Spatial complete transcription sequencing method suitable for tobacco petiole tissue and application

Abstract

The invention provides a spatial complete transcription sequencing method suitable for tobacco petiole tissues and application. According to the method, a tissue embedding method and a freezing slicing method suitable for the tobacco petioles are adopted, it is effectively guaranteed that tobacco petiole slices are uniform and flat, and the structure is kept complete; the pretreatment liquid suitable for being used before plant tissue permeabilization is adopted, plant tissue cell walls can be effectively subjected to enzymolysis, cell membrane permeability can be improved, and RNA can be better released; a treatment method suitable for in-situ polyadenylation of tobacco petiole tissues is adopted, so that in-situ polyadenylation can be effectively performed on non-coding RNA in a slice sample, and the capture efficiency of the non-coding RNA is improved; by adopting the method which is suitable for a space whole transcriptome sequencing process and can remove the rRNA from the cDNA library, the rRNA can be effectively removed from the library through a wet experiment end, the situation that the sequencing depth of other RNA types is reduced due to the fact that the amount of captured ribosome RNA is too high is avoided, and the data utilization rate is greatly increased. The method has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology and relates to a spatial full transcription sequencing method suitable for tobacco petiole tissue and its application. Background Art

[0002] Spatial transcriptomics is a technology used to study gene expression in different cell types and regions within tissues. In recent years, the rise of spatial transcriptomics has revealed the spatial background of gene expression in many mammals, such as identifying different cell populations and spatial structures in tumors to reveal their relationship with tumor development and treatment response. In addition, it has also been applied in botany, such as exploring the gene expression patterns in different regions of leaves during photosynthesis, but the current application scope of plants cannot be compared with that of mammals. The main reasons are: 1. Plant tissues usually have complex, multi-level differentiation structures, so it is difficult to prepare higher resolution and accurate samples. 2. The presence of plant cell walls has a certain impact on steps such as RNA diffusion and sample fixation, and special processing methods different from animal tissues are required. 3. The genomic data of many plant species are missing or incomplete, which also limits the scope and depth of application of spatial transcriptomics in plant tissue research to a certain extent.

[0003] At present, mature spatial transcriptomics methods are limited to measuring transcripts with polyadenylic acid, and are insensitive to many types of non-A-tailed RNA. It is known that 85% of the plant genome will be 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, while most other types of RNA molecules are non-coding RNA, which also has a very important regulatory effect on cell function and state. Expanding the scope of plant spatial transcriptomics to the whole transcriptome can understand the expression pattern of all plant genes under different conditions, observe the spatial distribution of regulatory RNA and its targets, and link non-host RNA with the transcription process of the host, thereby revealing the interaction relationship and multi-level regulatory network 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, and may also explore many unknown functional genes or regulatory factors, thereby expanding the understanding and application of plant gene functions. These new genes may affect plant metabolism, signal transduction, protein synthesis and other aspects, and have important biological and agricultural value.

[0004] At present, the application of spatial transcriptome in animal tissues is very mature, but the structure of animal and plant tissues is very different. Plant cells have characteristics such as cell walls, large vacuoles, and high water content in tissues, which makes it difficult to slice and permeabilize tissues. The same method as animal tissues cannot produce ideal results. Due to the defects in spatial transcriptome methods, spatial full transcriptome has not been well developed and applied in plants. In summary, it is urgent to develop an operation method and process for spatial full transcriptome of plant tissues.

[0005] Currently, the existing 10XVisium spatial transcriptome sequencing technology is widely used in the study of mammalian tissues. This technology can combine RNA sequencing and tissue imaging to achieve high-resolution spatial visualization and quantitative analysis of gene expression in tissues. However, this technology is developed for mammalian tissues, and the permeabilization agent with pepsin as the main component is not well applicable to plant tissues with cell walls. Moreover, this method is limited to the detection of transcripts with polyadenylation, and cannot explore some non-coding RNAs that play an important role in gene expression regulation, signal transduction, metabolic regulation and environmental response. Summary of the invention

[0006] In order to address the deficiencies of the prior art, based on a large amount of in-depth research and experiments, the present invention provides a set of spatial whole transcriptome experimental processes / methods suitable for tobacco petiole tissue. The method is simple, easy to use, accurate, and has comprehensive detection information. It can also be directly combined with the existing 10X Visium spatial transcriptome sequencing process, which can not only make up for the shortcomings of spatial transcriptome technology in the field of botany, but also obtain more detailed cell clustering effects and more comprehensive gene expression information with less sequencing amount.

[0007] The spatial whole transcriptome method applicable to tobacco petiole tissue proposed in the present invention refers to a method that can effectively solve the problems of difficult preparation of sample sections and incomplete permeabilization of tobacco petiole tissue due to the particularity of the samples during the spatial transcriptome sequencing experiment of tobacco petiole tissue on the basis of the conventional 10XVisium spatial transcriptome sequencing process, and the in situ polyadenylation method and rRNA removal method provided can effectively detect low-abundance non-coding RNA in tobacco petiole tissue on the basis of conventional spatial transcriptome sequencing, thereby realizing the method process of spatial whole transcriptome sequencing of tobacco petioles.

[0008] The method of the present invention is processed and pretreated through the steps of embedding tobacco petiole tissue samples, sample tissue slicing, tissue permeabilization, and in situ polyadenylation, and then through cDNA library construction and cDNA library quality inspection, spatial full transcriptome sequencing and analysis. In a specific embodiment, the method of the present invention includes: sample embedding pretreatment, sample embedding, sample tissue slicing, tissue slice fixation and HE staining, tissue slice scanning imaging, tissue permeabilization pretreatment, tissue permeabilization, in situ polyadenylation pretreatment, in situ polyadenylation, cDNA library construction, rRNA removal, library quality inspection, spatial full transcriptome sequencing and analysis.

[0009] Since tobacco petiole belongs to plant tissue, its tissue characteristics are very different from those of animal tissue. Tobacco petiole tissue has large vacuoles and cell walls. The use of conventional spatial transcriptome processes has problems such as difficulty in preparing sample slices and incomplete permeabilization. In order to solve the shortcomings of the prior art, the technical solution described in the present invention has made 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) infiltration before sample embedding, and the embedding and freezing conditions are adjusted to enhance tissue uniformity and overall support of the slices, thereby ensuring the integrity of the slices; before tissue permeabilization, a permeabilization pretreatment solution containing Tween 20 (Tween20) is used to loosen the cell wall structure and increase the permeability of the cell membrane, thereby effectively improving the release efficiency of RNA during permeabilization while ensuring the RNA location information; an in situ polyadenylation step is introduced into the process to achieve in situ capture of non-coding RNA in tobacco petiole tissue in the spatial process; before in situ polyadenylation, the tissue Pretreatment was carried out, and β-galactosidase was used to degrade and remove secondary metabolites in tobacco petioles, which effectively improved the polyadenylation efficiency of non-coding RNA in sample tissues; an rRNA removal step was introduced into the process, and the synthetic rRNA guide probe was assembled into a complex with the Cas9 enzyme, and then the complex was added to the reaction system to make it complementary to the target DNA sequence. The Cas9 enzyme positioned on the rRNA sequence and catalyzed the double-strand break of the target sequence, causing it to be cut into short fragments, thereby effectively achieving the purpose of removing the rRNA sequence.

[0010] The method of the present invention comprises the following steps:

[0011] Step 1: 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 2: Sample embedding: Gently wipe the surface of the pretreated tobacco petiole tissue described in step 1 to remove excess liquid, place the embedding mold horizontally on ice, inject the pre-cooled frozen section embedding agent OCT into 1 / 3 of the mold groove capacity, confirm the direction of tissue sectioning, embed OCT, and transfer horizontally to a -20℃ environment for freezing to ensure that OCT and tissue are frozen;

[0013] The OCT refers to an optimal cutting temperature compound commonly used for cryosection embedding. It is a common sample embedding agent used in cryosection experiments. Its main purpose is to support tissues during cryosectioning to increase tissue continuity and reduce wrinkles and fragmentation.

[0014] Step 3, sample tissue sectioning: Slice the embedded tissue sample in step 2, flatten it, place the sample on a common slide, immediately put your finger on the back of the slide for 5 seconds, use the finger temperature to thaw the slice, and make the tissue slice and the slide fully fit together;

[0015] Step 4, fixing and HE staining the tissue sections described in step 3;

[0016] Step 5, scanning and imaging the slices described in step 4;

[0017] Step 6. Tissue permeabilization pretreatment: stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, prepare the permeabilization pretreatment solution, add the permeabilization pretreatment solution to the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, stick a sealing strip, and place the Slide Cassette in the PCR instrument for pretreatment;

[0018] The Visium spatial gene expression slide is a highly transparent, chemically stable slide designed for Visium spatial transcriptome sequencing. The slide has millions of micro-reaction pools that can capture and locate RNA molecules in individual cells and tissues and match them with unique barcodes. Each reaction pool 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, accurate transcriptome data.

[0019] The Slide Cassette is a slide box, which is a container for fixing the Visium spatial gene expression slide containing samples. This slide box can wrap the slide well, and there are 8 hollow square holes on its surface, which correspond to the 8 sample areas on the slide. There is a detachable gasket between the slide box and the slide, and the detachable gasket also corresponds to the capture area on the slide. After the sample area of ​​the slide is matched with the holes of the slide box one by one, 8 well-sealed and leak-free holes can be formed to facilitate the addition of reagents to different samples.

[0020] The Slide Cassette sample wells refer to eight well-sealed, leak-free independent sample wells formed after the Slide Cassette glass box and the Visium spatial gene expression slide are installed, into which the required reagents can be added without contaminating other sample areas.

[0021] Step 7, tissue permeabilization: After pretreatment, carefully remove the pretreatment solution from the Slide Cassette sample wells, add washing buffer to each Slide Cassette sample well, remove after washing, resuspend and preheat the permease, add the permease to the well against the lower left corner of the Slide Cassette sample well, tap the Slide Cassette to make the permease evenly cover the tissue, attach the sealing strip, put the Slide Cassette containing the Visum spatial gene expression slides on the PCR instrument with the Slide Cassette adapter, cover the PCR instrument cover and incubate for permeabilization to optimize the selected permeabilization time, after permeabilization, remove the sealing strip, use a pipette to remove the permease in each well without touching the tissue, and add washing buffer to each well;

[0022] Step 8, in situ polyadenylation pretreatment: adding in situ polyadenylation pretreatment solution to each Slide Cassette sample well described in step 7, and removing the pretreatment solution after incubation;

[0023] Step 9, in situ polyadenylation: adding a wash buffer to each Slide Cassette sample well described in step 8, removing the buffer after incubation, adding a polyadenylate polymerase mixture to each Slide Cassette sample well, sealing the reaction chamber, removing the enzyme mixture after incubation, adding a wash buffer to each well for washing, and then discarding the wash buffer;

[0024] Step 10: cDNA library construction;

[0025] Step 11, rRNA removal: prepare an in vitro digestion mixture, mix the in vitro digestion mixture with the cDNA library in a molar ratio and incubate at 37°C, add proteinase K to the reaction and incubate at 56°C to allow Cas9 endonuclease to release DNA, and use 0.8×SPRI magnetic beads to purify and elute the sample;

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

[0027] Step 12: cDNA library quality inspection;

[0028] Step 13: Spatial whole transcriptome sequencing and analysis.

[0029] In the method of the present invention, an innovative embedding pretreatment method / step suitable for tobacco petiole tissue sections is proposed. Specifically, the tobacco petiole sample is pretreated by polyvinyl alcohol (PVA) infiltration before embedding.

[0030] In a specific embodiment, the tobacco is 90-100 days old, preferably, the tobacco is 90 days old; the petiole is the petiole at the connection between 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; the sampling method is cross-section sampling.

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

[0032] Wherein, 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 apparatus is 10 to 15 minutes, and preferably, the penetration time in the vacuum apparatus is 10 minutes.

[0034] In the method of the present invention, it is proposed to use the method suitable for processing tobacco petiole tissue sections in plant spatial whole transcriptome sequencing.

[0035] The present invention also provides a pretreatment solution suitable for tobacco petiole tissue permeabilization. The tobacco petiole tissue permeabilization pretreatment solution comprises, in addition to enzyme components used for conventional enzymatic hydrolysis of cell walls, Tween 20, and the concentration range of each component of the treatment solution is specified. The specific components of the tobacco petiole tissue permeabilization pretreatment solution further comprise: cellulase R10, macerate R10, pectinase, hemicellulase, and Tween 20.

[0036] Preferably, the formula of the tobacco petiole tissue permeabilization pretreatment solution is: 0.02M MES (2-(N-morpholino)ethanesulfonic acid), 0.02M KCl (potassium chloride), 0.1% (m / v) BSA (bovine serum albumin), 0.01M CaCl 2 (calcium chloride), 0.1M mannitol, 0.25-0.5% (m / v) cellulase R10, 0.05%-0.1% (m / v) maceration enzyme 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; preferably, the tobacco petiole tissue permeabilization pretreatment solution is formulated as 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.35% (m / v) cellulase R10, 0.1% (m / v) macerate R10, 0.1% (m / v) pectinase, 0.15% (m / v) hemicellulase, 0.15% (v / v) Tween 20, 0.4 U / μL RNase inhibitor.

[0037] The present invention innovatively proposes that the formula of tobacco petiole tissue permeabilization pretreatment solution contains Tween 20 component and its specific optimal concentration range, which is used to enhance cell membrane permeability, which is different from the existing technology of plant tissue enzymatic hydrolysis.

[0038] The present invention also provides application of the tobacco petiole tissue permeabilization pretreatment solution in tobacco petiole spatial full transcriptome sequencing.

[0039] The method of the present invention also proposes the use of a pretreatment solution suitable for in situ polyadenylation of tobacco petiole tissue, wherein the in situ polyadenylation pretreatment solution comprises: β-galactosidase and 1×PBS (phosphate buffered saline).

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

[0041] The aforementioned pretreatment liquid and pretreatment steps innovatively proposed for the first time in the method of the present invention are different from the existing technology.

[0042] The method of the present invention also includes an in situ polyadenylation step suitable for spatial full transcriptome sequencing of tobacco petioles.

[0043] The present invention also provides an in situ polyadenylation method / step (i.e., steps eight and nine) suitable for spatial full transcriptome sequencing of tobacco petioles, comprising the following specific steps:

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

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

[0046] Step (9.3), add polyadenylate 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 washing buffer to each well for washing.

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

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

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

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

[0053] The washing buffer in step (9.2) contains the following components (final concentration): 1× polyadenylate polymerase reaction buffer, 0.8 U / μL RNase inhibitor, ddH 2 O; Preferably, the poly(A) polymerase reaction buffer is a polymerase reaction buffer suitable for yeast origin.

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

[0055] The incubation conditions described in step (9.2) are room temperature, 30 seconds.

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

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

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

[0059] The incubation conditions in step (9.4) are 37°C for 20 min.

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

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

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

[0063] The method of the present invention includes a step of removing rRNA from a cDNA library, which is applicable to the whole transcriptome sequencing process of tobacco petiole space.

[0064] The present invention also provides a method / step (i.e., step 11) for removing rRNA from a cDNA library, which is applicable to the whole transcriptome sequencing process of tobacco petiole space. The method / step includes the following specific steps:

[0065] Step (11.1), prepare 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 release: After in vitro digestion, proteinase K is added to the reaction, and after incubation, Cas9 endonuclease releases DNA.

