Preparation method and application of space transcriptome of high-water-content sample

Abstract

The invention discloses a preparation method and application of a space transcriptome of a high-water-content sample. The preparation method comprises the following steps: sequentially carrying out stationary liquid fixation, gelatin replacement, embedding and slicing treatment on a high-water-content biological sample to obtain a sliced sample. According to the preparation method of the space transcriptome sample of the high-water-content organism, by optimizing the tissue treatment, embedding and slicing processes, the slice form integrity and RNA integrity are ensured, the requirement for space transcriptome sequencing is met, and the preparation method has wide applicability and is suitable for popularization and application. And reliable technical support is provided for application of space transcriptome sequencing in high-water-content samples.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for preparing spatial transcriptome of high-water-content samples and its application. Background Art

[0002] Spatial Transcriptomics (ST) is an emerging biotechnology aimed at combining the spatial location information of cells in tissues and gene expression information to analyze cell heterogeneity in a spatial context. Before spatial transcriptome sequencing experiments, the morphological integrity and RNA integrity of tissue sections are crucial, directly related to the accuracy of library construction, the reliability of sequencing results, and the repeatability of gene expression data. The RNA Integrity Number (RIN or RQN) is a key indicator for measuring the quality of sample RNA. A higher RNA Integrity Number (usually required to be ≥7) indicates a lower degree of RNA degradation, which can better bind to spatial transcriptome probes, thereby obtaining high-quality sequencing data.

[0003] Jellyfish, as a lower marine organism, belongs to the phylum Cnidaria. The most common moon jellyfish is widely distributed in the Atlantic, Pacific, Indian Oceans, etc., from 70° north latitude to 40° south latitude, and has important research value in the fields of genetics, ecology, and evolutionary biology. However, moon jellyfish tissues have characteristics such as high polysaccharide content, easy RNA degradation, and extremely high water content (about 95%-98%), which pose great challenges to their spatial transcriptomics research. Whether tissue sections with complete slice morphology and high RNA integrity can be obtained is a key technical issue in jellyfish spatial transcriptomics research. However, there has been no reported effective tissue sectioning scheme for high-water-content biological samples such as moon jellyfish while ensuring RNA integrity in the current field. Summary of the Invention

[0004] The first object of the present invention is to provide a method for preparing a spatial transcriptome sample of a high-water-content organism.

[0005] The second object of the present invention is to provide the application of the above method for preparing a spatial transcriptome sample of a high-water-content organism in spatial transcriptome sequencing.

[0006] The third object of the present invention is to provide a method for spatial transcriptome sequencing of a high-water-content organism.

[0007] In order to achieve the above objects, the technical solutions adopted by the present invention are as follows: In the first aspect of the present invention, a method for preparing a spatial transcriptome sample of a high-water-content organism is provided. The preparation method includes the following steps: sequentially fixing, gelatin replacement, embedding, and sectioning a high-water-content biological sample to obtain a section sample.

[0008] Preferably, the high water content biological sample refers to a biological sample with a water content of ≥70%, for example: biological samples with water contents of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%.

[0009] Preferably, the high water content biological sample includes at least one of jellyfish, sponge, comb jelly, seaweed, hydra, sea squirt larvae, bumblebee pupae samples.

[0010] Preferably, the sample is a section sample, for example, a tissue section sample.

[0011] Preferably, the jellyfish includes Aurelia aurita, Turritopsis dohrnii, Cyanea capillata, Margelopsis polypoides, Rhizostoma pulmo.

[0012] Preferably, the high water content biological sample includes the umbrella tissue sample of jellyfish, the strobila sample in the early stage of jellyfish development, and the ephyra sample in the early stage of jellyfish development.

[0013] Preferably, the fixing solution includes formaldehyde or paraformaldehyde.

[0014] Preferably, the mass fraction of the paraformaldehyde is 3%-5%, for example: it can be 3%, 4%, 5%.

[0015] Preferably, the fixing time of the fixing solution is 0.5h - 2h, for example: it can be 0.5h, 1h, 1.5h, 2h.

