Method for spatial transcriptome analysis based on ffpe samples

Abstract

This invention relates to the field of plant spatial transcriptomics technology, and particularly to a spatial transcriptomics analysis method based on FFPE samples. Based on plant FFPE samples, this invention simultaneously captures plant cell wall (and nucleus) and track line signals in a single staining and imaging process, providing precise cell contours for the plant while automatically registering the track lines with the expression matrix without manual integration, significantly improving the efficiency and accuracy of plant cellbin information acquisition.

Description

Technical Field

[0001] This invention relates to the field of plant spatial transcriptomics technology, and in particular to a spatial transcriptomics analysis method based on FFPE samples. Background Technology

[0002] Stereo-seq spatial transcriptome sequencing technology is a high-throughput spatiotemporal sequencing technology. It uses fixed probes on a microarray to capture nucleic acid information from tissues in situ. Two sequencing operations are performed to confirm the spatial location and corresponding expression levels of mRNA sequences. Simultaneously, microscopic imaging techniques are used to scan the microarray and image the tissue. Track lines (irregularly arranged latitude and longitude lines on the microarray surface, used for image stitching and registration) are used for registration with the expression matrix. It is now applied in multiple biological and medical fields and has become one of the mainstream methods for spatial transcriptome sequencing. It adds precise spatial information to traditional transcriptome sequencing, helping researchers identify the location of different cells within tissues. This will promote the understanding of individual cells and the interpretation of relationships between cell populations within tissues.

[0003] Current spatial transcriptomics technology primarily studies animal tissues. It can simultaneously identify probes on the cell nucleus and solid support surface using SS (ssDNAAssay Kit) fluorescent staining, thus obtaining the location of individual cells (since cells in animal tissues are usually relatively regular and similar in size, cell size and location can be predicted) and track line distribution. However, plant cells, due to their cell walls, chloroplasts, vacuoles, varying cell sizes, irregular shapes, non-centralized nuclei, and the presence of various secondary metabolites, cannot simultaneously obtain track lines and the location of individual cells using SS fluorescent staining alone. Therefore, it is necessary to use SS fluorescent staining to identify track lines, followed by FB28 (Fluorescent Brightener 28) fluorescent staining to identify individual plant cells, and then manually register the two images. This process is cumbersome and has poor compatibility.

[0004] In summary, the existing technology has the following drawbacks:

[0005] 1. Existing technology requires two fluorescent staining and imaging operations to indirectly achieve cell wall and expression matrix registration, which increases the difficulty of experimental operation and prolongs tissue exposure time, leading to nucleic acid degradation and reduced capture.

[0006] 2. The lack of track lines in the cell wall fluorescence staining image and the absence of reference objects in the tissue blank area resulted in excessive local errors in the image stitching results;

[0007] 3. Image registration and cell segmentation need to be done manually, which cannot be adapted to the automated standard analysis workflow (Stereo-seq Analysis Workflow, SAW). Cellbin expression matrix needs to be obtained manually, which is not conducive to product layout and promotion.

[0008] 4. Fresh samples have a high water content and require rapid OCT cryopreservation after sampling. This can easily lead to ice crystal formation within cells, causing tissue section fragmentation and damage to the cell wall fluorescence staining image (example: cell wall imaging of a frozen section of a fresh sample (Arabidopsis leaf)). Figure 7 (As shown); Frozen sections are relatively thick, generally 10-15 μm thick, making it difficult to distinguish the upper and lower sections of the cell wall, and they are prone to collapse after being broken, which is not conducive to the segmentation of plant cells. Summary of the Invention

[0009] In view of this, the present invention provides a spatial transcriptome analysis method based on FFPE samples. Based on plant FFPE samples, this invention captures plant cell wall and track line signals simultaneously in a single staining and imaging process, providing precise cell contours for the plant while automatically registering the track lines with the expression matrix, eliminating the need for manual integration and significantly improving the efficiency and accuracy of plant cellbin information acquisition.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] In a first aspect, the present invention provides an FB28 fluorescent dye solution, comprising the following components:

[0012] FB28 1 volume portion;

[0013] Nuclease-Free Water 99-999 parts by volume;

[0014] The FB28 fluorescent dye solution does not contain organic solvents; preferably, the organic solvents include ethanol.

[0015] More preferably, the FB28 fluorescent staining solution does not include RNase inhibitors.

[0016] Secondly, the present invention also provides a mixed staining reagent, comprising the FB28 fluorescent staining solution and ssDNA staining solution.

[0017] In some specific embodiments of the present invention, the ssDNA staining solution includes ssDNA reagent; the ssDNA staining solution does not include RNase inhibitors.

[0018] In some specific embodiments of the present invention, the ssDNA reagent is Qubit ssDNAHSReagent;

[0019] Preferably, the mixed staining reagent further includes a chip cleaning solution; the chip cleaning solution is 0.1×SSC buffer.

[0020] More preferably, the volume ratio of FB28 to Qubit ssDNAHS Reagent is (1-2):(1-5).

[0021] Thirdly, the present invention also provides a kit comprising the FB28 fluorescent staining solution or the mixed staining reagent.

[0022] Fourthly, the present invention also provides the application of the FB28 fluorescent staining solution, the mixed staining reagent, or the kit in tissue fluorescent staining;

[0023] Preferably, the tissue comprises a plant FFPE sample.

[0024] In some specific embodiments of the present invention, the application includes any one of the following:

[0025] (I) Stereo-seq transcriptome technology was carried out using plant FFPE samples;

[0026] (II) Single staining imaging captures fluorescence signals of the cell wall or cell nucleus and / or track line fluorescence signals on a single image;

[0027] (III) Automatic image registration of plant spatial transcriptome based on FFPE samples;

[0028] (IV) Adapt to existing SAW automated analysis workflows to quickly extract plant Cellbin expression matrices;

[0029] (V) Shorten the time to reduce RNA degradation and increase the amount of expression information captured; or

[0030] (VI) Improve the accuracy of Cellbin zoning.