[0068] Step (11.4), magnetic bead purification: Add SPRI magnetic beads to the reaction system, mix well and incubate, then place on a magnetic rack, wait until the solution is clear, discard the supernatant, add 80% ethanol and wash twice, with an interval of 30 seconds between each wash, and place at room temperature for no more than 2 minutes to allow the ethanol on the surface of the magnetic beads to evaporate, but do not allow it to dry too much, and finally elute with elution buffer (10mM Tris-HCl (pH7.5)).

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

[0070] Unidirectional guide RNA pool: 26 synthetic guide RNAs (13 18sRNAs and 13 28sRNAs) 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 into a unidirectional guide RNA pool with a final concentration of 100 μM. The 100 μM unidirectional guide RNA pool was then diluted to 10 μM for standby use. Specifically, the sequences of the 26 synthetic guide RNAs are shown in Appendix 10.

[0071] RNP (ribonucleoprotein complex) mixed solution: According to the sample volume, the ingredients include 10μM guide RNA, 20μM Cas9 nuclease, S. pyogenes (Cas9 nuclease), and 1×PBS. The guide RNA and Cas9 nuclease are added at equal concentrations, and the remaining volume is filled with 1×PBS. After mixing, incubate at room temperature for 10 minutes.

[0072] In vitro digestion mixture: According to the sample volume, the ingredients include 10×Cas9 nuclease reaction buffer (NEB / M0386M), 1μM Cas9 RNP and sample library. The final molar ratio of Cas9 RNP to sample library is 40:1. The remaining volume is filled with 10X Cas9 nuclease reaction buffer.

[0073] The reagent components described in step (11.1) were purchased from Tris HCl (Thermo / 15567027), EDTA (Thermo / 15575020), guide RNA (Sanggong Biosynthesis), Cas9 nuclease, S. pyogenes (NEB / M0386M), and Cas9 nuclease reaction buffer (NEB / M0386M).

[0074] The incubation temperature of the in vitro digestion reaction mixture in step (11.2) is 37° C. for 60 min.

[0075] The reagent components in step (11.3) were purchased from Proteinase K (Thermo / 25530049).

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

[0077] The incubation conditions in step (11.3) are 56°C for 10 min.

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

[0079] The incubation conditions in step (11.4) are specifically incubation at room temperature for 5 minutes.

[0080] The method of the present invention includes a step of spatial full transcriptome applicable to tobacco petiole tissue combined with a 10X Visium spatial transcriptome sequencing process.

[0081] In one embodiment of the present invention, a complete method flow for spatial full transcriptome of tobacco petiole tissue that can be combined with the 10XVisium 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 mold groove capacity.

[0088] f. Confirm the direction of tissue sections and proceed with OCT embedding.

[0089] g. Horizontally transfer to -20°C environment for freezing to ensure that OCT and tissue are frozen.

[0090] Step 2: Sample tissue section:

[0091] Slice the embedded tissue sample, flatten it, place the sample on a normal slide, and immediately put your finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that the tissue slice and the slide fit together fully.

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

[0093] Step 4: Slice scanning imaging

[0094] Step 5: Tissue Permeabilization:

[0095] a. Prepare tissue permeabilization pretreatment solution.

[0096] b. Add pretreatment solution into the wells one by one along the lower left corner of the sample well of Slide Cassette, tap Slide Cassette gently to make the treatment solution evenly cover the tissue, and attach the sealing strip.

[0097] c. Place the Slide Cassette in the PCR instrument for pretreatment.

[0098] d. After pretreatment, carefully remove the pretreatment solution from the wells, add wash buffer to each well, and remove after washing.

[0099] e. Resuspend and preheat the permeabilization enzyme.

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

[0101] g. Add permease to the sample well along the lower left corner to evenly cover the tissue without creating bubbles, tapping the SlideCassette to ensure even coverage.

[0102] h. Put a seal on the Slide Cassette, then place the Slide Cassette on a PCR instrument with a Slide Cassette adapter, close the PCR instrument lid and incubate to optimize the selected permeabilization time.

[0103] i. After the permeabilization time is completed, remove the Slide Cassette from the PCR instrument adapter and remove the sealing strip.

[0104] j. Use a pipette to remove the permease from each well without touching the tissue.

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

[0106] Step 6, in situ polyadenylation:

[0107] a. Pretreatment for removal of secondary metabolites: Add in situ polyadenylation pretreatment solution to each sample well and remove the pretreatment solution after incubation.

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

[0109] c. Add polyadenylate polymerase mixture to each sample well.

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

[0111] e. Add washing buffer to each well for washing.

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

[0113] Step 7: Library construction:

[0114] The cDNA library was constructed according to the 10X Visium spatial transcriptome sequencing process.

[0115] Step 8, rRNA removal:

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

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

[0118] c. DNA release: After in vitro digestion, proteinase K is added to the reaction and incubated to allow Cas9 endonuclease to release DNA.

[0119] d. Magnetic bead purification: Add SPRI magnetic beads to the reaction system, mix well and incubate, then place on a magnetic rack. When the solution is clear, discard the supernatant and add 80% ethanol to wash twice, with an interval of 30 seconds between each wash. Leave at room temperature for no more than 2 minutes to allow the ethanol on the surface of the magnetic beads to evaporate, but do not make it too dry. Finally, elute with elution buffer.

[0120] Step 9: cDNA library quality control

[0121] Step 10: Spatial transcriptome sequencing and analysis

[0122] In step 1a, the tobacco is 90 to 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 to 0.8 cm, preferably 0.5 cm; and the sampling method is cross-section sampling.

[0123] The pretreatment of the tobacco petiole tissue before embedding in step 1c is specifically as follows: placing the tobacco petiole tissue in PVA (PVA covers the tissue), and placing it in a vacuum apparatus to accelerate the penetration of PVA into the tissue. The PVA concentration is 25-35% (v / v), preferably 25% (v / v); the accelerated penetration time in the vacuum apparatus is 10-15 minutes, preferably 10 minutes.

[0124] As described in steps 1f-1g, place the embedding mold horizontally on ice at -20°C and freeze for 30 minutes.

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

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

[0127] The tissue permeabilization pretreatment solution in step 5a contains the following components (final concentration): 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1M mannitol, 0.25-0.5% (m / v) cellulase R10, 0.05%-0.1% (m / v) maceration enzyme 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; preferably, the tissue permeabilization pretreatment solution is formulated as 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.35% (m / v) cellulase R10, 0.1% (m / v) macerate R10, 0.1% (m / v) pectinase, 0.15% (m / v) hemicellulase, 0.15% (v / v) Tween 20, 0.4 U / μL RNase inhibitor.

[0128] The components described in step 5a were purchased from MES (sigma / 1266615-59-1), KCl (sigma / P9541-500G), BSA (Miltenyi / 130-091-376), CaCl 2(sigma / 793639-100G), cellulase R10 (Solarbio / C8260-10g), chromatase R10 (polyene / 9032-75-1), mannitol (Yishen / 60327ES76), pectinase (Solarbio / P8181-5G), hemicellulase (Solarbio / H8112-500g), Tween 20 (sigma / P9416-50ML), RNase inhibitor (Thermo / AM2694).

[0129] The pretreatment condition in step 5c is 37° C. pretreatment for 10 min.

[0130] The permeabilization steps described in steps 5d to 5k were all performed according to the routine operation of the 10X Visium spatial transcriptome sequencing instructions.

[0131] The in situ polyadenylation pretreatment solution in step 6a comprises the following components (final concentrations): (0.2-0.5)×β-galactosidase, 1×PBS; preferably, the final concentration of β-galactosidase is 0.2×.

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

[0133] The volume of polyadenylation solution added to each well in step 6a is 70 μL.

[0134] The incubation temperature in step 6b is room temperature for 10 min.

[0135] The washing buffer in step 6b contains the following components (final concentration): 1× polyadenylate polymerase reaction buffer, 0.8 U / μL RNase inhibitor, ddH 2 O; Preferably, the poly(A) polymerase reaction buffer is a polymerase reaction buffer suitable for yeast origin.

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

[0137] The volume of the wash buffer added to each well in step 6b is 100 μL.

[0138] The incubation condition in step 6b is room temperature for 30 seconds.

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

[0140] The components of the polyadenylate polymerase mixture in step 6c were purchased from polyadenylate polymerase (Thermo / 74225Z25KU), 5×polyadenylate polymerase reaction buffer (Thermo / 74225Z25KU), and ATP (Thermo / AM8110G), respectively.

[0141] The volume of the polyadenylate polymerase mixture added to each 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 0.1×SSC.

[0144] The volume of wash buffer added as described in step 6e was 100 μL.

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

[0146] The library construction steps described in step 7 were performed according to the routine operation of the 10X Visium spatial transcriptome sequencing instructions.

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

[0148] Unidirectional guide RNA pool: 26 synthetic guide RNAs (13 18sRNAs and 13 28sRNAs) were dissolved in IDTE buffer respectively to make the final concentration of each guide RNA 100 μM. 2 μl of each guide RNA was mixed into a unidirectional guide RNA pool with a final concentration of 100 μM. The 100 μM unidirectional guide RNA pool was then diluted to 10 μM for standby use. Specifically, the sequences of the 26 synthetic guide RNAs are shown in Table 10.

[0149] RNP (ribonucleoprotein complex) mixed solution: According to the sample volume, the ingredients include 10μM guide RNA, 20μM Cas9 nuclease, and 1×PBS. The guide RNA and Cas9 nuclease, S. pyogenes (Cas9 nuclease) are added at equal concentrations, and the remaining volume is filled with 1×PBS. After mixing, incubate at room temperature for 10 minutes.

[0150] In vitro digestion mixture: According to the sample volume, the ingredients include 10×Cas9 nuclease reaction buffer (NEB, M0386M), 1μM Cas9 RNP and sample library. The final molar ratio of Cas9 RNP to sample library is 40:1. The remaining volume is filled with 10X Cas9 nuclease reaction buffer.

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

[0152] The incubation temperature of the in vitro digestion reaction mixture in step 8 is 37° C. for 60 min.

[0153] The reagent components in step 8 were 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 in step 8 is 56° C., 10 min.

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

[0157] The incubation condition in step 8 is specifically incubating at room temperature for 5 minutes.

[0158] In a specific embodiment, the specific steps of the method of the present invention are:

[0159] 1. Sample embedding:

[0160] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0161] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

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

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

[0164] 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, being careful not to generate bubbles.

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

[0166] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0167] 2. Sample tissue sections:

[0168] The embedded tissue samples were sliced ​​to a thickness of 10 μm, flattened, and placed on a Visium spatial gene expression slide. Immediately, a finger was placed on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adhered to the slide.

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

[0170] The sequencing was performed according to the conventional 10X Visium spatial transcriptome sequencing process.

[0171] 4. Slice scanning imaging

[0172] 5. Tissue Permeabilization:

[0173] a. Prepare tissue permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.35% (m / v) cellulase R10, 0.1% (m / v) macerate R10, 0.1% (m / v) pectinase, 0.15% (m / v) hemicellulase, 0.15% (v / v) Tween 20, 0.4 U / μL RNase inhibitor.

[0174] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0175] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 10 min.

[0176] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0177] e. Resuspend the permease and equilibrate to 37°C for 15 min.

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

[0179] g. Add 70 μL of permease to 4 wells along the lower left corner, evenly covering the tissue without creating bubbles, and tap the SlideCassette to ensure even coverage.

[0180] h. Put a seal on the slide cassette, then place the slide cassette on a 37°C PCR instrument with an adapter, close the PCR instrument cover and incubate to optimize the selected permeabilization time.

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

[0182] j. Use a pipette to remove the permease from each well, being careful not to touch the tissue.

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

[0184] 6. In situ polyadenylation:

[0185] a. After permeabilization and washing, use a pipette to remove 0.1×SSC.

[0186] b. Pretreatment for removal of secondary metabolites: 250 U / mg β-galactosidase (Sigma, G6008-1KU) was diluted with 1×PBS to 40 U / μL enzyme solution, 70 μL of 0.2× β-galactosidase solution was added to each well, and the enzyme solution was removed after incubation at room temperature for 10 min.

[0187] c. Add 100 μL 1× washing buffer (20 μL 5× yPAP reaction buffer, 2 μL 40U / μL RNase inhibitor, 78 μL ddHO 2 O), and the buffer was removed after incubation at room temperature for 30 s.

[0188] d. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo / 74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using ThermoAM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddH 2 O to 75μl.

[0189] e. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 20 minutes before the enzyme mixture was removed.

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

[0191] g. Discard the 0.1×SSC in each well.

[0192] h. Then, follow the 10XVisium spatial transcriptome sequencing instructions to perform subsequent reverse transcription and cDNA amplification steps.

[0193] 7. Library construction:

[0194] The steps are the same as those 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] Unidirectional guide RNA pool: 26 synthetic guide RNAs (13 18sRNAs and 13 28sRNAs) were dissolved in IDTE buffer respectively to make the final concentration of each guide RNA 100 μM. 2 μL of each guide RNA was mixed into a guide RNA pool with a final concentration of 100 μM. The 100 μM guide RNA pool was then diluted to 10 μM for standby use. The sequences of the 26 synthetic guide RNAs are shown in SEQ ID NO.1-13 and sequence numbers SEQ ID NO.14-26 in Table 10.

[0199] RNP (ribonucleoprotein complex) mixture: according to the sample volume, the ingredients include 10μM guide RNA, 20μM Cas9 endonuclease, and 1×PBS, where guide RNA and Cas9 endonuclease are added at equal concentrations, and the remaining volume is filled with 1×PBS. After the RNP mixture is mixed, incubate at room temperature for 10 minutes.

[0200] In vitro digestion mixture: According to the sample volume, the ingredients include 10X Cas9 nuclease reaction buffer, 1μM Cas9 RNP and sample library. The final molar ratio of Cas9 RNP to sample library is 40:1. The remaining volume is filled with 10X Cas9 nuclease reaction buffer.

[0201] b. In vitro digestion:

[0202] The in vitro digestion reaction mixture was incubated at 37°C for 60 min.

[0203] c. DNA release:

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

[0205] d. Magnetic bead purification:

[0206] Add 0.8×SPRI magnetic beads to the reaction system, mix well and incubate at room temperature for 5 minutes, then place on a magnetic rack. When the solution is clear, discard the supernatant and add 80% ethanol to wash twice, with an interval of 30 seconds between each wash. Place at room temperature for no more than 2 minutes to allow the ethanol on the surface of the magnetic beads to evaporate, but do not make it too dry. Finally, elute with 20 μL elution buffer.

[0207] 9. Library quality inspection

[0208] 10. Spatial transcriptome sequencing and analysis

[0209] In a specific embodiment, the 26 synthetic guide RNAs are guide RNAs designed and synthesized according to the 18srRNA and 28srRNA fragment sequences, 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 used to remove rRNA contains the following components: IDTE buffer: 10mM Tris HCl, 0.1mM EDTA; unidirectional guide RNA pool: 26 synthetic guide RNAs were dissolved in IDTE buffer respectively, so that the final concentration of each guide RNA was 100μM, 2μL of each guide RNA was mixed into a guide RNA pool with a final concentration of 100μM, and then the 100μM guide RNA pool was diluted to 10μM for standby use; RNP (ribonucleoprotein complex) mixed solution: according to the sample volume, the ingredients include 10μM guide RNA, 20μM Cas9 nuclease, S.pyogenes (Cas9 nuclease), 1×PBS, wherein the guide RNA and Cas9 nuclease were added at equal concentrations, and the remaining volume was filled with 1×PBS, mixed and incubated at room temperature for 10min.