[0016] Preferably, after the fixation with the fixing solution and before the replacement with gelatin, there is also a step of washing the high water content biological sample fixed by the fixing solution, and the step includes: soaking the high water content biological sample fixed by the fixing solution in a buffer solution.

[0017] Preferably, the buffer solution includes PBS buffer solution.

[0018] Preferably, the soaking time is 4 - 12min, for example: it can be 4min, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min.

[0019] Preferably, the soaking time is 5 - 10min, for example: it can be 5min, 6min, 7min, 8min, 9min, 10min.

[0020] Preferably, the number of washing times is 1 - 5 times, such as 1 time, 2 times, 3 times, 4 times, 5 times.

[0021] Preferably, the number of washing times is 1 - 3 times.

[0022] Preferably, the gelatin replacement includes: soaking the high-water-content biological sample fixed with the fixing solution in a gelatin solution with a concentration of 0.08 - 0.15 g / mL.

[0023] Preferably, the concentration of the gelatin solution is 0.08 - 0.12 g / mL; for example, it can be 0.08 g / mL, 0.09 g / mL, 0.1 g / mL, 0.11 g / mL, 0.12 g / mL.

[0024] Preferably, the soaking time is 0.5 - 4 h, for example, it can be 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h.

[0025] Furthermore, the soaking time is 0.5 - 2 h. At this time, the jellyfish sample can not only ensure the spatial morphology but also ensure the nucleic acid quality.

[0026] Even further, the soaking time is 0.5 - 1.5 h.

[0027] Preferably, the soaking temperature is 0 - 6 °C, for example, it can be 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C.

[0028] Preferably, the embedding includes cryo-embedding.

[0029] Preferably, the embedding agent used for cryo-embedding includes OCT embedding agent.

[0030] Preferably, the sectioning is performed using a cryostat to cut the cryo-embedded tissue into section samples with a thickness of 8 - 12 μm (e.g., 10 μm).

[0031] Preferably, after the sectioning, it further includes the step of performing RNA quality inspection on the section samples. By introducing RNA quality inspection in the present invention and detecting through the RNA integrity number (RIN value), it is ensured that the RNA quality of the samples meets the requirements for spatial transcriptome detection.

[0032] Preferably, the method further includes the steps of performing H&E staining and slice permeabilization gradient fluorescence imaging test detection on the section samples.

[0033] Preferably, before the embedding, it further includes a step of quickly freezing the high-water-content biological sample after gelatin replacement; the quick freezing includes placing the fixed high-water-content biological sample at -76°C to -82°C and freezing for 25 - 35 minutes. Through the gelatin solution soaking and quick freezing techniques, the tissue shrinkage and fragmentation phenomena of high-water-content samples during embedding and sectioning are effectively reduced, ensuring the integrity of the section morphology, obtaining high-quality complete sections, and ensuring that the spatial distribution information of cells in the tissue can be accurately captured. In specific applications, samples that are very small and have a relatively stable sample morphology, such as the strobila and ephyra in the early stages of the Aurelia life cycle, do not require the quick freezing method to maintain the sample morphology unchanged due to their very small size, and the quick freezing step can be omitted in specific experiments.

[0034] In the second aspect of the present invention, it lies in providing the application of the spatial transcriptome sample of high-water-content organisms prepared by the preparation method of the first aspect of the present invention in spatial transcriptome detection.

[0035] Preferably, before spatial transcriptome detection, it further includes a step of performing RNA quality inspection on the spatial transcriptome sample.

[0036] Preferably, the sample is a section sample.

[0037] Preferably, before spatial transcriptome detection, it further includes steps of performing H&E staining and section permeabilization gradient fluorescence imaging test detection on the spatial transcriptome sample.

[0038] Preferably, before spatial transcriptome detection and after RNA quality inspection, it further includes steps of performing H&E staining and section permeabilization gradient fluorescence imaging test detection on the sample.

[0039] In the third aspect of the present invention, it lies in providing a method for spatial transcriptome detection of high-water-content organisms, and the method includes the following steps: performing spatial transcriptome detection on the sample prepared by the preparation method of the first aspect of the present invention.