[0031] In some specific embodiments of the present invention, multiple image information can be obtained by a single staining and photographing, which lowers the threshold of experimental operation, shortens the time, reduces RNA degradation, and increases the amount of expression information captured.

[0032] In some specific embodiments of the present invention, it can be adapted to the SAW automated analysis workflow, quickly generate the Cellbin expression matrix of plant samples, and preliminary analysis results, laying the foundation for the automated spatial transcriptome data analysis workflow of plants; reducing manual operation and improving the accuracy of Cellbin regionalization. Figure 4 This makes spatial clustering annotation results more reliable, helping researchers to uncover more accurate data results.

[0033] Fifthly, the present invention also provides a staining method for plant spatial transcriptomics technology, wherein a fixed tissue chip is stained with the FB28 fluorescent staining solution, the mixed staining reagent, or the kit described above.

[0034] The staining is performed exactly once.

[0035] In some specific embodiments of the present invention, the staining method,

[0036] The final working concentration of the FB28 fluorescent dye solution is 0.01% (v / v);

[0037] The staining time includes 2 to 5 minutes;

[0038] In some specific embodiments of the present invention, the staining method includes the following steps:

[0039] Plant FFPE sample paraffin-embedded blocks were prepared, sectioned, spread, retrieved, baked, and dewaxed. The tissue was then covered with 100 μL / chip using the FB28 fluorescent staining solution, the mixed staining reagent, or the kit described above. The staining was carried out at room temperature in the dark for 2 min. The chip was then washed twice with 100 μL / chip of 0.1×SSC (diluted from 20×SSC (AMBION, AM9770) with Nuclease-Free Water).

[0040] In a sixth aspect, the present invention also provides a method for obtaining image information for plant spatial transcriptomics technology, wherein FFPE samples of plants are processed, frozen sections are prepared, fixed, dewaxed, stained with the FB28 fluorescent staining solution, the mixed staining reagent or the kit, mounted, scanned and photographed to obtain image information.

[0041] The staining is performed exactly once;

[0042] The image information includes fluorescence signals from the plant's cell walls or nuclei and / or track line fluorescence signals.

[0043] In a seventh aspect, the present invention also provides a method for plant spatial transcriptome analysis, which is based on the above-mentioned method for obtaining image information for plant spatial transcriptome technology, and then performs tissue permeation, reverse transcription, removal, cDNA release and recovery, purification and amplification, library preparation, sequencing, obtaining expression matrix, and performing spatial transcriptome analysis.

[0044] In some specific embodiments of the present invention, the spatial transcriptome analysis includes a manual analysis process and / or an automated analysis process;

[0045] The automated analysis process includes, but is not limited to, the Stereo-seq Analysis Workflow;

[0046] In some specific embodiments of the present invention, the spatial transcriptome analysis includes:

[0047] The fluorescence staining image was obtained and automatically spliced ​​using StereoMap software based on the track line, and then registered with the expression matrix captured by Stereo-seq transcriptome technology.

[0048] Using a plant cell recognition model, precise cell delineation can be achieved through automatic identification based on cell wall or nucleus fluorescence staining patterns.

[0049] A mask segmentation pattern is generated based on cell outlines, and the Cellbin expression matrix is ​​finally obtained. Stereopy is used to complete the clustering results at single-cell resolution. Based on the cell outlines, the single-cell expression matrix is ​​divided to realize the single-cell resolution plant spatial transcriptome data analysis.

[0050] This invention provides a spatial transcriptome analysis method based on FFPE samples. This invention utilizes Stereo-seq transcriptome technology on plant FFPE samples, employing Fluorescent Brightener 28 fluorescent dye for staining and imaging. A single staining and imaging process can capture fluorescence signals from the cell wall or nucleus, as well as track line fluorescence signals, on a single image. It is also compatible with existing SAW automated analysis workflows to obtain plant cellbin information.

[0051] The beneficial effects of this invention include:

[0052] 1. Multiple image information can be obtained through a single staining and photographing, which lowers the threshold of experimental operation, shortens the time, reduces RNA degradation, and increases the amount of expression information captured;

[0053] 2. It can adapt to the SAW automated analysis workflow, quickly generate Cellbin expression matrices of plant samples, and preliminary analysis results, laying the foundation for automated spatial transcriptome data analysis workflows for plants;

[0054] 3. Reduce manual operations, improve the accuracy of Cellbin zoning, make spatial clustering annotation results more reliable, and help researchers discover more accurate data results. Attached Figure Description

[0055] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0056] Figure 1Demonstrates the Stereo-seq transcriptome technique workflow for plant FFPE samples;

[0057] Figure 2 This shows FFPE samples from Arabidopsis thaliana leaves;

[0058] Figure 3 This demonstrates the simultaneous capture of the fluorescent staining contours of plant cell walls and the fluorescence signals of track lines in a single imaging session.

[0059] Figure 4 This shows the results of automatic segmentation of plant tissue cells;

[0060] Figure 5 Show the gene counts results mapped back to the mask;

[0061] Figure 6 This demonstrates existing image stitching and registration modes;

[0062] Figure 7 Cell wall imaging of frozen sections of fresh samples (Arabidopsis thaliana leaves) in comparative examples;

[0063] Figure 8 In the illustrated example, cell wall imaging was performed on paraffin-embedded sections of Arabidopsis thaliana leaf FFPE samples;

[0064] Figure 9 The results of automatic regionalization of Arabidopsis thaliana leaves are shown. Among them, A is the cell wall fluorescence staining image of OCT frozen embedded section of fresh sample, and B is the cell wall fluorescence staining image of paraffin embedded section.

