006] In order to solve the problems existing in the prior art, on the basis of a large number of research and experiments, the present application provides a spatial whole transcriptome experimental process / method suitable for tobacco petiole tissues. The method is simple, easy to apply, accurate, comprehensive in detection information, and can be directly combined with the existing 10X Visium spatial transcriptome sequencing process. It can not only make up for the defects of spatial transcriptome technology in the field of botany, but also obtain more detailed cell clustering effect and more comprehensive gene expression information under less sequencing amount.

[0007] The spatial whole transcriptome method suitable for tobacco petiole tissues provided by the present application is a method which can effectively solve the problems of difficult sample slicing and incomplete permeation of tobacco petiole tissues in the process of spatial transcriptome sequencing experiment due to the particularity of the samples on the basis of the conventional 10XVisium spatial transcriptome sequencing process. The in situ polyadenylation method and rRNA removal method provided can effectively detect low-abundance non-coding RNAs in tobacco petiole tissues on the basis of conventional spatial transcriptome sequencing, and realize the method process of spatial whole transcriptome sequencing of tobacco petiole.

[0008] The method comprises the following steps: sample embedding pretreatment, sample embedding, sample tissue sectioning, tissue section fixation and HE staining, tissue section scanning imaging, tissue permeation pretreatment, tissue permeation, in situ polyadenylation pretreatment, in situ polyadenylation, cDNA library construction, rRNA removal, library quality inspection, spatial whole transcriptome sequencing and analysis.

[0009] Since the tobacco petiole belongs to plant tissue, the tissue characteristics are quite different from animal tissues, the tobacco petiole tissue has large vacuoles and cell walls, and the use of a conventional spatial transcriptome process has problems such as difficulty in preparing sample sections and incomplete permeation, in order to solve the problems in the prior art, the technical scheme of the present application makes a series of improvements on the basis of the conventional 10XVisium spatial transcriptome sequencing process: first, the tobacco petiole tissue is pretreated with PVA (polyvinyl alcohol) before sample embedding, and the embedding freezing conditions are adjusted to enhance the uniformity of the tissue and the overall support of the section, thereby ensuring the integrity of the section; before tissue permeation, a permeation pretreatment solution containing Tween 20 is used to loosen the structure of the cell wall and increase the permeability of the cell membrane, thereby effectively improving the release efficiency of RNA during permeation while ensuring the position information of RNA; an in situ polyadenylation step is introduced in the process to achieve in situ capture of non-coding RNA in the tobacco petiole tissue in the spatial process; the tissue is pretreated before in situ polyadenylation, and beta-galactosidase is used to degrade and remove the secondary metabolites in the tobacco petiole, thereby effectively improving the polyadenylation efficiency of non-coding RNA in the sample tissue; an rRNA removal step is introduced in the process, and the synthesized rRNA guide probe is assembled into a complex with Cas9 enzyme, and then the complex is added to the reaction system to make it complementary to the target DNA sequence, and the Cas9 enzyme is positioned on the rRNA sequence to catalyze the double-strand break of the target sequence, so that it is cut into short fragments, thereby effectively achieving the purpose of removing the rRNA sequence.

[0010] The method comprises the following steps:

[0011] Step one, sample embedding pretreatment: take fresh tobacco petiole tissue, gently wipe the surface to remove excess liquid, and pretreat the tobacco petiole tissue before embedding;

[0012] Step two, sample embedding: gently wipe the surface of the pretreated tobacco petiole tissue described in step one to remove excess liquid, place the embedding mold horizontally on ice, inject pre-cooled OCT freezing section embedding agent into 1 / 3 of the mold groove capacity, confirm the direction of the tissue section, perform OCT embedding, horizontally transfer to a -20℃ environment for freezing, and ensure that the OCT and tissue are frozen;

[0013] The OCT refers to an optimal cutting temperature compound commonly used for freezing section embedding, which is a commonly used sample embedding agent in freezing section experiments. The main purpose is to support the tissue during freezing section to increase the continuity of the tissue and reduce wrinkles and fragmentation.

[0014] Step three, sample tissue sectioning: section the embedded tissue sample described in step two, flatten, and place the sample on a regular glass slide. Immediately place your finger on the back of the slide for 5 seconds to thaw the section using the finger temperature, and make the tissue section fully adhere to the glass slide;

[0015] Step four, fix and HE stain the tissue section described in step three;

[0016] Step five, scan and image the section described in step four;

[0017] Step six, tissue permeation pretreatment: attach the tissue section to the Visium spatial gene expression glass slide at the specified position, install the Visium spatial gene expression glass slide in the Slide Cassette (glass slide box), configure the permeation pretreatment solution, and add the pretreatment solution to the hole from the lower left corner of the Slide Cassette sample hole, gently tap the Slide Cassette to evenly cover the tissue with the treatment solution, attach the sealing strip, and place the Slide Cassette in the PCR instrument for pretreatment;

[0018] The Visium spatial gene expression glass slide refers to a highly transparent and chemically stable glass slide designed specifically for Visium spatial transcriptome sequencing. This glass slide has millions of micro-reaction wells that can capture and locate RNA molecules in single cells and tissues and match them with unique barcodes. Each reaction well has a fixed position and size, which can be used to separate and label RNA molecules from different cells or regions and generate high-quality and accurate transcriptome data.

[0019] The Slide Cassette refers to a slide box, which is a container for fixing the Visium spatial gene expression slide containing samples. The slide box can well wrap the slide, and the surface of the slide box has 8 hollow square holes corresponding to 8 sample areas on the slide. The slide box and the slide contain a detachable gasket, which corresponds to the capture area on the slide. After the sample area of the slide is one-to-one corresponding to the hole of the slide box, the slide box is installed to form 8 sealed and leak-free holes to facilitate the addition of reagents to different samples.

[0020] The Slide Cassette sample hole refers to the 8 sealed and leak-free independent sample holes formed after the Slide Cassette slide box is installed with the Visium spatial gene expression slide. The required reagents can be added to the sample holes without contaminating other sample areas.

[0021] Step seven, tissue permeation: after the pretreatment is completed, the pretreatment liquid is carefully removed from the Slide Cassette sample hole, and the washing buffer is added to each Slide Cassette sample hole. After washing, the permeability enzyme is removed, the permeability enzyme is resuspended and preheated, and the permeability enzyme is added to the hole by sticking to the lower left corner of the Slide Cassette sample hole. Lightly tap the Slide Cassette to evenly cover the tissue with the permeability enzyme, stick the sealing strip, place the slide box Slide Cassette containing the Visum spatial gene expression slide on the PCR instrument with the Slide Cassette adapter, cover the PCR instrument cover and incubate for the optimized permeation time. After permeation is completed, remove the sealing strip, remove the permeability enzyme in each hole with a pipette without touching the tissue, and add washing buffer to each hole;

[0022] Step eight, in situ polyadenylation pretreatment: add in situ polyadenylation pretreatment liquid to each Slide Cassette sample hole described in step seven, and remove the pretreatment liquid after incubation;

[0023] Step nine, in situ polyadenylation: add washing buffer to each Slide Cassette sample hole described in step eight, remove the buffer after incubation, add polyadenylate polymerase mixture to each Slide Cassette sample hole, seal the reaction chamber, remove the enzyme mixture after incubation, and add washing buffer to each hole for washing, followed by discarding the washing buffer;

[0024] Step ten, cDNA library construction;

[0025] Step eleven, rRNA removal: prepare in vitro digestion mixture, mix the in vitro digestion mixture and the cDNA library in a molar ratio, incubate at 37 DEG C, add proteinase K in the reaction and incubate at 56 DEG C to release DNA from Cas9 endonuclease, and purify and elute the sample using 0.8x SPRI magnetic beads;

[0026] The molar ratio of Cas9 RNP in the in vitro digestion mixture to the cDNA library is 40:1.

[0027] Step twelve, cDNA library quality inspection;

[0028] Step thirteen, spatial whole transcriptome sequencing and analysis.

[0029] In the method, a pretreatment method / steps suitable for embedding tobacco petiole tissue sections is innovatively proposed. Specifically, the tissue is pretreated by polyvinyl alcohol (PVA) infiltration before embedding the tobacco petiole sample.

[0030] In specific embodiments, the tobacco is 90-100-day-old tobacco, preferably, the tobacco is 90-day-old; the petiole is the petiole at the junction of the leaf base and the stem, and the tissue length is about 0.5-0.8 cm, preferably, the tissue length is 0.5 cm; and the sampling method is transverse sampling.

[0031] The method is to place the tobacco petiole tissue in PVA (PVA covers the tissue) before OCT embedding, and accelerate the penetration of PVA into the tissue in a vacuum instrument.

[0032] The PVA is used at a concentration of 25-35% (v / v), preferably, the PVA is used at a concentration of 25% (v / v).

[0033] The penetration time in the vacuum instrument is 10-15 min, preferably, the penetration time in the vacuum instrument is 10 min.

[0034] In the method, the application of the tobacco petiole tissue section processing method in plant spatial whole transcriptome sequencing is proposed.

[0035] The application further provides a pretreatment solution suitable for tobacco petiole tissue permeation before permeation, and the components of the tobacco petiole tissue permeation pretreatment solution include enzymes used for conventional enzymatic cell wall digestion and Tween 20, and the concentration ranges of the components are specified. The specific components of the tobacco petiole tissue permeation pretreatment solution further include cellulase R10, dissociation enzyme R10, pectinase, hemicellulase, and Tween 20.

[0036] Preferably, the tobacco leaf petiole tissue permeabilization pretreatment liquid formula is: 0.02M MES (2-(N-morpholino) ethanesulfonic acid), 0.02M KCl (potassium chloride), 0.1% (m / v) BSA (bovine serum albumin), 0.01M CaCl2 (calcium chloride), 0.1M mannitol, 0.25-0.5% (m / v) cellulase R10, 0.05%-0.1% (m / v) macerozyme R10, 0.05%-0.2% (m / v) pectinase, 0.1%-0.2% (m / v) hemicellulase, 0.1-0.25% (v / v) Tween 20, 0.4U / μL RNAase inhibitor; preferably, the tobacco leaf petiole tissue permeabilization pretreatment liquid formula is 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.35% (m / v) cellulase R10, 0.1% (m / v) macerozyme R10, 0.1% (m / v) pectinase, 0.15% (m / v) hemicellulase, 0.15% (v / v) Tween 20, 0.4U / μL RNAase inhibitor.

[0037] The present application innovatively proposes that the tobacco leaf petiole tissue permeabilization pretreatment liquid formula contains a Tween 20 component and a specific optimal concentration range thereof, for enhancing cell membrane permeability, which is different from the existing technology of plant tissue enzymolysis.

[0038] The present application also provides application of the tobacco leaf petiole tissue permeabilization pretreatment liquid in tobacco leaf petiole spatial whole transcriptome sequencing.

[0039] The present application also proposes a pretreatment liquid suitable for tobacco leaf petiole tissue in situ polyadenylation in the method, and components of the in situ polyadenylation pretreatment liquid include: β-galactosidase and 1×PBS (phosphate buffer).

[0040] The in situ polyadenylation pretreatment liquid formula is: a final concentration of (0.2-0.5)×β-galactosidase solution (250U / mg β-galactosidase (Sigma / G6008-1KU) diluted with 1×PBS to 40U / μL stock solution); preferably, the in situ polyadenylation pretreatment liquid formula is: a final concentration of 0.2×β-galactosidase solution (250U / mg β-galactosidase (Sigma / G6008-1KU) diluted with 1×PBS to 40U / μL stock solution).

[0041] The foregoing pretreatment liquid and pretreatment step proposed in the method of the present application are innovative and different from the existing technology.

[0042] The method of the present application also includes an in situ polyadenylation step suitable for tobacco leaf petiole spatial whole transcriptome sequencing.

[0043] The present application also provides in situ polyadenylation methods / steps (i.e. steps eight and nine) suitable for whole transcriptome sequencing of tobacco petiole space, comprising the following specific steps:

[0044] Step (9.1), secondary metabolite removal pre-treatment: add the above-mentioned in situ polyadenylation pre-treatment solution to each Slide Cassette sample well, and remove the pre-treatment solution after incubation.

[0045] Step (9.2), in situ polyadenylation: add the washing buffer to each Slide Cassette sample well, and remove the buffer after incubation.

[0046] Step (9.3), add the polyadenylation polymerase mixture to each sample well.

[0047] Step (9.4), seal the reaction chamber, and remove the enzyme mixture after incubation.

[0048] Step (9.5), add the washing buffer to each well for washing.

[0049] Step (9.6), discard the washing buffer in each well.

[0050] The in situ polyadenylation pre-treatment solution in step (9.1) comprises the following components (final concentration): (0.2-0.5) x β-galactosidase (Sigma / G6008-1KU), 1 x PBS; preferably, the final concentration of β-galactosidase is 0.2 x.

[0051] The volume of the polyadenylation pre-treatment solution added to each well in step (9.1) is 70 μl.

[0052] The incubation temperature in step (9.1) is room temperature, 10 min.

[0053] The washing buffer in step (9.2) comprises the following components (final concentration): 1 x polyadenylation polymerase reaction buffer, 0.8 U / μL RNAse inhibitor, ddH2O; preferably, the polyadenylation polymerase reaction buffer is suitable for yeast-derived polymerase reaction buffer.

[0054] The volume of the washing buffer added to each well in step (9.2) is 100 μL.

[0055] The incubation condition in step (9.2) is room temperature, 30 s.

[0056] The polyadenylate polymerase mixture in step (9.3) comprises the following components (final concentration): 1x polyadenylate polymerase reaction buffer, 24 U / μL polyadenylate polymerase, 0.5 mM ATP, 1.5 U / μL RNAase inhibitor; preferably, the polyadenylate polymerase is a yeast-derived polymerase, the polyadenylate polymerase is purchased from Thermo / 74225Z25KU, the polyadenylate polymerase reaction buffer is suitable for yeast-derived polymerase reaction buffer, the polyadenylate polymerase reaction buffer is purchased from Thermo / 74225Z25KU, and the ATP is purchased from Thermo / AM8110G.

[0057] The ATP is adenosine triphosphate, a nucleotide commonly found in living organisms. In cellular life activities, ATP can store and release energy. RNA requires ATP to participate in the process of polyadenylation. ATP provides energy by constantly converting to ADP and drives the polymerase to add adenosine units to the 3' end of mRNA to form a poly(A) tail.

[0058] The volume of the polyadenylate polymerase mixture added per well in step (9.3) is 75 μL.

[0059] The incubation condition in step (9.4) is 37℃, 20 min.

[0060] The washing buffer added in step (9.5) is 0.1x SSC (sodium citrate buffer).

[0061] The volume of the washing buffer added in step (9.5) is 100 μL.

[0062] The washing time in step (9.5) is 10 s.

[0063] In the method of the present application, a step for removing rRNA from a cDNA library suitable for a tobacco leaf petiole spatial whole transcriptome sequencing process is included.

[0064] The present application also provides a method / step (i.e., step eleven) for removing rRNA from a cDNA library suitable for a tobacco leaf petiole spatial whole transcriptome sequencing process. The method comprises the following specific steps:

[0065] Step (11.1), preparation of reagents: IDTE buffer, one-way guide RNA pool, RNP (ribonucleoprotein complex) mixture, in vitro digestion mixture.

[0066] Step (11.2), in vitro digestion: incubate the prepared in vitro digestion mixture.

[0067] Step (11.3), DNA releasing: Proteinase K was added into the reaction after in vitro digestion, and then incubated. After incubation, Cas9 endonuclease was used to release DNA.

[0068] Step (11.4), magnetic bead purification: SPRI magnetic beads were added into the reaction, mixed and incubated. Then, the solution was placed on a magnetic stand until the solution was clear. The supernatant was discarded, and 80% ethanol was added for washing twice with an interval of 30 s. The magnetic beads were placed at room temperature for no more than 2 min to volatilize ethanol on the surface of the magnetic beads, but not too dry. Finally, elution buffer (10 mM Tris-HCl (pH 7.5)) was used for elution.

[0069] The specific components of the reagents prepared in step (11.1) are as follows (final concentration): IDTE buffer: 10 mM Tris-HCl, 0.1 mM EDTA.

[0070] One-way guide RNA pool: 26 synthesized guide RNAs (18sRNA 13, 28sRNA 13) were dissolved in IDTE buffer to make the final concentration of each guide RNA 100 μM. 2 μl of each guide RNA was mixed to make a one-way guide RNA pool with a final concentration of 100 μM. The 100 μM one-way guide RNA pool was diluted to 10 μM for use. Specifically, the sequences of the 26 synthesized guide RNAs are shown in Table 10.

[0071] RNP (ribonucleoprotein complex) mixture: according to the sample amount, the components include 10 μM guide RNA, 20 μM Cas9 nuclease, S. pyogenes (Cas9 endonuclease), and 1x PBS. The guide RNA and Cas9 endonuclease were added at the same concentration, and the rest of the volume was supplemented with 1x PBS. After mixing and incubating at room temperature for 10 min.

[0072] In vitro digestion mixture: according to the sample amount, the components include 10x Cas9 nuclease reaction buffer (NEB / M0386M), 1 μM Cas9 RNP, and sample library. The final molar ratio of Cas9 RNP to sample library is 40:1, and the rest of the volume is supplemented with 10X Cas9 nuclease reaction buffer.

[0073] The components of the reagents in step (11.1) were purchased from Tris HCl (Thermo / 15567027), EDTA (Thermo / 15575020), guide RNA (Shengong Biotechnology), Cas9 nuclease, S. pyogenes (NEB / M0386M), and Cas9 nuclease reaction buffer (NEB / M0386M).

[0074] The incubation temperature condition of the in vitro digestion reaction mixture in step (11.2) is 37℃, 60 min.

[0075] The reagent component in step (11.3) is purchased from protease K (Thermo / 25530049).

[0076] The amount of protease K added in step (11.3) is specifically 1 μL of protease K with a concentration of 20 mg / ml.

[0077] The incubation condition in step (11.3) is 56℃, 10 min.

[0078] The magnetic beads added in step (11.4) are 0.8x SPRI.

[0079] The incubation condition in step (11.4) is specifically 5 min of room temperature incubation.

[0080] In the method of the present application, a step of spatial whole transcriptome suitable for tobacco petiole tissue combined with 10X Visium spatial transcriptome sequencing process is included.

[0081] In one specific embodiment of the present application, a complete method process of spatial whole transcriptome suitable for tobacco petiole tissue combined with 10X Visium spatial transcriptome sequencing process includes the following specific steps:

[0082] Step 1, sample embedding:

[0083] a, take fresh tobacco petiole tissue.

[0084] b, gently wipe the surface to remove excess liquid.

[0085] c, pre-treat the tobacco petiole tissue before embedding.

[0086] d, after pretreatment, gently wipe the surface to remove excess liquid.

[0087] e, place the embedding mold horizontally on ice, and inject the pre-cooled embedding agent into 1 / 3 of the groove capacity of the mold.

[0088] f, confirm the direction of the tissue section, and perform OCT embedding.

[0089] g, horizontally transfer to a-20℃ environment for freezing, and ensure that the OCT and tissue are frozen.

[0090] Step 2, sample tissue sectioning:

[0091] Embedding tissue samples are sectioned, flattened, and placed on a regular glass slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the section using the finger temperature to allow the tissue section to fully adhere to the slide.

[0092] Step 3, Tissue section fixation and HE staining

[0093] Step 4, Section scanning imaging

[0094] Step 5, Tissue permeabilization:

[0095] a. Prepare the tissue permeabilization pre-treatment solution.

[0096] b. Add the pre-treatment solution to the sample well of the Slide Cassette, starting at the lower left corner and working your way across the well. Gently tap the Slide Cassette to ensure the pre-treatment solution evenly covers the tissue. Apply the sealing strip.

[0097] c. Place the Slide Cassette in the PCR machine for pre-treatment.

[0098] d. Once the pre-treatment is complete, carefully remove the pre-treatment solution from the well. Add the wash buffer to each well and remove after washing.

[0099] e. Resuspend and pre-warm the permeabilization enzyme.

[0100] f. Mount the Visium Spatial Gene Expression slide into the Slide Cassette.

[0101] g. Add the permeabilization enzyme to the sample well of the Slide Cassette, starting at the lower left corner and working your way across the well. Ensure the tissue is evenly covered without air bubbles. Gently tap the Slide Cassette to ensure even coverage.

[0102] h. Apply the sealing strip to the Slide Cassette and place it into the PCR machine with the Slide Cassette adapter. Close the PCR machine lid and incubate for the optimized permeabilization time.

[0103] i. Once the permeabilization time is complete, remove the Slide Cassette from the PCR machine adapter and remove the sealing strip.

[0104] j. Remove the permeabilization enzyme from each well using a pipette without touching the tissue.

[0105] k. Add the wash buffer to each well.

[0106] Step 6, In situ polyadenylation:

[0107] a. Secondary metabolite removal pretreatment: Add in situ polyadenylation pretreatment solution to each sample well, remove the pretreatment solution after incubation.

[0108] b. In situ polyadenylation: Add wash buffer to each sample well, remove the buffer after incubation.

[0109] c. Add poly(A) polymerase mixture to each sample well.

[0110] d. Seal the reaction chamber, remove the enzyme mixture after incubation.

[0111] e. Wash each well with wash buffer.

[0112] f. Discard the wash buffer in each well.

[0113] Step 7, Library construction:

[0114] Construct cDNA library according to 10X Visium Spatial Transcriptome Sequencing Protocol.

[0115] Step 8, rRNA removal:

[0116] a. Prepare reagents: IDTE buffer, one-way guide RNA pool, RNP (Ribonucleoprotein complex) mixture, in vitro digestion mixture.

[0117] b. In vitro digestion: Incubate the prepared in vitro digestion mixture.

[0118] c. DNA release: After in vitro digestion, add proteinase K to the reaction, incubate to release DNA from Cas9 endonuclease.