[0040] In this application, the spatial transcriptome detection refers to detecting the spatial position of transcripts (i.e., RNA), and common technical means in the art include: in-situ spatial transcriptome detection, including FISH, in-situ decoding techniques such as the Xenium platform of 10xGenomics, etc.; spatial transcriptome sequencing techniques, such as the spatial transcriptome sequencing platform of BGI Stomics, the Visium or Visium HD platform of 10xGenomics. The samples prepared by the present invention can be used for high-throughput spatial transcriptome detection. For example, using spatial transcriptome sequencing techniques, transcripts of 9303 genes can be detected.

[0041] Preferably, the spatial transcriptome sequencing is performed using the Stereo-Seq sequencing technology.

[0042] The beneficial effects of the present invention are as follows: The method for preparing a spatial transcriptome sample of a high-water-content organism provided by the present invention optimizes the tissue treatment, embedding, and sectioning processes to ensure the morphological integrity of the sections and the integrity of RNA, so as to meet the requirements of spatial transcriptome sequencing. Among them, the gelatin involved is a protein obtained by partial hydrolysis of collagen, and its molecules contain a large number of polar groups such as hydroxyl groups and carboxyl groups. At low temperatures, the hydrogen bonds and other interactions between these groups are enhanced, causing the gelatin molecules to form a tighter network structure, thereby forming a gel. After soaking treatment with gelatin, part of the water in the jellyfish can be replaced by gelatin. After the tissue is frozen, the solidified gelatin can provide the support required for sectioning, ensuring that the morphological integrity of the sample is not damaged during sectioning. Through the soaking of the gelatin solution and the quick-freezing technology, the tissue shrinkage and fragmentation phenomena of high-water-content samples during embedding and sectioning are effectively reduced, ensuring the morphological integrity of the sections, obtaining high-quality complete sections, and ensuring that the spatial distribution information of cells in the tissue can be accurately captured.

[0043] At the same time, the preparation method of the present invention also optimizes the steps of fixation, washing, and gelatin soaking, especially controls the operation details and adjusts the treatment duration, minimizing the degradation of section RNA to ensure that the RNA integrity number (RIN or RQN) ≥ 7, meeting the requirements of spatial transcriptome sequencing, ensuring that the sample RNA quality meets the spatial transcriptome sequencing standard, and improving the reliability and repeatability of experimental data.

[0044] The method of the present invention is not only applicable to jellyfish, but also can be extended to other high-water-content species or tissue samples (such as hydra, ascidian larvae, the hind legs of bumblebee pupae, etc.), with wide applicability. Through detailed operation procedures and parameter settings, this method ensures the repeatability and scalability of the experiment, providing reliable technical support for the application of spatial transcriptome sequencing in high-water-content samples. Description of the Drawings