[0065] Figure 10 Figures showing the staining results of FB28 fluorescent dye solution prepared with different configurations for FFPE sample sections of Arabidopsis thaliana leaf tissue;

[0066] Figure 11 This image shows the results of multichannel scanning of FFPE sample sections of tomato callus stained with mixed staining solution (FB28 & ssDNA). Detailed Implementation

[0067] This invention discloses a spatial transcriptome analysis method based on FFPE samples. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0068] The specific steps for extracting the Cellbin expression matrix in this embodiment of the invention are as follows:

[0069] 1. Based on Stereo-seq FFPE transcriptome technology, the paraffin-embedded blocks of plant FFPE samples were sectioned (5μm), spread (42℃, 30% ethanol room temperature pre-spreading), retrieved, baked (60℃, 30min~1h), and dewaxed (using environmentally friendly dewaxing agent Histo-clear (Hyde, H6025) at room temperature for 20min, twice; 100% ethanol and 96% ethanol for 5min each, twice; 90%, 80%, 70%, 50%, 30% ethanol and ddH2O for 2min each).

[0070] 2. Prepare plant cell wall fluorescent staining solution in advance. Take 1 μL of Fluorescent Brightener 28 (FB28, SIGMA-ALDRICH, 910090-50mL) into a 1.5mL brown centrifuge tube, add Nuclease-Free Water (NF water, Ambion, Cat. No. AM9937) to 1mL, vortex and mix well to dilute the stock solution 1000 times to the working concentration. Store at room temperature in the dark for a short period of time. (For other brands of plant cell wall calcium fluorescent white (CFW) fluorescent staining reagents, prepare the stock solution according to the corresponding instructions and dilute it to 1 / 1000 for the working concentration.)

[0071] 3. After dewaxing, blow dry the chip (carrier), add diluted FB28 fluorescent staining solution (1 / 1000 working concentration, 100μL / chip) to cover the tissue, stain at room temperature in the dark for 2 min, and wash the chip twice with 0.1×SSC (100μL / chip) (20×SSC, AMBION, AM9770 diluted solution).

[0072] 4. Dry the chip, add FFPE Mounting Medium (Stereo-seq FFPE Transcriptomics Reagent Kit (item number: 201SN114) from Shenzhen BGI Genomics Co., Ltd., reagent number 1000047466) to the center of the tissue (5 μL / chip), gently cover with a glass slide (avoid air bubbles), and prepare for microscope scanning.

[0073] 5. Transfer the carrier to the microscope scanning stage, turn on the fluorescence microscope control system, select epifluorescence, DAPI channel, create a new slice, create a map under 4x magnification, manually scan under 10x magnification, quickly add model points (1-2 focal points inside the tissue to ensure tissue clarity, 4 focal points evenly distributed around the edge of the chip outside the tissue to ensure track lines are clear), adjust the overall exposure (based on tissue exposure to avoid overexposure), and scan the entire chip.

[0074] 6. Use ImageStudio to complete image quality control, stitching, calibration, and segmentation (customizable cell segmentation models), and automatically upload the results to the SAW cloud platform to complete the Cellbin automated workflow analysis.

[0075] This invention, based on plant FFPE samples, simultaneously captures plant cell wall and track line signals in a single staining and imaging process (using the cell wall imaging of a paraffin-embedded section of an Arabidopsis leaf FFPE sample as an example, e.g.) Figure 8 As shown in the figure, it provides accurate cell outlines for plants and automatically registers them based on track lines and expression matrices without manual integration, greatly improving the efficiency and accuracy of obtaining plant cellbin information.

[0076] The Stereo-seq FFPE transcriptome technology of this invention improves the fit of plant tissues. By pre-treating samples such as FAA fixation and paraffin embedding, the water content of the tissue is reduced, making the plant tissue easier to section and requiring only one staining and imaging, which is conducive to the advancement of plant spatial transcriptome technology.

[0077] This invention is not limited to the dyes and concentrations used, nor is it limited to plant species or tissue types.

[0078] The registration and segmentation methods in this invention can be either automatic or manual.

[0079] The raw materials and reagents used in the spatial transcriptome analysis method based on FFPE samples provided by this invention are all commercially available.

[0080] The present invention will be further illustrated below with reference to the embodiments:

[0081] Example 1: Extraction of Cellbin Expression Matrix from Arabidopsis Leaf FFPE Samples

[0082] The flowchart of Stereo-seq transcriptome technology for plant FFPE samples is as follows: Figure 1 As shown, this embodiment uses Arabidopsis thaliana leaf FFPE samples as an example, according to... Figure 1 The technical process and the extraction of Cellbin expression matrix through offline data visualization analysis.

[0083] I. Experimental Procedure

[0084] 1. Sample preparation, slide preparation, slide spreading, slide retrieval and baking (Day 1)

[0085] (1) Turn on the tissue baking machine in advance, with a water bath temperature of 42-45℃;

[0086] (2) Install the PCR adapter on the PCR instrument in advance, set the slide baking program as shown in Table 1 (program selection: Incubate), and click run;

[0087] Table 1

[0088]

[0089] (3) Prepare a paraffin microtome, brush, tweezers and new blades; place 30% ethanol (300-400 mL) in an enzyme-free flared container (e.g., 1 mL or 200 μL pipette tip box; 30% ethanol can be left at room temperature for 1 day).

[0090] (4) Remove the FFPE wax block stored at 4℃ (e.g.) Figure 2 (As shown), place the tissue surface on an ice pack that has just been taken out at -20℃ for 5-10 minutes (or place the tissue surface on a cold table for 5-10 minutes).