[0119] d. Magnetic bead purification: Add SPRI magnetic beads to the reaction system, mix and incubate, then place on a magnetic stand until the solution is clear, discard the supernatant, wash twice with 80% ethanol, with an interval of 30s, and place at room temperature for no more than 2min, allowing the ethanol on the surface of the magnetic beads to evaporate, but not too dry, finally elute with elution buffer.

[0120] Step 9, cDNA library quality control

[0121] Step 10, Spatial Transcriptome Sequencing and Analysis

[0122] The tobacco in step 1a is 90-100 days old, preferably 90 days old; the petiole is the petiole at the junction of the leaf base and the stem; the length of the sampled tissue is about 0.5-0.8 cm, preferably 0.5 cm; and the sampling method is transverse sampling.

[0123] The pre-treatment of the tobacco leaf petiole tissue before embedding in step 1c is specifically as follows: the tobacco leaf petiole tissue is placed in PVA (PVA covers the tissue), and the PVA is accelerated to penetrate into the tissue in a vacuum instrument. The PVA concentration is 25-35% (v / v), preferably 25% (v / v); and the time for accelerating the penetration in the vacuum instrument is 10-15 min, preferably 10 min.

[0124] The condition for placing the embedding mold horizontally on ice in steps 1f-1g is freezing at -20°C for 30 min.

[0125] The ingredients in step 1 are purchased from PVA (sigma / 475904-M) and OCT (American Sakura / 4583) respectively.

[0126] The contents in steps 2-4 are all performed according to the routine operation of 10X Visium spatial transcriptome sequencing instructions.

[0127] The tissue permeation pre-treatment liquid formula in step 5a contains the following ingredients (final concentration): 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.25-0.5% (m / v) cellulase R10, 0.05%-0.1% (m / v) macerozyme R10, 0.05%-0.2% (m / v) pectinase, 0.1%-0.2% (m / v) hemicellulase, 0.1-0.25% (v / v) Tween 20, 0.4U / μL RNAase inhibitor; preferably, the tissue permeation pre-treatment liquid formula is 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.35% (m / v) cellulase R10, 0.1% (m / v) macerozyme R10, 0.1% (m / v) pectinase, 0.15% (m / v) hemicellulase, 0.15% (v / v) Tween 20, 0.4U / μL RNAase inhibitor.

[0128] The components in step 5a were purchased from MES (sigma / 1266615-59-1), KCl (sigma / P9541-500G), BSA (Mabec / 130-091-376), CaCl2(sigma / 793639-100G), Cellulase R10 (Solarbio / C8260-10g), Lyticase R10 (Polyene / 9032-75-1), Mannitol (Yisheng / 60327ES76), Pectinase (Solarbio / P8181-5G), Hemicellulase (Solarbio / H8112-500g), Tween 20 (sigma / P9416-50ML), RNase Inhibitor (Thermo / AM2694) respectively.

[0129] The pre-treatment condition in step 5c was pre-treatment at 37℃ for 10 min.

[0130] The permeabilization steps in steps 5d-5k were all performed according to the regular operation of 10X Visium Spatial Transcriptome Sequencing Instruction.

[0131] The in situ polyadenylation pre-treatment solution formula in step 6a contains the following components (final concentration): (0.2-0.5) x β-galactosidase, 1 x PBS; preferably, the final concentration of β-galactosidase is 0.2x.

[0132] The components in step 6a were purchased from β-galactosidase (Sigma / G6008-1KU), 1 x PBS (Thermo / 10010031) respectively.

[0133] The volume of polyadenylation treatment solution added per well in step 6a was 70 μL.

[0134] The incubation temperature in step 6b was room temperature, 10 min.

[0135] The washing buffer in step 6b contains the following components (final concentration): 1 x polyadenylation polymerase reaction buffer, 0.8 U / μL RNAse inhibitor, ddH2O; preferably, the polyadenylation polymerase reaction buffer is suitable for yeast-derived polymerase reaction buffer.

[0136] The washing buffer in step 6b was purchased from 5 x polyadenylation polymerase reaction buffer (Thermo / 74225Z25KU), RNAse inhibitor (Roche / 3335402001) respectively.

[0137] The volume of washing buffer added per well in step 6b was 100 μL.

[0138] The incubation condition in step 6b was room temperature, 30 s.

[0139] The poly A polymerase mixture in step 6c contains the following components (final concentration): 1x poly A polymerase reaction buffer, 24 U / µL poly A polymerase, 0.5 mM ATP, 1.5 U / µL RNase inhibitor; preferably, the poly A polymerase is a yeast-derived polymerase.

[0140] The poly A polymerase mixture components in step 6c are purchased from poly A polymerase (Thermo / 74225Z25KU), 5x poly A polymerase reaction buffer (Thermo / 74225Z25KU), ATP (Thermo / AM8110G), respectively.

[0141] The volume of the poly A polymerase mixture added per well in step 6c is 75 µL.

[0142] The incubation condition in step 6d is 37°C, 20 min.

[0143] The washing buffer added in step 6e is 0.1x SSC.

[0144] The volume of the washing buffer added in step 6e is 100 µL.

[0145] The washing time in step 6e is 10 s.

[0146] The library construction step in step 7 is performed according to the routine operation of the 10X Visium Spatial Transcriptome Sequencing Instruction.

[0147] The specific components of the preparation reagent in step 8a are as follows (final concentration): IDTE buffer: 10 mM TrisHCl, 0.1 mM EDTA.

[0148] The one-way guide RNA pool: 26 synthesized guide RNAs (18sRNA 13, 28sRNA 13) are dissolved in IDTE buffer respectively, so that the final concentration of each guide RNA is 100 µM, 2 µl of each guide RNA is mixed to form a 100 µM one-way guide RNA pool, and then the 100 µM one-way guide RNA pool is diluted to 10 µM for use. Specifically, the sequences of the 26 synthesized guide RNAs are shown in Table 10.

[0149] The RNP (Ribonucleoprotein Complex) mixture: according to the sample amount, the components include 10 µM guide RNA, 20 µM Cas9 endonuclease, 1x PBS, wherein the guide RNA and Cas9 nuclease, S. pyogenes (Cas9 endonuclease) are added at the same concentration, and the rest of the volume is supplemented with 1x PBS, and then mixed and incubated at room temperature for 10 min.

[0150] In vitro digestion mixture: according to sample volume configuration, the ingredients include 10x Cas9 nuclease reaction buffer (NEB, M0386M), 1 mM Cas9 RNP and sample library, the final molar ratio of Cas9 RNP to sample library is 40:1, and the rest of the volume is supplemented with 10X Cas9 nuclease reaction buffer.

[0151] The reagent components described in step 8a are purchased from Tris HCl (Thermo / 15567027), EDTA (Thermo / 15575020), guide RNA (Shenguo Biosynthesis), Cas9 nuclease, S. pyogenes (NEB / M0386M), and Cas9 nuclease reaction buffer (NEB / M0386M), respectively.

[0152] The in vitro digestion reaction mixture described in step 8 is incubated at 37°C for 60 min.

[0153] The reagent components described in step 8 are purchased from proteinase K (Thermo / 25530049).

[0154] The amount of proteinase K added in step 8 is specifically 1 μl of proteinase K with a concentration of 20 mg / ml.

[0155] The incubation condition described in step 8 is 56°C for 10 min.

[0156] The magnetic beads added in step 8 are 0.8x SPRI.

[0157] The incubation condition described in step 8 is specifically incubated at room temperature for 5 min.

[0158] In one specific embodiment, the specific steps of the method of the present application are:

[0159] 1. Sample embedding:

[0160] a. Take fresh tobacco petiole tissue, the tissue should not be too large, and make sure that the tissue can be placed into the mold.

[0161] b. Gently absorb the excess liquid such as tissue fluid with dust-free paper, and pay attention not to stick to impurities other than the tissue.

[0162] c. Place the tobacco petiole tissue into 25% PVA (v / v) (PVA covers the tissue), and place it into a vacuum instrument to accelerate the penetration of 25% PVA (v / v) into the tissue for 10 min.

[0163] d. Take the tissue out of the 25% PVA (v / v), and gently wipe off the excess PVA adhered to the surface.

[0164] e. Place the embedding mold horizontally on the table top (operate on ice), inject the pre-cooled embedding medium into the mold cavity to 1 / 3 of its volume, and be careful not to generate air bubbles.

[0165] f. Confirm the direction of the tissue section, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0166] g. Horizontally transfer to a -20°C environment and freeze for 30 min to ensure that the OCT and tissue are frozen.

[0167] 2. Sample tissue sectioning:

[0168] Slice the embedded tissue sample to a thickness of 10 pm, flatten it, and place the sample on the Visium Spatial Gene Expression Slide. Immediately place your finger on the back of the slide for 5 seconds to use the temperature of your finger to thaw the section and allow it to fully adhere to the slide.

[0169] 3. Tissue section fixation and HE staining:

[0170] Follow the conventional 10X Visium Spatial Transcriptome Sequencing workflow.

[0171] 4. Section scanning and imaging

[0172] 5. Tissue permeabilization:

[0173] a. Prepare the tissue permeabilization pretreatment solution, which contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.35% (m / v) cellulase R10, 0.1% (m / v) lyxase R10, 0.1% (m / v) pectinase, 0.15% (m / v) hemicellulase, 0.15% (v / v) Tween 20, and 0.4U / μL RNase inhibitor.

[0174] b. Place the tissue section on the designated position of the Visium Spatial Gene Expression Slide, install the Visium Spatial Gene Expression Slide in the Slide Cassette, and add 70μL of the permeabilization pretreatment solution to the lower left corner of the sample well, gently tap the Slide Cassette to ensure that the treatment solution evenly covers the tissue, and then apply the sealing strip.

[0175] c. Place the PCR instrument adapter in the PCR instrument, place the Slide Cassette on the adapter, and perform pretreatment at 37°C for 10 min.

[0176] d. After pre-treatment is complete, carefully remove the pre-treatment solution from the well, add 100 μΐ of 0.1 x SSC to each well, and remove the pre-treatment solution after 30 seconds.

[0177] e. Resuspend the permeabilization enzyme and equilibrate to 37°C for 15 minutes.

[0178] f. Place the Visium Spatial Gene Expression Slide into the Slide Cassette.

[0179] g. Add 70 μΐ of permeabilization enzyme to each of the four wells, touching the lower left corner, and evenly cover the tissue without creating air bubbles. Gently tap the Slide Cassette to ensure even coverage.

[0180] h. Place the sealing strip on the Slide Cassette, then place the Slide Cassette into the PCR machine with the adapter, cover the machine, and incubate the permeabilization optimization for the selected permeabilization time at 37°C.

[0181] i. After the permeabilization time is complete, remove the Slide Cassette from the PCR machine adapter and remove the sealing strip.

[0182] j. Remove the permeabilization enzyme from each well using a pipette, taking care not to touch the tissue.

[0183] k. Add 100 μΐ of 0.1 x SSC (sodium citrate buffer) to each well.

[0184] 6. In situ polyadenylation:

[0185] a. After permeabilization wash, remove the 0.1 x SSC using a pipette.

[0186] b. Secondary metabolite removal pre-treatment: Dilute 250 U / mg β-galactosidase (Sigma, G6008-1KU) to a 40 U / μΐ^ enzyme solution using 1 x PBS, add 70 μΐ of 0.2 x β-galactosidase solution to each well, and remove the enzyme solution after 10 minutes of incubation at room temperature.

[0187] c. Add 100 μΐ of 1 x wash buffer (20 μΐ of 5 x yPAP reaction buffer, 2 μΐ of RNAse inhibitor at 40 U / μΐ^, 78 μΐ of ddH2O), and remove the buffer after 30 seconds of incubation at room temperature.

[0188] d. Add 75 μL yPAP enzyme (polyadenylate polymerase, yeast-derived, Thermo / 74225Z25KU) mix (5x yPAP reaction buffer (final concentration 1x), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (use Thermo AM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), q.s. to 75 μL with ddH2O) to each sample well.

[0189] e. Then seal the reaction chamber and incubate the Slide Cassette at 37°C for 20 minutes, then remove the enzyme mix.

[0190] f. Add 100 μL 0.1x SSC to each well and wash for 10 s.

[0191] g. Discard the 0.1x SSC from each well.

[0192] h. Follow the subsequent reverse transcription and cDNA amplification steps according to the 10X Visium Spatial Transcriptome Sequencing Instructions.

[0193] 7. Library construction:

[0194] Follow the steps in the 10X Visium Spatial Transcriptome Sequencing Instructions.

[0195] 8. rRNA removal:

[0196] a. Reagent preparation:

[0197] IDTE buffer: 1 M Tris HCl (final concentration 10 mM), 0.5 M EDTA (final concentration 0.1 mM).

[0198] Single-direction guide RNA pool: 26 synthetic guide RNAs (18sRNA 13, 28sRNA 13) were dissolved in IDTE buffer respectively, so that the final concentration of each guide RNA was 100 μM, 2 μL of each guide RNA was mixed to form a guide RNA pool with a final concentration of 100 μM, and then the 100 μM guide RNA pool was diluted to 10 μM for use. The sequences of the 26 synthetic guide RNAs are shown in SEQ ID NO. 1-13 and SEQ ID NO. 14-26 in Table 10.

[0199] RNP (Ribonucleoprotein complex) mix: according to the sample volume, the components include 10 μM guide RNA, 20 μM Cas9 endonuclease, 1x PBS, wherein the guide RNA and Cas9 endonuclease are added at the same concentration, and the rest of the volume is supplemented with 1x PBS. After mixing the RNP mix, incubate at room temperature for 10 min.

[0200] In vitro digestion mixture: according to sample volume configuration, the components include 10X Cas9 nuclease reaction buffer, 1 μM Cas9 RNP and sample library, the final molar ratio of Cas9 RNP to sample library is 40:1, and the rest of the volume is supplemented with 10X Cas9 nuclease reaction buffer.

[0201] b. In vitro digestion:

[0202] Incubate the in vitro digestion reaction mixture at 37°C for 60 min.

[0203] c. DNA release:

[0204] After in vitro digestion, add 1 μL of proteinase K (20 mg / mL) to the reaction and incubate at 56°C for 10 min to release the DNA from the Cas9 endonuclease.

[0205] d. Magnetic bead purification:

[0206] Add 0.8X SPRI magnetic beads to the reaction system, mix well, incubate at room temperature for 5 min, then place in a magnetic stand, discard the supernatant when the solution is clear, wash twice with 80% ethanol, with an interval of 30 s, and place at room temperature for no more than 2 min to evaporate the ethanol on the surface of the magnetic beads, but do not dry too much, and finally elute with 20 μL of elution buffer.

[0207] 9. Library quality control

[0208] 10. Spatial transcriptome sequencing and analysis

[0209] In specific embodiments, the 26 synthetic guide RNAs are designed and synthesized according to the sequences of 18s rRNA and 28s rRNA fragments, and the guide RNA sequences include SEQ ID NO. 1-13 and sequence numbers SEQ ID NO. 14-26, see Table 10.

[0210] The solution for removing rRNA comprises the following components: IDTE buffer: 10 mM Tris HCl, 0.1 mM EDTA; single-direction guide RNA pool: 26 synthetic guide RNAs are respectively dissolved in IDTE buffer to have a final concentration of 100 μM of each guide RNA, 2 μL of each guide RNA is mixed to have a final concentration of 100 μM of guide RNA pool, and the 100 μM guide RNA pool is diluted to 10 μM for use; RNP (ribonucleoprotein complex) mixture: configured according to the sample amount, and the components comprise 10 μM guide RNA, 20 μM Cas9 nuclease, S. pyogenes (Cas9 endonuclease), and 1×PBS, wherein the guide RNA and the Cas9 endonuclease are added at the same concentration, and the rest of the volume is supplemented with 1×PBS, and the mixture is incubated at room temperature for 10 min.

[0211] In-vitro digestion mixture: configured according to the sample amount, and the components comprise 10×Cas9 nuclease reaction buffer (NEB / M0386M), 1 μM Cas9 RNP, and sample library, and the final molar ratio of Cas9 RNP to sample library is 40:1, and the rest of the volume is supplemented with 10X Cas9 nuclease reaction buffer; the proteinase K is added in an amount of 1 μL of proteinase K with a concentration of 20 mg / ml per reaction.

[0212] The application further provides a reagent / kit / product suitable for spatial whole transcriptome sequencing of tobacco petiole, which comprises a tobacco petiole tissue permeation pretreatment solution, an in-situ polyadenylation pretreatment solution, an in-situ polyadenylation washing buffer, a polyadenylate polymerase mixture, and / or an in-vitro digestion mixture.

[0213] Preferably, the tobacco petiole tissue permeation pretreatment solution comprises the following components: 0.02 M MES, 0.02 M KCl, 0.1 % (m / v) BSA, 0.01 M CaCl2, 0.1 M mannitol, 0.25-0.5 % (m / v) cellulase R10, 0.05 %-0.1 % (m / v) macerozyme R10, 0.05 %-0.2 % (m / v) pectinase, 0.1 %-0.2 % (m / v) hemicellulase, 0.1-0.25 % (v / v) Tween 20, and 0.4 U / μL RNase inhibitor.

[0214] The in-situ polyadenylation pretreatment solution comprises the following components: a solution of (0.2-0.5)×β-galactosidase (stock solution concentration: 40 U / μL) with a final concentration.

[0215] The in situ polyadenylation washing buffer comprises the following ingredients (final concentration): 1x Poly(A) Polymerase Reaction Buffer, 0.8 U / μL RNase Inhibitor, ddH2O.

[0216] The poly(A) polymerase mixture comprises the following ingredients (final concentration): 1x Poly(A) Polymerase Reaction Buffer, 24 U / μL Poly(A) Polymerase, 0.5 mM ATP, 1.5 U / μL RNase Inhibitor.

[0217] The in vitro digestion mixture comprises the following ingredients (final concentration): 10 μM single-direction guide RNA pool, 20 μM Cas9 nuclease, S. pyogenes, 1x PBS, 10x Cas9 nuclease reaction buffer, 20 mg / mL proteinase K.

[0218] The application also provides applications of the method, the pretreatment solution, the reagent / kit / product, etc. in plant spatial whole transcriptome sequencing, in tobacco petiole spatial whole transcriptome sequencing.

[0219] Compared with the prior art, the application has the beneficial effects including: an optimized method for freezing sectioning of plant tissues is designed, pretreatment of the tissues before embedding can effectively support the structure of the plant tissues, the optimal freezing time and temperature are set, which are suitable for plant tissues with large vacuoles and high water content, and the damage to the tissue structure during sectioning is avoided, and the shrinkage of the section caused by unsuitable freezing and temperature time is avoided. In the permeation process, pretreatment of the plant tissues before permeation can effectively destroy the cell wall structure, and at the same time, the permeability of the plant cell membrane is enhanced, which is helpful for the release of the subsequent permeated RNA, and solves the problem that the permeation effect of the plant tissues is poor due to the existence of the complete cell wall. After permeation, the step of in situ polyadenylation is introduced, all non-coding RNAs are polyadenylated, the capture of non-coding RNAs is realized, the number of detected non-coding RNAs (rRNA, tRNA, snoRNA, miRNA, snRNA and scaRNA) is effectively increased, the detection efficiency of non-coding RNAs is improved, and the purpose of detecting the spatial full transcriptome information of plants is achieved. At the same time, in this step, beta-galactosidase is used for in situ polyadenylation pretreatment of the sample, which can effectively degrade the secondary metabolites in the plant tissues to avoid the inhibition of the secondary metabolites in the plant tissues on the activity of the polyadenylic acid polymerase, and improve the in situ polyadenylation efficiency. After library construction, the ribosomal RNA removal step is set, which avoids the reduction of the sequencing depth of other RNA types due to the high amount of captured ribosomal RNA, and ensures the effectiveness of the overall data. In summary, the application provides an experimental process suitable for spatial transcriptome sequencing of plant tissues, which can effectively capture various non-coding RNAs, makes up for the defects of plant tissues in spatial transcriptome, and provides a basis for in-depth understanding of the mechanisms of plant growth and development, environmental adaptation and stress resistance, and mining of unknown functional genes or regulatory factors.

[0220] The overall process of the method of the application is improved on the basis of the conventional 10X Visium spatial transcriptome sequencing process, so that it can be applied to plant tissues. Therefore, for tobacco petiole tissues, the overall process of the application is an innovative method, as shown in the following figure: Figure 7 The sample embedding pretreatment, sample embedding, tissue permeation pretreatment, in situ polyadenylation pretreatment, in situ polyadenylation and rRNA removal in the application are steps improved on the basis of the conventional spatial transcriptome process. The innovative improvements of the application include:

[0221] Sample embedding pretreatment: a pretreatment step is added before sample embedding, which is an innovation of the application.

[0222] Sample embedding: the optimal conditions for embedding the tobacco petiole tissue sample are explored.

[0223] The tissue permeation pretreatment: Tween 20 component, and the concentration range of each component of the pretreatment solution is also limited by the present application after testing.

[0224] In situ polyadenylation pretreatment and in situ polyadenylation: this step is proposed by the present application, although polyadenylation is a conventional step in the whole transcriptome sequencing process, but it has not been applied to plant spatial transcriptome, and the conventional polyadenylation method suitable for animal tissues cannot be simply applied to plant tissues, so it does not belong to the prior art; in order to carry out in situ polyadenylation treatment in plant tissue spatial transcriptome, the present application carries out in situ polyadenylation pretreatment according to the characteristics of plant tissue, the pretreatment method is proposed by the present application, which includes the composition, concentration range and treatment condition of the pretreatment solution, which are determined by the present application after testing.

[0225] rRNA removal: although this method is a conventional step in the whole transcriptome sequencing process, the conventional step is to remove rRNA from the RNA step, but in this method, due to the limitation of spatial transcriptome experiment process, rRNA cannot be removed from the RNA step, and rRNA needs to be removed from the cDNA library, and the method described in the present application is the most efficient method for removing rRNA from the cDNA library, which does not belong to the conventional prior art.