[0045] The following further illustrates the present invention with reference to the drawings and embodiments, where: Figure 1 is the experimental flow chart in Embodiment 1 of the present invention; Figure 2 is the comparison diagram of the section morphology between other methods in the test example of the present invention and the method in Embodiment 1 of the present invention. Among them, A is the result diagram of the paraffin embedding section of the traditional method, showing serious tissue shrinkage; B is the detection result diagram of the section of the conventional frozen embedding, showing that the sample is easily broken; C is the result diagram of the agar replacement and infiltration wax embedding section, showing that it is difficult to section the soft tissue; D is the result diagram of the section embedded by the method in Embodiment 1; Figure 3 This is the microscopic image of continuous patch sections and H&E staining of jellyfish in the test example of the present invention. Here, each grid of the scale represents 1 mm; Figure 4 This is the original sample image of jellyfish 5 in the test example of the present invention; Figure 5 This is the quality inspection result image of jellyfish 5 in the test example of the present invention. Here, RNA Area is 84.1, RNA concentration is 91 ng / μL, rRNA Rstio (28S / 18S) is 0.9, RIN is 8.2, and Corr. Area 1 is 68.9; Figure 6 This is the original sample image of jellyfish 6 in the test example of the present invention; Figure 7 This is the quality inspection result image of jellyfish 6 in the test example of the present invention. Here, RNA Area is 135.8, RNA concentration is 146 ng / μL, rRNA Rstio (28S / 18S) is 0.8, RIN is 7.7, and Corr. Area 1 is 121.4; Figure 8 This is the original sample image of jellyfish 7 in the test example of the present invention; Figure 9 This is the quality inspection result image of jellyfish 7 in the test example of the present invention. Here, RNA Area is 59.5, RNA concentration is 64 ng / μL, rRNA Rstio (28S / 18S) is 1.0, RIN is 8.2, and Corr. Area 1 is 43.6; Figure 10 This is the test result image of permeabilization gradient fluorescence imaging of jellyfish sections in the test example of the present invention. Here, A is the sample morphology image of jellyfish 7 after embedding treatment, B is the H&E staining result image of jellyfish 7, C is the detection result image of permeabilization gradient fluorescence imaging patch of jellyfish 7, and D is the high-definition microscopic image of the detection result image of permeabilization gradient fluorescence imaging patch of jellyfish 7; Figure 11 This is the sample information image of "one divided into eight" of jellyfish 8 in the test example of the present invention. Here, the scale bar is 1 cm; Figure 12 This is the sample morphology image and H&E staining result image of jellyfish 8 after embedding treatment in the test example of the present invention. Here, A is the sample morphology image of jellyfish 8 after embedding treatment, B is the H&E staining result image of jellyfish 8, and the scale bar is 1 cm; Figure 13 This is the quality inspection result image of jellyfish 8 in the test example of the present invention; Figure 14This is the quality inspection peak result graph of jellyfish 8 in the test example of the present invention. Among them, A is the detection result graph of Ac8-1; B is the detection result graph of Ac8-2; C is the detection result graph of Ac8-3; D is the detection result graph of Ac8-4; E is the detection result graph of Ac8-5; F is the detection result graph of Ac8-6; G is the detection result graph of Ac8-7; H is the detection result graph of Ac8-8; Figure 15 This is the spatial transcriptome sequencing detection result graph in the test example of the present invention. Among them, A is the original morphology graph of jellyfish Ac8-3; B is the sample morphology graph and H&E staining result graph of jellyfish Ac8-3 after embedding treatment, and C is the quality inspection RIN detection result graph of jellyfish 8-3; Figure 16 This is the spatial group chip patch and H&E staining result graph of jellyfish Ac8-3 in the test example of the present invention; among them, A is the spatial group chip patch graph of jellyfish Ac8-3; B is the H&E staining result graph of jellyfish Ac8-3; Figure 17 This is the ssDNA fluorescence staining graph of jellyfish Ac8-3 in the test example of the present invention; Figure 18 This is the DNA detection result graph of jellyfish Ac8-3 in the test example of the present invention; Figure 19 This is the spatial transcriptome sequencing saturation statistical graph of jellyfish Ac8-3 in the test example of the present invention; Figure 20 This is the spatial gene expression distribution graph of jellyfish Ac8-3 in the test example of the present invention; Figure 21 This is the spatial Spots morphology distribution and UMAP clustering analysis graph of jellyfish Ac8-3 in the test example of the present invention. Among them, A is the spatial Spots morphology distribution graph; B is the UMAP clustering analysis graph; Figure 22 This is the process of preparing the transverse fission body and ephyra samples in the early stage of the Aurelia aurita development cycle in the test example of the present invention using the method in Example 2, as well as the microscopic graph of patch section H&E staining. Among them, A is the morphology graph of the transverse fission body and ephyra samples of Aurelia aurita, B is the morphology graph of the sample during the PFA fixation process, C is the morphology graph of the sample during the PBS washing process, and D is the morphology graph of the sample during the soaking and water replacement process with gelatin solution; Figure 23 This is the actual photo samples of the transverse fission body (early stage), transverse fission body (late stage), and ephyra of Aurelia aurita in the test example of the present invention and the corresponding microscopic graph of section H&E staining. Detailed implementation method

[0046] The concept of the present invention and the technical effects produced will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0047] For the experimental methods without specific conditions indicated in the following embodiments, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in this embodiment, unless otherwise specified, are reagents and materials obtained through commercial channels.