[0211] In vitro digestion mixture: according to the sample volume, the ingredients include 10×Cas9 nuclease reaction buffer (NEB / M0386M), 1μM Cas9 RNP and sample library. The final molar ratio of Cas9 RNP to sample library is 40:1, and the remaining volume is filled with 10X Cas9 nuclease reaction buffer; the amount of proteinase K added in each reaction is 1μL of proteinase K at a concentration of 20mg / ml.

[0212] The present invention also proposes a reagent / kit / product suitable for spatial full transcriptome sequencing of tobacco petioles, which includes a tobacco petiole tissue permeabilization pretreatment solution, an in situ polyadenylation pretreatment solution, an in situ polyadenylation washing buffer solution, a polyadenylic acid polymerase mixture, and / or an in vitro digestion mixture.

[0213] Preferably, the tobacco petiole tissue permeabilization pretreatment solution comprises the following components: 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1M mannitol, 0.25-0.5% (m / v) cellulase R10, 0.05%-0.1% (m / v) macerate 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.

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

[0215] The in situ polyadenylation washing buffer contains the following components (final concentration): 1×Poly (A) Polymerase Reaction buffer (polyadenylate polymerase reaction buffer), 0.8U / μL RNase inhibitor, ddH 2 O.

[0216] The poly(A) polymerase mixed solution comprises the following components (final concentration): 1×Poly(A) Polymerase Reaction buffer (poly(A) polymerase reaction buffer), 24U / μL Poly(A) Polymerase (poly(A) polymerase), 0.5mM ATP, and 1.5U / μL RNase inhibitor.

[0217] The in vitro digestion mixture contains the following components (final concentration): 10 μM unidirectional guide RNA pool, 20 μM Cas9 nuclease, S. pyogenes (Cas9 nuclease), 1×PBS, 10×Cas9 nuclease reaction buffer, and 20 mg / mL proteinase K.

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

[0219] Compared with the prior art, the beneficial effects of the present invention include: designing an optimization method for frozen sectioning of plant tissues, pre-treating the tissues before embedding, which can effectively support the plant tissue structure, setting the optimal freezing time and temperature, and being suitable for plant tissues with large vacuoles and high water content, avoiding damage to the tissue structure during sectioning and shrinkage of sections caused by inappropriate freezing and temperature and time. Pre-treating plant tissues before permeabilization during the permeabilization process can effectively destroy the cell wall structure, while enhancing the permeability of the plant cell membrane, which is conducive to the subsequent release of permeabilized RNA, and solves the current situation where the permeabilization effect of plant tissues is poor due to the intact presence of the cell wall. After permeabilization, an in situ polyadenylation step was introduced to polyadenylate all non-coding RNAs to achieve the capture of non-coding RNAs, which can effectively increase the number of non-coding RNAs (rRNA, tRNA, snoRNA, miRNA, snRNA, scaRNA) detected, improve the detection efficiency of non-coding RNAs, and achieve the purpose of detecting the full-transformation information of plant space. At the same time, β-galactosidase was used in this step to pre-treat the samples with in situ polyadenylation, which can effectively degrade secondary metabolites in plant tissues to avoid the inhibition of polyadenylic acid polymerase activity by plant secondary metabolites and improve the in situ polyadenylation efficiency. After library construction, a ribosomal RNA removal step was set up to avoid the reduction of sequencing depth of other RNA species due to excessive capture of ribosomal RNA, and ensure the validity of the overall data. In summary, the present invention provides a set of experimental processes suitable for spatial whole transcriptome sequencing of plant tissues, which can effectively capture a variety of non-coding RNAs, make up for the defects of plant tissues in spatial transcriptome, and provide a basis for in-depth understanding of the mechanisms of plant growth and development, environmental adaptation and stress resistance, and for exploring unknown functional genes or regulatory factors.

[0220] The overall process of the method of the present invention 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 tissue, the overall process of the present invention is an innovative method, such as Figure 7 As shown, sample embedding pretreatment, sample embedding, tissue permeabilization pretreatment, in situ polyadenylation pretreatment, in situ polyadenylation, and rRNA removal are specifically improved steps based on the conventional spatial transcriptome process in the present invention. The innovative improvements of the present invention include:

[0221] Sample embedding pretreatment: A pretreatment step is added before sample embedding, and this pretreatment method is innovatively proposed by the present invention.

[0222] Sample embedding: The optimal conditions for embedding tobacco petiole tissue samples were explored.

[0223] Tissue permeabilization pretreatment: Tween 20 component, and the concentration range of each component of the pretreatment solution is also limited by the present invention after testing.

[0224] In situ polyadenylation pretreatment and in situ polyadenylation: This step is innovatively proposed by the present invention. Although polyadenylation is a conventional step in the whole transcriptome sequencing process, it has not yet been applied to the plant spatial transcriptome. The conventional polyadenylation method applicable to animal tissues cannot be simply applied to plant tissues, so it does not belong to the prior art; in order to perform in situ polyadenylation treatment in the plant tissue spatial transcriptome, the present invention performs in situ polyadenylation pretreatment according to the characteristics of plant tissues. This pretreatment method is innovatively proposed by the present invention, which includes the composition of the pretreatment solution, the concentration range, and the treatment conditions, all of which are determined by the present invention 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. In this method, due to the limitation of the spatial transcriptome experimental process, rRNA cannot be removed from the RNA step, and rRNA needs to be removed from the cDNA library. After experimental testing by the present invention, the method described in the present invention is the most efficient method for removing rRNA from the cDNA library, and does not belong to the conventional existing technology.

[0226] The present invention innovatively proposes a spatial whole transcriptome sequencing method suitable for tobacco petiole tissue, which can effectively solve the problems of difficult preparation of sample sections and incomplete permeabilization of tobacco petioles due to the particularity of samples during spatial transcriptome sequencing experiments. The in situ polyadenylation method and rRNA removal method provided in the present invention can effectively detect low-abundance non-coding RNA in tobacco petioles on the basis of spatial transcriptome sequencing, and realize spatial whole transcriptome sequencing of tobacco petioles. The method is simple, easy to use, accurate, and has comprehensive detection information. It can also be directly combined with the 10XVisium spatial transcriptome sequencing process, and has long-term stability. It can not only make up for the shortcomings of spatial transcriptome technology in the field of botany, but also obtain more detailed cell clustering effects and more comprehensive gene expression information with a smaller sequencing amount. BRIEF DESCRIPTION OF THE DRAWINGS

[0227] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0228] Figure 1 This is a picture of tissue section quality inspection under an optical microscope of Example 1 of the present invention.

[0229] Figure 2 This is a picture of the Agilent2100 RNA quality inspection results of tissue sections in Example 1 of the present invention.

[0230] Figure 3 This is the quality inspection picture of Agilent4150 cDNA library in Example 9 of the present invention.

[0231] Figure 4 This is a picture of the quality inspection of the Agilent 4150 cDNA product in Example 10 of the present invention.

[0232] Figure 5 This is the quality inspection picture of Agilent4150 cDNA library in Example 10 of the present invention.

[0233] Figure 6 This is a distribution diagram of different non-coding RNA capture at 1050G sequencing volume in an embodiment of the present invention.

[0234] Figure 7 The figure is a schematic diagram of the working process of the method of the present invention. DETAILED DESCRIPTION

[0235] The present invention is further described in detail with reference to the following specific examples. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0236] Example 1

[0237] 1. Sample embedding:

[0238] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0239] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0240] c. Place tobacco petiole tissue in 25% PVA (v / v) (PVA covers the tissue), and place in a vacuum apparatus for 10 minutes to accelerate the penetration of 25% PVA (v / v) into the tissue.

[0241] d. Take the tissue out of 25% PVA (v / v) and gently wipe off the excess PVA adhering 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 mold groove capacity, being careful not to generate bubbles.

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

[0244] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0245] 2. Sample tissue sections:

[0246] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0247] The results are as follows Figure 1 , Figure 2 Table 1: Through the technical solution of the present invention, 25% PVA (v / v) was pre-infiltrated into the tissue when the sample was embedded, which could provide good support for the tissue structure. The tissue sections were observed to be relatively uniform and flat under an optical microscope, and the tissue structure was basically intact without large cavities ( Figure 1 , Table 1); and the sections were subjected to RNA extraction quality inspection, and the RIN value was higher than 7 ( Figure 2 , Table 1), indicating that using 25% PVA (v / v) to pretreat the tissue during the embedding process can not only maintain the basic morphological structure of the plant slices, but also has no effect on the tissue RNA. The technical solution of the present 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, and it should be able to be placed in the mold.

[0251] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

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

[0253] d. Take the tissue out of 30% PVA (v / v) and gently wipe off the 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, being careful not to generate bubbles.

[0255] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0256] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0257] 2. Sample tissue sections:

[0258] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0259] The results are shown in Table 1: Through the technical scheme of the present invention, 30% PVA is allowed to penetrate into the tissue in advance when the sample is embedded, which can provide good support for the tissue structure. The tissue sections are observed to be relatively uniform and flat under an optical microscope, and the tissue structure is basically intact without large voids. The sections are subjected to RNA extraction quality inspection, and the RIN value is higher than 7 (Table 1), indicating that the use of 30% PVA to pretreat 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 scheme of the present invention has achieved the expected technical effect.

[0260] Example 3

[0261] 1. Sample embedding:

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

[0263] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0264] c. Place tobacco petiole tissue in 35% PVA (v / v) (PVA covers the tissue), and place in a vacuum apparatus for 15 minutes to accelerate the penetration of 35% PVA into the tissue.

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

[0266] 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, being careful not to generate bubbles.

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

[0268] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0269] 2. Sample tissue sections:

[0270] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0271] The results are shown in Table 1: Through the technical scheme of the present invention, 35% PVA is pre-infiltrated into the tissue when the sample is embedded, which can provide good support for the tissue structure. The tissue sections are observed to be relatively uniform and flat under an optical microscope, and the tissue structure is basically intact without large voids. The sections are subjected to RNA extraction quality inspection, and the RIN value is higher than 7 (Table 1), indicating that the use of 35% PVA to pretreat 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 scheme of the present invention has achieved the expected technical effect.

[0272] Example 4

[0273] The results of Examples 1, 2, and 3 all show that when the sample is embedded, 25-35% PVA is allowed to penetrate into the tissue in advance, which can provide good support for the tissue structure and maintain the basic morphological structure of the plant slices, and will not affect the tissue RNA. After comparison of Examples 1 to 3, although Examples 2 and 3 can play the same supporting role, they consume more PVA than Example 1 in terms of the amount of PVA used and the processing time. Example 1 is more conducive to saving time and cost, so the scheme of Example 1 is 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, and it should be able to be placed in the mold.

[0276] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

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

[0278] d. Take the tissue out of 25% PVA (v / v) and gently wipe off the excess PVA adhering to the surface.

[0279] 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, being careful not to generate bubbles.

[0280] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0281] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0282] 2. Sample tissue sections:

[0283] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

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

[0285] The steps are the same as those in the 10X Visium spatial transcriptome sequencing instructions.

[0286] 4. Slice scanning imaging

[0287] 5. Tissue Permeabilization Optimization:

[0288] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.5% cellulase R10 (m / v), 0.1% macerate R10 (m / v), 0.2% pectinase (m / v), 0.2% hemicellulase (m / v), 0.25% Tween 20 (v / v), 0.4 U / μL RNase inhibitor.

[0289] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0290] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 10 min.

[0291] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0292] e. Then, follow the 10X Visium spatial transcriptome sequencing instructions for subsequent permeabilization optimization.

[0293] The results are shown in Table 2. The scanning imaging results of the permeabilization optimization slices show that, through the technical solution of the present invention, the fluorescence intensity of the six slices treated for 3 to 30 minutes is clearly distinguished, the fluorescence signal is strong enough to be distinguished from the background noise, there is no overexposure, the fluorescence signal is evenly distributed in the sample area, there is no obvious signal gradient or local loss phenomenon, and the permeabilization requirements are met (Table 2). The optimal permeabilization time is finally judged to be 18 minutes. It shows that the use of the formula pretreatment solution before permeabilization can effectively enzymatically decompose the cell wall, and can enhance the permeability of the plant cell membrane to facilitate the subsequent release of permeabilized RNA, meet the subsequent processing conditions, and the technical solution of the present invention has achieved the desired technical effect.

[0294] Example 5

[0295] 1. Sample embedding:

[0296] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0297] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

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

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

[0300] 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, being careful not to generate bubbles.

[0301] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0302] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0303] 2. Sample tissue sections:

[0304] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

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

[0306] The steps are the same as those in the 10X Visium spatial transcriptome sequencing instructions.

[0307] 4. Slice scanning imaging

[0308] 5. Tissue Permeabilization Optimization:

[0309] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.35% cellulase R10 (m / v), 0.1% macerate R10 (m / v), 0.1% pectinase (m / v), 0.15% hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4 U / μL RNase inhibitor.

[0310] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0311] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 10 min.

[0312] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0313] e. Then, follow the 10X Visium spatial transcriptome sequencing instructions for subsequent permeabilization optimization.

[0314] The results are shown in Table 2. The scanning imaging results of the permeabilization optimization slices show that, through the technical solution of the present invention, the fluorescence intensity of the six slices treated for 3 to 30 minutes is clearly distinguished, the fluorescence signal is strong enough to be distinguished from the background noise, there is no overexposure, the fluorescence signal is evenly distributed in the sample area, there is no obvious signal gradient or local loss phenomenon, and the permeabilization requirements are met (Table 2). The optimal permeabilization time is finally judged to be 18 minutes. It shows that the use of the formula pretreatment solution before permeabilization can effectively enzymatically decompose the cell wall, and can enhance the permeability of the plant cell membrane to facilitate the subsequent release of permeabilized RNA, meet the subsequent processing conditions, and the technical solution of the present invention has achieved 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 should be able to be placed in the mold.

[0318] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0319] c. Place tobacco petiole tissue in 25% PVA (v / v) (PVA covers the tissue), and place in a vacuum apparatus for 10 minutes to accelerate the penetration of 25% PVA (v / v) into the tissue.

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

[0321] 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, being careful not to generate bubbles.

[0322] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

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

[0324] 2. Sample tissue sections:

[0325] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

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

[0327] The steps are the same as those in the 10X Visium spatial transcriptome sequencing instructions.

[0328] 4. Slice scanning imaging

[0329] 5. Tissue Permeabilization Optimization:

[0330] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.25% cellulase R10 (m / v), 0.05% macerate R10 (m / v), 0.05% pectinase (m / v), 0.1% hemicellulase (m / v), 0.1% Tween 20 (v / v), 0.4 U / μL RNase inhibitor.

[0331] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0332] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 15 minutes.

[0333] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0334] e. Then, follow the 10X Visium spatial transcriptome sequencing instructions for subsequent permeabilization optimization.

[0335] The results are shown in Table 2. The scanning imaging results of the permeabilization optimization slices show that, through the technical solution of the present invention, the fluorescence intensity of the six slices treated for 3 to 30 minutes is clearly distinguished, the fluorescence signal is strong enough to be distinguished from the background noise, there is no overexposure, the fluorescence signal is evenly distributed in the sample area, there is no obvious signal gradient or local loss phenomenon, and the permeabilization requirements are met (Table 2). The optimal permeabilization time is finally judged to be 18 minutes. It shows that the use of the formula pretreatment solution before permeabilization can effectively enzymatically decompose the cell wall, and can enhance the permeability of the plant cell membrane to facilitate the subsequent release of permeabilized RNA, meet the subsequent processing conditions, and the technical solution of the present invention has achieved the desired technical effect.