[0226] The spatial whole transcript sequencing method suitable for tobacco petiole tissue proposed by the present application can effectively solve the problems of sample slicing difficulty and incomplete permeation caused by the particularity of tobacco petiole in the spatial transcriptome sequencing experiment process, and the in situ polyadenylation method and the rRNA removal method provided in the present application can effectively detect low-abundance non-coding RNA in tobacco petiole on the basis of spatial transcriptome sequencing, realize spatial whole transcriptome sequencing of tobacco petiole, the method is simple, easy to apply, accurate, and can directly combine with 10XVisium spatial transcriptome sequencing process, has long-term stability, can not only make up for the defects of spatial transcriptome technology in the field of botany, but also can obtain more detailed cell clustering effect and more comprehensive gene expression information under less sequencing amount. BRIEF DESCRIPTION OF DRAWINGS

[0227] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiment or prior art description will be briefly introduced below, and obviously, the drawings in the following description can obtain other drawings without creative labor for those skilled in the art.

[0228] Figure 1 The tissue slice quality inspection picture of Example 1 of the present application under an optical microscope.

[0229] Figure 2 Agilent 2100 RNA quality control result picture of tissue section of Example 1 of the present application.

[0230] Figure 3 Agilent 4150 cDNA library quality control picture of Example 9 of the present application.

[0231] Figure 4 Agilent 4150 cDNA product quality control picture of Example 10 of the present application.

[0232] Figure 5 Agilent 4150 cDNA library quality control picture of Example 10 of the present application.

[0233] Figure 6 Different non-coding RNA capture distribution diagram under 50G sequencing amount of Example 105 of the present application.

[0234] Figure 7 Schematic diagram of the method workflow of the present application. DETAILED DESCRIPTION

[0235] The present application will be further described in conjunction with the following specific examples. The process, conditions, experimental methods, etc. for implementing the present application are the general knowledge and common sense in the art, and the present application does not have special limitations.

[0236] Example 1

[0237] 1. Sample embedding:

[0238] a. Take fresh tobacco petiole tissue, the tissue should not be too large, and ensure that the tissue can be placed into the mold.

[0239] b. Gently absorb the excess liquid such as tissue fluid with dust-free paper, and pay attention not to adhere to impurities other than the tissue.

[0240] c. Put the tobacco petiole tissue into 25% PVA (v / v) (PVA covers the tissue), and put it into a vacuum instrument to accelerate the penetration of 25% PVA (v / v) into the tissue for 10 min.

[0241] d. Take the tissue out of 25% PVA (v / v), and gently wipe off the excess PVA adhered to the surface.

[0242] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into 1 / 3 of the volume of the mold groove, and pay attention not to generate bubbles.

[0243] f. Confirm the orientation of the tissue section, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that the OCT fully covers the tissue surface.

[0244] g. Transfer horizontally to a -20°C environment and freeze for 30 minutes to ensure OCT and tissue freezing.

[0245] 2. Sample tissue sections:

[0246] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a regular glass slide. Immediately, a finger was placed on the back of the slide for 5 seconds to thaw the slice using the warmth of the finger, allowing it to fully adhere to the slide.

[0247] The results are as follows Figure 1 , Figure 2 Table 1: Through the technical solution of this invention, 25% PVA (v / v) is pre-infiltrated into the tissue during sample embedding, which can provide good support for the tissue structure. Under an optical microscope, the tissue sections are observed to be relatively uniform and flat, and the tissue structure is basically intact, without large cavities. Figure 1 (Table 1); and RNA extraction quality control was performed on the slides, with RIN values ​​higher than 7 ( Figure 2 Table 1 shows that using 25% PVA (v / v) to pretreat tissues during embedding not only maintains the basic morphological structure of plant slices but also does not affect tissue RNA. The technical solution of this invention has achieved the expected technical effect.

[0248] Example 2

[0249] 1. Sample embedding:

[0250] a. Take fresh tobacco petiole tissue. The tissue should not be too large, just large enough to fit into the mold.

[0251] b. Gently blot away excess liquid, such as tissue fluid, with lint-free paper, being careful not to let any impurities outside the tissue adhere to it.

[0252] c. Place the tobacco petiole tissue in 30% PVA (v / v) (PVA covers the tissue), and place it in a vacuum chamber for 10 minutes to accelerate the penetration of 30% PVA into the tissue.

[0253] d. Remove the tissue from the 30% PVA (v / v) solution and gently wipe away any excess PVA adhering to the surface.

[0254] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into 1 / 3 of the mold groove capacity, taking care not to generate air bubbles.

[0255] f. Confirm the good organization section direction, use tweezers to put the prepared tissue into the mold with OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0256] g. Transfer horizontally to a -20°C environment and freeze for 30 min to ensure that the OCT and tissue are frozen.

[0257] 2. Sample tissue sectioning:

[0258] Slice the embedded tissue sample with a thickness of 10 μm, flatten it, and place the sample on a regular glass slide. Immediately place your finger on the back of the slide for 5 seconds to use the finger temperature to thaw the section and make it fully adhere to the slide.

[0259] The results are shown in Table 1: By the technical scheme of the present application, 30% PVA is pre-permeated into the tissue during embedding, which can provide good support to the tissue structure. The tissue section is observed to be relatively uniform and flat under an optical microscope, and the tissue structure is basically intact without large cavities. The RNA extraction quality test of the section shows that the RIN value is higher than 7 (Table 1), indicating that the use of 30% PVA for pretreatment of the tissue during embedding can maintain the basic morphological structure of the plant section, and has no effect on the tissue RNA. The technical scheme of the present application achieves the desired technical effect.

[0260] Example 3

[0261] 1. Sample embedding:

[0262] a. Take fresh tobacco petiole tissue, and the tissue should not be too large to ensure that it can be placed in the mold.

[0263] b. Gently absorb the excess liquid such as tissue fluid with a dust-free paper, and pay attention not to stick to impurities other than the tissue.

[0264] c. Put the tobacco petiole tissue into 35% PVA (v / v) (PVA covers the tissue), and put it into a vacuum instrument to accelerate the penetration of 35% PVA into the tissue for 15 min.

[0265] d. Take the tissue out of the 35% PVA (v / v), and gently wipe off the excess PVA adhered to the surface.

[0266] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into the mold groove to 1 / 3 of the capacity, and pay attention not to generate bubbles.

[0267] f. Confirm the orientation of the tissue section, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that the OCT fully covers the tissue surface.

[0268] g. Transfer horizontally to a -20°C environment and freeze for 30 minutes to ensure OCT and tissue freezing.

[0269] 2. Sample tissue sections:

[0270] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a regular glass slide. Immediately, a finger was placed on the back of the slide for 5 seconds to thaw the slice using the warmth of the finger, allowing it to fully adhere to the slide.

[0271] The results are shown in Table 1: Through the technical solution of this invention, the sample is pre-embedded with 35% PVA, which can provide good support for the tissue structure. Under an optical microscope, the tissue sections are relatively uniform and flat, and the tissue structure is basically intact without large cavities. Furthermore, the RNA extraction quality inspection of the sections showed that the RIN value was higher than 7 (Table 1). This indicates that the use of 35% PVA to pre-treat the tissue during the embedding process can maintain the basic morphological structure of the plant sections and will not affect the tissue RNA. The technical solution of this invention has achieved the expected technical effect.

[0272] Example 4

[0273] The results of Examples 1, 2, and 3 all show that pre-inserting 25-35% PVA into the tissue during embedding can provide good support for the tissue structure, maintain the basic morphological structure of the plant slices, and have no effect on the tissue RNA. After comparing Examples 1 and 3, although Examples 2 and 3 can provide the same support, they consume more PVA and take longer to process than Example 1. Example 1 is more advantageous in saving time and cost. Therefore, the scheme of Example 1 was adopted to further carry out subsequent experimental steps.

[0274] 1. Sample embedding:

[0275] a. Take fresh tobacco petiole tissue. The tissue should not be too large, just large enough to fit into the mold.

[0276] b. Gently blot away excess liquid, such as tissue fluid, with lint-free paper, being careful not to let any impurities outside the tissue adhere to it.

[0277] c. Place the tobacco petiole tissue in 25% PVA (v / v) (PVA completely covers the tissue), and place it in a vacuum chamber for 10 minutes to accelerate the penetration of 25% PVA (v / v) into the tissue.

[0278] d. Remove the tissue from the 25% PVA (v / v) and gently wipe off the excess PVA adhered to the surface.

[0279] e. Place the embedding mold horizontally on the table (operate on ice), inject the pre-cooled embedding medium into the mold groove to 1 / 3 of the volume, and pay attention not to generate bubbles.

[0280] f. Confirm the direction of the tissue section, place the prepared tissue into the mold containing OCT using tweezers, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0281] g. Horizontally transfer to a -20 °C environment and freeze for 30 min to ensure that the OCT and tissue are frozen.

[0282] 2. Sample tissue sectioning:

[0283] Slice the embedded tissue sample to a thickness of 10 pm, flatten it, and place the sample on a regular glass slide. Immediately place your finger on the back of the slide for 5 seconds to use the temperature of your finger to thaw the section and make it fully adhere to the slide.

[0284] 3. Tissue section fixation and HE staining:

[0285] The procedure is the same as the 10X Visium Spatial Transcriptome Sequencing Instruction Manual.

[0286] 4. Slice scanning imaging

[0287] 5. Tissue permeabilization optimization:

[0288] a. Prepare the permeabilization pretreatment solution, which contains 0.02 M MES, 0.02 M KCl, 0.1% (m / v) BSA, 0.01 M CaCl2, 0.1 M mannitol, 0.5% cellulase R10 (m / v), 0.1% lyase R10 (m / v), 0.2% pectinase (m / v), 0.2% hemicellulase (m / v), 0.25% Tween 20 (v / v), and 0.4 U / μL RNase inhibitor.

[0289] b. Place the tissue section on the specified position of the Visium Spatial Gene Expression Slide, install the Visium Spatial Gene Expression Slide in the Slide Cassette, and add 70 μL of the permeabilization pretreatment solution to the lower left corner of the sample well in the Slide Cassette. Gently tap the Slide Cassette to ensure that the treatment solution evenly covers the tissue, and then attach the sealing strip.

[0290] c. Place the PCR instrument adapter into the PCR instrument, place the Slide Cassette onto the adapter, and pre-treat at 37°C for 10 min.

[0291] d. After pre-treatment, carefully remove the pre-treatment solution from the wells, and add 100 μL of 0.1x SSC to each well. After 30 s, remove the pre-treatment solution.

[0292] e. Subsequently, follow the 10X Visium Spatial Transcriptome Sequencing Instructions for subsequent permeabilization optimization.

[0293] The results are shown in Table 2. The permeabilization optimization section scanning imaging results show that, by the technical scheme of the present application, the fluorescence intensity of the six sections treated for 3-30 min is relatively obvious, the fluorescence signal is strong enough to distinguish from the background noise, there is no excessive exposure, the fluorescence signal is uniformly distributed in the sample area, there is no obvious signal gradient or local missing phenomenon, which meets the permeabilization requirements (Table 2), and the optimal permeabilization time is finally judged to be 18 min. It is shown that the use of the pre-treatment solution before permeabilization can effectively enzymatically hydrolyze the cell wall, and can enhance the permeability of the plant cell membrane, which is helpful for the release of the subsequent permeabilized RNA, meets the subsequent processing conditions, and the technical scheme of the present application achieves the desired technical effect.

[0294] Example 5

[0295] 1. Sample embedding:

[0296] a. Take fresh tobacco petiole tissue, and the tissue should not be too large, so as to ensure that the tissue can be placed into the mold.

[0297] b. Gently absorb the excess liquid such as tissue fluid with a dust-free paper, and pay attention to not adhere to impurities other than the tissue.

[0298] c. Place the tobacco petiole tissue into 25% PVA (v / v) (PVA covers the tissue), and place it into a vacuum instrument to accelerate the penetration of 25% PVA (v / v) into the tissue for 10 min.

[0299] d. Take the tissue out of the 25% PVA (v / v), and gently wipe off the excess PVA adhered to the surface.

[0300] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into the mold groove to 1 / 3 of the capacity, and pay attention to not generate bubbles.

[0301] f. Confirm the tissue section direction, place the prepared tissue into the mold containing OCT with tweezers, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0302] g. Transfer to -20°C environment and freeze for 30 min to ensure OCT and tissue freezing.

[0303] 2. Sample tissue sectioning:

[0304] Section the embedded tissue sample at 10 pm thickness, flatten and place the sample on a regular glass slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the section using the finger temperature to ensure the section is fully adhered to the slide.

[0305] 3. Tissue section fixation and HE staining:

[0306] Follow the same procedure as the 10X Visium Spatial Transcriptomics protocol.

[0307] 4. Section scanning and imaging

[0308] 5. Tissue permeabilization optimization:

[0309] a. Prepare the permeabilization pre-treatment solution containing 0.02 M MES, 0.02 M KCl, 0.1% (m / v) BSA, 0.01 M CaCl2, 0.1 M Mannitol, 0.35% Cellulase R10 (m / v), 0.1% Macerozyme R10 (m / v), 0.1% Pectinase (m / v), 0.15% Hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4 U / pL RNase inhibitor.

[0310] b. Place the tissue section on the Visium Spatial Gene Expression slide at the designated location, install the Visium Spatial Gene Expression slide into the Slide Cassette, and add 70 pL of the permeabilization pre-treatment solution to the well, starting from the lower left corner of the well. Gently tap the Slide Cassette to ensure the solution evenly covers the tissue, and apply the sealing strip.

[0311] c. Place the PCR instrument adapter into the PCR instrument, and place the Slide Cassette onto the adapter. Pre-treat at 37°C for 10 min.

[0312] d. After the pre-treatment is complete, carefully remove the pre-treatment solution from the well, and add 100 pL of 0.1x SSC to each well. After 30 s, remove the pre-treatment solution.

[0313] e. Follow the subsequent permeabilization optimization procedure in the 10X Visium Spatial Transcriptomics protocol.

[0314] The results are shown in Table 2. The permeation optimization section scanning imaging results show that, by the technical scheme of the application, the fluorescence intensity of the six sections treated for 3-30 min is relatively obvious, the fluorescence signal is strong enough, can be distinguished from the background noise, there is no excessive exposure, the fluorescence signal is uniformly distributed in the sample area, there is no obvious signal gradient or local missing phenomenon, which meets the permeation requirements (Table 2), and the final judgment is that the best permeation time is 18 min. It is proved that the use of the pretreatment liquid before permeation can effectively enzymolyze the cell wall, and can enhance the permeability of the plant cell membrane, which is helpful for the release of the subsequent permeated RNA, meets the subsequent processing conditions, and the technical scheme of the application achieves the desired technical effect.

[0315] Example 6

[0316] 1. Sample embedding:

[0317] a. Take fresh tobacco petiole tissue, the tissue should not be too large, and it can be placed in the mold.

[0318] b. Gently absorb the excess liquid such as tissue fluid with a dust-free paper, and pay attention to not adhere to impurities outside the tissue.

[0319] c. Put the tobacco petiole tissue into 25% PVA (v / v) (PVA covers the tissue), and put it into a vacuum instrument to accelerate the penetration of 25% PVA (v / v) into the tissue for 10 min.

[0320] d. Take the tissue out of the 25% PVA (v / v), and gently wipe off the excess PVA adhered to the surface.

[0321] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into the mold groove to 1 / 3 of the capacity, and pay attention to not generate bubbles.

[0322] f. Confirm the direction of the tissue section, use tweezers to put the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0323] g. Horizontally transfer to a-20℃ environment, and freeze for 30 min to ensure that the OCT and the tissue are frozen.

[0324] 2. Sample tissue sectioning:

[0325] Section the embedded tissue sample with a thickness of 10 μm, flatten the sample, and place the sample on a common glass slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the section using the finger temperature, so that the section and the glass slide are fully adhered together.

[0326] 3. Tissue section fixation and HE staining:

[0327] Steps same as 10X Visium Spatial Transcriptome Sequencing Instructions.

[0328] 4. Slice scanning imaging

[0329] 5. Tissue permeabilization optimization:

[0330] a. Configure the permeabilization pretreatment solution, which contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.25% cellulase R10 (m / v), 0.05% macerozyme R10 (m / v), 0.05% pectolase (m / v), 0.1% hemicellulase (m / v), 0.1% Tween 20 (v / v), and 0.4U / μL RNase inhibitor.

[0331] b. Paste the tissue slice on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, and add 70μL of the permeabilization pretreatment solution to the hole from the lower left corner of the sample hole of the Slide Cassette, gently tap the Slide Cassette to make the treatment solution evenly cover the tissue, and paste the sealing strip.

[0332] c. Place the PCR instrument adapter in the PCR instrument, place the Slide Cassette on the adapter, and pre-treat at 37℃ for 15min.

[0333] d. After the pretreatment is completed, carefully remove the pretreatment solution from the hole, and add 100μL of 0.1x SSC to each hole, and remove the pretreatment solution after 30s.

[0334] e. Then follow the 10X Visium Spatial Transcriptome Sequencing Instructions for subsequent permeabilization optimization.

[0335] The results are shown in Table 2. The slice scanning imaging results of the permeabilization optimization show that, through the technical scheme of the present application, the fluorescence intensity of the six slices treated for 3-30min is relatively obvious, the fluorescence signal is strong enough, can be distinguished from the background noise, there is no excessive exposure, the fluorescence signal is uniformly distributed in the sample area, there is no obvious signal gradient or local missing phenomenon, and the permeabilization requirement is met (Table 2). The optimal permeabilization time is finally judged to be 18min. It is shown that the use of the pretreatment solution before permeabilization can effectively enzymatically hydrolyze the cell wall, and can enhance the permeability of the plant cell membrane, which is helpful for the release of the subsequent permeabilized RNA, meets the subsequent treatment conditions, and the technical scheme of the present application achieves the desired technical effect.

[0336] Example 7

[0337] The results of Examples 4, 5 and 6 show that the use of a permeation pretreatment solution including 0.25-0.5% cellulase R10 (m / v), 0.05%-0.1% macerase R10 (m / v), 0.05%-0.2% pectinase (m / v), 0.1%-0.2% hemicellulase (m / v) and 0.1-0.25% Tween 20 (v / v) before permeation can effectively enzymatically hydrolyze the plant cell wall and increase the permeability of the plant cell membrane, which is helpful for the subsequent release of permeated RNA. The optimized fluorescence capture area shows that the use of the permeation pretreatment solution has no effect on the RNA of the tissue. After comparison, although Examples 4 and 6 can have the same pretreatment effect, the enzyme consumption and treatment time are greater than those of Example 5. Therefore, the scheme of Example 5 is adopted for further subsequent experimental steps.

[0338] 1. Sample embedding:

[0339] a. Fresh tobacco petiole tissue is taken. The tissue should not be too large, and it should be ensured that the tissue can be placed into the mold.

[0340] b. The excess liquid, such as tissue fluid, is gently absorbed with a dust-free paper. Attention should be paid to not adhere to impurities other than the tissue.

[0341] c. The tobacco petiole tissue is placed in 25% PVA (v / v) (PVA covers the tissue), and is placed in a vacuum instrument to accelerate the penetration of 25% PVA (v / v) into the tissue for 10 min.

[0342] d. The tissue is taken out of the 25% PVA (v / v), and the excess PVA adhered to the surface is gently wiped off.

[0343] e. The embedding mold is placed horizontally on the table (operated on ice), and the pre-cooled embedding agent is injected into the mold groove to 1 / 3 of the capacity. Attention should be paid to not generate bubbles.

[0344] f. The direction of the tissue section is confirmed, the prepared tissue is placed into the mold containing OCT using tweezers, and the exposed tissue surface is completely covered with OCT. If the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0345] g. The horizontal transfer is performed to a-20°C environment, and the freezing is performed for 30 min to ensure that the OCT and the tissue are frozen.

[0346] 2. Sample tissue sectioning:

[0347] The embedded tissue sample is sectioned to a thickness of 10 μm, and is flattened. The sample is placed on a common glass slide, and the finger is immediately placed on the back of the glass slide for 5 seconds to thaw the section using the temperature of the finger, so that the section and the glass slide are fully adhered together.

[0348] 3. Tissue section fixation and HE staining:

[0349] Steps are the same as 10X Visium Spatial Transcriptome Sequencing Instructions.

[0350] 4. Section scanning imaging

[0351] 5. Tissue permeabilization:

[0352] a. Prepare the permeabilization pre-treatment solution, which contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.35% cellulase R10 (m / v), 0.1% lyticase R10 (m / v), 0.1% pectinase (m / v), 0.15% hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4U / μL RNase inhibitor.

[0353] b. Paste the tissue section on the Visium Spatial Gene Expression Slide at the specified position, install the Visium Spatial Gene Expression Slide in the Slide Cassette, and add 70μL of permeabilization pre-treatment solution to the well from the lower left corner of the sample hole, gently tap the Slide Cassette to evenly cover the tissue with the treatment solution, and paste the sealing strip.

[0354] c. Place the PCR instrument adapter in the PCR instrument, place the Slide Cassette on the adapter, and pre-treat at 37℃ for 10min.

[0355] d. After the pre-treatment is complete, carefully remove the pre-treatment solution from the well, and add 100μL of 0.1x SSC to each well, and remove the pre-treatment solution after 30s.

[0356] e. Resuspend and equilibrate the permeabilization enzyme to 37℃ for 15min.

[0357] f. Place the Visium Spatial Gene Expression Slide in the Slide Cassette.

[0358] g. Add 70μL of permeabilization enzyme to the 4 wells from the lower left corner, evenly cover the tissue without air bubbles, and gently tap the Slide Cassette to ensure uniform coverage.