[0048] Experimental materials: Tissue fixative (containing 4% paraformaldehyde by mass fraction, brand: meilunbio, catalog number: MA0192-2); Nuclease-free water (Nuclease-free Water, brand: TRAN, catalog number: GI101-02); 20× buffer (20× PBS Buffer, brand: Sangon Biotech, product number: B548117-0500); Gelatin powder (Gelatin from cold water fish skin, brand: SIGMA, catalog number: G7041-100G).

[0049] Example 1 A method for preparing a spatial transcriptome sample of a high-water-content organism This example provides a method for preparing a spatial transcriptome sample of a high-water-content organism (taking Aurelia aurita as an example). The flow chart is as Figure 1 shown, and the specific steps are as follows: 1. Preparation of the spatio-temporal transcriptome sample of Aurelia aurita (1) Excise the oral arms of Aurelia aurita Select a small sample of Aurelia aurita with a complete and clean body and an umbrella diameter of about 2 cm (stop feeding 24 hours in advance to ensure the sample quality). Invert it (with the umbrella top down) and flatten it in a culture dish, and add seawater just enough to cover the jellyfish. Use a sterile tool to excise the oral arms of Aurelia aurita, and retain the umbrella tissue.

[0050] (2) Fix the jellyfish sample after excising the oral arms Immerse the jellyfish sample after excising the oral arms in a fixative solution (4% paraformaldehyde (PFA)). The volume ratio of the fixative to the sample is 15:1, and fix it on ice for 1 h (0.5 h - 2 h is acceptable). The fixation step aims to maintain the tissue morphology and prevent RNA degradation.

[0051] (3)Washing Transfer the fixed jellyfish sample to a Petri dish, invert and flatten it, and add 1×PBS buffer to soak for 5 min for washing (room temperature, the liquid volume should be enough to cover the sample) (if the sample is large, washing can be repeated 3 times, 10 min each time, to thoroughly remove the residual fixative).

[0052] (4)Soaking in gelatin solution Transfer the washed jellyfish sample to a gelatin solution with a concentration of 0.1 g / mL (10% gelatin solution) (preparation of gelatin solution: preheat the water bath to 65 - 70 °C. Weigh 1 g of gelatin powder, add 10 mL of deionized nuclease-free water, seal and heat in a water bath for 6 hours, and mix well to obtain a 10% gelatin solution (m:v = 1:10)) and soak it.

[0053] The detailed steps of soaking are as follows: Make an aluminum foil box according to the size of the jellyfish, invert the sample into it and flatten it; pour in the 10% gelatin solution cooled to room temperature, and place it in a 4 °C refrigerator to soak for 2 h (0.5 h - 4 h is acceptable).

[0054] (5)Quick-freezing the sample After soaking in the gelatin solution, adjust the aluminum foil box containing the sample to a horizontal and flattened state, slowly suck out all the gelatin solution with a pipette, and keep the sample flat during the process (after sucking out the solution, the edges of the aluminum foil box can be flattened to facilitate subsequent removal), and then transfer the sample to a -80 °C refrigerator to quick-freeze for 30 min to avoid ice crystal formation.

[0055] (6)Embedding Pour 1 / 3 of the OCT embedding medium into a transparent embedding mold in advance and place it horizontally on dry ice. When the embedding medium gradually solidifies, carefully take out the quick-frozen sample from the aluminum foil box (the operation is carried out in a -25 °C environment), quickly transfer it to the transparent embedding mold containing the OCT embedding medium, adjust the position of the sample to ensure it is horizontally placed, then continue to fill it with OCT embedding medium, and mark the position of the sample on the embedding box, and place the embedding box on dry ice or in a -80 °C refrigerator environment to freeze into a block.

[0056] (7)Frozen sectioning Adjust the temperature of the cryostat to -25 °C, and use the cryostat to cut the embedded tissue into 10 -μm thick sections. The embedded block after sectioning needs to be immediately transferred to a -80 °C refrigerator for storage for subsequent experiments.