[0091] (5) Fix the wax block base on the microtome sample head, adjust the position of the wax block so that the blade is at a 5-degree angle to the surface of the wax block; if the tissue is deeply embedded, the old blade can be used to trim the tissue first, and a new blade can be used for sectioning after the tissue is exposed.

[0092] (6) Adjust the section thickness. For ordinary tissues, 5μm is recommended. When slicing, hold the brush in your left hand and rotate the microtome wheel with your right hand to slice continuously. Discard the first slice and carefully divide the second slice. After trimming, attach the slice to the N-carrier chip area of ​​the Stereo-seq chip.

[0093] Note: Paraffin sections here have two sides, A and B. The side that is against the blade is side B (smooth and reflective); the side facing the operator is side A (non-reflective). When spreading the section, side B should be in contact with the liquid. After picking up the section, attach it to the surface of the chip and glass slide to prevent it from falling off.

[0094] (7) Trim the second slice to about 1cm×1cm in size and carefully transfer it to the surface of 30% ethanol with a brush; pick up the slice with a clean ordinary glass slide and transfer it to a preheated water bath, making sure to keep side B in contact with the water surface (this step is used to assist in transferring the slice to the water bath and ensure that the slide is spread smoothly. If an experienced technician is operating this step, this step can be skipped).

[0095] (8) Carefully observe the state of the tissue section. The tissue section needs to be completely flat without wrinkles. If the spreading effect is not good, the water bath temperature can be increased by 0.5 to 1°C and the observation can continue until the tissue is completely flat. If there are signs of melting of the wax around the tissue, it means that the water bath temperature is too high. You can stop heating and reduce the water bath temperature by 0.5 to 1°C, and immediately retrieve the section.

[0096] (9) After the tissue section is completely flattened, take out the Stereo-seq chip N carrier, record the chip number, and be careful not to touch the chip surface; gently press a corner of the flattened section with a brush so that the tissue can cover the chip part on the Stereo-seq chip N carrier, and then pick up the section.

[0097] Note: The tissue region attached to the N-carrier of the Stereo-seq chip needs to have obvious tissue features and be located as centrally as possible.

[0098] (10) Wipe away the liquid on the back of the carrier and around the chip with a lint-free paper, place it flat on the adapter, click edit and then click next step, skip 42℃∞, bake at 42℃ for 3 hours and then bake at 37℃ overnight.

[0099] 2. Dewaxing and rehydration (day 2)

[0100] (1) Turn on the sheet spreading and baking machine or sheet baking machine in advance, and the baking temperature is 60℃;

[0101] (2) Remove the Stereo-seq chip N carrier and place it in a 60℃ wafer oven for 30 min to 1 h.

[0102] (3) Prepare 10 staining jars in the fume hood: 2 x Histo-clear (H6025) dewaxing reagent, 2 x 100% ethanol, 2 x 96% ethanol, 1 x 90% ethanol, 1 x 80% ethanol, 1 x 70% ethanol, 1 x 50% ethanol, 1 x 30% ethanol, and 1 x ddH2O;

[0103] (4) Dewaxing: Place the baked Stereo-seq chip N carrier into Histo-clear ① (① represents one of the two staining tanks) and incubate at room temperature for 20 min; then take out the Stereo-seq chip N carrier and place it into Histo-clear ② (② represents the other of the two staining tanks besides ①) and incubate at room temperature for 20 min;

[0104] (5) Remove the Stereo-seq chip N carrier, use a lint-free paper to absorb the excess Histo-clear, and place it in 100% ethanol ① for 5 min, 100% ethanol ② for 5 min, 96% ethanol ① for 5 min, 96% ethanol ② for 5 min, 90% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min, 50% ethanol for 2 min, 30% ethanol for 2 min, and ddH2O for 2 min in sequence. Remove it and absorb the excess moisture.

[0105] 3. Dyeing and photography (Day 2)

[0106] (1) Cell wall staining

[0107] 1) Prepare fluorescent staining solution: Prepare a stock solution of Fluorescent Brightener 28 (FB28, SIGMA-ALDRICH, 910090-50mL) in advance, and dilute it 100 times with NF water (Nuclease Free Water, Ambion, Cat. No. AM9937) to the working concentration. It can be stored at room temperature in the dark for a short period of time.

[0108] 2) Staining: After dewaxing, the surface of the chip on the carrier is dried. 0.01×FB28 fluorescent staining solution is dropped onto the chip (100μL / chip) to completely cover the tissue area. Stain at room temperature in the dark for 2 min. The chip is then washed twice with 0.1×SSC (100μL / chip) (diluted from 20×SSC (AMBION, AM9770) with Nuclease-Free Water).

[0109] (2) Photographing the film.

[0110] 1) Mounting: Dry the chip, then drop 5 μL of mounting medium FFPE Mounting Medium (Stere-seq FFPE transcriptome reagent kit (item number: 201SN114) from Shenzhen BGI Genomics Co., Ltd., reagent number 1000047466) into the center of the tissue, and slowly lower the coverslip to avoid generating air bubbles.

[0111] 2) Imaging: Referencing the "Stereo-seq Spatial Multi-omics Technology Imaging Guide" published by Shenzhen BGI Genomics Co., Ltd., epifluorescence (black and white) and DAPI channel scanning imaging were used. The specific steps were as follows: Transfer the carrier to the microscope scanning stage, turn on the fluorescence microscope control system, select epifluorescence, DAPI channel, create a new section, create a map under 4x magnification, manually scan under 10x magnification, quickly add model points (1-2 focal points within the tissue to ensure tissue clarity, and 4 focal points evenly distributed around the chip edge outside the tissue to ensure track lines are clear), adjust the overall exposure (based on tissue exposure to avoid overexposure), and scan the entire chip.