[0359] h. Paste the sealing strip on the Slide Cassette, then place the Slide Cassette in the PCR instrument with the adapter at 37℃, cover the PCR instrument cover, and incubate the permeabilization optimization selected permeabilization time.

[0360] i. After the permeation time is complete, remove the Slide Cassette from the PCR instrument adapter and remove the sealing strip.

[0361] j. Remove the permeation enzyme from each well with a pipette, taking care not to touch the tissue.

[0362] k. Add 100 μΐ of 0.1 x SSC (sodium citrate buffer) to each well.

[0363] 6. In situ polyadenylation:

[0364] a. After the permeation wash, remove the 0.1 x SSC with a pipette.

[0365] b. Secondary metabolite removal pretreatment: Dilute 250 U / mg β-galactosidase (Sigma, G6008-1 KU) to 40 U / μL enzyme solution with 1 x PBS, add 70 μΐ of 0.2 x β-galactosidase solution to each well, and remove the enzyme solution after 10 min of incubation at room temperature.

[0366] c. Add 100 μΐ of 1 x wash buffer (20 μΐ of 5 x yPAP reaction buffer, 2 μΐ of RNase inhibitor at 40 U / μl, 78 μΐ of nuclease-free H2O), and remove the buffer after 30 s of incubation at room temperature.

[0367] d. Add 75 μΐ of yPAP enzyme (yeast-derived poly(A) polymerase, Thermo 74225Z25 KU) mix (5 x yPAP reaction buffer (final concentration 1 x), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (use Thermo AM8110G, final concentration 0.5 mM), 40 U / μL RNAse inhibitor (final concentration 1.5 U / μL), and ddH2O to 75 μΐ to each sample well.

[0368] e. Then seal the reaction chamber, and incubate the Slide Cassette at 37 °C for 20 min, and then remove the enzyme mix.

[0369] f. Add 100 μΐ of 0.1 x SSC to each well for 10 s of washing.

[0370] g. Discard the 0.1 x SSC from each well.

[0371] h. Follow the subsequent reverse transcription and cDNA amplification steps according to the 10X Visium Spatial Transcriptome Sequencing Instructions.

[0372] The results are shown in Table 3. The quality inspection results after cDNA amplification show that the quality of cDNA and library meets the requirements of space transcriptome sequencing. The non-coding RNA capture efficiency is significantly improved compared with Comparative Examples 30-31 under 50G sequencing amount (Table 3), indicating that the use of 0.2 x beta-galactosidase can effectively degrade secondary metabolites in plant tissues, improve in situ polyadenylation efficiency, meet the subsequent processing conditions, and the technical scheme of the present application achieves the expected technical effect.

[0373] Example 8

[0374] 1. Sample embedding:

[0375] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be ensured that the tissue can be placed in the mold.

[0376] b. Gently absorb the excess liquid such as tissue fluid with dust-free paper. Be careful not to stick impurities other than the tissue.

[0377] c. Put the tobacco petiole tissue into 25% PVA (v / v) (PVA covers the tissue), and put it into a vacuum instrument to accelerate the penetration of 25% PVA (v / v) into the tissue for 10 min.

[0378] d. Take the tissue out of the 25% PVA (v / v), and gently wipe off the excess PVA adhered to the surface.

[0379] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into the mold groove to 1 / 3 of the capacity. Be careful not to generate bubbles.

[0380] f. Confirm the direction of the tissue section, and use tweezers to put the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface. If the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0381] g. Horizontally transfer to a-20℃ environment and freeze for 30 min to ensure that the OCT and tissue are frozen.

[0382] 2. Sample tissue sectioning:

[0383] Slice the embedded tissue sample with a thickness of 10 μm, flatten it, and place the sample on a regular glass slide. Immediately place your finger on the back of the slide for 5 seconds to use the finger temperature to thaw the section and make it fully adhere to the slide.

[0384] 3. Tissue section fixation and HE staining:

[0385] The steps are the same as the 10X Visium space transcriptome sequencing instructions.

[0386] 4. Slice scanning imaging

[0387] 5. Tissue permeabilization:

[0388] a. Prepare the permeabilization pre-treatment solution containing 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M Mannitol, 0.35% Cellulase R10 (m / v), 0.1% Macerozyme R10 (m / v), 0.1% Pectolase (m / v), 0.15% Hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4U / μL RNase inhibitor.

[0389] b. Place the tissue section on the Visium Spatial Gene Expression Slide at the designated location, place the Visium Spatial Gene Expression Slide in the Slide Cassette, add 70μL of the permeabilization pre-treatment solution to the well, starting at the lower left corner of the well, tap the Slide Cassette to ensure the solution covers the tissue evenly, and apply the sealing strip.

[0390] c. Place the PCR instrument adapter in the PCR instrument, place the Slide Cassette on the adapter, and pre-treat at 37°C for 10 minutes.

[0391] d. After the pre-treatment is complete, carefully remove the pre-treatment solution from the well, add 100μL of 0.1x SSC to each well, and remove the pre-treatment solution after 30 seconds.

[0392] e. Resuspend the permeabilization enzymes and equilibrate to 37°C for 15 minutes.

[0393] f. Place the Visium Spatial Gene Expression Slide in the Slide Cassette.

[0394] g. Add 70μL of the permeabilization enzymes to the four wells, starting at the lower left corner, and ensure the solution covers the tissue evenly without air bubbles, tap the Slide Cassette to ensure the solution covers the tissue evenly.

[0395] h. Apply the sealing strip to the Slide Cassette, then place the Slide Cassette in the PCR instrument with the adapter at 37°C, cover the PCR instrument, and incubate the tissue for the permeabilization optimization selected permeabilization time.

[0396] i. After the permeabilization time is complete, remove the Slide Cassette from the PCR instrument adapter and remove the sealing strip.

[0397] j. Remove the permeabilization enzymes from each well using a pipette, taking care not to touch the tissue.

[0398] k. Add 100 μL of 0.1x SSC (sodium citrate buffer) to each well.

[0399] 6. In situ polyadenylation:

[0400] a. After permeabilization wash, use a pipette to remove 0.1x SSC.

[0401] b. Secondary metabolite removal pretreatment: Dilute 250 U / mg β-galactosidase (Sigma, G6008-1KU) to 40 U / μL enzyme solution with 1x PBS, add 70 μL of 0.5x β-galactosidase solution to each well, and remove the enzyme solution after incubation at room temperature for 10 min.

[0402] c. Add 100 μL of 1x wash buffer (20 μL of 5x yPAP reaction buffer, 2 μl of 40 U / μL RNase inhibitor, 78 μL of nuclease-free H2O), and remove the buffer after incubation at room temperature for 30 s.

[0403] d. Add 75 μL of yPAP enzyme (yeast-derived poly(A) polymerase, Thermo 74225Z25KU) mixture (5x yPAP reaction buffer (final concentration 1x), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (use Thermo AM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), and supplement with ddH2O to 75 μL to each sample well.

[0404] e. Then seal the reaction chamber, and incubate the Slide Cassette at 37°C for 20 min, and then remove the enzyme mixture.

[0405] f. Add 100 μL of 0.1x SSC to each well and wash for 10 s.

[0406] g. Discard 0.1x SSC in each well.

[0407] h. Subsequently, follow the subsequent reverse transcription and cDNA amplification steps according to the 10X Visium Spatial Transcriptome Sequencing Instructions.

[0408] The results are shown in Table 3. The quality inspection results after cDNA amplification show that the quality of cDNA and the library meets the requirements of spatial transcriptome sequencing, and the capture efficiency of non-coding RNA is significantly improved compared with Comparative Examples 30-31 under the condition of 50G sequencing amount (Table 3), indicating that the use of 0.5x β-galactosidase can effectively degrade the secondary metabolites in plant tissues, improve the in situ polyadenylation efficiency, meet the subsequent processing conditions, and the technical scheme of the present application achieves the expected technical effect.

[0409] Example 9

[0410] Both Example 7 and Example 8 show that using a pre-treatment solution containing (0.2-0.5) x β-galactosidase before in situ polyadenylation can effectively degrade secondary metabolites in plant tissues, improving the efficiency of in situ polyadenylation. After comparison, Example 8 can also degrade secondary metabolites, but the efficiency is not significantly different from Example 7. The lower dosage of Example 7 is more conducive to cost savings, so the scheme of Example 7 is adopted for further subsequent experimental steps.

[0411] 1. Sample embedding:

[0412] a. Take fresh tobacco petiole tissue, the tissue should not be too large, and ensure that the tissue can be placed in the mold.

[0413] b. Gently absorb the excess liquid such as tissue fluid with dust-free paper, and pay attention not to stick to impurities outside the tissue.

[0414] c. Put the tobacco petiole tissue into 25% PVA (v / v) (PVA covers the tissue), and put it into a vacuum instrument to accelerate the penetration of 25% PVA (v / v) into the tissue for 10 minutes.

[0415] d. Take the tissue out of the 25% PVA (v / v), and gently wipe off the excess PVA adhered to the surface.

[0416] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into the mold groove to 1 / 3 of the capacity, and pay attention not to generate bubbles.

[0417] f. Confirm the direction of the tissue section, and use tweezers to put the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, you can add more OCT to ensure that the OCT fully covers the tissue surface.

[0418] g. Horizontally transfer to a -20°C environment and freeze for 30 minutes to ensure that the OCT and tissue are frozen.

[0419] 2. Sample tissue sectioning:

[0420] Slice the embedded tissue sample to a thickness of 10 μm, flatten it, and place the sample on a regular glass slide. Immediately place your finger on the back of the slide for 5 seconds to use the finger temperature to thaw the section and make it fully adhere to the slide.

[0421] 3. Tissue section fixation and HE staining:

[0422] The steps are the same as the 10X Visium spatial transcriptome sequencing instructions.

[0423] 4. Slice scanning imaging

[0424] 5. Tissue permeabilization:

[0425] a. Prepare the permeabilization pre-treatment solution with the following formula: 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M Mannitol, 0.35% Cellulase R10 (m / v), 0.1% Macerozyme R10 (m / v), 0.1% Pectolase (m / v), 0.15% Hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4U / μL RNase inhibitor.

[0426] b. Place the tissue slice on the Visium Spatial Gene Expression Slide at the designated position, install the Visium Spatial Gene Expression Slide in the Slide Cassette, add 70μL of the permeabilization pre-treatment solution to the well from the lower left corner, tap the Slide Cassette to ensure the solution covers the tissue evenly, and apply the sealing strip.

[0427] c. Place the PCR instrument adapter in the PCR instrument, and place the Slide Cassette on the adapter, and pre-treat at 37℃ for 10min.

[0428] d. After the pre-treatment, carefully remove the pre-treatment solution from the well, and add 100μL of 0.1x SSC to each well, and remove the pre-treatment solution after 30s.

[0429] e. Resuspend the permeabilization enzyme and equilibrate to 37℃ for 15min.

[0430] f. Place the Visium Spatial Gene Expression Slide in the Slide Cassette.

[0431] g. Add 70μL of the permeabilization enzyme to the four wells from the lower left corner, and ensure the tissue is evenly covered without air bubbles, and tap the Slide Cassette to ensure the even coverage.

[0432] h. Apply the sealing strip to the Slide Cassette, and then place the Slide Cassette in the PCR instrument with the adapter at 37℃, cover the PCR instrument, and incubate the tissue for the permeabilization optimization selected permeabilization time.

[0433] i. After the permeabilization time, remove the Slide Cassette from the PCR instrument adapter, and remove the sealing strip.

[0434] j. Use a pipette to remove the permeabilization enzyme from each well, and ensure the tissue is not touched.

[0435] k. Add 100 pL 0.1x SSC (sodium citrate buffer) to each well.

[0436] 6. In situ polyadenylation:

[0437] a. After permeabilization wash, pipette 0.1x SSC away.

[0438] b. Secondary metabolite removal pretreatment: Dilute 250 U / mg b-galactosidase (Sigma, G6008-1KU) to 40 U / pL enzyme solution with 1x PBS, add 70 pL 0.2x b-galactosidase solution to each well, and remove the enzyme solution after 10 min incubation at room temperature.

[0439] c. Add 100 pL 1x wash buffer (20 pL 5x yPAP reaction buffer, 2 pL RNase inhibitor at 40 U / pL, 78 pL ddH2O), and remove the buffer after 30 s incubation at room temperature.

[0440] d. Add 75 pL yPAP enzyme (yeast-derived poly(A) polymerase, Thermo 74225Z25KU) mix (5x yPAP reaction buffer (final concentration 1x), 600 U / pL yPAP enzyme (final concentration 24 U / pL), 10 mM ATP (use Thermo AM8110G, final concentration 0.5 mM), 40 U / pL RNase inhibitor (final concentration 1.5 U / pL), and supplement with ddH2O to 75 pL to each sample well.

[0441] e. Then seal the reaction chamber, and incubate the Slide Cassette at 37 °C for 20 min, and then remove the enzyme mix.

[0442] f. Add 100 pL 0.1x SSC to each well and wash for 10 s.

[0443] g. Discard 0.1x SSC from each well.

[0444] h. Follow the subsequent reverse transcription and cDNA amplification steps according to the 10X Visium Spatial Transcriptome Sequencing Instructions.

[0445] 7. Library construction:

[0446] Follow the steps in the 10X Visium Spatial Transcriptome Sequencing Instructions.

[0447] 8. rRNA removal:

[0448] a. Reagent preparation:

[0449] IDTE buffer: 1M Tris HC1 (final concentration 10 mM), 0.5M EDTA (final concentration 0.1 mM).

[0450] One-way guide RNA pool: 26 synthetic guide RNAs (18sRNA 13, 28sRNA 13) were dissolved in IDTE buffer respectively, so that the final concentration of each guide RNA was 100 μM, 2 μL of each guide RNA was mixed to form a 100 μM one-way guide RNA pool, and the 100 μM one-way guide RNA pool was diluted to 10 μM for use. The sequences of the 26 synthetic guide RNAs are shown in Table 10.

[0451] RNP (Ribonucleoprotein complex) mixture: according to the sample amount, the components include 10 μM guide RNA, 20 μM Cas9 endonuclease, and 1x PBS, wherein the guide RNA and Cas9 endonuclease are added at the same concentration, and the rest of the volume is supplemented with 1x PBS. After the RNP mixture is mixed, it is incubated at room temperature for 10 min.

[0452] In vitro digestion mixture: according to the sample amount, the components include 10X Cas9 nuclease reaction buffer, 1 μM Cas9 RNP and sample library, and the final molar ratio of Cas9 RNP to sample library is 40:1, and the rest of the volume is supplemented with 10X Cas9 nuclease reaction buffer.

[0453] b. In vitro digestion:

[0454] The in vitro digestion reaction mixture is incubated at 37°C for 60 min.

[0455] c. DNA release:

[0456] After in vitro digestion, 1 μL of proteinase K (20 mg / mL) is added to the reaction, and incubated at 56°C for 10 min to release the DNA from the Cas9 endonuclease.

[0457] d. Magnetic bead purification:

[0458] 1x SPRI magnetic beads are added to the reaction system, mixed and incubated at room temperature for 5 min, then placed in a magnetic stand until the solution is clear, the supernatant is discarded, washed twice with 80% ethanol, with an interval of 30 s, and placed at room temperature for no more than 2 min to evaporate the ethanol on the surface of the magnetic beads, but not too dry, and finally eluted with 20 μL of elution buffer.

[0459] 9. Library quality control

[0460] 10. Spatial transcriptome sequencing and analysis

[0461] The results are as follows Figure 3As shown in Table 4, by the technical scheme of the present application, the library quality inspection result after removing ribosomal RNA shows that, in addition to the main fragment of 400-500 bp of the library, there is also a small fragment below 200 bp ( Figure 3 ), the plant spatial whole transcriptome sequencing result shows that, non-coding RNA (rRNA, tRNA, snoRNA, miRNA, snRNA, scaRNA) is successfully detected, the capture rate of rRNA is less than 10%, and the capture efficiency of the rest of the low-abundance non-coding RNA is significantly improved compared with Comparative Examples 32-33 (Table 4), and the purpose of plant spatial whole transcriptome is preliminarily achieved. The technical scheme of the present application achieves the expected technical effect.

[0462] Example 10

[0463] 1. Sample embedding:

[0464] a. Take fresh tobacco petiole tissue, and the tissue should not be too large, so as to ensure that the tissue can be placed into the mold.

[0465] b. Gently absorb the excess liquid such as tissue fluid with dust-free paper, and pay attention to not adhere to impurities other than the tissue.

[0466] c. Put the tobacco petiole tissue into 25% PVA (v / v) (PVA covers the tissue), and put it into a vacuum instrument to accelerate the penetration of 25% PVA (v / v) into the tissue for 10 min.

[0467] d. Take the tissue out of the 25% PVA (v / v), and gently wipe off the excess PVA adhered to the surface.

[0468] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into the mold groove to 1 / 3 of the capacity, and pay attention to not generate bubbles.

[0469] f. Confirm the direction of the tissue section, use tweezers to put the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0470] g. Horizontally transfer to a-20℃ environment, and freeze for 30 min to ensure that the OCT and the tissue are frozen.

[0471] 2. Sample tissue sectioning:

[0472] Slice the embedded tissue sample with a thickness of 10 μm, flatten it, and place the sample on a common glass slide. Immediately place your finger on the back of the slide for 5 seconds to use the temperature of the finger to thaw the section and make it fully adhere to the slide.

[0473] 3. Tissue section fixation and HE staining:

[0474] Steps are the same as the 10X Visium Spatial Transcriptome Sequencing Instruction.

[0475] 4. Slice Scanning Imaging

[0476] 5. Tissue Permeabilization:

[0477] a. Prepare the permeabilization pre-treatment solution, which contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M Mannitol, 0.35% Cellulase R10 (m / v), 0.1% Macerozyme R10 (m / v), 0.1% Pectolase (m / v), 0.15% Hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4U / μL RNase Inhibitor.

[0478] b. Paste the tissue slice on the Visium Spatial Gene Expression Slide at the designated position, install the Visium Spatial Gene Expression Slide in the Slide Cassette, add 70μL of the permeabilization pre-treatment solution to the well from the lower left corner of the well, tap the Slide Cassette to ensure the solution evenly covers the tissue, and paste the sealing strip.

[0479] c. Place the PCR instrument adapter in the PCR instrument, place the Slide Cassette on the adapter, and pre-treat at 37℃ for 10min.

[0480] d. After the pre-treatment is complete, carefully remove the pre-treatment solution from the well, add 100μL of 0.1x SSC to each well, and remove the pre-treatment solution after 30s.

[0481] e. Resuspend and equilibrate the permeabilization enzymes to 37℃ for 15min.

[0482] f. Place the Visium Spatial Gene Expression Slide in the Slide Cassette.

[0483] g. Add 70μL of permeabilization enzymes to the four wells from the lower left corner, evenly cover the tissue without air bubbles, and tap the Slide Cassette to ensure uniform coverage.

[0484] h. Paste the sealing strip on the Slide Cassette, then place the Slide Cassette in the PCR instrument with the adapter at 37℃, cover the PCR instrument, and incubate the permeabilization for the selected permeabilization time.

[0485] i. After the permeabilization time is complete, remove the Slide Cassette from the PCR instrument adapter and remove the sealing strip.

[0486] j. Remove the permeabilization enzyme from each well with a pipette, taking care not to touch the tissue.

[0487] k. Add 100 pL of 0.1 x SSC (sodium citrate buffer) to each well.

[0488] 6. In situ polyadenylation:

[0489] a. After permeabilization wash, remove 0.1 x SSC with a pipette.

[0490] b. Secondary metabolite removal pretreatment: Dilute 250 U / mg b-galactosidase (Sigma, G6008-1KU) to 40 U / pL enzyme solution with 1 x PBS, add 70 pL of 0.2 x b-galactosidase solution to each well, and remove the enzyme solution after 10 min incubation at room temperature.

[0491] c. Add 100 pL of 1 x wash buffer (20 pL of 5 x yPAP reaction buffer, 2 pL of RNAse inhibitor at 40 U / pL, 78 pL of ddH2O), and remove the buffer after 30 s incubation at room temperature.

[0492] d. Add 75 pL of yPAP enzyme (yeast-derived poly(A) polymerase, Thermo 74225Z25KU) mix (5 x yPAP reaction buffer (final concentration 1 x), 600 U / pL yPAP enzyme (final concentration 24 U / pL), 10 mM ATP (use Thermo AM8110G, final concentration 0.5 mM), 40 U / pL RNAse inhibitor (final concentration 1.5 U / pL), and supplement with ddH2O to 75 pL to each sample well.

[0493] e. Then seal the reaction chamber, and incubate the Slide Cassette at 37 °C for 20 min, and then remove the enzyme mix.

[0494] f. Add 100 pL of 0.1 x SSC to each well for 10 s wash.

[0495] g. Discard the 0.1 x SSC from each well.

[0496] h. Follow the subsequent reverse transcription and cDNA amplification steps according to the 10X Visium Spatial Transcriptomics Sequencing Instructions.

[0497] 7. Library construction:

[0498] Follow the steps as per the 10X Visium Spatial Transcriptomics Sequencing Instructions.

[0499] 8. rRNA removal:

[0500] a. Reagent preparation:

[0501] IDTE buffer: 1M Tris HC1 (final concentration 10 mM), 0.5M EDTA (final concentration 0.1 mM).

[0502] Single guide RNA pool: 26 synthetic guide RNAs (18sRNA 13, 28sRNA 13) were dissolved in IDTE buffer respectively, so that the final concentration of each guide RNA was 100 μM, 2 μL of each guide RNA was mixed to form a single guide RNA pool with a final concentration of 100 μM, and then the 100 μM single guide RNA pool was diluted to 10 μM for use. The sequences of the 26 synthetic guide RNAs are shown in Table 10.