[0057] (8)RNA quality control Take an appropriate amount of tissue samples continuously from the frozen sections; subsequently, use the RNeasy FFPE Kit to extract and detect RNA. Among them, skip the dewaxing step in the kit and directly perform RNA extraction from the lysis operation according to the instructions; use a bioanalyzer to detect the integrity of RNA and obtain the RNA Integrity Number (RIN) value to evaluate the RNA quality.

[0058] 2. Sample analysis (1)H&E staining The specific operation steps of H&E staining are carried out according to the standard frozen section H&E staining process, which are briefly described as follows: After the frozen sections are naturally thawed at room temperature, successively carry out methanol fixation, wash methanol with deionized water, hematoxylin staining (staining time is 1 - 2 min), running water rinsing, eosin staining (staining time is 1 - 2 min), dehydration, clearing and mounting.

[0059] (2)Slice permeabilization gradient fluorescence imaging test Take 4 consecutive slices of 1 cm × 1 cm and stick them onto the permeabilization chip in sequence, and observe the tissue morphology through gradient fluorescence imaging.

[0060] Example 2 Preparation method of spatial transcriptome samples for high - water - content samples in the early stage of the Aurelia aurita development cycle This example provides a method for preparing spatial transcriptome samples of high - water - content samples using the strobila and ephyra samples in the early stage of the Aurelia aurita development cycle. The preparation flow chart is similar to the one in Example 1 (as Figure 1 shown), and the specific steps are as follows: 1. Preparation of spatial transcriptome samples of Aurelia aurita strobila and ephyra (1)Fix the strobila and ephyra samples Immerse the Aurelia aurita strobila and ephyra samples in a fixative solution (4% paraformaldehyde (PFA)) for 0.5 h. The fixation step aims to maintain the tissue morphology and prevent RNA degradation.

[0061] (2)Washing Transfer the fixed Aurelia aurita strobila and ephyra samples to a culture dish, and add 1×PBS buffer to soak for 5 min for washing.

[0062] (3)Soak in gelatin solution Transfer the washed Aurelia aurita strobila and ephyra samples to a gelatin solution with a concentration of 0.1 g / mL (10% gelatin solution), and place them in a 4℃ refrigerator to soak for 0.5 h.

[0063] (4)Embedding Pour 1 / 3 of the OCT embedding medium into a transparent embedding mold in advance and place it horizontally on dry ice. When the embedding medium gradually solidifies, quickly transfer the sample into the transparent embedding mold filled with OCT embedding medium, adjust the position of the sample to ensure it is placed horizontally, then continue to fill it with OCT embedding medium, mark the position of the sample on the embedding cassette, and place the embedding cassette on dry ice or in a -80°C refrigerator environment to freeze it into a block.

[0064] (5)Frozen section Adjust the temperature of the cryostat to -25°C, and use the cryostat to cut the embedded tissue into 10-μm-thick sections. The embedded block after sectioning needs to be immediately transferred to a -80°C refrigerator for storage for subsequent experiments.

[0065] 2. Sample analysis Perform sample analysis using H&E staining: The specific operation steps of H&E staining are carried out according to the standard H&E staining procedure for frozen sections, which are briefly described as follows: After the frozen sections are naturally thawed at room temperature, they are successively subjected to methanol fixation, deionized water washing of methanol, hematoxylin staining (staining time is 1-2 min), running water rinsing, eosin staining (staining time is 1-2 min), dehydration, clearing, and mounting.

[0066] Test example 1. Embedding and frozen section processing of the umbrella tissue of Aurelia aurita Taking Aurelia aurita 1, Aurelia aurita 2, Aurelia aurita 3, and Aurelia aurita 4 as research objects, they are respectively processed by traditional paraffin embedding, conventional frozen embedding, agar replacement and infiltration wax embedding, and the preparation method of the Aurelia aurita sample in Example 1.

[0067] The traditional paraffin embedding treatment method is as follows: a) Fixation: Fix the tissue sample with 4% paraformaldehyde at 4°C overnight to maintain the tissue morphology.