[0336] Example 7

[0337] The results of Examples 4, 5, and 6 all show that before permeabilization, using a permeabilization pretreatment solution including 0.25-0.5% cellulase R10 (m / v), 0.05%-0.1% maceration enzyme 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) to treat tissue sections can effectively enzymatically hydrolyze plant cell walls, enhance the permeability of plant cell membranes, and facilitate the subsequent release of permeabilized RNA. The fluorescence capture area after permeabilization optimization shows that the use of the permeabilization pretreatment solution will not affect tissue RNA. After comparison of Examples 4 to 6, although Examples 4 and 6 can play the same pretreatment role, they consume more enzymes and treatment time than Example 5. Example 5 is more conducive to saving time and cost, so the scheme of Example 5 is adopted to further carry out subsequent experimental steps.

[0338] 1. Sample embedding:

[0339] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0340] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0341] c. Place tobacco petiole tissue in 25% PVA (v / v) (PVA covers the tissue), and place in a vacuum apparatus for 10 minutes to accelerate the penetration of 25% PVA (v / v) into the tissue.

[0342] d. Take the tissue out of 25% PVA (v / v) and gently wipe off the excess PVA adhering to the surface.

[0343] 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, being careful not to generate bubbles.

[0344] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0345] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0346] 2. Sample tissue sections:

[0347] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

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

[0349] The steps are the same as those in the 10X Visium spatial transcriptome sequencing instructions.

[0350] 4. Slice scanning imaging

[0351] 5. Tissue Permeabilization:

[0352] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.35% cellulase R10 (m / v), 0.1% macerate R10 (m / v), 0.1% pectinase (m / v), 0.15% hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4 U / μL RNase inhibitor.

[0353] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0354] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 10 min.

[0355] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0356] e. Resuspend the permease and equilibrate to 37°C for 15 min.

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

[0358] g. Add 70 μL of permease to 4 wells along the lower left corner, evenly covering the tissue without creating bubbles, and tap the SlideCassette to ensure even coverage.

[0359] h. Put a seal on the slide cassette, then place the slide cassette on a 37°C PCR instrument with an adapter, close the PCR instrument cover and incubate to optimize the selected permeabilization time.

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

[0361] j. Use a pipette to remove the permease from each well, being careful not to touch the tissue.

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

[0363] 6. In situ polyadenylation:

[0364] a. After permeabilization and washing, use a pipette to remove 0.1×SSC.

[0365] b. Pretreatment for removal of secondary metabolites: 250 U / mg β-galactosidase (Sigma, G6008-1KU) was diluted with 1×PBS to 40 U / μL enzyme solution, 70 μL of 0.2× β-galactosidase solution was added to each well, and the enzyme solution was removed after incubation at room temperature for 10 min.

[0366] c. Add 100 μL 1× washing buffer (20 μL 5× yPAP reaction buffer, 2 μL 40 U / μl RNase inhibitor, 78 μl nuclease-free H 2 O), and the buffer was removed after incubation at room temperature for 30 s.

[0367] d. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×)), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using ThermoAM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddHO to each sample well. 2 O to 75 μL.

[0368] e. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 20 minutes before the enzyme mixture was removed.

[0369] f. Add 100 μL 0.1x SSC to each well and wash for 10 seconds.

[0370] g. Discard the 0.1X SSC in each well.

[0371] h. Then, follow the 10XVisium spatial transcriptome sequencing instructions to perform subsequent reverse transcription and cDNA amplification steps.

[0372] The results are shown in Table 3. The quality inspection results after cDNA amplification showed that the quality of cDNA and library met the requirements of spatial transcriptome sequencing. The capture efficiency of non-coding RNA was significantly improved compared with that of comparative examples 30 to 31 at a sequencing volume of 50G (Table 3), indicating that the use of 0.2×β-galactosidase can effectively degrade secondary metabolites in plant tissues, improve the in situ polyadenylation efficiency, meet the subsequent processing conditions, and the technical solution of the present invention has achieved 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 able to be placed in the mold.

[0376] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0377] c. Place tobacco petiole tissue in 25% PVA (v / v) (PVA covers the tissue), and place in a vacuum apparatus for 10 minutes to accelerate the penetration of 25% PVA (v / v) into the tissue.

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

[0379] 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, being careful not to generate bubbles.

[0380] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

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

[0382] 2. Sample tissue sections:

[0383] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

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

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

[0386] 4. Slice scanning imaging

[0387] 5. Tissue Permeabilization:

[0388] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.35% cellulase R10 (m / v), 0.1% macerate R10 (m / v), 0.1% pectinase (m / v), 0.15% hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4 U / μL RNase inhibitor.

[0389] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0390] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 10 min.

[0391] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0392] e. Resuspend the permease and equilibrate to 37°C for 15 min.

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

[0394] g. Add 70 μL of permease to 4 wells along the lower left corner, evenly covering the tissue without creating bubbles, and tap the SlideCassette to ensure even coverage.

[0395] h. Put a seal on the slide cassette, then place the slide cassette on a 37°C PCR instrument with an adapter, close the PCR instrument cover and incubate to optimize the selected permeabilization time.

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

[0397] j. Use a pipette to remove the permease from each well, being careful 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 and washing, use a pipette to remove 0.1×SSC.

[0401] b. Pretreatment for removal of secondary metabolites: 250 U / mg β-galactosidase (Sigma, G6008-1KU) was diluted with 1×PBS to 40 U / μL enzyme solution, 70 μL of 0.5× β-galactosidase solution was added to each well, and the enzyme solution was removed after incubation at room temperature for 10 min.

[0402] c. Add 100 μL 1× washing buffer (20 μL 5× yPAP reaction buffer, 2 μL 40U / μL RNase inhibitor, 78 μL nuclease-free H 2 O), and the buffer was removed after incubation at room temperature for 30 s.

[0403] d. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×)), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using ThermoAM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddHO to each sample well. 2 O to 75 μL.

[0404] e. The reaction chamber was then sealed, the Slide Cassette was incubated at 37°C for 20 min, and the enzyme mixture was then removed.

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

[0406] g. Discard the 0.1X SSC in each well.

[0407] h. Then, follow the 10XVisium spatial transcriptome sequencing instructions to perform subsequent reverse transcription and cDNA amplification steps.

[0408] The results are shown in Table 3. The quality inspection results after cDNA amplification showed that the quality of cDNA and library met the requirements of spatial transcriptome sequencing. The capture efficiency of non-coding RNA at 50G sequencing volume was significantly improved compared with that of comparative examples 30 to 31 (Table 3), indicating that the use of 0.5×β-galactosidase can effectively degrade secondary metabolites in plant tissues, improve the in situ polyadenylation efficiency, meet the subsequent processing conditions, and the technical solution of the present invention has achieved the expected technical effect.

[0409] Example 9

[0410] The results of Examples 7 and 8 both show that the use of a pretreatment solution containing (0.2-0.5)×β-galactosidase before in situ polyadenylation can effectively degrade secondary metabolites in plant tissues and improve the efficiency of in situ polyadenylation. After comparison of Examples 7 and 8, although Example 8 can play a role in degrading secondary metabolites, its efficiency is not significantly different from that of Example 7, and the dosage is lower than that of Example 7, which is more conducive to cost savings. Therefore, the scheme of Example 7 is adopted to further carry out subsequent experimental steps.

[0411] 1. Sample embedding:

[0412] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0413] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0414] c. Place tobacco petiole tissue in 25% PVA (v / v) (PVA covers the tissue), and place in a vacuum apparatus for 10 minutes to accelerate the penetration of 25% PVA (v / v) into the tissue.

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

[0416] 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, being careful not to generate bubbles.

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

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

[0419] 2. Sample tissue sections:

[0420] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

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

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

[0423] 4. Slice scanning imaging

[0424] 5. Tissue Permeabilization:

[0425] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.35% cellulase R10 (m / v), 0.1% macerate R10 (m / v), 0.1% pectinase (m / v), 0.15% hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4 U / μL RNase inhibitor.

[0426] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0427] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 10 min.

[0428] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0429] e. Resuspend the permease and equilibrate to 37°C for 15 min.

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

[0431] g. Add 70 μL of permease to 4 wells along the lower left corner, evenly covering the tissue without creating bubbles, and tap the SlideCassette to ensure even coverage.

[0432] h. Put a seal on the slide cassette, then place the slide cassette on a 37°C PCR instrument with an adapter, close the PCR instrument cover and incubate to optimize the selected permeabilization time.

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

[0434] j. Use a pipette to remove the permease from each well, being careful not to touch the tissue.

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

[0436] 6. In situ polyadenylation:

[0437] a. After permeabilization and washing, use a pipette to remove 0.1×SSC.

[0438] b. Pretreatment for removal of secondary metabolites: 250 U / mg β-galactosidase (Sigma, G6008-1KU) was diluted with 1×PBS to 40 U / μL enzyme solution, 70 μL of 0.2× β-galactosidase solution was added to each well, and the enzyme solution was removed after incubation at room temperature for 10 min.

[0439] c. Add 100 μL 1× washing buffer (20 μL 5× yPAP reaction buffer, 2 μL 40U / μL RNase inhibitor, 78 μL ddHO 2 O), and the buffer was removed after incubation at room temperature for 30 s.

[0440] d. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×)), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using ThermoAM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddHO to each sample well. 2 O to 75 μL.

[0441] e. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 20 minutes before the enzyme mixture was removed.

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

[0443] g. Discard the 0.1X SSC in each well.

[0444] h. Then, follow the 10XVisium spatial transcriptome sequencing instructions to perform subsequent reverse transcription and cDNA amplification steps.

[0445] 7. Library construction:

[0446] The steps are the same as those in the 10X Visium spatial transcriptome sequencing instructions.

[0447] 8. rRNA removal:

[0448] a. Reagent preparation:

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

[0450] Unidirectional guide RNA pool: 26 synthetic guide RNAs (13 18sRNAs and 13 28sRNAs) 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 into a unidirectional guide RNA pool with a final concentration of 100 μM. The 100 μM unidirectional guide RNA pool was then diluted to 10 μM for standby use. The sequences of the 26 synthetic guide RNAs are shown in Appendix 10.

[0451] RNP (ribonucleoprotein complex) mixture: according to the sample volume, the ingredients include 10μM guide RNA, 20μM Cas9 endonuclease, and 1×PBS, where guide RNA and Cas9 endonuclease are added at equal concentrations, and the remaining volume is filled with 1×PBS. After the RNP mixture is mixed, incubate at room temperature for 10 minutes.

[0452] In vitro digestion mixture: According to the sample volume, the ingredients include 10X Cas9 nuclease reaction buffer, 1μM Cas9 RNP and sample library. The final molar ratio of Cas9 RNP to sample library is 40:1. The remaining volume is filled with 10X Cas9 nuclease reaction buffer.

[0453] b. In vitro digestion:

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

[0455] c. DNA release:

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

[0457] d. Magnetic bead purification:

[0458] Add 1×SPRI magnetic beads to the reaction system, mix well and incubate at room temperature for 5 minutes, then place on a magnetic rack. When the solution is clear, discard the supernatant and add 80% ethanol to wash twice, with an interval of 30 seconds between the two times. Leave at room temperature for no more than 2 minutes to allow the ethanol on the surface of the magnetic beads to evaporate, but do not make it too dry. Finally, elute with 20μL elution buffer.

[0459] 9. Library quality inspection

[0460] 10. Spatial transcriptome sequencing and analysis

[0461] The results are as follows Figure 3 As shown in Table 4, the quality inspection results of the library after removing ribosomal RNA by the technical solution of the present invention show that in addition to the 400-500 bp main fragment of the library, there are also small fragments of less than 200 bp ( Figure 3 ), the results of plant space full transcriptome sequencing showed that non-coding RNA (rRNA, tRNA, snoRNA, miRNA, snRNA, scaRNA) was successfully detected, the rRNA capture rate was less than 10%, and the capture efficiency of the remaining low-abundance non-coding RNAs was significantly improved compared with comparative examples 32 to 33 (Table 4), preliminarily achieving the purpose of plant space full transcriptome sequencing. The technical solution of the present invention has achieved the desired technical effect.

[0462] Example 10

[0463] 1. Sample embedding:

[0464] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0465] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0466] c. Place tobacco petiole tissue in 25% PVA (v / v) (PVA covers the tissue), and place in a vacuum apparatus for 10 minutes to accelerate the penetration of 25% PVA (v / v) into the tissue.

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

[0468] 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, being careful not to generate bubbles.

[0469] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

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

[0471] 2. Sample tissue sections:

[0472] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

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

[0474] The steps are the same as those in the 10X Visium spatial transcriptome sequencing instructions.

[0475] 4. Slice scanning imaging

[0476] 5. Tissue Permeabilization:

[0477] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.35% cellulase R10 (m / v), 0.1% macerate R10 (m / v), 0.1% pectinase (m / v), 0.15% hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4 U / μL RNase inhibitor.

[0478] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0479] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 10 min.

[0480] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0481] e. Resuspend the permease and equilibrate to 37°C for 15 min.

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

[0483] g. Add 70 μL of permease to 4 wells along the lower left corner, evenly covering the tissue without creating bubbles, and tap the SlideCassette to ensure even coverage.

[0484] h. Put a seal on the slide cassette, then place the slide cassette on a 37°C PCR instrument with an adapter, close the PCR instrument cover and incubate to optimize the selected permeabilization time.

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

[0486] j. Use a pipette to remove the permease from each well, being careful not to touch the tissue.

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

[0488] 6. In situ polyadenylation:

[0489] a. After permeabilization and washing, use a pipette to remove 0.1×SSC.

[0490] b. Pretreatment for removal of secondary metabolites: 250 U / mg β-galactosidase (Sigma, G6008-1KU) was diluted with 1×PBS to 40 U / μL enzyme solution, 70 μL of 0.2× β-galactosidase solution was added to each well, and the enzyme solution was removed after incubation at room temperature for 10 min.

[0491] c. Add 100 μL 1× washing buffer (20 μL 5× yPAP reaction buffer, 2 μL 40U / μL RNase inhibitor, 78 μL ddHO 2 O), and the buffer was removed after incubation at room temperature for 30 s.

[0492] d. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×)), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using ThermoAM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddHO to each sample well. 2 O to 75 μL.

[0493] e. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 20 minutes before the enzyme mixture was removed.

[0494] f. Add 100 μL 0.1x SSC to each well and wash for 10 seconds.

[0495] g. Discard the 0.1X SSC in each well.

[0496] h. Then, follow the 10XVisium spatial transcriptome sequencing instructions to perform subsequent reverse transcription and cDNA amplification steps.

[0497] 7. Library construction:

[0498] The steps are the same as those in the 10X Visium spatial transcriptome sequencing instructions.

[0499] 8. rRNA removal:

[0500] a. Reagent preparation:

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

[0502] Unidirectional guide RNA pool: 26 synthetic guide RNAs (13 18sRNAs and 13 28sRNAs) 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 into a unidirectional guide RNA pool with a final concentration of 100 μM. The 100 μM unidirectional guide RNA pool was then diluted to 10 μM for standby use. The sequences of the 26 synthetic guide RNAs are shown in Appendix 10.

[0503] RNP (ribonucleoprotein complex) mixture: according to the sample volume, the ingredients include 10μM guide RNA, 20μM Cas9 endonuclease, and 1×PBS, where guide RNA and Cas9 endonuclease are added at equal concentrations, and the remaining volume is filled with 1×PBS. After the RNP mixture is mixed, incubate at room temperature for 10 minutes.

[0504] In vitro digestion mixture: According to the sample volume, the ingredients include 10X Cas9 nuclease reaction buffer, 1μM Cas9 RNP and sample library. The final molar ratio of Cas9 RNP to sample library is 40:1. The remaining volume is filled with 10X Cas9 nuclease reaction buffer.