[0503] RNP (Ribonucleoprotein complex) mixture: according to the sample amount, the components include 10 μM guide RNA, 20 μM Cas9 endonuclease, and 1x PBS, wherein the guide RNA and Cas9 endonuclease are added at the same concentration, and the rest of the volume is supplemented with 1x PBS. After mixing the RNP mixture, incubate at room temperature for 10 min.

[0504] In vitro digestion mixture: according to the sample amount, the components include 10X Cas9 nuclease reaction buffer, 1 μM Cas9 RNP and sample library, and the final molar ratio of Cas9 RNP to sample library is 40:1, and the rest of the volume is supplemented with 10X Cas9 nuclease reaction buffer.

[0505] b. In vitro digestion:

[0506] Incubate the in vitro digestion reaction mixture at 37°C for 60 min.

[0507] c. DNA release:

[0508] After in vitro digestion, add 1 μL of proteinase K (20 mg / mL) to the reaction, and incubate at 56°C for 10 min to release the DNA from the Cas9 endonuclease.

[0509] d. Magnetic bead purification:

[0510] Add 0.8x SPRI magnetic beads to the reaction system, mix well, and incubate at room temperature for 5 min, then place it in a magnetic stand, wait for the solution to be clear, discard the supernatant, wash twice with 80% ethanol, with an interval of 30 s, and place it at room temperature for no more than 2 min to evaporate the ethanol on the surface of the magnetic beads, but do not dry too much, and finally elute with 20 μL of elution buffer.

[0511] 9. Library quality control

[0512] 10. Spatial transcriptome sequencing and analysis

[0513] The results are as follows Figure 4 , 5 As shown in Tables 6 and 4, the cDNA quality control results obtained using the technical solution of this invention show that the main peak of the cDNA product is at 1283 bp (…). Figure 4 The library quality control results showed that the main peak of the library was between 400 and 500 bp. Figure 5 Plant spatial whole transcriptome sequencing results showed that non-coding RNAs (rRNA, tRNA, snoRNA, miRNA, snRNA, scaRNA) were successfully detected. The rRNA capture rate was less than 10%, and the capture efficiency of the other low-abundance non-coding RNAs was significantly improved compared with comparative examples 32-33. Figure 6 This invention has initially achieved the goal of complete spatial rotation of plants. The technical solution of this invention has achieved the expected technical effects.

[0514] Comparative Example 1

[0515] This comparative example first uses the standard embedding and slicing procedures in the 10X Visium spatial transcriptome sequencing manual to prepare tissue samples from tobacco petioles.

[0516] 1. Sample embedding

[0517] a. Take fresh tobacco petiole tissue. The tissue should not be too large, just large enough to fit into the mold.

[0518] b. Gently blot away excess liquid, such as tissue fluid, with lint-free paper, being careful not to let any impurities outside the tissue adhere to it.

[0519] c. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into 1 / 3 of the mold groove capacity, taking care not to generate air bubbles.

[0520] d. Confirm the orientation of the tissue section, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that the OCT fully covers the tissue surface.

[0521] e. Transfer horizontally to dry ice and freeze for 30 minutes to ensure that the OCT and tissue are completely frozen.

[0522] 2. Sample tissue sections

[0523] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a regular glass slide. Immediately, a finger was placed on the back of the slide for 5 seconds to thaw the slice using the warmth of the finger, allowing it to fully adhere to the slide.

[0524] The results are shown in Table 5. Under optical microscope, it was observed that the tissue section had serious shrinkage, the parenchyma was damaged, there was local fracture, and a large number of tissue cavities appeared, which could not guarantee the original complete structure of the plant tissue.

[0525] Comparative Example 2

[0526] In this comparative example, the conventional embedding and sectioning process in the 10X Visium Spatial Transcriptome Sequencing Instructions was used for tissue sample preparation of tobacco petiole, and the freezing time was shortened to 20 min, and the other steps were consistent with those of Comparative Example 1.

[0527] 1. Sample embedding

[0528] a. Fresh tobacco petiole tissue was taken, and the tissue should not be too large, so that it could be placed in the mold.

[0529] b. The excess liquid such as tissue fluid was gently absorbed with a dust-free paper, and attention should be paid to not adhere to impurities other than the tissue.

[0530] c. The embedding mold was placed horizontally on the table (operated on ice), and the pre-cooled embedding agent was injected into the mold groove at 1 / 3 of the capacity, and attention should be paid to not generate bubbles.

[0531] d. The direction of the tissue section was confirmed, the prepared tissue was placed into the mold containing OCT using tweezers, and the exposed tissue surface was completely covered with OCT; if the tissue was large, more OCT could be added to ensure that the OCT fully covered the tissue surface.

[0532] e. It was transferred horizontally to dry ice for freezing for 20 min to ensure that the OCT and the tissue were completely frozen.

[0533] 2. Sample tissue sectioning

[0534] The embedded tissue sample was sectioned to a thickness of 10 pm, flattened, and placed on a conventional glass slide. The finger was immediately placed on the back of the slide for 5 seconds to thaw the section using the finger temperature, so that it was fully adhered to the glass slide.

[0535] The results are shown in Table 5. Under optical microscope, it was observed that the tissue section had serious shrinkage, the parenchyma was damaged, there was local fracture, and a large number of tissue cavities appeared, which could not guarantee the original complete structure of the plant tissue.

[0536] Comparative Example 3

[0537] In this comparative example, the conventional embedding and sectioning process in the 10X Visium Spatial Transcriptome Sequencing Instructions was used for tissue sample preparation of tobacco petiole, and the freezing time was shortened to 10 min, and the other steps were consistent with those of Comparative Example 1.

[0538] 1. Sample embedding

[0539] a. Take fresh tobacco leaf petiole tissue, the tissue should not be too large, and make sure that the tissue can be placed into the mold.

[0540] b. Gently absorb the excess liquid such as tissue fluid with a dust-free paper, and make sure that no impurities other than the tissue are attached.

[0541] c. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into 1 / 3 of the capacity of the mold groove, and make sure that no bubbles are generated.

[0542] d. Confirm the direction of the tissue section, and use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0543] e. Horizontally transfer to dry ice for 10 min to ensure that the OCT and the tissue are completely frozen.

[0544] 2. Sample tissue sectioning

[0545] Section the embedded tissue sample with a thickness of 10 pm, flatten the sample, and place the sample on a common glass slide, immediately place a finger on the back of the slide for 5 seconds, use the finger temperature to thaw the section, and make it fully adhere to the glass slide.

[0546] The results are shown in Table 5. Under the optical microscope, it was observed that the tissue section still had a shrinkage phenomenon, the tissue had local fractures, and a large number of tissue cavities appeared, and the original complete structure of the plant tissue could not be guaranteed.

[0547] Comparative Example 4

[0548] This comparative example uses the conventional embedding and sectioning process in the 10X Visium spatial transcriptome sequencing instructions to prepare the tobacco leaf petiole tissue sample, and the freezing time is shortened to 5 min, and the remaining steps are consistent with those of Comparative Example 1.

[0549] 1. Sample embedding

[0550] a. Take fresh tobacco leaf petiole tissue, the tissue should not be too large, and make sure that the tissue can be placed into the mold.

[0551] b. Gently absorb the excess liquid such as tissue fluid with a dust-free paper, and make sure that no impurities other than the tissue are attached.

[0552] c. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into 1 / 3 of the capacity of the mold groove, and make sure that no bubbles are generated.

[0553] d. Confirm the direction of the tissue section, use tweezers to put the prepared tissue into the mold with OCT, and then use OCT to completely cover the exposed tissue surface. If the tissue is large, you can add more OCT to ensure that the OCT fully covers the tissue surface.

[0554] e. Transfer horizontally to dry ice and freeze for 5 min to ensure that the OCT and tissue are completely frozen.

[0555] 2. Sample tissue sectioning

[0556] Slice the embedded tissue sample with a thickness of 10 μm, flatten it, and place the sample on a regular slide. Immediately place your finger on the back of the slide for 5 seconds to thaw the slice using the temperature of your finger, and make it fully adhere to the slide.

[0557] The results are shown in Table 5. Freezing for 5 min did not completely freeze the tissue, and the section was shriveled, making it impossible to observe the tissue morphology.

[0558] Comparative Example 5

[0559] In this comparative example, the embedding temperature was increased to -20°C, and the freezing time was extended to 1 h to ensure that the OCT and tissue were completely frozen. The remaining steps were consistent with Comparative Example 1.

[0560] 1. Sample embedding

[0561] a. Take fresh tobacco leaf petiole tissue, and make sure the tissue is not too large so that it can be placed in the mold.

[0562] b. Gently absorb the excess liquid, such as tissue fluid, with a dust-free paper, and make sure not to stick to impurities other than the tissue.

[0563] c. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into the mold groove to 1 / 3 of the capacity, making sure not to generate bubbles.

[0564] d. Confirm the direction of the tissue section, use tweezers to put the prepared tissue into the mold with OCT, and then use OCT to completely cover the exposed tissue surface. If the tissue is large, you can add more OCT to ensure that the OCT fully covers the tissue surface.

[0565] e. Transfer horizontally to a -20°C environment and freeze for 1 h to ensure that the OCT and tissue are completely frozen.

[0566] 2. Sample tissue sectioning

[0567] Slice the embedded tissue sample with a thickness of 10 μm, flatten it, and place the sample on a regular slide. Immediately place your finger on the back of the slide for 5 seconds to thaw the slice using the temperature of your finger, and make it fully adhere to the slide.

[0568] The results are shown in Table 5. The tissue sections were still observed to have shrinkage under an optical microscope, the parenchyma was damaged, and there were tissue cavities, which failed to guarantee the original intact structure of the plant tissue.

[0569] Comparative Example 6

[0570] In this comparative example, the freezing time was shortened to 45 min to guarantee OCT and tissue freezing, and the other steps were the same as in Comparative Example 5.

[0571] 1. Sample embedding

[0572] a. Fresh tobacco petiole tissue was taken, and the tissue should not be too large, so as to guarantee that the tissue can be placed into the mold.

[0573] b. The excess liquid, such as tissue fluid, was gently absorbed with a dust-free paper, and attention should be paid to not adhere to impurities outside the tissue.

[0574] c. The embedding mold was placed horizontally on the table top (operating on ice), and the pre-cooled embedding agent was injected into the mold groove to a capacity of 1 / 3, and attention should be paid to not generate bubbles.

[0575] d. The direction of the tissue section was confirmed, the prepared tissue was placed into the mold containing OCT using tweezers, and the exposed tissue surface was completely covered with OCT; if the tissue was large, more OCT could be added to ensure that the OCT fully covered the tissue surface.

[0576] e. It was horizontally transferred to a -20°C environment, and was frozen for 45 min to ensure OCT and tissue freezing.

[0577] 2. Sample tissue sectioning

[0578] The embedded tissue sample was sectioned to a thickness of 10 μm, and was flattened, and the sample was placed on a common glass slide, and the finger was immediately placed on the back of the slide for 5 seconds, the section was thawed by the finger temperature, and the section was fully adhered to the glass slide.

[0579] The results are shown in Table 5. The tissue shrinkage was reduced under an optical microscope, but the parenchyma was still damaged, and there were tissue cavities, which failed to guarantee the original intact structure of the plant tissue.

[0580] Comparative Example 7

[0581] In this comparative example, the freezing time was shortened to 30 min to guarantee OCT and tissue freezing, and the other steps were the same as in Comparative Example 5.

[0582] 1. Sample embedding

[0583] a. Fresh tobacco petiole tissue was taken, and the tissue should not be too large, so as to guarantee that the tissue can be placed into the mold.

[0584] b. Gently use a clean paper to suck up the excess liquid, such as tissue fluid, etc., and make sure not to stick to the impurities outside the tissue.

[0585] c. Place the embedding mold horizontally on the table (operate on ice), inject the pre-cooled embedding agent into the mold groove capacity of 1 / 3, and make sure not to produce bubbles.

[0586] d. Confirm the direction of the tissue section, use tweezers to put the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is larger, you can add more OCT to ensure that the OCT fully covers the tissue surface.

[0587] e. Transfer horizontally to a -20°C environment and freeze for 30 min to ensure that the OCT and tissue are frozen.

[0588] 2. Sample tissue sectioning

[0589] Slice the embedded tissue sample with a thickness of 10 μm, flatten it, and place the sample on a regular glass slide. Immediately place your finger on the back of the slide for 5 seconds to use the finger temperature to thaw the section and make it fully adhere to the slide.

[0590] The results are shown in Table 5. Under the optical microscope, it was observed that the tissue had a small amount of shrinkage, and the tissue still had cavities after sectioning, which did not well guarantee the original complete structure of the plant tissue.

[0591] Comparative Example 8

[0592] In this comparative example, the tobacco petiole tissue was embedded with OCT before embedding, and then placed in a vacuum instrument to accelerate penetration using negative pressure for 10 min. The remaining steps were the same as those in Comparative Example 7.

[0593] 1. Sample embedding

[0594] a. Take fresh tobacco petiole tissue, and make sure the tissue is not too large to ensure that it can be placed in the mold.

[0595] b. Gently use a clean paper to suck up the excess liquid, such as tissue fluid, etc., and make sure not to stick to the impurities outside the tissue.

[0596] c. Take the tissue out of the glycerol and gently wipe off the excess glycerol adhering to the surface.

[0597] d. Place the embedding mold horizontally on the table (operate on ice), inject the pre-cooled embedding agent into the mold groove capacity of 1 / 3, and make sure not to produce bubbles.

[0598] e. Confirm the direction of the tissue section, use tweezers to put the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is larger, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0599] f. Put into a vacuum instrument for 10 min to promote the penetration of OCT into the tissue.

[0600] g. Horizontally transfer to a -20°C environment and freeze for 30 min to ensure that the OCT and tissue are frozen.

[0601] 2. Sample tissue sectioning

[0602] Slice the embedded tissue sample with a thickness of 10 μm, flatten it, and place the sample on a regular glass slide. Immediately place your finger on the back of the slide for 5 seconds to use the finger temperature to thaw the section and make it fully adhere to the slide.

[0603] The results are shown in Table 6. Under optical microscopy, it was observed that the tissue had a small amount of shrinkage, and after sectioning, the tissue still had cavities, which did not well guarantee the original complete structure of the plant tissue.

[0604] Comparative Example 9

[0605] In this comparative example, the tobacco petiole tissue was pretreated by soaking in 10% glycerol before embedding, and was placed in a vacuum instrument to accelerate penetration using negative pressure for 10 min. The remaining steps were the same as in Comparative Example 7.

[0606] 1. Sample embedding

[0607] a. Take fresh tobacco petiole tissue, which should not be too large, to ensure that the tissue can be placed in the mold.

[0608] b. Gently absorb the excess liquid such as tissue fluid with a dust-free paper, and pay attention not to stick to impurities other than the tissue.

[0609] c. Place the tobacco petiole tissue in 10% glycerol (glycerol covering the tissue), and place it in a vacuum instrument for 10 min to accelerate the penetration of 10% glycerol into the tissue.

[0610] d. Take the tissue out of the glycerol and gently wipe off the excess glycerol adhering to the surface.

[0611] e. Place the embedding mold horizontally on the table (operate on ice), inject the pre-cooled embedding agent into the mold groove to 1 / 3 of the capacity, and pay attention not to generate bubbles.

[0612] f. Confirm the direction of the tissue section, use tweezers to put the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is larger, you can add more OCT to ensure that the OCT fully covers the tissue surface.

[0613] g. Transfer horizontally to a -20°C environment and freeze for 30 min to ensure that the OCT and tissue are frozen.

[0614] 2. Sample tissue sectioning

[0615] Slice the embedded tissue sample to a thickness of 10 μm, flatten it, and place the sample on a regular slide. Immediately place your finger on the back of the slide for 5 seconds to use the finger temperature to thaw the section and make it fully adhere to the slide.

[0616] The results are shown in Table 6. Under optical microscopy, it was observed that the tissue had a small amount of shrinkage, the cell morphology was irregular after sectioning, the thin-walled tissue was damaged by extrusion, and the tissue had cavities, which failed to well preserve the original structure of the plant tissue.

[0617] Comparative Example 10

[0618] In this comparative example, the tobacco petiole tissue was pretreated by soaking in 10% PEG before embedding, and was placed in a vacuum instrument to accelerate the penetration of 10% PEG for 10 min. The remaining steps were the same as those in Comparative Example 7.

[0619] 1. Sample embedding

[0620] a. Take fresh tobacco petiole tissue, which should not be too large, to ensure that the tissue can be placed in the mold.

[0621] b. Gently absorb the excess liquid such as tissue fluid with dust-free paper, and pay attention not to stick to impurities other than the tissue.

[0622] c. Place the tobacco petiole tissue in 10% PEG (PEG covers the tissue), and place it in a vacuum instrument to accelerate the penetration of 10% PEG into the tissue for 10 min.

[0623] d. Take the tissue out of the 10% PEG and gently wipe off the excess PEG adhering to the surface.

[0624] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into the mold to 1 / 3 of the groove capacity, and pay attention not to generate bubbles.

[0625] f. Confirm the direction of the tissue section, use tweezers to put the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is larger, you can add more OCT to ensure that the OCT fully covers the tissue surface.

[0626] g. Horizontally transfer to -20 °C environment, freeze for 30 min, ensure OCT and tissue freeze.

[0627] 2. Sample tissue sectioning

[0628] Slice the embedded tissue sample, thickness 10 μm, flatten, place the sample on a regular glass slide, immediately place a finger on the back of the slide for 5 seconds, use the finger temperature to thaw the section, and make it fully adhere to the slide.

[0629] The results are shown in Table 6. Under optical microscope, the tissue was observed to have wrinkles, the tissue morphology was irregular after sectioning, the tissue had a tendency to accumulate downward, and the structure and morphology of the plant tissue could not be well maintained.

[0630] Comparative Example 11

[0631] In this comparative example, the tobacco petiole tissue was pretreated by soaking in 20% PEG before embedding, and was placed in a vacuum instrument to accelerate the penetration by negative pressure for 10 min. The remaining steps were the same as those in Comparative Example 7.

[0632] 1. Sample embedding

[0633] a. Take fresh tobacco petiole tissue, the tissue should not be too large, and make sure that the tissue can be placed into the mold.

[0634] b. Gently absorb the excess liquid such as tissue fluid with a dust-free paper, and pay attention not to stick impurities other than the tissue.

[0635] c. Place the tobacco petiole tissue into 20% PEG (PEG covers the tissue), and place it in a vacuum instrument to accelerate the penetration of 20% PEG into the tissue for 10 min.

[0636] d. Take the tissue out of the 20% PEG, and gently wipe off the excess PEG adhered to the surface.

[0637] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into the mold groove to 1 / 3 of the capacity, and pay attention not to generate bubbles.

[0638] f. Confirm the direction of the tissue sectioning, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0639] g. Horizontally transfer to -20 °C environment, freeze for 30 min, ensure OCT and tissue freeze.

[0640] 2. Sample tissue sectioning

[0641] The embedded tissue sample is sliced, with a thickness of 10 μm, flattened, and the sample is placed on a common glass slide, a finger is immediately placed on the back of the slide for 5 seconds, the slice is thawed using the finger temperature, and the slice is fully adhered to the glass slide.

[0642] The results are shown in Table 6. Under optical microscopy, the tissue was observed to have wrinkles, the tissue morphology was irregular, the tissue had a downward stacking tendency, and the structure and morphology of the plant tissue could not be well maintained.

[0643] Comparative Example 12

[0644] In this comparative example, the tobacco petiole tissue is pretreated by soaking in 30% PEG before embedding, and is placed in a vacuum instrument to accelerate the penetration using negative pressure for 10 min, and the remaining steps are the same as in Comparative Example 7.

[0645] 1. Sample embedding

[0646] a. Fresh tobacco petiole tissue is taken, and the tissue should not be too large, so that it can be placed in the mold.

[0647] b. The excess liquid, such as tissue fluid, is gently absorbed with a dust-free paper, and attention should be paid to not adhere to impurities other than the tissue.

[0648] c. The tobacco petiole tissue is placed in 30% PEG (PEG covers the tissue), and is placed in a vacuum instrument to accelerate the penetration of 30% PEG into the tissue for 15 min.

[0649] d. The tissue is taken out of the 30% PEG, and the excess PEG adhering to the surface is gently wiped off.

[0650] e. The embedding mold is placed horizontally on the table (operated on ice), and the pre-cooled embedding agent is injected into the mold groove to 1 / 3 of the capacity, and attention should be paid to not generate bubbles.

[0651] f. The direction of the tissue slice is confirmed, the prepared tissue is placed in the mold containing OCT using tweezers, and the exposed tissue surface is completely covered with OCT; if the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0652] g. It is horizontally transferred to a -20°C environment, and is frozen for 30 min to ensure that the OCT and the tissue are frozen.

[0653] 2. Sample tissue slicing

[0654] The embedded tissue sample is sliced, with a thickness of 10 μm, flattened, and the sample is placed on a common glass slide, a finger is immediately placed on the back of the slide for 5 seconds, the slice is thawed using the finger temperature, and the slice is fully adhered to the glass slide.

[0655] The results are shown in Table 6. Under optical microscope, it was observed that the tissue was still shrunk, the tissue structure was deformed, and there was a tendency to accumulate downwardly, and the structure and morphology of the plant tissue could not be well maintained.

[0656] Comparative Example 13

[0657] In this comparative example, the tobacco petiole tissue was pretreated by soaking in 40% PEG before embedding, and was placed in a vacuum instrument to accelerate the penetration by negative pressure for 15 min, and the remaining steps were the same as those in Comparative Example 7.

[0658] 1. Sample embedding

[0659] a. Fresh tobacco petiole tissue was taken, and the tissue should not be too large, so as to ensure that the tissue could be placed into the mold.

[0660] b. The excess liquid such as tissue fluid was gently absorbed with a dust-free paper, and attention should be paid to not adhere to impurities outside the tissue.