[0068] b) Washing: Wash the sample with 1×PBS buffer for 30 minutes to remove the residual fixative.

[0069] c) Gradient dehydration: Immerse the sample in ethanol solutions of different concentrations (30%, 50%, 70%, 85%, 95%) in turn, 1 hour for each step, and slowly shake on a shaker to gradually remove the water in the tissue.

[0070] d) Clearing treatment: Transfer the sample to n-butanol and soak it 3 times, 1 hour each time, to make the tissue transparent.

[0071] e) Infiltration with wax and embedding: Place the sample in paraffin at 65°C for 3 hours, change the paraffin every 1 hour, and finally perform paraffin embedding.

[0072] f) Sectioning: Use a microtome to cut the embedded tissue into thin slices 10 μm thick (any thickness between 10 - 20 μm is acceptable).

[0073] The conventional cryo - embedding treatment method is as follows: a) Fixation: Fix the tissue sample with 4% paraformaldehyde on ice for 2 hours to maintain the tissue morphology.

[0074] b) Washing: Wash the sample with 1×PBS buffer for 5 minutes to remove the residual fixative.

[0075] c) Sucrose sedimentation: Immerse the sample in 30% sucrose solution for 2 hours to enhance the tissue's frost resistance.

[0076] d) Embedding: Use OCT embedding medium to embed the tissue.

[0077] e) Sectioning: Cut the embedded tissue into thin slices 10 μm thick (any thickness between 10 - 20 μm is acceptable) on a cryostat microtome.

[0078] The agar replacement and wax - impregnation embedding treatment is as follows: a) Gradient dehydration: Immerse the sample in 80%, 85%, 90%, and 95% ethanol successively, with each step lasting 5 minutes.

[0079] b) Agarose replacement: Immerse the dehydrated tissue sample in 3% agarose solution, embed it into a block, and place it at 4°C to cool and solidify, replacing the traditional agarose protective layer.

[0080] c) Clearing treatment: Transfer the sample to xylene and soak it 2 - 3 times, 5 minutes each time, to make the tissue transparent and replace the ethanol.

[0081] d) Wax - impregnation and embedding: Place the sample in paraffin at 65°C for 3 hours, changing the paraffin every 1 hour, and finally perform paraffin embedding.

[0082] e) Sectioning: Use a microtome to cut the embedded tissue into thin slices 10 μm thick (any thickness between 10 - 20 μm is acceptable).

[0083] The comparison of sectioning effects is as Figure 2 shown. It can be seen from the figure that during the traditional paraffin - embedding process (as shown in Figure 2 Figure A in it), the high - temperature environment during the wax - impregnation operation can cause tissue shrinkage and damage to the morphological structure. This shrinkage not only destroys the integrity of the sample but also leads to severe deformation of the tissue structure, resulting in the distortion or even loss of spatial position information. For spatial transcriptome sequencing, the morphological integrity of the sample and the accuracy of spatial position information are crucial, and the paraffin - embedding method is difficult to meet these requirements when dealing with samples with high water content; the conventional cryo - embedding (such as Figure 2Figure B (as shown in Figure B) is prone to sample fragmentation during the sectioning operation, and this fragmentation cannot meet the strict requirements of spatial transcriptome sequencing for sample integrity. In addition, the formation of ice crystals will also damage the cell structure, further affecting the integrity of RNA, which is not conducive to spatial transcriptome sequencing. Therefore, it is difficult to obtain complete and high-quality sections; using agar treatment (such as Figure 2 The jellyfish shown in Figure C) although its morphology did not change significantly after overnight wax impregnation, but during the subsequent paraffin embedding process, the tissue showed a softer state, resulting in incomplete and continuous sections being unable to be obtained during sectioning. This is mainly because agarose failed to effectively support the tissue structure after being soaked in high-temperature paraffin, and the jellyfish tissue itself has a high water content and a soft texture, making the tissue prone to local deformation and softening during the wax penetration and solidification process. The softened tissue is prone to breakage and shrinkage when cut by the blade of the microtome, and complete sections cannot be formed, affecting subsequent spatial transcriptome analysis. And the method of the present invention, on the basis of conventional cryoembedding, innovatively adopts the gelatin solution soaking optimization technique (such as Figure 2 Figure D) effectively guarantees the morphological integrity of the sections and the clarity of the tissue structure.