[0112] 4. Decrosslinking and fixation (Day 2)

[0113] (1) Load the PCR adapter into the PCR instrument in advance, set the cross-linking removal program as shown in Table 2 (program selection: Incubate), and click run;

[0114] Table 2

[0115]

[0116] (2) After imaging, immerse the chip carrier covered with a cover glass in a slide box containing 0.1×SSC, move it up and down to let the cover glass fall off naturally, clean the sealing liquid on the chip surface, wipe the liquid around the chip with a lint-free paper to keep the chip surface moist.

[0117] (3) Assemble the handheld carrier according to Chapter 2 of the "Stereo-seq Chip Carrier and Accessories Instruction Manual" (Document No.: STOG00003) issued by Shenzhen Huada Sanjian Qifa Technology Co., Ltd., and fix the carrier on the carrier;

[0118] (4) Add 200 μL of FFPE Decrosslinking Reagent (Shenzhen BGI Genomics Co., Ltd. Stereo-seq FFPE Transcriptome Reagent Kit (Catalog No.: 201SN114) reagent, 1000047464) to the corresponding wells of the carrier chip, rinse the chip and discard the liquid; add 400 μL of FFPE Decrosslinking Reagent again, seal the wells with the sealing membrane; place the carrier on the adapter, click edit and then click next step, skip the 37℃∞, 95℃ reaction for 30 min;

[0119] (5) After the reaction is complete, remove the carrier, carefully move it to the nearest platform, tear off the sealing film, aspirate the reaction liquid, remove the carrier, and place the chip carrier into a glass slide box (or 50mL centrifuge tube) containing pre-cooled methanol, and fix at -20℃ for 20min.

[0120] (6) After fixation is complete, transfer the slide box to the fume hood, take out the chip carrier, use lint-free paper to absorb excess methanol on the back of the carrier and around the chip, and ensure that there is no liquid residue in the gaps between the chips; place the carrier upright on the slide staining rack, and ventilate in the fume hood for 4 to 6 minutes to allow the methanol to evaporate fully.

[0121] 5. Permeation treatment (day 2)

[0122] (1) Prepare 2 mL of 0.01 N HCl in advance, and prepare 10× permeabilization reagent stock solution PR Enzyme (Shenzhen Huada Sanjian Qifa Technology Co., Ltd. Stereo-seqFFPE transcriptome reagent kit (catalog number: 201SN114) matching reagent, 1000028500); prepare 1× permeabilization reagent working solution (prepare fresh for use): dilute 20 μL of 10× permeabilization reagent stock solution with 0.01 N HCl to 200 μL and place on ice;

[0123] (2) Load the adapter into the PCR instrument in advance, set the permeabilization program as shown in Table 3 (program selection: Incubate), and click run;

[0124] Table 3

[0125]

[0126] (3) Replace the new gasket and clamp, and assemble the handheld carrier; drop 1× permeation reagent working solution into one corner of the chip, 200μL / well, to ensure that the liquid completely covers the chip, and seal the well with the sealing film; when the PCR instrument reaches 37℃∞, place the carrier on the adapter, click edit and then click next step to skip 37℃∞, and react at 37℃ for 30min.

[0127] (4) After the reaction is complete, remove the carrier, carefully move it to the nearest test plate, tear off the sealing film, and aspirate the 1× permeation working solution; wash once with 0.1×SSC (diluted from 20×SSC (AMBION, AM9770) with Nuclease-Free Water, containing 5% RI (Shenzhen BGI Sanjian Qifa Technology Co., Ltd. Stereo-seq FFPE transcriptome reagent kit (catalog number: 201SN114) reagent, 1000028499), 200μL / well.

[0128] 6. MIX reaction (day 3)

[0129] (1) While waiting for permeabilization, prepare the RT Mix (the matching reagent of the Stereo-seqFFPE transcriptome reagent kit (catalog number: 201SN114) from Shenzhen BGI Genomics Co., Ltd.) according to Table 4 and place it on ice;

[0130] Table 4

[0131]

[0132] (2) Load the adapter into the PCR instrument in advance, set the reverse transcription program as shown in Table 5 (program selection: Incubate), and click run;

[0133] Table 5

[0134]

[0135] (3) Add RT Mix dropwise from one corner of the chip, 200 μL / well, to ensure that the liquid completely covers the chip. Seal the well with the sealing film, place the carrier on the adapter, and react overnight at 42°C.

[0136] 7. cDNA Release and Recovery (Day 3)

[0137] (1) Remove the download from the adapter, discard the RT Mix, and rinse twice with NF water, 200μL / well, being careful not to poke the chip surface;

[0138] (2) Prepare the cDNA Release Mix (the matching reagent of the Stereo-seqFFPE transcriptome reagent kit (catalog number: 201SN114) from Shenzhen BGI Genomics Co., Ltd.) according to Table 6, and place it at room temperature;

[0139] Table 6

[0140]

[0141] (3) Load the adapter into the PCR instrument in advance, set the cDNA Release reaction program as shown in Table 7 (program selection: Incubate), and click Run;

[0142] Table 7

[0143]

[0144] (4) Add cDNA Release Mix, 400 μL / well, seal the well with sealing membrane, and then place it on the adapter and react at 55℃ for 5 h;

[0145] (5) After the reaction is complete, the liquid in the reaction well is completely recycled into a new 2.0 mL centrifuge tube; NF water is added to clean the reaction well chip (350 μL / well) and collected into the same 2.0 mL centrifuge tube.