[0661] c. The tobacco petiole tissue was placed into 40% PEG (PEG covered the tissue), and was placed into a vacuum instrument to accelerate the penetration of 40% PEG into the tissue for 10 min.

[0662] d. The tissue was taken out of the 40% PEG, and the excess PEG adhered to the surface was gently wiped off.

[0663] e. The embedding mold was placed horizontally on the table (operated on ice), and the pre-cooled embedding agent was injected into the mold groove to a capacity of 1 / 3, and attention should be paid to not generate bubbles.

[0664] f. The direction of the tissue section was confirmed, the prepared tissue was placed into the mold containing OCT using tweezers, and the exposed tissue surface was completely covered with OCT; if the tissue was large, more OCT could be added to ensure that the OCT fully covered the tissue surface.

[0665] g. It was horizontally transferred to a-20°C environment, and was frozen for 30 min to ensure that the OCT and the tissue were frozen.

[0666] 2. Sample tissue sectioning

[0667] The embedded tissue sample was sectioned to a thickness of 10 μm, and was flattened, and the sample was placed on a common glass slide, and the finger was immediately placed on the back of the slide for 5 seconds, and the section was thawed by the temperature of the finger, so that the section and the glass slide were fully adhered together.

[0668] The results are shown in Table 6. Under optical microscope, it was observed that the tissue morphology was relatively complete, the parenchyma structure was well maintained, and the structure and morphology of the plant tissue could be maintained. However, subsequent RNA extraction and quality inspection showed that the RNA RIN value was lower than 7, and there was degradation, which did not meet the requirements of subsequent experiments.

[0669] Comparative Example 14

[0670] In this comparative example, the tobacco petiole tissue was pre-treated by soaking in 10% PVA (v / v) before embedding and was put into a vacuum instrument to accelerate the penetration of 10% PVA into the tissue under negative pressure for 10 min. The remaining steps were the same as those in Comparative Example 7.

[0671] 1. Sample embedding

[0672] a. Fresh tobacco petiole tissue was taken. The tissue should not be too large, and it should be ensured that the tissue can be placed into the mold.

[0673] b. The excess liquid, such as tissue fluid, was gently absorbed with a dust-free paper. Attention should be paid to not adhere to impurities other than the tissue.

[0674] c. The tobacco petiole tissue was put into 10% PVA (v / v) (PVA covered the tissue), and was put into a vacuum instrument to accelerate the penetration of 10% PVA into the tissue for 10 min.

[0675] d. The tissue was taken out of 10% PVA (v / v), and the excess PVA adhered to the surface was gently wiped off.

[0676] e. The embedding mold was placed horizontally on the table (operated on ice), and the pre-cooled embedding agent was injected into the mold to a capacity of 1 / 3. Attention should be paid to not generate air bubbles.

[0677] f. The direction of the tissue section was confirmed, the prepared tissue was put into the mold containing OCT using tweezers, and the exposed tissue surface was completely covered with OCT. If the tissue was large, more OCT could be added to ensure that the OCT fully covered the tissue surface.

[0678] g. The sample was transferred horizontally to a -20°C environment and was frozen for 30 min to ensure that the OCT and the tissue were frozen.

[0679] 2. Sample tissue sectioning

[0680] The embedded tissue sample was sectioned to a thickness of 10 μm, was flattened, and was placed on a common glass slide. The finger was immediately placed on the back of the slide for 5 seconds to thaw the section using the temperature of the finger and to make the section fully adhere to the glass slide.

[0681] The results are shown in Table 6. Under an optical microscope, it was observed that the tissue was shrunk, the tissue structure was deformed, and there was a tendency to accumulate downward. The structure and morphology of the plant tissue were not well maintained.

[0682] Comparative Example 15

[0683] In this comparative example, the tobacco petiole tissue was pre-treated by soaking in 20% PVA (v / v) before embedding and was put into a vacuum instrument to accelerate the penetration of 10% PVA into the tissue under negative pressure for 10 min. The remaining steps were the same as those in Comparative Example 7.

[0684] 1. Sample embedding

[0685] a. Take fresh tobacco petiole tissue, the tissue should not be too large, and make sure that the tissue can be placed into the mold.

[0686] b. Gently absorb the excess liquid, such as tissue fluid, with a dust-free paper, and make sure that no impurities other than the tissue are adhered.

[0687] c. Place the tobacco petiole tissue into 20% PVA (v / v) (PVA covers the tissue), and place it into a vacuum instrument to accelerate the penetration of 20% PVA into the tissue for 10 min.

[0688] d. Take the tissue out of the 20% PVA (v / v), and gently wipe off the excess PVA adhered to the surface.

[0689] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into the mold to fill 1 / 3 of the groove capacity, and make sure that no bubbles are generated.

[0690] f. Confirm the direction of the tissue section, and use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, more OCT can be added to ensure that the OCT fully covers the tissue surface.

[0691] g. Horizontally transfer to a -20°C environment, and freeze for 30 min to ensure that the OCT and the tissue are frozen.

[0692] 2. Sample tissue sectioning

[0693] Section the embedded tissue sample, with a thickness of 10 μm, and flatten the sample, and place the sample on a common glass slide, and immediately place a finger on the back of the glass slide for 5 seconds, and use the temperature of the finger to thaw the section, and make it fully adhere to the glass slide.

[0694] The results are shown in Table 6. Under the optical microscope, it is observed that the tissue still has a little shrinkage, the tissue structure is deformed, and has a tendency to accumulate downward, and the structure and morphology of the plant tissue are not well maintained.

[0695] Comparative Example 16

[0696] In this comparative example, the tobacco petiole tissue is pretreated by soaking in 40% PVA (v / v) before embedding, and is placed into a vacuum instrument to accelerate the penetration by negative pressure for 15 min, and the remaining steps are the same as those in Comparative Example 7.

[0697] 1. Sample embedding

[0698] a. Take fresh tobacco petiole tissue, the tissue should not be too large, and make sure that the tissue can be placed into the mold.

[0699] b. Gently blot away excess liquid, such as tissue fluid, with lint-free paper, being careful not to let any impurities outside the tissue adhere to it.

[0700] c. Place the tobacco petiole tissue in 40% PVA (v / v) (PVA covers the tissue), and place it in a vacuum apparatus for 15 minutes to accelerate the penetration of 30% PVA into the tissue.

[0701] d. Remove the tissue from 40% PVA (v / v) and gently wipe off any excess PVA adhering to the surface.

[0702] e. Place the embedding mold horizontally on the table (operate on ice), and inject the pre-cooled embedding agent into 1 / 3 of the mold groove capacity, taking care not to generate air bubbles.

[0703] f. Confirm the orientation of the tissue section, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to completely cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that the OCT fully covers the tissue surface.

[0704] g. Transfer horizontally to a -20°C environment and freeze for 30 minutes to ensure OCT and tissue freezing.

[0705] 2. Sample tissue sections

[0706] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a regular glass slide. Immediately, a finger was placed on the back of the slide for 5 seconds to thaw the slice using the warmth of the finger, allowing it to fully adhere to the slide.

[0707] The results are shown in Table 6. Under an optical microscope, the tissue was observed to be shrunken, deformed, and porous, failing to maintain the structure and morphology of the plant tissue.

[0708] Comparative Example 17

[0709] In this comparative example, after the sections were qualified, a tissue permeability optimization step was performed, which was the same as the 10X Visium spatial transcriptome sequencing instruction manual.

[0710] The results are shown in Table 7. The scanning imaging results of the permeabilized sections showed that the fluorescence intensity of the six sections processed for 3–30 min was weak and the differences were not significant, making it impossible to determine the optimal permeabilization time and failing to meet the conditions for subsequent processing.

[0711] Comparative Example 18

[0712] In this comparative example, the permeation optimization steps prioritize the use of a permeation pretreatment solution to improve permeation efficiency, followed by permeation treatment with 10X permeation enzyme. The remaining steps are the same as in comparative example 17.

[0713] The permeabilization optimization steps are as follows:

[0714] a. Prepare the permeabilization pretreatment solution, which contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 2% cellulase R10 (m / v), 0.4% lyticase R10 (m / v), 1% pectinase (m / v), and 0.4U / μL RNase inhibitor.

[0715] b. Paste the tissue section on the Visium Spatial Gene Expression Slide at the specified position, install the Visium Spatial Gene Expression Slide in the Slide Cassette, and add 70μL of the permeabilization pretreatment solution to the hole from the lower left corner of the sample hole of the Slide Cassette, gently pat the Slide Cassette to make the treatment solution evenly cover the tissue, and paste the sealing strip.

[0716] c. Place the PCR instrument adapter in the PCR instrument, place the Slide Cassette on the adapter, and pretreat at 37℃ for 15min.

[0717] d. After the pretreatment is completed, carefully remove the pretreatment solution from the hole, and add 100μL of 0.1x SSC to each hole. After 30s, remove the pretreatment solution.

[0718] e. Then follow the 10X Visium Spatial Transcriptome Sequencing Instructions for subsequent permeabilization optimization.

[0719] The results are shown in Table 7. The permeabilization optimization section scanning imaging results show that the fluorescence intensity of the six sections treated for 3-30min is weak, and only the middle part of the tissue has weak fluorescence, so the optimal permeabilization time cannot be determined, and the subsequent processing conditions are not met.

[0720] Comparative Example 19

[0721] In this comparative example, hemicellulase is added to the permeabilization pretreatment solution, and the remaining steps are the same as those in Comparative Example 18.

[0722] The permeabilization optimization steps are as follows:

[0723] a. Prepare the permeabilization pretreatment solution, which contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 2% cellulase R10 (m / v), 0.4% lyticase R10 (m / v), 1% pectinase (m / v), and 0.4U / μL RNase inhibitor.

[0724] b. Place the tissue sections on the Visium Spatial Gene Expression Slide at the designated locations. Install the Visium Spatial Gene Expression Slide into the Slide Cassette. Add 70 μΐ of the permeabilization pre-treatment solution to the well, starting at the lower left corner of the well. Gently tap the Slide Cassette to ensure the solution evenly covers the tissue. Apply the sealing strip.

[0725] c. Place the PCR instrument adapter into the PCR instrument. Place the Slide Cassette onto the adapter. Pre-treat at 37 °C for 15 min.

[0726] d. After the pre-treatment, carefully remove the pre-treatment solution from the well. Add 100 μΐ of 0.1 x SSC to each well. After 30 s, remove the pre-treatment solution.

[0727] e. Follow the 10X Visium Spatial Transcriptome Sequencing Instructions for subsequent permeabilization optimization.

[0728] The results are shown in Table 7. The permeabilization optimization section scanning imaging results showed that the fluorescence intensity of the six sections treated for 3-30 min was weak, but the tissue fluorescence range was expanded. The optimal permeabilization time could not be determined, and the subsequent processing conditions were not met.

[0729] Comparative Example 20

[0730] In this comparative example, the enzyme concentration in the permeabilization pre-treatment solution formula was reduced, and the other steps were the same as those in Comparative Example 18.

[0731] The permeabilization optimization steps are as follows:

[0732] a. Prepare the permeabilization pre-treatment solution, which contains 0.02 M MES, 0.02 M KCl, 0.1% (m / v) BSA, 0.01 M CaCl2, 0.1 M mannitol, 1% cellulase R10 (m / v), 0.2% lyticase R10 (m / v), 0.5% pectinase (m / v), 0.5% hemicellulase (m / v), and 0.4 U / μΐ of RNAse inhibitor.

[0733] b. Place the tissue sections on the Visium Spatial Gene Expression Slide at the designated locations. Install the Visium Spatial Gene Expression Slide into the Slide Cassette. Add 70 μΐ of the permeabilization pre-treatment solution to the well, starting at the lower left corner of the well. Gently tap the Slide Cassette to ensure the solution evenly covers the tissue. Apply the sealing strip.

[0734] c. Place the PCR instrument adapter into the PCR instrument, place the Slide Cassette onto the adapter, and pre-treat at 37 °C for 15 min.

[0735] d. After pre-treatment is complete, carefully remove the pre-treatment solution from the well, and add 100 μL of 0.1x SSC to each well. Remove the pre-treatment solution after 30 s.

[0736] e. Subsequent permeabilization optimization was performed according to the 10X Visium Spatial Transcriptome Sequencing Instructions.

[0737] The results are shown in Table 7. Permeabilization optimization section scanning imaging results show that the fluorescence intensity of the six sections treated for 3-30 min is slightly stronger than that of Comparative Example 19, and the fluorescence range basically covers the entire tissue. Permeabilization for 18 min has the strongest fluorescence brightness, but is still weaker than the conventional fluorescence brightness.

[0738] Comparative Example 21

[0739] This comparative example continues to reduce the enzyme concentration in the permeabilization pre-treatment solution formula, and the remaining steps are the same as those of Comparative Example 18.

[0740] The permeabilization optimization steps are as follows:

[0741] a. Prepare the permeabilization pre-treatment solution, which contains 0.02 M MES, 0.02 M KCl, 0.1% (m / v) BSA, 0.01 M CaCl2, 0.1 M mannitol, 0.5% cellulase R10 (m / v), 0.1% lyticase R10 (m / v), 0.2% pectinase (m / v), 0.2% hemicellulase (m / v), and 0.4 U / μL RNase inhibitor.

[0742] b. Place the tissue section on the Visium spatial gene expression slide at the specified position, install the Visium spatial gene expression slide in the Slide Cassette (slide box), and add 70 μL of permeabilization pre-treatment solution to the well from the lower left corner of the Slide Cassette sample well, gently tap the Slide Cassette to evenly cover the tissue with the treatment solution, and attach the sealing strip.

[0743] c. Place the PCR instrument adapter into the PCR instrument, place the Slide Cassette onto the adapter, and pre-treat at 37 °C for 15 min.

[0744] d. After pre-treatment is complete, carefully remove the pre-treatment solution from the well, and add 100 μL of 0.1x SSC to each well. Remove the pre-treatment solution after 30 s.

[0745] e. Follow the subsequent permeabilization optimization according to the 10X Visium Spatial Transcriptomics Sequencing Instructions.

[0746] The results are shown in Table 7. The permeabilization optimization section scanning imaging results show that the fluorescence intensity of the six sections treated for 3-30 min is slightly stronger than that of Comparative Example 20, and the fluorescence range basically covers the entire tissue. Permeabilization for 18 min has the strongest fluorescence brightness, but is still weaker than the conventional fluorescence brightness.

[0747] Comparative Example 22

[0748] In this comparative example, Tween 20 is added to the permeabilization pretreatment liquid formula, and the remaining steps are the same as those of Comparative Example 21.

[0749] The permeabilization optimization steps are as follows:

[0750] a. Configure the permeabilization pretreatment liquid, which contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.5% cellulase R10 (m / v), 0.1% lyticase R10 (m / v), 0.2% pectinase (m / v), 0.2% hemicellulase (m / v), 1% Tween 20 (v / v), and 0.4U / μL RNase inhibitor.

[0751] b. Place the tissue section on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, and add 70μL of the permeabilization pretreatment liquid to the hole, gently tap the Slide Cassette to make the treatment liquid evenly cover the tissue, and then place the sealing strip.

[0752] c. Place the PCR instrument adapter in the PCR instrument, place the Slide Cassette on the adapter, and pretreat at 37℃ for 15 min.

[0753] d. After the pretreatment is completed, carefully remove the pretreatment liquid from the hole, and add 100μL of 0.1x SSC to each hole. After 30s, remove the pretreatment liquid.

[0754] e. Follow the subsequent permeabilization optimization according to the 10X Visium Spatial Transcriptomics Sequencing Instructions.

[0755] The results are shown in Table 7. The permeabilization optimization section scanning imaging results show that the fluorescence intensity of the six sections treated for 3-30 min is slightly stronger than that of Comparative Example 20, and the fluorescence range basically covers the entire tissue. Permeabilization for 18 min has the strongest fluorescence brightness, but is still weaker than the conventional fluorescence brightness.

[0756] Comparative Example 23

[0757] This comparative example reduces the concentration of Tween 20 in the permeabilization pretreatment solution formulation, and the remaining steps are the same as those of Comparative Example 22.

[0758] The permeabilization optimization steps are as follows:

[0759] a. Prepare the permeabilization pretreatment solution, which contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.5% cellulase R10 (m / v), 0.1% lyticase R10 (m / v), 0.2% pectinase, 0.2% hemicellulase (m / v), 0.5% Tween 20 (v / v), and 0.4U / μL RNase inhibitor.

[0760] b. Place the tissue section on the Visium Spatial Gene Expression Slide at the designated position, install the Visium Spatial Gene Expression Slide in the Slide Cassette, and add 70μL of the permeabilization pretreatment solution to the well, starting from the lower left corner of the well, and gently tap the Slide Cassette to ensure that the solution evenly covers the tissue. Then, apply the sealing strip.

[0761] c. Place the PCR instrument adapter in the PCR instrument, and place the Slide Cassette on the adapter. Then, pre-treat at 37℃ for 15min.

[0762] d. After the pre-treatment is complete, carefully remove the pretreatment solution from the well, and add 100μL of 0.1x SSC to each well. After 30s, remove the pretreatment solution.

[0763] e. Then, follow the 10X Visium Spatial Transcriptome Sequencing Instructions for subsequent permeabilization optimization.

[0764] The results are shown in Table 7. The permeabilization optimization section scanning imaging results show that the fluorescence intensity of the six sections treated for 3-30min is slightly stronger than that of Comparative Example 21, and the fluorescence range basically covers the entire tissue. The fluorescence intensity is the strongest when the permeabilization time is 18min, but it is still weaker than the conventional fluorescence intensity.

[0765] Comparative Example 24

[0766] This comparative example adds an in situ polyadenylation step after permeabilization to capture non-coding RNA, and the remaining steps are the same as those of the 10X Visium Spatial Transcriptome Sequencing Instructions.

[0767] The in situ polyadenylation steps are as follows:

[0768] a. After permeabilization and washing, use a pipette to remove the 0.1x SSC.

[0769] b. Add 80 μL of Poly(A) Polymerase mix (NEB / MT0004) (E. coli Poly(A) Polymerase 5 U / μL (0.125 U / μL final), 10X EPAP Buffer (0.5X final), 10 mM ATP (0.5 mM final), 25 mM MnCl2(1.25 mM final), 40 U / μL RNase Inhibitor (1.5 U / μL final), ddH2O to 80 μL) to each sample well.

[0770] c. Then seal the reaction chamber and incubate the Slide Cassette at 37 °C for 30 minutes, then remove the enzyme mix.

[0771] d. Add 100 μL of 0.1x SSC to each well for 10 s wash.

[0772] e. Discard the 0.1X SSC from each well.

[0773] f. Follow the subsequent reverse transcription and library construction steps according to the 10X Visium Spatial Transcriptome Sequencing Instructions. The results are shown in Table 8. No microRNA was captured and the rest of the non-coding RNA content was very low at 50G sequencing depth.

[0774] Comparative Example 25

[0775] In this comparative example, the poly(A) polymerase was changed, and the rest of the steps were the same as in Comparative Example 24.

[0776] The in situ polyadenylation step was as follows:

[0777] a. After permeabilization wash, aspirate 0.1X SSC with a pipette.

[0778] b. Add 80 μL of Poly(A) Polymerase mix (Thermo / AM1350) (E. coli Poly(A) Polymerase 2 U / μL (0.125 U / μL final), 5X EPAP Buffer (0.5X final), 10 mM ATP (0.5 mM final), 25 mM MnCl2(1.25 mM final), 40 U / μL RNase Inhibitor (1.5 U / μL final), ddH2O to 80 μL) to each sample well.

[0779] c. Then seal the reaction chamber and incubate the Slide Cassette at 37 °C for 30 minutes, then remove the enzyme mix.

[0780] d. Add 100 μL of 0.1x SSC to each well for 10 s wash.

[0781] e. Discard 0.1X SSC from each well.

[0782] f. Follow subsequent reverse transcription and library construction steps according to the 10X Visium Spatial Transcriptome Sequencing Instructions. Results are shown in Table 8. No microRNA was captured and the rest of the non-coding RNA content was very low at 50G sequencing depth.

[0783] Comparative Example 26

[0784] In this comparative example, the poly(A) polymerase was replaced with a yeast-derived polymerase, and the rest of the steps were the same as in Comparative Example 24. The in situ polyadenylation step was as follows:

[0785] a. After the permeabilization wash, aspirate 0.1X SSC with a pipette.

[0786] b. Add 75 μΐ of yPAP enzyme (yeast-derived poly(A) polymerase, Thermo / 74225Z25KU) mix (5X yPAP reaction buffer (final concentration 1X), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (use NEB / P0756S, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), and ddH2O to 75 μΐ to each sample well.

[0787] c. Then seal the reaction chamber and incubate the Slide Cassette at 37 °C for 20 minutes, then remove the enzyme mix.

[0788] d. Add 100 μΐ of 0.1x SSC to each well and wash for 10 s.

[0789] e. Discard 0.1X SSC from each well.

[0790] f. Follow subsequent reverse transcription and library construction steps according to the 10X Visium Spatial Transcriptome Sequencing Instructions. Results are shown in Table 8. Non-coding RNA, including microRNA, was captured but at a low level at 50G sequencing depth.

[0791] Comparative Example 27

[0792] In this comparative example, the ATP was replaced, and the rest of the steps were the same as in Comparative Example 26.

[0793] The in situ polyadenylation step was as follows:

[0794] a. After the permeabilization wash, aspirate 0.1X SSC with a pipette.

[0795] b. Add 100 pL 1 x wash buffer (20 pL 5 x yPAP reaction buffer, 2 pL 40 U / pL RNase inhibitor, 78 pL nuclease-free H2O), remove buffer after 30 s incubation at room temperature.