[0505] b. In vitro digestion:

[0506] The in vitro digestion reaction mixture was incubated at 37°C for 60 min.

[0507] c. DNA release:

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

[0509] d. Magnetic bead purification:

[0510] Add 0.8×SPRI magnetic beads to the reaction system, mix well and incubate at room temperature for 5 minutes, then place on a magnetic rack. When the solution is clear, discard the supernatant and add 80% ethanol to wash twice, with an interval of 30 seconds between the two times. Leave at room temperature for no more than 2 minutes to allow the ethanol on the surface of the magnetic beads to evaporate, but do not make it too dry. Finally, elute with 20μL elution buffer.

[0511] 9. Library quality inspection

[0512] 10. Spatial transcriptome sequencing and analysis

[0513] The results are as follows Figure 4 , 5 , 6 and Table 4, through the technical solution of the present invention, the cDNA quality inspection results show that the main peak of the cDNA product is 1283bp ( Figure 4 ), the library quality inspection results showed that the main peak of the library was 400~500bp ( Figure 5 ), the results of plant spatial whole transcriptome sequencing showed that non-coding RNA (rRNA, tRNA, snoRNA, miRNA, snRNA, scaRNA) was successfully detected, the rRNA capture rate was less than 10%, and the capture efficiency of the remaining low-abundance non-coding RNAs was significantly improved compared with comparative examples 32 to 33 ( Figure 6 ), the purpose of full spatial rotation of plants is preliminarily achieved. The technical solution of the present invention has achieved the expected technical effect.

[0514] Comparative Example 1

[0515] In this comparative example, the conventional embedding and slicing process in the 10X Visium spatial transcriptome sequencing instruction manual was first used to prepare tissue samples of tobacco petioles.

[0516] 1. Sample embedding

[0517] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0518] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[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, being careful not to generate bubbles.

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

[0521] e. Transfer horizontally to dry ice and freeze for 30 minutes to ensure that both 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 common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0524] The results are shown in Table 5. Under an optical microscope, it was observed that the tissue sections were severely shrunken, the thin-walled tissue was damaged, there were local fractures, and a large number of tissue cavities appeared, which failed to ensure the original integrity of the plant tissue structure.

[0525] Comparative Example 2

[0526] In this comparative example, the conventional embedding and slicing process in the 10X Visium spatial transcriptome sequencing instruction manual was used to prepare tissue samples from tobacco petioles, and the freezing time was shortened to 20 minutes. The remaining steps were consistent with comparative example 1.

[0527] 1. Sample embedding

[0528] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0529] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0530] 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, being careful not to generate bubbles.

[0531] d. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0532] e. Transfer horizontally to dry ice and freeze for 20 minutes to ensure that both OCT and tissue are completely frozen.

[0533] 2. Sample tissue sections

[0534] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0535] The results are shown in Table 5. Under an optical microscope, it was observed that the tissue sections were severely shrunken, the thin-walled tissue was damaged, there were local fractures, and a large number of tissue cavities appeared, which failed to ensure the original integrity of the plant tissue structure.

[0536] Comparative Example 3

[0537] In this comparative example, the conventional embedding and slicing process in the 10X Visium spatial transcriptome sequencing instruction manual was used to prepare tissue samples from tobacco petioles, and the freezing time was shortened to 10 minutes. The remaining steps were consistent with comparative example 1.

[0538] 1. Sample embedding

[0539] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0540] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[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 mold groove capacity, being careful not to generate bubbles.

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

[0543] e. Transfer horizontally to dry ice and freeze for 10 minutes to ensure that both OCT and tissue are completely frozen.

[0544] 2. Sample tissue sections

[0545] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0546] The results are shown in Table 5. The tissue sections were observed to shrink under an optical microscope, with local tissue fractures and a large number of tissue cavities, which failed to ensure the original integrity of the plant tissue structure.

[0547] Comparative Example 4

[0548] In this comparative example, the conventional embedding and slicing process in the 10X Visium spatial transcriptome sequencing instruction manual was used to prepare tissue samples from tobacco petioles, and the freezing time was shortened to 5 minutes. The remaining steps were consistent with comparative example 1.

[0549] 1. Sample embedding

[0550] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0551] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[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 mold groove capacity, being careful not to generate bubbles.

[0553] d. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

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

[0555] 2. Sample tissue sections

[0556] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0557] The results are shown in Table 5. The tissue was not completely frozen after freezing for 5 min, and the slices were wrinkled, making it impossible to observe the tissue morphology.

[0558] Comparative Example 5

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

[0560] 1. Sample embedding

[0561] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0562] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0563] 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, being careful not to generate bubbles.

[0564] d. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

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

[0566] 2. Sample tissue sections

[0567] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0568] The results are shown in Table 5. The tissue sections were observed to shrink under an optical microscope, the thin-walled tissue was damaged, and there were tissue cavities, which failed to ensure the original integrity of the plant tissue.

[0569] Comparative Example 6

[0570] In this comparative example, the freezing time is shortened to 45 minutes to ensure OCT and tissue freezing, and the remaining steps are the same as those in comparative example 5.

[0571] 1. Sample embedding

[0572] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0573] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0574] 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, being careful not to generate bubbles.

[0575] d. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0576] e. Horizontally transfer to -20℃ environment and freeze for 45min to ensure that OCT and tissue are frozen.

[0577] 2. Sample tissue sections

[0578] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0579] The results are shown in Table 5. The optical microscope showed that the tissue shrinkage was reduced, but the parenchyma was still damaged, and there were tissue cavities, which failed to ensure the original integrity of the plant tissue.

[0580] Comparative Example 7

[0581] In this comparative example, the freezing time is shortened to 30 minutes to ensure OCT and tissue freezing, and the remaining steps are the same as those in comparative example 5.

[0582] 1. Sample embedding

[0583] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0584] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0585] 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, being careful not to generate bubbles.

[0586] d. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0587] e. Transfer horizontally to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0588] 2. Sample tissue sections

[0589] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0590] The results are shown in Table 5. A small amount of tissue shrinkage was observed under an optical microscope, and there were still cavities in the tissue after sectioning, which failed to well maintain the original intact 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 apparatus to accelerate penetration using negative pressure for 10 minutes. The remaining steps were the same as those in comparative example 7.

[0593] 1. Sample embedding

[0594] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0595] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0596] c. Remove the tissue from 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), and inject the pre-cooled embedding agent into 1 / 3 of the mold groove capacity, taking care not to generate bubbles.

[0598] e. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0599] f. Place the sample in a vacuum apparatus and draw the blood for 10 minutes to promote the penetration of OCT into the tissue.

[0600] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0601] 2. Sample tissue sections

[0602] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0603] The results are shown in Table 6. A small amount of tissue shrinkage was observed under an optical microscope, and there were still cavities in the tissue after sectioning, which failed to well maintain the original intact structure of the plant tissue.

[0604] Comparative Example 9

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

[0606] 1. Sample embedding

[0607] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0608] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

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

[0610] d. Remove the tissue from 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), and inject the pre-cooled embedding agent into 1 / 3 of the mold groove capacity, being careful not to generate bubbles.

[0612] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0613] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0614] 2. Sample tissue sections

[0615] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0616] The results are shown in Table 6. A small amount of tissue shrinkage was observed under an optical microscope, and the cell morphology was irregular after sectioning. The parenchyma tissue was squeezed and damaged, and there were cavities in the tissue, which failed to well maintain the original intact structure of the plant tissue.

[0617] Comparative Example 10

[0618] In this comparative example, the tobacco petiole tissue was pretreated by soaking it in 10% PEG before embedding, and was placed in a vacuum apparatus to accelerate penetration by negative pressure for 10 minutes. The remaining steps were the same as those in comparative example 7.

[0619] 1. Sample embedding

[0620] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0621] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

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

[0623] d. Remove the tissue from 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 1 / 3 of the mold groove capacity, being careful not to generate bubbles.

[0625] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0626] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0627] 2. Sample tissue sections

[0628] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0629] The results are shown in Table 6. Under an optical microscope, it was observed that the tissue was wrinkled, and after sectioning, the tissue morphology was irregular, and the tissue tended to pile up downward, and the structure and morphology of the plant tissue were not well maintained.

[0630] Comparative Example 11

[0631] In this comparative example, the tobacco petiole tissue was pretreated by soaking it in 20% PEG before embedding, and was placed in a vacuum apparatus to accelerate the penetration by negative pressure for 10 minutes. 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 it should be able to be placed in the mold.

[0634] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

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

[0636] d. Remove the tissue from the 20% PEG and gently wipe off the excess PEG adhering to the surface.

[0637] 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, being careful not to generate bubbles.

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

[0639] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0640] 2. Sample tissue sections

[0641] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0642] The results are shown in Table 6. Under an optical microscope, it was observed that the tissue was wrinkled, the tissue morphology was irregular, the tissue had a tendency to pile up downward, and the structure and morphology of the plant tissue were not well maintained.

[0643] Comparative Example 12

[0644] In this comparative example, the tobacco petiole tissue was pretreated by soaking it in 30% PEG before embedding, and was placed in a vacuum apparatus to accelerate penetration by negative pressure for 10 minutes. The remaining steps were the same as those in comparative example 7.

[0645] 1. Sample embedding

[0646] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0647] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0648] c. Place tobacco petiole tissue in 30% PEG (PEG covers the tissue), and place in a vacuum apparatus for 15 minutes to accelerate the penetration of 30% PEG into the tissue.

[0649] d. Remove the tissue from the 30% PEG and gently wipe off the excess PEG adhering to the surface.

[0650] 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, being careful not to generate bubbles.

[0651] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0652] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0653] 2. Sample tissue sections

[0654] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0655] The results are shown in Table 6. Under an optical microscope, it was observed that the tissue was still wrinkled, the tissue structure was deformed, and there was a tendency to pile up downwards, and the structure and morphology of the plant tissue were not well maintained.

[0656] Comparative Example 13

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

[0658] 1. Sample embedding

[0659] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0660] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0661] c. Place tobacco petiole tissue in 40% PEG (PEG covers the tissue), and place in a vacuum apparatus for 10 minutes to accelerate the penetration of 40% PEG into the tissue.

[0662] d. Remove the tissue from the 40% PEG and gently wipe off the excess PEG adhering to the surface.

[0663] 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, being careful not to generate bubbles.

[0664] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0665] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0666] 2. Sample tissue sections

[0667] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

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

[0669] Comparative Example 14

[0670] In this comparative example, the tobacco petiole tissue was pretreated by soaking it in 10% PVA (v / v) before embedding, and was placed in a vacuum apparatus to accelerate penetration by negative pressure for 10 minutes. The remaining steps were the same as those in comparative example 7.

[0671] 1. Sample embedding

[0672] a. Take fresh tobacco petiole tissue. The tissue should not be too large, and it should be able to be placed in the mold.

[0673] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

[0674] c. Place tobacco petiole tissue in 10% PVA (v / v) (PVA covers the tissue), and place it in a vacuum apparatus for 10 minutes to accelerate the penetration of 10% PVA into the tissue.

[0675] d. Take the tissue out of 10% PVA (v / v) and gently wipe off the excess PVA adhering to the surface.

[0676] 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, being careful not to generate bubbles.

[0677] f. Confirm the direction of tissue slicing, use tweezers to place the prepared tissue into the mold containing OCT, and then use OCT to thoroughly cover the exposed tissue surface; if the tissue is large, add more OCT to ensure that OCT fully covers the tissue surface.

[0678] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0679] 2. Sample tissue sections

[0680] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0681] The results are shown in Table 6. Under an optical microscope, it was observed that the tissue was wrinkled, the tissue structure was deformed, and there was a tendency to pile up downwards, and 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 pretreated by soaking it in 20% PVA (v / v) before embedding, and was placed in a vacuum apparatus to accelerate penetration by negative pressure for 10 minutes. 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 it should be able to be placed in the mold.

[0686] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

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

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

[0689] 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, being careful not to generate bubbles.

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

[0691] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0692] 2. Sample tissue sections

[0693] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0694] The results are shown in Table 6. The tissue was still slightly wrinkled and deformed under an optical microscope, with a tendency to pile up downwards, and the structure and morphology of the plant tissue were not well maintained.

[0695] Comparative Example 16

[0696] In this comparative example, the tobacco petiole tissue was pretreated by soaking it in 40% PVA (v / v) before embedding, and was placed in a vacuum apparatus to accelerate penetration by negative pressure for 15 minutes. The remaining steps were 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 it should be able to be placed in the mold.

[0699] b. Gently use dust-free paper to absorb excess liquid, such as tissue fluid, etc., and be careful not to allow impurities other than the tissue to adhere.

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

[0701] d. Take the tissue out of 40% PVA (v / v) and gently wipe off the 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, being careful not to generate bubbles.

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

[0704] g. Horizontally transfer to -20℃ environment and freeze for 30 minutes to ensure that OCT and tissue are frozen.

[0705] 2. Sample tissue sections

[0706] The embedded tissue sample was sliced ​​to a thickness of 10 μm, flattened, and placed on a common slide. Immediately place a finger on the back of the slide for 5 seconds to thaw the slice using the finger temperature so that it fully adheres to the slide.

[0707] The results are shown in Table 6. Under an optical microscope, it was observed that the tissue was wrinkled, the tissue structure was deformed, there were cavities, and the structure and morphology of the plant tissue were not well maintained.

[0708] Comparative Example 17

[0709] In this comparative example, after the slices are qualified, the tissue permeabilization optimization step is performed, and the permeabilization optimization step is 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 permeabilization optimization slices showed that the fluorescence intensity of the six slices treated for 3 to 30 min was weak and the difference was not large, so the optimal permeabilization time could not be determined and the subsequent treatment conditions were not met.

[0711] Comparative Example 18

[0712] In the permeabilization optimization step of this comparative example, the permeabilization pretreatment solution is preferentially used to improve the permeabilization efficiency, and then 10X permeabilization enzyme is used for permeabilization treatment. The remaining steps are the same as those of comparative example 17.

[0713] The steps for permeabilization optimization are as follows:

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

[0715] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0716] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 15 minutes.

[0717] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0718] e. Then, follow the 10X Visium spatial transcriptome sequencing instructions for subsequent permeabilization optimization.

[0719] The results are shown in Table 7. The scanning imaging results of the permeabilization optimization slices showed that the fluorescence intensity of the six slices treated for 3 to 30 minutes was weak, and only the middle part of the tissue had weak fluorescence. It was impossible to determine the optimal permeabilization time and the subsequent processing conditions were not met.

[0720] Comparative Example 19

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

[0722] The steps for permeabilization optimization are as follows:

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

[0724] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0725] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 15 minutes.

[0726] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0727] e. Then, follow the 10X Visium spatial transcriptome sequencing instructions for subsequent permeabilization optimization.

[0728] The results are shown in Table 7. The scanning imaging results of the permeabilization optimization slices showed that the fluorescence intensity of the six slices treated for 3 to 30 minutes was weak, but the tissue fluorescence range was expanded. It was still impossible to determine the optimal permeabilization time, and the subsequent treatment conditions were not met.

[0729] Comparative Example 20

[0730] In this comparative example, the enzyme concentration in the permeabilization pretreatment solution formula is reduced, and the remaining steps are the same as those in comparative example 18.

[0731] The steps for permeabilization optimization are as follows:

[0732] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2, 0.1 M mannitol, 1% cellulase R10 (m / v), 0.2% macerate R10 (m / v), 0.5% pectinase (m / v), 0.5% hemicellulase (m / v), 0.4 U / μL RNase inhibitor.

[0733] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0734] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 15 minutes.