[0146] 8. Purification and Amplification (Day 3)

[0147] (1) Remove the magnetic beads 30 minutes in advance and allow them to equilibrate to room temperature; if a white precipitate is observed in the cDNA recovery solution, it can be dissolved at 55°C, and after returning to room temperature, 1.0× magnetic bead cDNA purification can be performed:

[0148] 1) Mix the recovered liquid (approximately 750 μL) from the previous step with the magnetic beads that have been equilibrated at room temperature at a volume ratio of 1:1, shake to mix, and incubate at room temperature for 10 min;

[0149] 2) After instantaneous centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 3 minutes until the liquid is clear;

[0150] 3) After the liquid has clarified, carefully remove the supernatant with a pipette (if there is foam on the cap, remove the foam);

[0151] 4) Hold the centrifuge tube on the magnetic rack, add 1.5 mL of 80% ethanol (use freshly prepared 80% ethanol equilibrated to room temperature), rotate the centrifuge tube on the magnetic rack until all the magnetic beads are attracted to the tube wall on the side closest to the magnetic rack, then rotate the centrifuge tube again to allow the magnetic beads to re-adhere to the other side to rinse them. Let stand for 30 seconds, then carefully aspirate and discard the supernatant. The pipette tip should be used on the tube wall away from the magnetic rack; do not aspirate or stir the magnetic beads. (If there is foam on the tube cap, it is recommended to clean it with 80% ethanol).

[0152] 5) Repeat step 4) once;

[0153] 6) Keep the centrifuge tubes on the magnetic rack and air dry at room temperature for 5-8 minutes, until the surface of the magnetic beads is no longer reflective and cracked;

[0154] 7) Add 44 μL of NF water to dissolve, shake to mix, let stand at room temperature for 5 min, centrifuge briefly, let stand on a magnetic rack for 3-5 min, until the liquid becomes clear;

[0155] 8) Transfer the supernatant (~42 μL cDNA) to a new 0.2 mL PCR tube;

[0156] (2) If the recovered sample is less than 42 μL, make up the difference with NF water;

[0157] (3) Prepare PCR Mix (the reagents that come with the Stereo-seqFFPE transcriptome reagent kit (catalog number: 201SN114) from Shenzhen BGI Genomics Co., Ltd.) according to Table 8, totaling 100 μL;

[0158] Table 8

[0159]

[0160] (4) Centrifuge briefly and amplify according to the PCR procedure in Table 9;

[0161] Table 9

[0162]

[0163] (5) Prepare the Qubit dsDNA HS Assay Kit (Invitrogen, Cat. No. Q32854) according to Table 10;

[0164] Table 10

[0165]

[0166] (6) After shaking and mixing, take 1 μL of PCR product, use the Qubit dsDNA HS Kit to detect and record the concentration. The DNA concentration is usually higher than 10 ng / μL.

[0167] (7) The PCR product was purified using 1.0× magnetic beads;

[0168] 1) Mix the PCR product (100 μL) with the magnetic beads that have been equilibrated at room temperature at a ratio of 1:1, vortex to mix, and incubate at room temperature for 10 min;

[0169] 2) After brief centrifugation, place the PCR tube on a magnetic rack and let it stand for 3 minutes until the liquid becomes clear, then remove the supernatant.

[0170] 3) Hold the centrifuge tube on the magnetic rack and add 200 μL of 80% ethanol to rinse (freshly prepared and equilibrated to room temperature 80% ethanol). Rinse the magnetic beads by rotating the centrifuge tube on the magnetic rack. Let stand for 30 seconds, then carefully aspirate and discard the supernatant. The pipette tip should be used on the tube wall away from the magnetic rack; do not aspirate or stir the magnetic beads.

[0171] 4) Repeat step 3) once;

[0172] 5) Keep the centrifuge tubes on the magnetic rack, open the lid, and air dry at room temperature for 5-8 minutes until the surface of the magnetic beads is no longer reflective and cracked;

[0173] 6) Add 42 μL of TE buffer (pH 8.0) to reconstitute the sample, vortex to mix, and let stand at room temperature for 5 min. Centrifuge briefly, and let stand on a magnetic rack for 3–5 min. Once the liquid is clear, transfer the supernatant (~40 μL) to a new 1.5 mL centrifuge tube. (This step can be stopped; the sample can be stored at -20°C.)

[0174] (8) Take 1 μL of cDNA sample (after purification), use the Qubit dsDNA HS Kit to detect the concentration and record it;

[0175] 9. Library preparation and sequencing

[0176] For detailed instructions on subsequent library construction, please refer to the "Stereo-seq 16Barcode Library Construction Reagent V1.0 User Manual" (Document No.: STOG00007) published by Shenzhen BGI Genomics Co., Ltd.

[0177] 10. Visualization and analysis of offline data

[0178] ImageStudio is used to perform image quality control, stitching, calibration, and segmentation (with customizable cell segmentation models), and the results are automatically uploaded to the SAW cloud platform for Cellbin automated workflow analysis.

[0179] (1) Cell wall fluorescence staining images were obtained using Arabidopsis leaf FFPE samples. The images were automatically spliced ​​using StereoMap software based on track lines and registered with the expression matrix captured by Stereo-seq transcriptome technology.

[0180] (2) Using the open-source program Cellpose, import the cell wall fluorescence staining image of Arabidopsis leaf FFPE sample and perform cell division (parameter settings: model=cyto2, calibrate=80, keep the other parameters as default).

[0181] (3) Generate mask segmentation graphics based on cell outlines, finally obtain the Cellbin expression matrix, and use Stereopy to complete the single-cell resolution clustering results.

[0182] II. Results and Discussion

[0183] The results in the offline data visualization experiment (1) are as follows: Figure 3 As shown in the figure, it can be seen from the figure that, based on FFPE samples, a single image can simultaneously capture the fluorescent staining contours of plant cell walls and the chip surface track lines that are registered with the expression matrix.