[0796] c. Add 75 pL yPAP enzyme (Yeast-derived poly(A) polymerase, Thermo / 74225Z25KU) mix (5 x yPAP reaction buffer (final concentration 1 x), 600 U / pL yPAP enzyme (final concentration 24 U / pL), 10 mM ATP (use Thermo / AM8110G, final concentration 0.5 mM), 40 U / pL RNAse inhibitor (final concentration 1.5 U / pL), q.s. to 75 pL with ddH2O) to each sample well.

[0797] d. Then seal the reaction chamber and incubate the Slide Cassette at 37 °C for 20 min, then remove the enzyme mix.

[0798] e. Add 100 pL 0.1 x SSC wash to each well for 10 s.

[0799] f. Discard 0.1 x SSC from each well.

[0800] g. Follow the subsequent reverse transcription and library construction steps as per the 10X Visium Spatial Transcriptome Sequencing Instructions. The results are shown in Table 8. Non-coding RNAs including microRNAs can be captured at 50G sequencing depth, but at lower levels.

[0801] Comparative Example 28

[0802] This comparative example uses a buffer containing 10 mM EDTA for secondary metabolite pre-treatment prior to the in situ polyadenylation step. The rest of the steps are the same as in Comparative Example 27.

[0803] The in situ polyadenylation step is as follows:

[0804] a. After permeabilization wash, pipette 0.1 x SSC away.

[0805] b. Secondary metabolite removal pre-treatment: add 10 mM EDTA buffer per well, remove pre-treatment buffer after 10 min incubation at room temperature.

[0806] c. Add 100 pL 1 x wash buffer (20 pL 5 x yPAP reaction buffer, 2 pL 40 U / pL RNase inhibitor, 78 pL nuclease-free H2O), remove buffer after 30 s incubation at room temperature.

[0807] d. To each sample well, add 75 μL of yPAP enzyme (yeast-derived poly(A) polymerase, Thermo 74225Z25KU) mix (5x yPAP reaction buffer (final concentration 1x), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (use Thermo / AM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), q.s. to 75 μL with ddH2O.

[0808] e. Then seal the reaction chamber and incubate the Slide Cassette at 37 °C for 20 minutes, then remove the enzyme mix.

[0809] f. Add 100 μL of 0.1x SSC to each well for 10 s.

[0810] g. Discard the 0.1X SSC from each well.

[0811] h. Follow the subsequent reverse transcription and library construction steps as per the 10X Visium Spatial Transcriptome Sequencing Instructions. Results are shown in Table 8. Non-coding RNAs including microRNAs were captured at 50G sequencing depth, but at low levels.

[0812] Comparative Example 29

[0813] This comparative example used a buffer containing 5 mM DTT for secondary metabolite pre-treatment prior to the in situ polyadenylation step, and the remaining steps were the same as Comparative Example 27.

[0814] The in situ polyadenylation step was as follows:

[0815] a. After permeabilization wash, pipette 0.1x SSC away.

[0816] b. Secondary metabolite removal pre-treatment: Add buffer containing 5 mM DTT per well, incubate at room temperature for 10 min, then remove the pre-treatment buffer.

[0817] c. Add 100 μL of 1x wash buffer (20 μL of 5x yPAP reaction buffer, 2 μL of 40 U / μL RNase inhibitor, 78 μL of nuclease-free H2O), incubate at room temperature for 30 s, then remove the buffer.

[0818] d. To each sample well, add 75 μL of yPAP enzyme (yeast-derived poly(A) polymerase, Thermo 74225Z25KU) mix (5x yPAP reaction buffer (final concentration 1x), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (use Thermo AM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), add ddH2O to 75 μL.

[0819] e. Then seal the reaction chamber and incubate the Slide Cassette at 37 °C for 20 min, then remove the enzyme mix.

[0820] f. Add 100 μL of 0.1x SSC to each well and wash for 10 s.

[0821] g. Discard the 0.1x SSC from each well.

[0822] h. Follow the remaining steps of reverse transcription and library construction according to the 10X Visium Spatial Transcriptome Sequencing Instructions. The results are shown in Table 8. Non-coding RNAs including microRNAs can be captured at 50G sequencing depth, but the content is low.

[0823] Comparative Example 30

[0824] This comparative example uses β-galactosidase for secondary metabolite pre-treatment, and the remaining steps are the same as those of Comparative Example 27.

[0825] The in situ polyadenylation step is as follows:

[0826] a. After permeabilization wash, remove 0.1x SSC with a pipette.

[0827] b. Secondary metabolite removal pre-treatment: Dilute 250 U / mg β-galactosidase (Sigma, G6008-1KU) to 40 U / μL enzyme solution with 1x PBS, add 70 μL of 0.05x β-galactosidase solution to each well, and remove the enzyme solution after incubation at room temperature for 10 min.

[0828] c. Add 100 μL of 1x wash buffer (20 μL of 5x yPAP reaction buffer, 2 μL of 40 U / μL RNase inhibitor, 78 μL of nuclease-free H2O), and remove the buffer after incubation at room temperature for 30 s.

[0829] d. To each sample well, add 75 μL of yPAP enzyme (yeast-derived poly(A) polymerase, Thermo 74225Z25KU) mix (5x yPAP reaction buffer (final concentration 1x), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (use Thermo AM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), add ddH2O to 75 μL.

[0830] e. Then seal the reaction chamber, incubate the Slide Cassette at 37 °C for 20 min, then remove the enzyme mix.

[0831] f. Add 100 μL of 0.1x SSC to each well and wash for 10 s.

[0832] g. Discard the 0.1X SSC from each well.

[0833] h. Follow the subsequent reverse transcription and library construction, quality control, etc. steps according to the 10X Visium Spatial Transcriptome Sequencing Instructions.

[0834] The results are shown in Table 8. The non-coding RNA capture efficiency was improved compared to Comparative Examples 24-29 at a sequencing amount of 50G.

[0835] Comparative Example 31

[0836] In this comparative example, the working concentration of β-galactosidase was increased, and the other steps were the same as those in Comparative Example 27.

[0837] The in situ polyadenylation step is as follows:

[0838] a. After permeabilization and washing, use a pipette to remove the 0.1x SSC.

[0839] b. Secondary metabolite removal pretreatment: Dilute 250 U / mg of β-galactosidase (Sigma, G6008-1KU) to 40 U / μL of enzyme solution with 1x PBS, add 70 μL of 0.1x β-galactosidase solution to each well, and remove the enzyme solution after incubation at room temperature for 10 min.

[0840] c. Add 100 μL of 1x wash buffer (20 μL of 5x yPAP reaction buffer, 2 μL of 40 U / μL RNAse inhibitor, 78 μL of nuclease-free H2O), and remove the buffer after incubation at room temperature for 30 s.

[0841] d. Add 75 μL yPAP enzyme (yeast-derived poly(A) polymerase, Thermo 74225Z25KU) mix (5x yPAP reaction buffer (final concentration 1x), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (use Thermo AM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), q.s. to 75 μL with ddH2O) to each sample well.

[0842] e. Then seal the reaction chamber and incubate the Slide Cassette at 37 °C for 20 minutes, then remove the enzyme mix.

[0843] f. Add 100 μL 0.1x SSC to each well and wash for 10 s.

[0844] g. Discard the 0.1x SSC from each well.

[0845] h. Follow the subsequent reverse transcription and library construction, quality control, etc. steps according to the 10X Visium Spatial Transcriptome Sequencing Instructions.

[0846] The results are shown in Table 8. The non-coding RNA capture efficiency was improved compared to Comparative Examples 24-29 at a 50G sequencing amount.

[0847] Comparative Example 32

[0848] This comparative example is the same as Example 7 from tissue section to library construction step, and after sequencing, the ribosomal RNA data is removed by bioinformatics analysis means.

[0849] The results are shown in Table 9. After removing the ribosomal RNA data, only the data noise is reduced, and the number of non-coding RNA detections is not significantly improved.

[0850] Comparative Example 33

[0851] This comparative example uses the existing commercial SEQuoia RiboDepletion ribosomal RNA removal kit (Bio-Rad / 17006487) to remove the ribosomal RNA from the library constructed in Example 7 of this study.

[0852] The results are shown in Table 9.

[0853] Table 1 Examples 1-3

[0854]

[0855]

[0856] Table 2 Examples 4-6

[0857]

[0858]

[0859] Table 3 Examples 7-8

[0860]

[0861] Table 4 Examples 9-10

[0862]

[0863]

[0864] The application provides a spatial whole transcriptome sequencing experiment process suitable for tobacco petioles, which comprises a freezing sectioning method suitable for tobacco petioles, a pretreatment liquid suitable for plant tissue permeation, a treatment method suitable for in situ polyadenylation of tobacco petiole tissue, and a method suitable for removing rRNA from a cDNA library in the spatial whole transcriptome sequencing process. The above-mentioned methods of the application can be directly grafted into a conventional 10X Visium spatial transcriptome sequencing process, fill the blank of the current spatial whole transcriptome in the field of plants, and help to deeply understand the mechanisms of plant growth and development, environmental adaptation and stress resistance.

[0865] The application designs a freezing section method suitable for tobacco petiole, a pretreatment liquid suitable for tobacco petiole tissue permeation, an in situ polyadenylation method suitable for tobacco petiole tissue, and a method suitable for space full transcriptome sequencing process which can remove rRNA from a cDNA library. The application designs a freezing section method suitable for tobacco petiole, which is specifically as follows: before OCT embedding, the tobacco petiole tissue is placed in 25% PVA (v / v) (PVA is above the tissue), and is placed in a vacuum instrument to accelerate the penetration of PVA into the tissue for 10 min, and then OCT is used for embedding, and the embedding freezing condition is horizontal placement for 30 min in a-20 DEG C environment; the application designs a pretreatment liquid suitable for tobacco petiole tissue permeation, and the formula of the pretreatment liquid is specifically as follows (final concentration): 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.35% cellulase R10 (m / v), 0.1% macerozyme R10 (m / v), 0.1% pectinase (m / v), 0.15% hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4U / μL RNAase inhibitor, and the pretreatment condition is 37 DEG C pretreatment for 10 min; the application designs an in situ polyadenylation method suitable for tobacco petiole tissue, and the method is specifically as follows: the pretreatment liquid composition (final concentration) is 20U / μL beta-galactosidase solution, and the pretreatment condition is incubation at room temperature for 10 min. The in situ polyadenylation mixed liquid composition (final concentration) is 1x yPAP reaction buffer, 24U / μL yPAP enzyme (yeast-derived polyadenylate polymerase, Thermo / 74225Z25KU), 0.5mM ATP (Thermo / AM8110G), 1.5U / μL RNAase inhibitor, and the treatment condition is incubation at 37 DEG C for 20 min; the application designs a method suitable for space full transcriptome sequencing process which can remove rRNA from a cDNA library, and the method is specifically as follows: the IDTE buffer composition (final concentration) is 10mM Tris HCl, and the final concentration of EDTA is 0.1mM. The one-way guide RNA pool: 26 synthesized guide RNAs (18sRNA 13, 28sRNA 13) are dissolved and diluted to 10uM with IDTE buffer, and the sequences of the 26 guide RNAs are specifically shown in the table. The RNP mixed liquid composition: 10uM guide RNA, 20uM Cas9 endonuclease, 1x PBS, wherein the guide RNA and the Cas9 endonuclease are added at the same concentration, and the rest of the volume is supplemented with 1x PBS. After the RNP mixed liquid is mixed, it is incubated at room temperature for 10 min.The in vitro digestion mixture composition: 10x Cas9 nuclease reaction buffer, 1 μM Cas9 RNP and sample library, the final molar ratio of Cas9 RNP to sample library is 40:1, the rest of the volume is supplemented with 10x Cas9 nuclease reaction buffer, and the in vitro digestion reaction condition is 37℃ incubation for 60 min. The specific method of DNA release is: adding proteinase K with a final concentration of 20 mg / mL in the reaction, and incubating at 56℃ for 10 min. The specific fragment purification is: using 0.8x SPRI magnetic beads for purification.

[0866] In order to design a method and process suitable for whole transcriptome sequencing experiment of tobacco petiole, the application attempts to design in situ polyadenylation step on the basis of spatial transcriptome, and optimizes the basic frozen section, tissue permeation and other steps in view of the characteristics of plant tissue, such as cell wall, high water content, high content of secondary metabolites and the like, and in order to ensure the effectiveness of the subsequent analysis data, rRNA removal processing is designed on the wet experimental end, and a series of comparative experiments are carried out based on the above steps, and the above method is evaluated by the indexes of slice quality, permeation optimization result, cDNA quality, library quality, non-coding RNA capture efficiency and the like, wherein the detection standard is that: the plant tissue slice morphology should be uniform and flat, the tissue structure is basically intact, and there is no large cavity; the fluorescence intensity of the 6 slices of permeation optimization is relatively clear, and there is no diffuse blur, and the best permeation time can be clearly judged; the cDNA fragment length is 900-1200 bp, and the concentration is greater than 5 ng / μL; the library fragment length is 400-450 bp, and the concentration is greater than 10 ng / μL; the rRNA capture efficiency of the cDNA library is less than 10% under the condition of 50G sequencing amount, and the low-abundance non-coding RNA capture efficiency is improved.

[0867] In order to obtain a spatial whole transcriptome sequencing experiment method suitable for tobacco petiole in the present application, a series of explorations are carried out from embedding frozen section, tissue permeation treatment, in situ polyadenylation and rRNA removal and other aspects. Through consulting literature and multiple verifications, the content in the present application is finally obtained. The method and results are shown in Table 5 as shown in Comparative Examples 1-16. First, by Comparative Example 1, the embedding frozen section of the tobacco petiole tissue frozen section sample is prepared by using the current conventional frozen section embedding frozen condition, and the results are shown in Table 5: under the conventional frozen section operation condition (-80℃ frozen for 30 min), the section morphology presents serious tissue shrinkage, the parenchyma is damaged, there is local fracture, and a large number of tissue cavities appear, which cannot guarantee the original complete structure of the plant tissue, that is, the position of the cells and intracellular nucleic acids in the tissue has been moved, and the original position information has been lost, so the spatial transcriptome experiment cannot be carried out. It is speculated that the ice crystal growth rate in the tissue is too fast due to the improper freezing temperature and time of the plant tissue with high water content, which squeezes the surrounding cell structure, causing deformation or rupture of the cell wall, cell membrane and interstitial space, resulting in shrinkage or deformation of the sample tissue. Generally, adjusting the embedding freezing temperature and time can improve this situation, so Comparative Examples 2-4 adjust the embedding freezing time to 5-10 min, and the results show that under the condition of-80℃ freezing temperature, shortening the freezing time, the section morphology still has serious shrinkage and tissue cavity phenomenon, and there is no obvious improvement, and qualified sections cannot be prepared for experiment. Comparative Examples 5-7 adjust the embedding freezing temperature to-20℃, and the freezing time is adjusted to 30 min-1h with the temperature, and the results show that increasing the freezing temperature and slowing down the ice crystal formation rate can improve the shrinkage of the tissue section, and the-20℃ freezing temperature for 30 min is the best embedding condition, but there are still a small amount of wrinkles and cavities formed by the destruction of the parenchyma, which cannot guarantee the best complete morphology of the section, and the section quality is unqualified. The reason is that there are large vacuoles in the plant tissue, and the water content in the tissue is not uniform. When the tissue is frozen, the water in some areas may be frozen into ice crystals quickly, causing water in other areas to gather to these areas, damaging the parenchyma and forming cavities. Or there may be air bubbles in the tissue sample, which will expand during frozen section and interact with the surrounding ice crystals, causing the sample to have cavities.

[0868] Table 5 Comparative Examples 1-7

[0869]

[0870]

[0871] By comparing the results of comparative examples 1-7, it can be seen that embedding pretreatment of the tissue can be an effective way to solve the problem by enhancing sample uniformity and removing air bubbles in the tissue. Therefore, comparative examples 8-16 attempt to soak the sample in different pretreatment agents and use the negative pressure of the vacuum instrument to promote the penetration of the pretreatment agent into the tissue. The vacuum negative pressure penetration method not only facilitates the penetration of the freezing medium into the interior of the sample, reduces the water content, enhances the uniformity of the sample, but also effectively removes air bubbles in the tissue and reduces cavities. Comparative examples 8-9 use conventional tissue protectants OCT and 10% glycerol, both of which are penetrated for 10 minutes under negative pressure in the vacuum instrument. The results show that the tissue has a small amount of shrinkage and the structure has cavities. The glycerol pretreated sample has strong penetration and good frost resistance due to the low viscosity of glycerol itself, which leads to the use of glycerol as a pretreatment agent that cannot provide good support for the tissue morphology. As shown by the section results, the cell morphology is irregular, the thin-walled tissue is damaged by extrusion, resulting in cavities, which cannot meet the requirements of maintaining the integrity of the tissue structure. This method cannot solve the problem of section preparation. PEG is also a commonly used tissue protectant in biology, which has strong penetration regulation ability and adhesion, and can form a mutual solution with water. Therefore, it can effectively avoid the problems of tissue sample swelling, deformation and mechanical damage in the process of freezing preservation and other experiments, thereby maintaining the integrity of the tissue structure and the morphological characteristics. In the present application, comparative examples 10-13 use different concentrations of PEG as embedding pretreatment agents. The results show that 10%-30% PEG as an embedding pretreatment agent has poor support after negative pressure penetration in the vacuum, the tissue morphology is irregular, and the section has a downward stacking tendency, which is unqualified. The 40% PEG has good support, the thin-walled tissue structure is well maintained, there are no large cavities, and it basically meets the section requirements, but subsequent RNA quality inspection of the tissue section shows that the RIN value is 5.7 and the 28S / 18S is 0.54, indicating that the tissue RNA is degraded, which cannot meet the requirements of subsequent experiments. The reason may be that although high concentration PEG has good support for tobacco leaf petiole tissue structure, on the other hand, high concentration PEG also has a certain destructive effect on RNA molecular structure, such as causing RNA chain breakage and base deletion, thereby affecting the integrity and stability of tissue RNA, which cannot meet the requirements of subsequent experiments. Next, the present application attempts to use different concentrations of PVA as embedding pretreatment agents. PVA is a stable, non-toxic water-soluble polymer, which is commonly used as a thickening agent, emulsifying agent, anti-crystallization agent, etc. in industry. Compared with glycerol, PVA has greater viscosity, which can reduce the activity of water molecules in the tissue and slow down the formation speed of ice crystals. Its use in negative pressure penetration into the tissue can reduce the size and number of ice crystals formed in the tissue, protect the tissue from mechanical damage, and PVA has good penetration regulation ability, which can effectively alleviate the extrusion and deformation of the sample during the freeze-thaw cycle.Therefore, comparative examples 14-16 respectively use 10%, 20%, and 40% PVA as embedding pretreatment agent, and vacuum infiltration in a vacuum instrument for 10-15 min. The results show that the tissue structure pretreated by 10% and 20% PVA is deformed, and the tissue morphology cannot be well maintained. This may be due to the low PVA concentration, and the ice crystals generated by the combination of PVA and water cause damage to the parenchyma, resulting in tissue shrinkage and failure to protect and support the tissue morphology, and thus unable to complete the qualified section preparation. Examples 1-3 attempt to use 25-35% PVA to pretreat the tissue, and the results show that the tissue section is relatively uniform and flat, the tissue structure is basically intact, and there is no large cavity. Subsequent quality inspection of the tissue section shows that the RIN value is greater than 7, and the 28S / 18S value is greater than 0.7. Comparative example 16 uses 40% PVA for pretreatment, and the tissue edge has shrinkage, the tissue structure is deformed, and there is a cavity. It is speculated that the viscosity of 40% PVA is high, and the negative pressure cannot well penetrate into the tissue, so it cannot achieve the role of supporting the tissue morphology. After comprehensive evaluation of the cost and operation time, the present application determines to use 25% PVA as the embedding pretreatment agent, and vacuum infiltration in a vacuum instrument for 10 min pretreatment before embedding sectioning, so as to achieve the best sectioning effect of tobacco petiole tissue.