[0735] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0736] e. Then, follow the 10X Visium spatial transcriptome sequencing instructions for subsequent permeabilization optimization.

[0737] The results are shown in Table 7. The scanning imaging results of the permeabilization optimized slices showed that the fluorescence intensity of the six slices treated for 3 to 30 minutes was slightly stronger than that of Comparative Example 19, and the fluorescence range basically covered the entire tissue. The fluorescence brightness was the strongest at 18 minutes of permeabilization, but still weaker than the conventional fluorescence brightness.

[0738] Comparative Example 21

[0739] This comparative example continues to reduce the enzyme concentration in the permeabilization pretreatment solution formula, and the remaining steps are the same as comparative example 18.

[0740] The steps for permeabilization optimization are as follows:

[0741] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.5% cellulase R10 (m / v), 0.1% macerate R10 (m / v), 0.2% pectinase (m / v), 0.2% hemicellulase (m / v), 0.4 U / μL RNase inhibitor.

[0742] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0743] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 15 minutes.

[0744] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0745] e. Then, follow the 10X Visium spatial transcriptome sequencing instructions for subsequent permeabilization optimization.

[0746] The results are shown in Table 7. The scanning imaging results of the permeabilization optimized slices showed that the fluorescence intensity of the six slices treated for 3 to 30 minutes was slightly stronger than that of Comparative Example 20, and the fluorescence range basically covered the entire tissue. The fluorescence brightness was the strongest at 18 minutes of permeabilization, but still weaker than the conventional fluorescence brightness.

[0747] Comparative Example 22

[0748] In this comparative example, Tween 20 was added to the permeabilization pretreatment solution formula, and the remaining steps were the same as those in comparative example 21.

[0749] The steps for permeabilization optimization are as follows:

[0750] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.5% cellulase R10 (m / v), 0.1% macerate R10 (m / v), 0.2% pectinase (m / v), 0.2% hemicellulase (m / v), 1% Tween 20 (v / v), 0.4 U / μL RNase inhibitor.

[0751] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0752] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 15 minutes.

[0753] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0754] e. Then, follow the 10X Visium spatial transcriptome sequencing instructions for subsequent permeabilization optimization.

[0755] The results are shown in Table 7. The scanning imaging results of the permeabilization optimization slices showed that the fluorescence intensity of the six slices treated for 3 to 30 min was weak, and the optimal permeabilization time could not be determined, and the subsequent processing conditions were not met.

[0756] Comparative Example 23

[0757] In this comparative example, the concentration of Tween 20 in the permeabilization pretreatment solution is reduced, and the remaining steps are the same as those in comparative example 22.

[0758] The steps for permeabilization optimization are as follows:

[0759] a. Prepare permeabilization pretreatment solution, the formula contains 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1 M mannitol, 0.5% cellulase R10 (m / v), 0.1% macerate R10 (m / v), 0.2% pectinase, 0.2% hemicellulase (m / v), 0.5% Tween 20 (v / v), 0.4 U / μL RNase inhibitor.

[0760] b. Stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in the Slide Cassette, add 70 μL of permeabilization pretreatment solution into the wells in sequence along the lower left corner of the sample well of the Slide Cassette, tap the Slide Cassette to make the treatment solution evenly cover the tissue, and stick a sealing strip.

[0761] c. Place the PCR adapter in the PCR instrument and the Slide Cassette on the adapter. Pre-treat at 37°C for 15 minutes.

[0762] d. After pretreatment, carefully remove the pretreatment solution from the wells, add 100 μL 0.1x SSC to each well, and remove the pretreatment solution after 30 seconds.

[0763] e. Then, follow the 10X Visium spatial transcriptome sequencing instructions for subsequent permeabilization optimization.

[0764] The results are shown in Table 7. The scanning imaging results of the permeabilization optimized slices showed that the fluorescence intensity of the six slices treated for 3 to 30 minutes was slightly stronger than that of Comparative Example 21, and the fluorescence range basically covered the entire tissue. The fluorescence brightness was the strongest at 18 minutes of permeabilization, but still weaker than the conventional fluorescence brightness.

[0765] Comparative Example 24

[0766] In this comparative example, an in situ polyadenylation step is added after permeabilization to achieve the capture of non-coding RNA, and the remaining steps are the same as the 10XVisium spatial transcriptome sequencing instructions.

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

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

[0769] b. Add 80 μL of Poly(A) polymerase mixture (BioLab / MT0004) (E. coli Poly(A) Polymerase 5U / μL (final concentration 0.125U / μL), 10× EPAP buffer (final concentration 0.5×), 10 mM ATP (final concentration 0.5 mM), 25 mM MnCl to each sample well. 2 (final concentration 1.25 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddH 2 0 to 80 μL).

[0770] c. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 30 minutes before the enzyme mixture was removed.

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

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

[0773] f. Then, the reverse transcription and library construction steps were performed according to the 10XVisium spatial transcriptome sequencing instructions. The results are shown in Table 8. MicroRNAs were not captured at 50G sequencing volume, and the content of other non-coding RNAs was extremely low.

[0774] Comparative Example 25

[0775] In this comparative example, poly(A) polymerase is replaced, and the remaining steps are the same as those in comparative example 24.

[0776] The steps for in situ polyadenylation are as follows:

[0777] a. After permeabilization and washing, use a pipette to remove 0.1×SSC.

[0778] b. Add 80 μL Poly(A) polymerase mixture (Thermo / AM1350) (E. coli Poly(A) Polymerase 2U / μL (final concentration 0.125U / μL), 5× EPAP buffer (final concentration 0.5×), 10 mM ATP (final concentration 0.5 mM), 25 mM MnCl to each sample well. 2 (final concentration 1.25 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddH 2 0 to 80 μL).

[0779] c. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 30 minutes before the enzyme mixture was removed.

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

[0781] e. Discard the 0.1X SSC in each well.

[0782] f. Then, the reverse transcription and library construction steps were performed according to the 10XVisium spatial transcriptome sequencing instructions. The results are shown in Table 8. MicroRNAs were not captured at 50G sequencing volume, and the content of other non-coding RNAs was extremely low.

[0783] Comparative Example 26

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

[0785] a. After permeabilization and washing, use a pipette to remove 0.1×SSC.

[0786] b. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo / 74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×)), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using NEB / P0756S, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL) to each sample well, and fill with ddH2O to 75 μL.

[0787] c. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 20 minutes before the enzyme mixture was removed.

[0788] d. Add 100 μL 0.1x SSC to each well and wash for 10 seconds.

[0789] e. Discard the 0.1X SSC in each well.

[0790] f. Then, the reverse transcription and library construction steps were performed according to the 10XVisium spatial transcriptome sequencing instructions. The results are shown in Table 8. Non-coding RNA including microRNA can be captured at a sequencing volume of 50G, but the content is low.

[0791] Comparative Example 27

[0792] In this comparative example, ATP is replaced, and the remaining steps are the same as those in comparative example 26.

[0793] The steps for in situ polyadenylation are as follows:

[0794] a. After permeabilization and washing, use a pipette to remove 0.1×SSC.

[0795] b. Add 100 μL 1× washing buffer (20 μL 5× yPAP reaction buffer, 2 μL 40 U / μL RNase inhibitor, 78 μL nuclease-free H 2 O), and the buffer was removed after incubation at room temperature for 30 s.

[0796] c. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo / 74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×)), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using Thermo / AM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddH 2 O to 75 μL.

[0797] d. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 20 minutes before the enzyme mixture was removed.

[0798] e. Add 100 μL 0.1x SSC to each well and wash for 10 seconds.

[0799] f. Discard the 0.1X SSC in each well.

[0800] g. Then, the reverse transcription and library construction steps were performed according to the 10X Visium spatial transcriptome sequencing instructions. The results are shown in Table 8. Non-coding RNA including microRNA can be captured at a sequencing volume of 50G, but the content is low.

[0801] Comparative Example 28

[0802] In this comparative example, a buffer containing 10 mM EDTA was used for pretreatment of secondary metabolites before the in situ polyadenylation step, and the remaining steps were the same as those in Comparative Example 27.

[0803] The steps for in situ polyadenylation are as follows:

[0804] a. After permeabilization and washing, use a pipette to remove 0.1×SSC.

[0805] b. Pretreatment for removal of secondary metabolites: Add 10 mM EDTA buffer to each well, incubate at room temperature for 10 min, and then remove the pretreatment buffer.

[0806] c. Add 100 μL 1× washing buffer (20 μL 5× yPAP reaction buffer, 2 μL 40 U / μL RNase inhibitor, 78 μL nuclease-free H 2 O), and the buffer was removed after incubation at room temperature for 30 s.

[0807] d. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×)), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using Thermo / AM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddH 2 O to 75 μL.

[0808] e. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 20 minutes before the enzyme mixture was removed.

[0809] f. Add 100 μL 0.1x SSC to each well and wash for 10 seconds.

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

[0811] h. Then, the reverse transcription and library construction steps were performed according to the 10X Visium spatial transcriptome sequencing instructions. The results are shown in Table 8. Non-coding RNA including microRNA can be captured at a sequencing volume of 50G, but the content is low.

[0812] Comparative Example 29

[0813] In this comparative example, a buffer containing 5 mM DTT was used to pretreat the secondary metabolites before the in situ polyadenylation step, and the remaining steps were the same as those in Comparative Example 27.

[0814] The steps for in situ polyadenylation are as follows:

[0815] a. After permeabilization and washing, use a pipette to remove 0.1×SSC.

[0816] b. Pretreatment for removal of secondary metabolites: Add a buffer containing 5 mM DTT to each well, incubate at room temperature for 10 min, and then remove the pretreatment buffer.

[0817] c. Add 100 μL 1× washing buffer (20 μL 5× yPAP reaction buffer, 2 μL 40 U / μL RNase inhibitor, 78 μL nuclease-free H 2 O), and the buffer was removed after incubation at room temperature for 30 s.

[0818] d. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×)), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using ThermoAM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddH 2 O to 75 μL.

[0819] e. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 20 minutes before the enzyme mixture was removed.

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

[0821] g. Discard the 0.1X SSC in each well.

[0822] h. Then, the reverse transcription and library construction steps were performed according to the 10X Visium spatial transcriptome sequencing instructions. The results are shown in Table 8. Non-coding RNA including microRNA can be captured at a sequencing volume of 50G, but the content is low.

[0823] Comparative Example 30

[0824] In this comparative example, β-galactosidase was used for pretreatment of secondary metabolites, and the remaining steps were the same as those in comparative example 27.

[0825] The steps for in situ polyadenylation are as follows:

[0826] a. After permeabilization and washing, use a pipette to remove 0.1×SSC.

[0827] b. Pretreatment for removal of secondary metabolites: 250 U / mg β-galactosidase (Sigma, G6008-1KU) was diluted with 1×PBS to 40 U / μL enzyme solution, 70 μL of 0.05× β-galactosidase solution was added to each well, and the enzyme solution was removed after incubation at room temperature for 10 min.

[0828] c. Add 100 μL 1× washing buffer (20 μL 5× yPAP reaction buffer, 2 μL 40U / μL RNase inhibitor, 78 μL nuclease-free H 2 O), and the buffer was removed after incubation at room temperature for 30 s.

[0829] d. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×)), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using ThermoAM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddHO to each sample well. 2 O to 75 μL.

[0830] e. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 20 minutes before the enzyme mixture was removed.

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

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

[0833] h. Then, follow the 10X Visium spatial transcriptome sequencing instructions to perform subsequent reverse transcription, library construction, quality inspection and other steps.

[0834] The results are shown in Table 8. At a sequencing volume of 50G, the non-coding RNA capture efficiency is improved compared with Comparative Examples 24 to 29.

[0835] Comparative Example 31

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

[0837] The steps for in situ polyadenylation are as follows:

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

[0839] b. Pretreatment for removal of secondary metabolites: 250 U / mg β-galactosidase (Sigma, G6008-1KU) was diluted with 1×PBS to 40 U / μL enzyme solution, 70 μL of 0.1× β-galactosidase solution was added to each well, and the enzyme solution was removed after incubation at room temperature for 10 min.

[0840] c. Add 100 μL 1× washing buffer (20 μL 5× yPAP reaction buffer, 2 μL 40 U / μL RNase inhibitor, 78 μL nuclease-free H 2 O), and the buffer was removed after incubation at room temperature for 30 s.

[0841] d. Add 75 μL of yPAP enzyme (polyadenylate polymerase from yeast, Thermo74225Z25KU) mixture (5×yPAP reaction buffer (final concentration 1×), 600 U / μL yPAP enzyme (final concentration 24 U / μL), 10 mM ATP (using ThermoAM8110G, final concentration 0.5 mM), 40 U / μL RNase inhibitor (final concentration 1.5 U / μL), supplemented with ddH 2 O to 75 μL.

[0842] e. The reaction chamber was then sealed and the Slide Cassette was incubated at 37°C for 20 minutes before the enzyme mixture was removed.

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

[0844] g. Discard the 0.1X SSC in each well.

[0845] h. Then, follow the 10X Visium spatial transcriptome sequencing instructions to perform subsequent reverse transcription, library construction, quality inspection and other steps.

[0846] The results are shown in Table 8. At a sequencing volume of 50G, the non-coding RNA capture efficiency is improved compared with Comparative Examples 24 to 29.

[0847] Comparative Example 32

[0848] The steps from tissue sectioning to library construction in this comparative example are the same as those in Example 7, and the ribosomal RNA data are eliminated by bioinformatics analysis after sequencing.

[0849] The results are shown in Table 9. After the ribosomal RNA data were removed, only the data noise was reduced, and the number of detections of other non-coding RNAs did not increase significantly.

[0850] Comparative Example 33

[0851] In this comparative example, the existing commercial SEQuoia RiboDepletion ribosomal RNA removal kit (Bio-Rad / 17006487) was used to remove 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 to 3

[0854]

[0855]

[0856] Table 2 Examples 4 to 6

[0857]

[0858]

[0859] Table 3 Examples 7 to 8

[0860]

[0861] Table 4 Examples 9 to 10

[0862]

[0863]

[0864] The present invention provides a spatial whole transcriptome sequencing experimental process suitable for tobacco petioles, including a frozen section method suitable for tobacco petioles, a pretreatment solution suitable for plant tissue permeabilization, a treatment method suitable for in situ polyadenylation of tobacco petiole tissue, and a method for removing rRNA from a cDNA library suitable for the spatial whole transcriptome sequencing process. The above methods of the present invention can be directly grafted into the conventional 10X Visium spatial transcriptome sequencing process, filling the gap in the current spatial whole transcriptome in the field of plants, and helping to deeply understand the mechanisms of plant growth and development, environmental adaptation and stress resistance.