[0184] The results in the offline data visualization experiment (2) are as follows: Figure 4 As shown in the figure, the plant cell recognition model, based on the cell wall fluorescence staining pattern, can automatically identify and accurately delineate cell regions. That is, the CellPose open-source program can quickly, efficiently, and accurately identify the outline of plant cells in FFPE samples and generate a mask file.

[0185] The results in the offline data visualization experiment (3) are as follows: Figure 5 As shown in the figure, single-cell expression matrix based on cell contour regions enables single-cell resolution analysis of plant spatial transcriptome data.

[0186] Comparative Example 1: Cellbin Expression Matrix Extracted from Fresh Arabidopsis Leaf Samples

[0187] Currently, plant samples are used for space transcriptomics experiments via cryopreservation and embedding of fresh tissue. First, SS (ssDNAAssayKit) fluorescent dye is used for staining, and the track lines on the surface of the Stereo-seq chip are scanned and identified under FITC excitation light. Second, FB28 (FluorescentBrightener28) fluorescent dye is used for staining, and the cell wall fluorescence signal is scanned and identified under DAPI excitation light to obtain cell outlines. The images from these two fluorescent staining and imaging processes need to be stitched together to indirectly achieve image registration (image stitching and registration mode is as follows). Figure 6The process, including cell segmentation, was followed to obtain the Cellbin-level expression matrix. This comparative example uses fresh Arabidopsis leaf samples. After OCT cryopreservation, the Cellbin expression matrix was extracted through two staining processes including fluorescence staining and imaging.

[0188] 1. Sample processing

[0189] Under laboratory conditions, fresh Arabidopsis leaf samples were rapidly cryopreserved using OCT (Sakura, USA) to prevent tissue necrosis and subsequent nucleic acid degradation.

[0190] 2. Frozen slicing

[0191] Turn on the cryostat in advance, set the chamber to pre-cool to -20℃ and the sample head to pre-cool to -15℃; replace the blade, and place the sample holder and forceps in the chamber for pre-cooling; take the tissue block out of the -80℃ freezer and place it in the cryostat to equilibrate for 3-5 minutes; take a small amount of OCT, attach the tissue block to it and freeze it quickly to the sample holder, insert the sample head with the cut side facing up, adjust the angle of the section, and perform sectioning; quickly remove excess OCT to expose the tissue, adjust the section thickness to 10μm, and rotate the rocker at a suitable and uniform speed to obtain the tissue section;

[0192] 3. Tissue mounting and fixation: Slowly flatten the tissue section on the slide stage with a soft brush, avoiding contact with the tissue area as much as possible; align the chip (Stereo-seq chip T-carrier) face down, at a height of more than 2 cm above the section, and quickly drop it vertically to allow the section to adhere to the chip surface. Quickly lift the chip and observe the adhesion; transfer the chip with the attached tissue to a metal plate preheated at 37℃, face up, and bake for 3 minutes (time must be strictly controlled); then quickly transfer it to precooled methanol at -20℃ for 30-40 minutes for fixation.

[0193] 4. Fluorescent staining

[0194] Remove the fixed tissue microarray from the methanol, blot dry the back and surrounding area of ​​the microarray with lint-free paper, and proceed with staining after the methanol has evaporated and the microarray surface has dried. Prepare ssDNA and FB28 fluorescent staining solutions according to Table 11. First, add ssDNA fluorescent staining solution (100 μL / microarray) to cover the tissue on the microarray, stain at room temperature in the dark for 5 min, slowly aspirate the liquid from the microarray surface, and wash the microarray once with 0.1×SSC (100 μL / microarray; containing 5% RI, RNase Inhibitor). Then, add FB28 fluorescent staining solution (100 μL / microarray; working final concentration 0.01%) to cover the tissue on the microarray, stain at room temperature in the dark for 3 min, slowly aspirate the liquid from the microarray surface, and wash the microarray once again with 0.1×SSC (100 μL / microarray; containing 5% RI), keeping the microarray surface moist.

[0195] Table 11

[0196]

[0197] 5. Scan and take a picture

[0198] Add 15 μL of Image Reagent (containing RI, 5%, a reagent that comes with the old version of the Stereo-seq transcriptome reagent kit (catalog number: 201ST114)) to ensure that there is no obvious liquid bulge on the chip surface, and slowly transfer it to the microscope scanning stage; refer to the epifluorescence (black and white) section of the "Motic Fluorescence Microscope Operation Guide", first select the FITC channel to scan the ssDNA staining results, and then keep the chip position unchanged and switch to the DAPI channel to scan the FB28 staining results.

[0199] 6. The procedures for tissue permeation, reverse transcription, removal, release and recovery, purification and amplification, library construction and sequencing shall be completed in accordance with the instruction manual of the "Stereo-seq Transcriptome Reagent Kit" (Shenzhen BGI Genomics Co., Ltd., item number: 201ST114);

[0200] 7. After obtaining the expression matrix, it can be used through... Figure 6 The process shown involves manually registering multiple layers based on the double-stained tissue outline using ImageJ, and performing cell region division using the open-source program Cellpose. Fluorescent staining images of cell walls from fresh plant samples and FFPE samples from the examples are imported respectively, and cell region division is performed with the same parameters (parameter settings: model=cyto2, calibrate=80, and other parameters are kept at default), thereby completing the Cellbin expression matrix extraction and analysis.