[0872] Table 6 comparative examples 8-16

[0873]

[0874]

[0875] Next, Comparative Examples 17-23 were used to explore a series of tobacco petiole permeation conditions. Comparative Example 17 used the conventional 10X Visium spatial transcriptome sequencing instruction flow to optimize the permeation of tobacco petiole tissue. The results showed that the fluorescence intensity of the six slices treated for 3-30 min was weak, and there was little difference, so the optimal permeation time could not be determined, and the subsequent processing conditions were not met. The reason may be that plant cells are composed of cell walls and plasma membranes and other structures, which make plant cells more hard and difficult to permeate than animal cells. When performing tissue permeation, the cell wall needs to be first destroyed or penetrated, so that the exogenous enzyme can contact the cell membrane for membrane permeation. This process is more difficult for plant tissues. Therefore, Comparative Examples 18-21 used different formulations and concentrations of pretreatment liquids to enzymatically digest the plant cell wall before permeation. Comparative Example 18 used a conventional plant cell wall enzymatic digestion formulation to pretreat the tobacco petiole. After permeation optimization, the results showed that the fluorescence intensity of the six slices treated for 3-30 min was weak, and only the middle part of the tissue had weak fluorescence, so the optimal permeation time could not be determined, and the subsequent processing conditions were not met. The reason may be that the conventional pretreatment liquid is mainly used for tissue protoplast separation, and its purpose is only to obtain protoplasts from a large amount of plant tissue, not to obtain as many or all protoplasts as possible from the tissue. This enzyme solution cannot have good enzymatic effect on all parts of the plant petiole. The main components of the cell wall in the middle phloem region are cellulose and pectin, so the enzyme solution formulation may have good enzymatic effect on the cell wall in this region. The main components of the cell wall in other parts contain hemicellulose. Therefore, Comparative Example 19 added 1% hemicellulase (m / v) to the original formulation. The permeation optimization results showed that the tissue fluorescence range was expanded, i.e., the addition of hemicellulase improved the problem of incomplete slice information, but the fluorescence intensity of the six slices treated for 3-30 min was weak, indicating that the detected RNA was very low, and the fluorescence intensity was close to zero, so the optimal permeation time could not be determined. Continuing the experiment would risk incomplete information detection and low RNA content leading to library construction failure. According to the conventional enzymatic effect, the enzyme species and concentration of this formulation can effectively enzymatically digest the cell wall of plant tissue. Therefore, it is speculated that for the slice tissue, the enzymatic concentration is too strong, the cell wall is completely digested, and the protoplast is damaged, causing the intracellular nucleic acid to flow out, resulting in nucleic acid loss during the washing process, low nucleic acid content, and low fluorescence intensity. Cell fragmentation also causes loss of positional information, affecting the subsequent determination of the source location and spatial distribution of RNA. Therefore, Comparative Examples 20-21 reduced the concentration of the enzyme solution to reduce the damage of the enzyme solution to the sample cells. The permeation optimization results showed that the fluorescence intensity of the six slices treated for 3-30 min was stronger than that of Comparative Example 19, and the fluorescence range basically covered the entire tissue. The fluorescence intensity was the strongest at 18 min of permeation, but it was still weaker than the conventional fluorescence intensity.In order to retain the spatial position information of the protoplast, the pretreatment process only makes the cell wall structure loose instead of completely enzymolysis of the cell wall, resulting in poor permeation effect of pepsin in the presence of part of cell wall components during the permeation process, and there is a risk of nucleic acid degradation by unreasonably prolonging the permeation time or changing the concentration of the protease, therefore, the cell membrane is pretreated before permeation, Tween 20 is a non-ionic surfactant with good permeability and dispersion performance, and is often used as a detergent, but it can also destroy the phospholipid bilayer structure on the cell membrane, so that the cell membrane becomes more loose, thereby improving the efficiency of the exogenous material into. Therefore, comparative examples 22-23 try to add Tween 20 in the pretreatment solution, and the cell membrane is permeable to a certain extent while the cell wall is enzymolysed, thereby improving the permeability efficiency of the subsequent pepsin. In comparative example 22, 1% Tween 20 (v / v) is added to the enzymolysis solution, and the results show that the fluorescence intensity of the six slices treated for 3-30 min is weak, and the optimal permeation time cannot be judged, which does not meet the subsequent processing conditions. In comparative example 23, the concentration of Tween 20 is reduced, and the results show that the fluorescence intensity of the six slices treated for 3-30 min is slightly stronger than that of comparative example 21, but still weaker than the conventional fluorescence brightness. Therefore, it can be known that too high concentration of Tween 20 can also cause cell membrane rupture and lead to loss of nucleic acid outflow, but appropriate amount of Tween 20 can obviously improve the permeation efficiency, therefore, examples 4-6 continue to explore the concentration of each component of the pretreatment solution formula, and the permeation optimization results of the three groups of formula show that the fluorescence intensity of the six slices treated for 3-30 min is obviously distinguished, the fluorescence signal is strong enough, can be distinguished from the background noise, there is no excessive exposure, the fluorescence signal is uniformly distributed in the sample area, there is no obvious signal gradient or local missing phenomenon, and the slice quality is qualified, and the optimal permeation time is 18 min. After comprehensive evaluation of the cost and operation time, the permeation pretreatment solution formula of the application is determined as 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.35% cellulase R10 (m / v), 0.1% macerozyme R10 (m / v), 0.1% pectinase (m / v), 0.15% hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4U / μL RNase inhibitor, and the pretreatment condition is 37°C incubation for 10 min.

[0876] Table 7 comparative examples 17-23

[0877]

[0878]

[0879]

[0880] Next, comparative examples 24-31 were used to explore the method conditions for in situ polyadenylation. Comparative example 24 used a conventional E. coli-derived poly(A) polymerase (Bio-Rad / MT0004) mixture to incubate at 37°C for 30 min to in situ polyadenylate non-coding RNA. The results showed that the quality of the cDNA and library after in situ polyadenylation met the requirements of space transcriptome sequencing, but the non-coding RNA capture efficiency was low at 50G sequencing amount, and miRNA was not captured. Comparative example 25 replaced another brand of E. coli-derived poly(A) polymerase (Thermo, AM1350) on the market to perform in situ polyadenylation, and the results were similar to comparative example 24. E. coli-derived poly(A) polymerase is more suitable for A tail addition to RNA of mammalian tissues due to its specific source. Yeast-derived poly(A) polymerase can be used for RNA samples of various cell types and species, and has a faster reaction rate, which can complete the addition of poly(A) tail on non-coding RNA in a shorter time, shortening the experimental time. Therefore, comparative examples 26-27 replaced the poly(A) polymerase with yeast-derived polymerase and used different brands of ATP on the market, and the results showed that the quality of the cDNA and library after in situ polyadenylation in the two experimental groups met the requirements of space transcriptome sequencing, the non-coding RNA capture efficiency was relatively improved compared with comparative examples 24-25 at 50G sequencing amount, and a small amount of miRNA was captured. Comparative example 27 also captured a small amount of scaRNA, but it was still far below the ideal capture rate.

[0881] The temperature of 37°C increases the risk of RNA degradation, so it is necessary to ensure the enzyme activity of the polymerase as much as possible to efficiently complete the A addition. It is well known that some amino acid residues of the protease catalytic center and substrate binding site affect enzyme activity and substrate specificity, and Met, Cys and His, Trp, Tyr amino acid residues are extremely easy to oxidize, thereby causing protein denaturation or affecting enzyme activity. A large amount of secondary metabolites can be stored in plant protoplasts, and the vascular tissue at the petiole is the center of iron transport in plants. The oxidizability of secondary metabolites and the inhibition of metal ions will greatly limit the activity of the protease. In comparative examples 28-29, the buffer containing metal ion chelators and reducing agents was used to pretreat the tissue to reduce the influence of metal ions and the oxidizability of secondary metabolites on the activity of the poly(A) polymerase. The results show that the cDNA and library quality after in situ polyadenylation of the two experimental groups meet the requirements of space transcriptome sequencing, but there is no obvious difference in the capture efficiency of non-coding RNA under 50G sequencing amount compared with comparative examples 26-27. At present, there are some literatures reporting that the secondary metabolite jasmonic acid in plants has a certain inhibitory effect on the activity of the poly(A) polymerase. When the plant body is subjected to external mechanical damage, the key enzyme 12-oxidase (LOX) in the jasmonic acid synthesis pathway is activated, thereby producing a large amount of 12-oxides. These 12-oxides can be converted into jasmonic acid through a series of enzyme catalytic reactions. Therefore, the mechanical damage such as cutting of the tobacco petiole can promote the synthesis of jasmonic acid, increase the content of the secondary metabolite jasmonic acid in the sample, and affect the activity of the poly(A) polymerase, which may be the main reason for reducing the polyadenylation efficiency of non-coding RNA in plant tissue. β-galactosidase is a kind of hydrolase that can catalyze the hydrolysis reaction of β-galactoside to decompose the substrate into galactose and glucose, and is commonly used for lactose hydrolysis. Although there is no β-galactoside bond in jasmonic acid, it can recognize the part of the bond stickiness in the jasmonic acid molecule to break it into 7-hydroxy-12-oxo jasmonic acid and galactose, so as to degrade and inactivate the jasmonic acid. Therefore, in comparative examples 30-31, different concentrations of β-galactosidase solution are used for incubation pretreatment before in situ polyadenylation. The results show that the non-coding RNA capture efficiency of the two experimental groups under 50G sequencing amount is improved compared with comparative examples 24-29. Examples 7-8 continue to explore the optimal addition concentration of β-galactosidase. The results show that the cDNA and library quality after in situ polyadenylation of the two experimental groups meet the requirements of space transcriptome sequencing, and the non-coding RNA capture efficiency under 50G sequencing amount is obviously improved compared with comparative examples 30-31, but there is no obvious difference in the capture efficiency between examples 7 and 8. After comprehensive evaluation of the cost and operation time, the in situ polyadenylation treatment solution of the present application is determined to be 0.2x β-galactosidase solution, the pretreatment temperature is room temperature, and the pretreatment time is 10 min.

[0882] Table 8 Comparative Examples 24-31

[0883]

[0884]

[0885]

[0886] By comparing Examples 24-31, it can be seen that rRNA accounts for more than 20% of the total RNA capture rate. rRNA is the member with the highest RNA abundance, has a relatively large molecular weight and is not metabolically active, so the transcriptome noise (invalid information) is basically all from rRNA. The purpose of sequencing is to obtain more biological information, but this most abundant member of RNA can only provide very little information on transcripts, and too much detection of rRNA can mask the expression of other low-abundance RNA species; therefore, rRNA removal is usually performed before sequencing to improve the effectiveness of the overall data. There are basically two ways to remove rRNA, one is to remove rRNA in the sequencing results through bioinformatics analysis, and to retain other valid data. Comparative Example 32 removes rRNA through bioinformatics means, and the results show that the rRNA content is finally reduced to 3.2% through bioinformatics means. Although the rRNA content is significantly reduced, the capture efficiency of other low-abundance non-coding RNA is not significantly improved, so this method can only be used to remove data noise and cannot improve the data amount of low-abundance non-coding RNA after sequencing is completed. Therefore, Comparative Example 33 uses a commercially available SEQuoia RiboDepletion ribosomal RNA removal kit (Bio-Rad / 17006487) to remove rRNA, and the results show that the rRNA capture rate is significantly reduced, and the capture efficiency of other low-abundance non-coding RNA is also correspondingly increased, but the capture efficiency of rRNA is still more than 10%.

[0887] Table 9 Comparative Examples 32-33

[0888]

[0889]

[0890] In summary, the present application provides a spatial whole transcriptome sequencing method suitable for tobacco petiole tissue. By comparing examples 1-7, it is determined that when the tobacco leaf is frozen sectioned, the embedding freezing conditions are-20℃, and the freezing time is 30 min, which can effectively improve the tobacco petiole sectioning shrinkage; by comparing examples 8-16, the pretreatment method of tobacco petiole tissue before embedding is evaluated, and by combining examples 1-3, it is determined that the optimal use concentration of PVA is 25%, and the vacuum instrument treatment time is 10 min, which can provide good support for the structure of tobacco petiole section and maintain the uniformity and integrity of the section. By comparing examples 17-23, the pretreatment method of the sample tissue before permeabilization is evaluated, and by combining examples 4-6, it is determined that the optimal permeabilization pretreatment liquid formula is (final concentration) 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.35% cellulase R10 (m / v), 0.1% lyticase R10 (m / v), 0.1% pectinase (m / v), 0.15% hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4U / μL RNase inhibitor, and the pretreatment condition is 37℃ incubation for 10 min, which can effectively enzymatically hydrolyze the plant cell wall to increase the cell membrane permeability and achieve the best permeabilization effect; by comparing examples 24-31, the method of in situ polyadenylation is evaluated, and it is determined that the use of yeast-derived poly(A) polymerase and ATP (Thermo / AM8110G) has the highest in situ polyadenylation efficiency, and by combining examples 7-8, it is determined that the use of 0.2x beta-galactosidase solution for pretreatment of the tissue can effectively reduce the influence of secondary metabolites in the plant tissue on the activity of poly(A) polymerase and improve the in situ polyadenylation efficiency. The results of comparative examples 32-33 show that the removal of rRNA by bioinformatics and the use of existing kits on the market cannot simultaneously reduce the capture efficiency of rRNA by 10% or less and improve the capture efficiency of the remaining low-abundance non-coding RNA, and by combining examples 9-10, it is determined that the method of synthesizing guide RNA combined with Cas9 enzyme by using 18s rRNA and 28s rRNA fragment sequences can effectively remove ribosomal RNA from the library and improve the capture efficiency of low-abundance non-coding RNA under the same sequencing amount, and it is determined that the use of 0.8x SPRI magnetic beads for purification can reduce the generation of small fragments in the library.

[0891] Table 10: Guide RNA sequence

[0892]

[0893]

[0894]

[0895] Wherein, m is 2-methoxy modification, * is thio modification, and "A", "G", "C", "U" represent adenine, guanine, cytosine and uracil respectively.

[0896] Reference: [1] Jasmonic acid interacts with abscisic acid to regulate plant responses to water stress conditions

[0897] The protection scope of the present application is not limited to the above-mentioned embodiments. Any changes and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the present application are included in the present application and are protected by the appended claims.

Claims

1. A spatial whole transcriptome sequencing method suitable for tobacco petiole tissue, characterized by, The method refers to effectively detecting low-abundance non-coding RNA in tobacco petiole tissue on the basis of conventional spatial transcriptome sequencing, and realizes spatial whole transcriptome sequencing of tobacco petiole; After the steps of tobacco petiole tissue sample embedding, sample tissue sectioning, tissue permeation, in situ polyadenylation processing and pretreatment, cDNA library construction, cDNA library quality inspection, spatial whole transcriptome sequencing and analysis are carried out; The pretreatment step before the tissue permeation includes using a tissue permeation pretreatment solution, and the tissue permeation pretreatment solution contains the following components, all in the form of final concentration: 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.25-0.5% (m / v) cellulase R10, 0.05%-0.1% (m / v) lyase R10, 0.05%-0.2% (m / v) pectinase, 0.1%-0.2% (m / v) hemicellulase, 0.1-0.25% (v / v) Tween 20, and 0.4U / μL RNase inhibitor.

2. The method of claim 1, wherein, The method comprises the following steps: Step one, sample embedding pretreatment: take fresh tobacco petiole tissue, wipe the surface to remove excess liquid, and pretreat the tobacco petiole tissue before embedding; Step two, sample embedding: wipe the surface of the pretreated tobacco petiole tissue in step one to remove excess liquid, place the embedding mold horizontally on ice, inject pre-cooled OCT freezing section embedding agent into 1 / 3 of the volume of the mold groove, confirm the tissue section direction, perform OCT embedding, and horizontally transfer to a-20℃ environment for freezing to ensure that the OCT and the tissue are frozen; Step three, sample tissue sectioning: section the embedded tissue sample in step two, flatten the sample, place the sample on a common glass slide, immediately press the back of the glass slide with a finger for 5 seconds, use the finger temperature to thaw the section, and make it fully adhere to the glass slide; Step four, fix and HE stain the tissue section in step three; Step five, scan and image the section in step four; Step six, tissue permeation pretreatment: adhere the tissue section to the specified position of the Visium spatial gene expression glass slide, install the Visium spatial gene expression glass slide in a glass slide box, configure the permeation pretreatment solution, add the permeation pretreatment solution to the hole in the glass slide box from the lower left corner of the sample hole, gently pat the glass slide box to make the treatment solution evenly cover the tissue, attach a sealing strip, and place the glass slide box containing the Visum spatial gene expression glass slide in a PCR instrument for pretreatment; Step seven, tissue permeation: after the pretreatment is completed, the pretreatment liquid is removed from the sample well of the slide box, a washing buffer is added to each well, and after washing, the permeation enzyme is resuspended and preheated, the permeation enzyme is added to the well by sticking to the lower left corner of the sample well of the slide box, the slide box is tapped to evenly cover the tissue with the permeation enzyme, a sealing strip is attached, the slide box containing the Visum spatial gene expression slide is placed on the PCR instrument with an adapter, the PCR instrument cover is covered and incubated for permeation optimization for a selected permeation time, after permeation is completed, the sealing strip is removed, the permeation enzyme in each well is removed with a pipette, and a washing buffer is added to each well; Step eight, in situ polyadenylation pretreatment: a pretreatment liquid for in situ polyadenylation is added to each sample well of the slide box described in step seven, and after incubation, the pretreatment liquid is removed; Step nine, in situ polyadenylation: a washing buffer is added to each sample well described in step eight, and after incubation, the washing buffer is removed, a poly A polymerase mixture of yeast origin is added to each sample well of the slide box, the reaction chamber is sealed, after incubation, the enzyme mixture is removed, a washing buffer is added to each well for washing, and then the washing buffer is discarded; Step ten, cDNA library construction; Step eleven, rRNA removal: a digestion mixture is prepared, the cDNA library described in step ten is mixed with the digestion mixture in proportion, and incubated at 37℃, proteinase K is added to the reaction, and incubation is performed at 56℃ to release DNA from Cas9 endonuclease, and the sample is purified and eluted using 0.8×SPRI magnetic beads; Step twelve, cDNA library quality inspection; Step thirteen, spatial whole transcriptome sequencing and analysis.

3. The method of claim 2, wherein, The method comprises a tobacco petiole tissue embedding pretreatment step; the embedding pretreatment in step one is that the tissue is immersed in 25%-35% (v / v) polyvinyl alcohol PVA before embedding, and the PVA is allowed to fully penetrate into the tissue by using a vacuum instrument to create negative pressure for 10-15 min; and / or, In step two, the embedding condition is freezing at-20℃ for 30 min; and / or, In step six, the tobacco petiole tissue permeation pretreatment liquid is used for pretreatment at 37℃ for 10-15 min in a PCR instrument before conventional tissue permeation; and / or, The method comprises an in situ polyadenylation pretreatment step; in step eight, a secondary metabolite removal pretreatment liquid is used for room temperature treatment for 10 min before the in situ polyadenylation step, the pretreatment liquid is a 0.2-0.5×β-galactosidase solution with a final concentration, and the stock solution concentration of the β-galactosidase is 40 U / μL; and / or, The method comprises an in situ polyadenylation step; in step nine, 1× washing buffer is added to each sample well, the washing buffer is removed after incubation at room temperature for 30 s, a yeast-derived poly A polymerase mixture is added to each sample well, and the mixture is removed after incubation at 37℃ for 20 min; and / or, The method comprises the step of removing rRNA from the cDNA library; in step eleven, a single-directional guide RNA pool containing 26 synthetic guide RNAs is mixed with the Cas9 enzyme and the sample cDNA library into an in vitro digestion mixture, incubated at 37℃ for 60 min, then proteinase K is added, incubated at 56℃ for 10 min to promote DNA release, and finally 0.8×SPRI is used for magnetic bead purification to separate the target cDNA library fragments excluding rRNA.

4. The method of claim 2, wherein, The tobacco petiole tissue permeabilization pretreatment liquid comprises the following components at the following final concentrations: 0.02M MES, 0.02M KCl, 0.1% BSA, 0.01M CaCl2, 0.1M mannitol, 0.25-0.5% (m / v) cellulase R10, 0.05%-0.1% (m / v) macerozyme R10, 0.05%-0.2% (m / v) pectinase, 0.1%-0.2% (m / v) hemicellulase, 0.1-0.25% (v / v) Tween 20, and 0.4U / μL RNase inhibitor; and / or, The in situ polyadenylation pretreatment liquid comprises the following components: 250 U / mg β-galactosidase is prepared into a stock solution of 40U / μL with 1×PBS, and the final use concentration of the pretreatment liquid is 0.2-0.5×β-galactosidase solution; and / or, The washing buffer in step nine comprises the following components: 1×polyadenylate polymerase reaction buffer, 0.8U / μL RNase inhibitor, and ddH2O; and / or, The polyadenylate polymerase mixture in step nine comprises the following components: 1×polyadenylate polymerase reaction buffer, 24U / μL polyadenylate polymerase, 0.5mM ATP, and 1.5U / μL RNase inhibitor.

5. The method of claim 3, wherein, The 26 synthetic guide RNAs in step eleven are guide RNAs designed and synthesized according to the sequences of 18s rRNA and 28s rRNA fragments, and the guide RNA sequences are shown in SEQ ID NO. 1-26; and / or, the in vitro digestion mixture comprises the following components: IDTE buffer: 10mM Tris HCl, 0.1mM EDTA; and a single-directional guide RNA pool.

6. A kit / reagent for tobacco petiole space full transcript sequencing, characterized by, The kit / reagent comprises a tobacco petiole tissue permeabilization pretreatment liquid, an in situ polyadenylation pretreatment liquid, an in situ polyadenylation washing buffer, a polyadenylate polymerase mixture, and / or an in vitro digestion mixture; The tobacco leaf petiole tissue permeabilization pretreatment solution comprises the following components: 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.25-0.5% (m / v) cellulase R10, 0.05%-0.1% (m / v) macerase R10, 0.05%-0.2% (m / v) pectinase, 0.1%-0.2% (m / v) hemicellulase, 0.1-0.25% (v / v) Tween 20, 0.4U / μL RNase inhibitor; The in situ polyadenylation pretreatment solution comprises the following components: 0.2-0.5×β-galactosidase solution with a final concentration, and the stock concentration of the β-galactosidase is 40U / μL; The in situ polyadenylation washing buffer comprises the following components: 1×polyadenylate polymerase reaction buffer, 0.8U / μL RNase inhibitor, and ddH2O; The polyadenylate polymerase mixture comprises the following components: 1×polyadenylate polymerase reaction buffer, 24U / μL polyadenylate polymerase, 0.5mM ATP, and 1.5U / μL RNase inhibitor; The in vitro digestion mixture comprises the following components: 10μM single-direction guide RNA pool, 20μM Cas9 endonuclease, 1×PBS, 10×Cas9 nuclease reaction buffer, and 20mg / ml proteinase K.

7. A pretreatment liquid characterized in that, The pretreatment solution comprises: The tobacco leaf petiole tissue permeabilization pretreatment solution comprises the following components: 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.25-0.5% (m / v) cellulase R10, 0.05%-0.1% (m / v) macerase R10, 0.05%-0.2% (m / v) pectinase, 0.1%-0.2% (m / v) hemicellulase, 0.1-0.25% (v / v) Tween 20, and 0.4U / μL RNase inhibitor; The in situ polyadenylation pretreatment solution comprises the following components: 0.2-0.5×β-galactosidase solution with a final concentration, and the stock concentration of the β-galactosidase is 40U / μL.

8. Use of the method according to any one of claims 1-5, the kit / reagent according to claim 6, the pretreatment solution according to claim 7 in plant spatial whole transcriptome sequencing, and in tobacco leaf petiole spatial whole transcriptome sequencing.

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