[0865] The present invention discloses a method for freezing tobacco petioles, a pretreatment solution before permeabilization of tobacco petioles tissue, an in situ polyadenylation method for tobacco petioles tissue, and a method for removing rRNA from a cDNA library suitable for a spatial whole transcriptome sequencing process. The method for freezing tobacco petioles is specifically as follows: before OCT embedding, the tobacco petiole tissue is placed in 25% PVA (v / v) (PVA covers the tissue), placed in a vacuum apparatus for 10 minutes to accelerate the penetration of PVA into the tissue, and then embedded with OCT. The embedding and freezing conditions are horizontal placement in a -20°C environment for 30 minutes; the formula of the pretreatment solution for permeabilization of tobacco petioles is specifically as follows (final concentration): 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2, 0.1M mannitol, 0.35% cellulase R10 (m / v), 0.1% macerate 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 pretreatment for 10min; the method designed by the present invention is suitable for in situ polyadenylation of tobacco petiole tissue, specifically: pretreatment liquid composition (final concentration): 20U / μL β-galactosidase solution, pretreatment conditions: incubation at room temperature for 10min. In situ polyadenylation mixture composition (final concentration): 1×yPAP reaction buffer, 24U / μLyPAP enzyme (yeast-derived polyadenylate polymerase, Thermo / 74225Z25KU), 0.5mMATP (Thermo / AM8110G), 1.5U / μL RNase inhibitor, treatment conditions are incubation at 37°C for 20min; The method designed by the present invention for removing rRNA from a cDNA library suitable for a spatial full transcriptome sequencing process is specifically as follows: IDTE buffer composition (final concentration): 10mM Tris HCl, final concentration 0.1mM EDTA. Unidirectional guide RNA pool: 26 synthetic guide RNAs (13 18sRNAs and 13 28sRNAs) are dissolved and diluted to 10μM with IDTE buffer, and the specific sequences of the 26 guide RNAs are shown in the attached table. RNP mixture composition: 10μM guide RNA, 20μM Cas9 nuclease, 1×PBS, where guide RNA and Cas9 nuclease are added at equal concentrations, and the remaining volume is filled with 1×PBS. RNP mixture is mixed and incubated at room temperature for 10 minutes. In vitro digestion mixture composition: 10×Cas9 nuclease reaction buffer, 1μM Cas9RNP and sample library, the final molar ratio of Cas9 RNP to sample library is 40:1, and the remaining volume is filled with 10×Cas9 nuclease reaction buffer. The in vitro digestion reaction conditions are incubation at 37°C for 60 minutes. The specific method for DNA release is: add proteinase K at a final concentration of 20mg / mL to the reaction and incubate at 56°C for 10 minutes. Fragment purification is specifically: purification using 0.8×SPRI magnetic beads.

[0866] In order to design a method and process for whole transcriptome sequencing experiments of tobacco petioles, the present invention attempts to design an in situ polyadenylation step based on the spatial transcriptome, and optimizes the basic steps of frozen sectioning and tissue permeabilization in view of the characteristics of plant tissues such as cell walls, high water content, and high secondary metabolite content. At the same time, to ensure the validity of the data for subsequent analysis, rRNA removal processing is designed at the wet experimental end, and a series of comparative experiments are carried out based on the above steps to compare the section quality, permeabilization optimization results, cDNA quality, library quality, and non-coding RNA capture efficiency. The above method was evaluated based on indicators such as rate, among which the detection standards were: the morphology of plant tissue sections should be uniform and flat, the tissue structure should be basically intact, and there should be no large voids; the fluorescence intensity of the six sections with optimized permeabilization is relatively clear, without diffusion and blur, and the optimal permeabilization time can be clearly judged; the cDNA fragment length is 900-1200bp, and the concentration is greater than 5ng / μL; the library fragment length is 400-450bp, and the concentration is greater than 10ng / μL; the rRNA capture efficiency of the cDNA library is less than 10% at a sequencing volume of 50G, and the capture efficiency of low-abundance non-coding RNA is improved at the same time.

[0867] In order to obtain a spatial full transcriptome sequencing experimental method suitable for tobacco petioles in the present invention, a series of explorations were carried out from the aspects of embedding frozen sections, tissue permeabilization, in situ polyadenylation and rRNA removal. The content of the present invention was finally obtained by consulting literature and multiple verifications. The method and results are shown in Table 5 below for comparative examples 1 to 16. First, by comparing Example 1, the embedding and freezing conditions of conventional frozen sections were used to prepare frozen section samples of tobacco petiole tissue, and the results are shown in Table 5: Comparative Example 1 Under conventional frozen section operation conditions (freezing at -80°C for 30min), the section morphology showed severe tissue shrinkage, the thin-walled tissue was destroyed, there were local fractures, and a large number of tissue cavities appeared, and the original intact structure of the plant tissue was not guaranteed, that is, the position of the cells and intracellular nucleic acids in the tissue moved, and the original position information was lost, so the spatial transcriptome experiment could not be carried out. It is speculated that it may be due to the high water content of the plant tissue, the rapid freezing temperature and the inappropriate freezing time, which led to the rapid growth rate of ice crystals in the tissue, squeezing the surrounding cell structure, causing the cell wall, cell membrane, gap, etc. to deform or rupture, so that the sample tissue appeared to shrink or deform. Usually, adjusting the embedding freezing temperature and time can improve this situation. Therefore, in Comparative Examples 2 to 4, the embedding freezing time is adjusted to 5 to 10 minutes. The results show that when the freezing temperature is -80°C, the freezing time is shortened, and the slice morphology still has serious shrinkage and tissue cavitation, and there is no obvious improvement. It is still impossible to prepare qualified slices for experiments. Comparative Examples 5 to 7 adjust the embedding freezing temperature to -20°C, and the freezing time is adjusted to 30min to 1h with the temperature. The results show that increasing the freezing temperature and slowing down the rate of ice crystal formation during freezing can improve the shrinkage of tissue slices. The embedding condition of -20°C freezing for 30min is the best, but there are still a small amount of shrinkage and cavities formed by the destruction of thin-walled tissue, which cannot ensure the best complete morphology of the slice, and the slice quality is unqualified. The reason for the analysis may be that there are large vacuoles in the plant tissue itself, and the water content inside the tissue is uneven. When freezing and slicing, the water in some areas may be frozen into ice crystals faster, thereby causing the water in other areas to gather in these areas, destroying the thin-walled tissue to form cavities. Or there may be bubbles of air in the tissue sample, which expand and interact with surrounding ice crystals when frozen and sectioned, resulting in voids in the sample.

[0868] Table 5 Comparative Examples 1 to 7

[0869]

[0870]

[0871] By comparing the results of Examples 1 to 7, it can be seen that pre-treating the tissue during embedding by enhancing the uniformity of the sample and removing bubbles in the tissue may be an effective way to solve the problem. Therefore, in Comparative Examples 8 to 16, different pre-treatment agents were tried to soak the sample and the negative pressure of the vacuum instrument was used to promote the penetration of the pre-treatment agent into the tissue. The vacuum negative pressure penetration method is not only conducive to the penetration of the freezing medium into the sample, reducing the water content, and enhancing the uniformity of the sample, but also can effectively remove bubbles in the tissue and reduce cavities. Comparative Examples 8 to 9 use conventional tissue protective agents OCT and 10% glycerol, both of which are infiltrated under negative pressure in the vacuum instrument for 10 minutes. The results all show that the tissue has a small amount of shrinkage and there are cavities in the structure. The samples pre-treated with glycerol have low viscosity, strong permeability and good antifreeze properties, resulting in the use of glycerol as a pre-treatment agent. It cannot form a good support for the tissue morphology. For example, the section results show that the cell morphology is irregular, the thin-walled tissue is squeezed and damaged, 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 protective agent in biology. It has strong osmotic regulation ability and adhesion, and can form a miscible solution with water. Therefore, it can effectively avoid the expansion, deformation and mechanical damage of tissue samples during experiments such as cryopreservation, thereby maintaining the integrity and morphological characteristics of tissue structure. In the present invention, comparative examples 10 to 13 respectively use different concentrations of PEG as an embedding pretreatment agent. The results show that after negative pressure infiltration in a vacuum with 10% to 30% PEG as an embedding pretreatment agent, the supporting force is poor, the tissue morphology is irregular, and the slices have a downward accumulation trend, and the slices are unqualified; 40% PEG has better supporting force, the thin-walled tissue structure is well maintained, there is no large cavity, and the slice requirements are basically met, but the subsequent RNA quality inspection of the tissue slices shows that the RIN value is 5.7, and the 28S / 18S is 0.54. The tissue RNA is degraded and does not meet the subsequent experimental requirements. The reason may be that although high concentrations of PEG have a good supporting ability for the tissue structure of tobacco petioles, on the other hand, PEG at high concentrations will also have a certain destructive effect on the RNA molecular structure, such as causing RNA chain breaks and base deletions, thereby affecting the integrity and stability of tissue RNA and failing to meet subsequent experimental requirements. Next, the present invention attempts to use different concentrations of PVA as an embedding pretreatment agent. PVA is a stable, non-toxic water-soluble polymer that is commonly used in industry as a thickener, emulsifier, anti-crystallization agent, etc. It has a greater viscosity than glycerol in nature, which can reduce the activity of water molecules in tissues and slow down the formation rate of ice crystals. Using negative pressure to penetrate into tissues may reduce the size and number of ice crystals formed in tissues, protect tissues from mechanical damage, and PVA has good osmotic regulation ability, which may effectively alleviate the extrusion and deformation of samples during freeze-thaw cycles.Therefore, comparative examples 14 to 16 used 10%, 20%, and 40% PVA as embedding pretreatment agents, respectively, and negative pressure infiltration was performed in a vacuum apparatus for 10 to 15 minutes. The results showed that the tissue structure was deformed after pretreatment with 10% and 20% PVA, and the tissue morphology could not be well maintained. This may be due to the low concentration of PVA, which caused ice crystals to damage the parenchyma when combined with water, resulting in wrinkled tissue, failing to protect and support the tissue morphology, and failing to complete qualified slice preparation. Embodiments 1 to 3 tried to use 25 to 35% PVA to pretreat the tissue. The results showed that the tissue sections were relatively uniform and flat, the tissue structure remained basically intact, and there were no large cavities. The tissue sections were subsequently inspected for quality. The results showed that the RIN values ​​were all greater than 7, and the 28S / 18S values ​​were all greater than 0.7. After the comparative embodiment 16 was pretreated with 40% PVA, the tissue edge was wrinkled, the tissue structure was deformed, and there were cavities. It is speculated that the viscosity of 40% PVA is relatively high, and the negative pressure failed to make it penetrate into the tissue well, so it failed to achieve the effect of supporting the tissue morphology. After comprehensively evaluating the cost and operation time, the present invention determined that 25% PVA was used as the embedding pretreatment agent, and the embedding and slicing were performed after the pretreatment of negative pressure infiltration for 10 minutes in a vacuum instrument to achieve the best slicing effect of tobacco petiole tissue.

[0872] Table 6 Comparative Examples 8 to 16

[0873]

[0874]

[0875] Next, a series of explorations were conducted on the permeabilization conditions of tobacco petioles in Comparative Examples 17 to 23. Comparative Example 17 used the conventional 10X Visium spatial transcriptome sequencing instruction manual process to optimize the permeabilization of tobacco petiole tissue. The results showed that the fluorescence intensity of the six slices treated for 3 to 30 minutes was weak, with little difference, and the optimal permeabilization time could not be determined, and the subsequent treatment conditions were not met. The reason may be that plant cells are composed of structures such as cell walls and plasma membranes, which make plant cells harder and less permeable than animal cells. When performing tissue permeabilization, the cell wall must first be destroyed or penetrated before the exogenous enzyme can contact the cell membrane for membrane permeabilization. This process is more difficult for plant tissues. Therefore, in Comparative Examples 18 to 21, pretreatment solutions of different formulations and concentrations were used to enzymatically hydrolyze the plant cell walls before permeabilization. In Comparative Example 18, a conventional plant cell wall enzymatic hydrolysis formula was used to pretreat the tobacco petioles. After permeabilization optimization, the results showed that the fluorescence intensity of the six slices treated for 3 to 30 minutes was weak, and only the middle part of the tissue had weak fluorescence. It was impossible to determine the optimal permeabilization time, and the subsequent treatment conditions were not met. The reason may be that conventional pretreatment liquid is mainly used for tissue protoplast separation, and its purpose is only to obtain protoplasts from a large number of plant tissues, rather than to obtain as many or all protoplasts as possible in the tissue. This enzymolysis solution cannot have a good enzymolysis 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 enzymolysis solution formula may have a good enzymolysis effect on the cell wall in this region. The main components of the cell walls in other parts all contain hemicellulose, so Comparative Example 19 added 1% hemicellulase (m / v) on the basis of the original formula. The permeabilization optimization results showed that the tissue fluorescence range was expanded, that is, the addition of hemicellulase improved the problem of incomplete slice information, but the fluorescence intensity of the six slices treated for 3 to 30 minutes was weak, indicating that very little RNA was detected, and the fluorescence brightness was close to the optimal permeabilization time and could not be determined. Continuing the experiment would result in incomplete information detection and the risk of library construction failure due to low RNA amount. According to the conventional enzymolysis effect, the type and concentration of the formula enzyme can effectively enzymolyze the cell wall of plant tissue, so it is speculated that the enzymolysis concentration is too strong for the sliced ​​tissue, the cell wall is completely enzymolyzed, and the protoplast is damaged, resulting in the outflow of intracellular nucleic acid. The nucleic acid loss during the cleaning process causes low nucleic acid content and low fluorescence intensity, and cell fragmentation will also cause the position information to be lost, affecting the subsequent judgment of the RNA source position and spatial distribution. Therefore, Comparative Examples 20 to 21 reduce the concentration of the enzymolysis solution, reduce the damage of the enzymolysis solution to the sample cells, and the permeabilization optimization results show that the fluorescence intensity of the six slices treated for 3 to 30 minutes is stronger than that of Comparative Example 19, and the fluorescence range basically covers the entire tissue. The fluorescence brightness is the strongest after permeabilization for 18 minutes, but it is still weaker than the conventional fluorescence brightness.In order to retain the spatial position information of the protoplast, the pretreatment process only loosens the cell wall structure instead of completely enzymolyzing the cell wall, resulting in poor permeabilization effect of pepsin in the presence of some cell wall components during the permeabilization process, and unlimited extension of the permeabilization time or change of the concentration of the protease has the risk of nucleic acid degradation, so it is attempted to pretreat the cell membrane before permeabilization. Tween 20 is a nonionic surfactant with good permeability and dispersibility. It is often used as a detergent, but it can also destroy the phospholipid bilayer structure on the cell membrane, making the cell membrane looser, thereby improving the efficiency of exogenous substances entering. Therefore, comparative examples 22 to 23 attempt to add Tween 20 to the pretreatment solution, and perform a certain degree of permeation on the cell membrane while enzymolyzing the cell wall, thereby improving the permeation efficiency of subsequent pepsin. Comparative example 22, 1% Tween 20 (v / v) was added to the enzymolysis solution, and the results showed that the fluorescence intensity of the six slices treated for 3 to 30 min was weak, and the optimal permeation time could not be determined, and the subsequent treatment conditions were not met. Comparative Example 23, the concentration of Tween 20 added was reduced. The results showed that the fluorescence intensity of the six slices treated for 3 to 30 minutes was slightly stronger than that of Comparative Example 21, but still weaker than the conventional fluorescence brightness. It can be seen that too high a concentration of Tween 20 added may also cause cell membrane fragmentation and nucleic acid outflow loss, but adding an appropriate amount of Tween 20 can significantly improve the permeabilization efficiency. Therefore, Examples 4 to 6 continued to explore the concentration of each component of the pretreatment solution formula. The permeabilization optimization results of the three groups of formulas all showed that the fluorescence intensity of the six slices treated for 3 to 30 minutes was clearly distinguished, the fluorescence signal was strong enough to be distinguished from the background noise, there was no overexposure, the fluorescence signal was evenly distributed in the sample area, there was no obvious signal gradient or local loss phenomenon, the slice quality was qualified, and the optimal permeabilization time was finally judged to be 18 minutes. After comprehensively evaluating the cost and operation time, the present invention determines that the permeabilization pretreatment solution formula is 0.02MMES, 0.02M KCl, 0.1% (m / v) BSA, and 0.01M CaCl. 2 , 0.1 M mannitol, 0.35% cellulase R10 (m / v), 0.1% macerate R10 (m / v), 0.1% pectinase (m / v), 0.15% hemicellulase (m / v), 0.15% Tween 20 (v / v), 0.4 U / μL RNase inhibitor, and the pretreatment conditions were incubation at 37°C for 10 min.