[0201] Comparison of effects:

[0202] Comparative Example: Fresh samples have a high water content, requiring rapid OCT cryopreservation after sampling. This can easily lead to ice crystal formation within cells, causing tissue section fragmentation and damage to cell wall fluorescence staining. An example of cell wall imaging from a frozen section of a fresh sample (Arabidopsis leaf) is provided below. Figure 7 As shown; frozen sections are relatively thick, typically 10–15 μm, making it difficult to distinguish the upper and lower cell wall sections, and they are prone to collapse after breakage, which is not conducive to plant cell segmentation. This invention, based on plant FFPE samples, captures plant cell wall and track line signals simultaneously in a single staining and imaging process. Taking the cell wall imaging of a paraffin-embedded section of an Arabidopsis leaf FFPE sample as an example, as shown... Figure 8 As shown, it provides accurate cell outlines for plants while automatically registering them based on track lines and expression matrices, eliminating the need for manual integration and greatly improving the efficiency and accuracy of plant cellbin information acquisition.

[0203] The results of automatic zoning of Arabidopsis leaves are as follows: Figure 9 As shown, A is the cell wall fluorescence staining image of the OCT frozen-embedded section of the fresh sample in the example, and B is the cell wall fluorescence staining image of the paraffin-embedded section in the comparative example. A comparison of the two images shows that the vascular and mesophyll cell division results of the FFPE sample are significantly better than those of the fresh sample.

[0204] Example 2: Staining of plant FFPE samples with FB28 fluorescent dye prepared using different formulations.

[0205] Control group: Using the method of Example 1, based on 5μm paraffin sections of Arabidopsis thaliana leaf FFPE tissue, FB28 fluorescent staining solution prepared using the old configuration [FB28 powder (MS4040, 916.98 g / mol), dissolved in anhydrous ethanol to prepare a 2 mg / mL stock solution, and finally diluted with Nuclease-Free Water (Ambion, Cat. No. AM9937) to a working concentration of 0.01%] was used for staining.

[0206] Experimental group: Using the method of Example 1, based on 5μm paraffin sections of Arabidopsis thaliana leaf FFPE tissue, the FB28 staining solution of the present invention [stock solution (SIGMA-ALDRICH, 910090-50mL), diluted 100 times with Nuclease Free Water to the working concentration] was used for staining and imaging.

[0207] The results are as follows Figure 10 As shown, it is evident that the old staining method left significant residue of fluorescent dye on the chip, resulting in strong background noise that interfered with cell segmentation.

[0208] Example 3: Staining of plant FFPE samples with mixed staining solution (FB28 & ssDNA)

[0209] Using the method in Example 1, 5μm sections of tomato callus FFPE paraffin-embedded blocks were stained with a mixed staining solution of FB28 and ssDNA (Table 12) to achieve multi-channel scanning imaging in a single staining process. This not only reduces the tissue staining time and the risk of slide detachment, but also enables the capture of multi-layer natively registered images of cell walls and nuclei in the same field of view, enriching image information and providing more dimensions for multimodal data analysis.

[0210] Table 12

[0211]

[0212] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. FB28 fluorescent dye solution, characterized in that, It includes the following components: FB28 1 volume portion; Nuclease-Free Water 99-999 parts by volume; The FB28 fluorescent dye solution does not contain organic solvents; preferably, the organic solvents include ethanol.

2. A mixed staining reagent, characterized in that, Includes the FB28 fluorescent staining solution and ssDNA staining solution as described in claim 1.

3. The mixed staining reagent as described in claim 2, characterized in that, The ssDNA staining solution includes ssDNA reagent; the ssDNA staining solution does not include RNase inhibitors.

4. The mixed staining reagent as described in claim 3, characterized in that, The ssDNA reagent is Qubit ssDNAHSReagent, and the volume ratio of FB28 to Qubit ssDNAHS Reagent is (1-2):(1-5).

5. A reagent kit, characterized in that, Includes the FB28 fluorescent dye solution as described in claim 1 or the mixed staining reagent as described in any one of claims 2 to 4.

6. The application of the FB28 fluorescent staining solution as described in claim 1, the mixed staining reagent as described in any one of claims 2 to 4, or the kit as described in claim 5 in tissue fluorescent staining; Preferably, the tissue comprises a plant FFPE sample.

7. The application as described in claim 6, characterized in that, Including any of the following: (I) Spatial transcriptome studies using plant FFPE samples; (II) Single staining imaging captures fluorescence signals of the cell wall or cell nucleus and / or track line fluorescence signals on a single image; (III) Automatic image registration of plant spatial transcriptome based on FFPE samples; (IV) Adapt to existing SAW automated analysis workflows to quickly extract plant Cellbin expression matrices; (V) Shorten the time to reduce RNA degradation and increase the amount of expression information captured; or (VI) Improve the accuracy of Cellbin zoning.

8. A staining method for plant spatial transcriptomics technology, characterized in that, The fixed tissue chip was stained with the FB28 fluorescent staining solution as described in claim 1, the mixed staining reagent as described in any one of claims 2 to 4, or the kit as described in claim 5. The staining is performed exactly once.

9. The staining method as described in claim 8, characterized in that, The final working concentration of the FB28 fluorescent dye solution is 0.01% (v / v); The staining time includes 2 to 5 minutes; Preferably, the staining method includes the following steps: Plant FFPE sample paraffin-embedded blocks were prepared, sectioned, spread, retrieved, baked, and dewaxed. Then, the tissue was covered with 100 μL / chip of FB28 fluorescent staining solution as described in claim 1, mixed staining reagent as described in any one of claims 2 to 4, or kit as described in claim 5. The staining was carried out at room temperature in the dark for 2 min. The chip was then washed twice with 0.1×SSC at 100 μL / chip.

10. A method for acquiring image information for plant spatial transcriptomics technology, characterized in that, Plant FFPE samples were processed, frozen, sectioned, fixed, dewaxed, stained with FB28 fluorescent staining solution as described in claim 1, mixed staining reagent as described in any one of claims 2 to 4, or kit as described in claim 5, mounted, scanned and photographed to obtain image information; The staining is performed exactly once; The image information includes fluorescence signals from the plant's cell walls or nuclei and / or track line fluorescence signals.

Images