[0876] Table 7 Comparative Examples 17 to 23

[0877]

[0878]

[0879]

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

[0881] A temperature of 37°C will increase the risk of RNA degradation, so the enzymatic activity of the polymerase should be guaranteed as much as possible so that it can efficiently complete the addition of A. It is well known that some amino acid residues in the catalytic center and substrate binding site of the protease affect the enzyme activity and substrate specificity, and amino acid residues such as Met, Cys and His, Trp, and Tyr are all easily oxidized, thereby causing protein denaturation or affecting enzyme activity. A large amount of secondary metabolites are stored in plant protoplasts, and the vascular tissue at the petiole is the center of plant iron transport; the oxidizing property of secondary metabolites and the inhibition of metal ions will greatly limit the activity of the protease. Comparative Examples 28 to 29 pre-treat the tissue with a buffer containing a metal ion chelator and a reducing agent to reduce the influence of the oxidizing property of metal ions and secondary metabolites on the activity of poly(A) polymerase. The results show that the quality of the cDNA and library after in situ polyadenylation in the two experimental groups meets the requirements of spatial transcriptome sequencing, but the capture efficiency of non-coding RNA at a sequencing amount of 50G is not significantly different from that of Comparative Examples 26 to 27. At present, there are literature reports that the plant secondary metabolite jasmonic acid has a certain inhibitory effect on the activity of poly(A) polymerase [1]. 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, which can be converted into jasmonic acid through a series of enzyme-catalyzed reactions. Therefore, mechanical damage such as cutting of tobacco petioles will promote the synthesis of jasmonic acid, resulting in an increase in the content of secondary metabolite jasmonic acid in the sample, thereby affecting the activity of poly(A) polymerase, which may be the main reason for reducing the efficiency of polyadenylation of non-coding RNA in plant tissues. β-galactosidase is a hydrolase that can catalyze the hydrolysis reaction of β-galactoside and decompose the substrate into galactose and glucose. It is often used for lactose hydrolysis. Although there is no β-galactosidic bond in jasmonic acid, it can recognize the viscosity of some bonds in the jasmonic acid molecule and break it into 7-hydroxy-12-oxojasmonic acid and galactose, thereby degrading and inactivating jasmonic acid. Therefore, in contrast to Examples 30 to 31, different concentrations of β-galactosidase solutions were used for incubation pretreatment before in situ polyadenylation. The results showed that the non-coding RNA capture efficiency of the two experimental groups at 50G sequencing volume was improved compared with that of Comparative Examples 24 to 29. Examples 7 to 8 continued to explore the optimal addition concentration of β-galactosidase. The results showed that the cDNA and library quality after in situ polyadenylation in the two experimental groups met the requirements of spatial transcriptome sequencing, and the non-coding RNA capture efficiency was significantly improved compared with Comparative Examples 30 to 31 at 50G sequencing volume, but there was no significant difference in capture efficiency between Examples 7 and 8. After comprehensively evaluating the cost and operation time, the present invention determined that the concentration of the in situ polyadenylation treatment solution was 0.2×β-galactosidase solution, the pretreatment temperature was room temperature, and the pretreatment time was 10min.

[0882] Table 8 Comparative Examples 24 to 31

[0883]

[0884]

[0885]

[0886] By comparing Examples 24 to 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, its molecular weight is relatively large and its metabolism is inactive, so the transcriptome noise (invalid information) is basically all derived from rRNA. The purpose of sequencing is to obtain more biological information, but rRNA, the most abundant member in RNA, can only provide very little information on transcripts, and too much rRNA detection will 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 from the sequencing results by bioinformatics analysis, retaining other valid data. Comparative Example 32 is to remove rRNA by bioinformatics means. The results show that the rRNA content is finally reduced to 3.2% by bioinformatics means. Although the rRNA content is significantly reduced, the capture efficiency of other low-abundance non-coding RNAs is not significantly improved. Therefore, this method can only be used to remove data noise, and the amount of data of low-abundance non-coding RNA cannot be increased after sequencing. Therefore, in Comparative Example 33, the commercially available SEQuoia RiboDepletion ribosomal RNA removal kit (Bio-Rad / 17006487) was used to remove rRNA. The results showed that the rRNA capture rate was significantly reduced, and the capture efficiency of other low-abundance non-coding RNAs was also increased accordingly, but the rRNA capture efficiency was still above 10%.

[0887] Table 9 Comparative Examples 32 to 33

[0888]

[0889]

[0890] In summary, the present invention provides a spatial full transcriptome sequencing method suitable for tobacco petiole tissue. By comparing Examples 1 to 7, it is concluded that when tobacco leaves are frozen and sliced, the embedding and freezing conditions are determined to be -20°C and frozen for 30 minutes, which can effectively improve the shrinkage of tobacco petiole slices; Comparing Examples 8 to 16, the method of pretreating tobacco petiole tissue before embedding is evaluated. Combined with Comparing Examples 1 to 3, it is determined that the optimal concentration of PVA is 25%, and the vacuum instrument processing time is 10 minutes, which can provide good support for the tissue structure of tobacco petiole slices and keep the slices uniform and intact. Comparing Examples 17 to 23, the pretreatment method of sample tissue before permeabilization is evaluated. Combined with Examples 4 to 6, it is determined that the optimal permeabilization pretreatment solution formula is (final concentration) 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl 2 , 0.1M mannitol, 0.35% cellulase R10 (m / v), 0.1% macerator 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 incubation at 37°C for 10min, which can effectively enzymatically hydrolyze the plant cell wall and increase the permeability of the cell membrane to achieve the best permeabilization effect; Comparative Examples 24 to 31, the in situ polyadenylation method was evaluated, and it was determined that the in situ polyadenylation efficiency using yeast-derived poly(A) polymerase and ATP (Thermo / AM8110G) was the highest. Combined with Examples 7 to 8, it was determined that the use of 0.2×β-galactosidase solution to pretreat the tissue can effectively reduce the effect of secondary metabolites in the plant tissue on the activity of poly(A) polymerase and improve the in situ polyadenylation efficiency. Comparative results of Examples 32 to 33 show that the results obtained by removing rRNA from the bioinformatics end and using commercially available kits cannot simultaneously reduce the rRNA capture efficiency by less than 10% and improve the capture efficiency of other low-abundance non-coding RNAs. Combined with Examples 9 to 10, it was determined that the synthesis of guide RNA through 18srRNA and 28srRNA fragment sequences combined with the Cas9 enzyme to cut and degrade the double-stranded DNA formed by reverse transcription of ribosomal RNA in the library, thereby removing ribosomal RNA from the library, can effectively remove rRNA in the library, and can improve the capture efficiency of low-abundance non-coding RNA at the same sequencing amount, and it was determined that the use of 0.8×SPRI magnetic beads for purification can reduce the generation of small fragments in the library.

[0891] Table 10: Guide RNA sequences

[0892]

[0893]

[0894]

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

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

[0897] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A spatial full transcriptome sequencing method suitable for tobacco petiole tissue, characterized in that: The method is to effectively detect low-abundance non-coding RNA in tobacco petiole tissue on the basis of conventional spatial transcriptome sequencing, and realize spatial full transcriptome sequencing of tobacco petiole; After embedding of tobacco petiole tissue samples, sample tissue sectioning, tissue permeabilization, in situ polyadenylation steps and pretreatment, and then cDNA library construction and cDNA library quality inspection, spatial whole transcriptome sequencing and analysis were performed.

2. The method according to claim 1, characterized in that The method comprises the following steps: Step 1: Sample embedding pretreatment: Take fresh tobacco petiole tissue, wipe the surface to remove excess liquid, and pretreat the tobacco petiole tissue before embedding; Step 2, sample embedding: wipe the surface of the pretreated tobacco petiole tissue described in step 1 to remove excess liquid, place the embedding mold horizontally on ice, inject the pre-cooled frozen section embedding agent OCT into 1 / 3 of the mold groove capacity, confirm the direction of tissue sectioning, embed OCT, and transfer horizontally to -20℃ environment for freezing to ensure that OCT and tissue are frozen; Step 3, sample tissue sectioning: Slice the embedded tissue sample in step 2, flatten it, place the sample on a common slide, immediately put your finger on the back of the slide and press for 5 seconds, using the temperature of your finger to thaw the slice so that it fits the slide fully; Step 4, fixing and HE staining the tissue sections described in step 3; Step 5, scanning and imaging the slices described in step 4; Step 6, tissue permeabilization pretreatment: stick the tissue slices on the specified position of the Visium spatial gene expression slide, install the Visium spatial gene expression slide in a slide box, prepare the permeabilization pretreatment solution, add the permeabilization pretreatment solution to the wells in sequence along the lower left corner of the sample well of the slide box, tap the slide box to make the treatment solution evenly cover the tissue, stick a sealing strip, and place the slide box containing the Visium spatial gene expression slide in the PCR instrument for pretreatment; Step 7, tissue permeabilization: After the pretreatment is completed, remove the pretreatment solution from the sample well of the slide box, add washing buffer to each well, remove it after washing, resuspend and preheat the permease, add the permease to the well close to the lower left corner of the sample well of the slide box, tap the slide box gently to make the permease evenly cover the tissue, affix the sealing strip, put the slide box containing the Visum spatial gene expression slides on the PCR instrument with an adapter, cover the PCR instrument cover and incubate for permeabilization to optimize the selected permeabilization time, after the permeabilization is completed, remove the sealing strip, use a pipette to remove the permease in each well, and add washing buffer to each well; Step 8, in situ polyadenylation pretreatment: adding in situ polyadenylation pretreatment solution to each glass slide box sample well described in step 7, and removing the pretreatment solution after incubation; Step 9, in situ polyadenylation: adding a wash buffer to each slide box sample well described in step 8, removing the wash buffer after incubation, adding a polyadenylic acid polymerase mixture to each slide box sample well, sealing the reaction chamber, removing the enzyme mixture after incubation, adding a wash buffer to each well for washing, and then discarding the wash buffer; Step 10: cDNA library construction; Step 11, rRNA removal: prepare an in vitro digestion mixture, mix the cDNA library described in step 10 with the in vitro digestion mixture in proportion, and incubate at 37°C, add proteinase K to the reaction, incubate at 56°C to allow Cas9 endonuclease to release DNA, and purify and elute the sample using 0.8×SPRI magnetic beads; Step 12: cDNA library quality inspection; Step 13: Spatial whole transcriptome sequencing and analysis.

3. The method according to claim 2, characterized in that The method comprises: a tobacco petiole tissue embedding pretreatment step; in the embedding pretreatment step 1, the tissue is immersed in 25% to 35% (v / v) polyvinyl alcohol (PVA) and vacuumed for 10 to 15 minutes using a vacuum instrument to allow the PVA to fully penetrate into the tissue before embedding; and / or, In the step 2, the embedding condition is freezing at -20°C for 30 minutes; and / or, The method comprises: a pretreatment step before permeabilization of tobacco petiole tissue; in the step 6, before conventional tissue permeabilization, the tobacco petiole tissue permeabilization pretreatment solution is used for pretreatment in a PCR instrument at 37°C for 10 to 15 minutes, wherein the tissue permeabilization pretreatment solution comprises the following components, all in final concentration: 0.02M MES, 0.02M KCl, 0.1% (m / v) BSA, 0.01M CaCl2, 0.1M mannitol, 0.25 to 0.5% (m / v) cellulase R10, 0.05% to 0.1% (m / v) macerate R10, 0.05% to 0.2% (m / v) pectinase, 0.1% to 0.2% (m / v) hemicellulase, 0.1 to 0.25% (v / v) Tween 20, 0.4U / μL RNase inhibitor; and / or, The method comprises: an in situ polyadenylation pretreatment step; in the step eight, before the in situ polyadenylation step, a secondary metabolite removal pretreatment solution is used for treatment at room temperature for 10 minutes, the pretreatment solution is a β-galactosidase solution with a final concentration of (0.2-0.5)×, and the mother solution concentration of the β-galactosidase is 40U / μL; and / or, The method comprises: an in situ polyadenylation step; in the step nine, adding 1× washing buffer to each sample well, incubating at room temperature for 30 seconds, then removing the washing buffer, adding a polyadenylic acid polymerase mixture from yeast to each sample well, incubating at 37° C. for 20 minutes, then removing the mixture; and / or, The method comprises: a step of removing rRNA from a cDNA library; in the step eleven, a unidirectional guide RNA pool containing 26 synthetic guide RNAs is mixed with a Cas9 enzyme and a sample cDNA library to form an in vitro digestion mixture, incubated at 37° C. for 60 minutes, then proteinase K is added, and incubated at 56° C. for 10 minutes to promote DNA release, and finally 0.8×SPRI is used for magnetic bead purification to separate target cDNA library fragments other than rRNA.

4. The method according to claim 3, characterized in that The tobacco petiole tissue permeabilization pretreatment solution comprises the following components (final concentration): 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) macerate 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; and / or, The in situ polyadenylation pretreatment solution comprises the following components: 250 U / mg β-galactosidase is prepared into a 40 U / μL stock solution with 1×PBS, and the final concentration of the pretreatment solution is (0.2-0.5)×β-galactosidase solution; and / or, In the step nine, the washing buffer comprises the following components: 1× polyadenylic acid polymerase reaction buffer, 0.8 U / μL RNase inhibitor, ddH2O; and / or, In the step nine, the poly(A) polymerase mixture comprises the following components: 1× poly(A) polymerase reaction buffer, 24 U / μL poly(A) polymerase, 0.5 mM ATP, and 1.5 U / μL RNase inhibitor.

5. The method as claimed in claim 3, characterized in that In the step eleven, the 26 synthetic guide RNAs are guide RNAs designed and synthesized according to the 18srRNA and 28srRNA fragment sequences, 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, 0.1mMEDTA; unidirectional guide RNA pool.

6. A kit / reagent for spatial full transcriptome sequencing of tobacco petioles, characterized in that: The kit / reagent comprises a tobacco petiole tissue permeabilization pretreatment solution, an in situ polyadenylation pretreatment solution, an in situ polyadenylation washing buffer solution, a polyadenylate polymerase mixed solution, and / or an in vitro digestion mixed solution.

7. The kit / reagent according to claim 6, characterized in that The tobacco 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) macerate 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; and / or, The in situ polyadenylation pretreatment solution comprises the following components: a final concentration of (0.2-0.5)×β-galactosidase solution, 1×PBS, wherein the concentration of the β-galactosidase mother solution is 40U / μL; and / or, The in situ polyadenylation washing buffer comprises the following components: 1× polyadenylate polymerase reaction buffer, 0.8 U / μL RNase inhibitor, ddH2O; and / or, The poly(A) polymerase mixture comprises the following components: 1× poly(A) polymerase reaction buffer, 24 U / μL poly(A) polymerase, 0.5 mM ATP, 1.5 U / μL RNase inhibitor; and / or, The in vitro digestion mixture contains the following components: 10 μM unidirectional guide RNA pool, 20 μM Cas9 endonuclease, 1×PBS, 10×Cas9 nuclease reaction buffer, and 20 mg / ml proteinase K.

8. A pretreatment solution, characterized in that The pretreatment solution comprises: A tobacco petiole tissue permeabilization pretreatment solution comprising 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) macerate 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; and / or, The in situ polyadenylation pretreatment solution comprises the following components: a final concentration of (0.2-0.5)×β-galactosidase solution, wherein the concentration of the β-galactosidase mother solution is 40 U / μL.

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

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