[0084] 2. Staining of serial complete sections of the umbrella tissue of Aurelia aurita Taking Aurelia aurita 4 in the embedding and cryosection treatment of the umbrella tissue of Aurelia aurita as a sample, serial sections were taken from the umbrella top (1 section) to the endoderm (4 sections) and then to the gonad (3 sections), the section thickness was 10 μm, and section mounting was carried out every 300 μm.

[0085] After the sections were stained with H&E, as Figure 3 shown, it can be clearly observed that the microscopic structure of the sections obtained by the method of the present invention is complete and the staining effect is clear.

[0086] 3. Detection of RNA integrity of the umbrella tissue of Aurelia aurita Taking Aurelia aurita 5, 6, and 7 and processing them according to the preparation method of the spatial transcriptome sample of Aurelia aurita in Example 1. Since there are differences in the sizes of Aurelia aurita, corresponding different duration combinations were set for PFA soaking and gelatin solution soaking.

[0087] Aurelia aurita 5 has a diameter of about 1 cm, corresponding to 0.5 h of PFA treatment + 5 min of PBS washing + 0.5 h of gelatin solution soaking; Aurelia aurita 6 has a diameter of about 1.8 cm, corresponding to 1.5 h of PFA treatment + 5 min of PBS washing + 1 h of gelatin solution soaking; Aurelia aurita 7 has a diameter of about 2 cm, corresponding to 1.5 h of PFA treatment + 5 min of PBS washing + 4 h of gelatin solution soaking.

[0088] The cryosections of the embedded Aurelia aurita 5, 6, and 7 were prepared, and the integrity of the sections was observed. At the same time, the RNA integrity of the sections was detected separately.

[0089] The results are as Figures 4 - 9 shown. As can be seen from the figure, complete sections can be obtained when the jellyfish embedded by different soaking duration combinations are sectioned. The RNA integrity of the sections was detected separately, and considerable RIN values were obtained. Among them, the RIN value of jellyfish 5 was 8.2, the RIN value of jellyfish 6 was 7.7, and the RIN value of jellyfish 7 was 8.2. The results show that the RNA integrity number (RIN value) of the samples processed by the method of the present invention is high, and the RIN values are all ≥7, meeting the requirements of spatial transcriptome sequencing, ensuring that the RNA quality of the samples meets the spatial transcriptome sequencing standards, and improving the reliability and repeatability of experimental data.

[0090] 4. Permeabilization gradient fluorescence imaging test of the sections of the umbrella tissue of Aurelia aurita The sample of Aurelia aurita 7 (with an RIN value of 8.20 during RNA integrity detection and complete section morphology) was selected for the permeabilization gradient fluorescence imaging test. At the same time, the cryosections of the sample of Aurelia aurita 7 were subjected to H&E staining.

[0091] The results are as Figure 10 shown. Four consecutive 1 cm×1 cm sections of Aurelia aurita 7 were successively attached to the permeabilization chip (as shown in Figure C of Figure 10 ). Through gradient fluorescence imaging observation, the tissue morphology of the sample of jellyfish 7 was found to be complete and clear at 18 minutes. Figure 10 The high-definition microscopic image in Figure D of Figure 10 showed the same texture characteristics as the H&E staining results shown in Figure B of

[0092] 5. Soaking duration gradient treatment and RNA integrity detection of the sections of the umbrella tissue of Aurelia aurita The sample of Aurelia aurita 8 (with a diameter of about 4 cm) was selected, and its umbrella tissue was evenly divided into eight parts (as shown in Figure 11 ). It was processed according to the preparation method of the spatial and temporal transcript sample of Aurelia aurita in Example 1. To explore the optimal treatment duration for protecting the RNA integrity of the sections, gradient combination treatments were performed on the soaking durations of the eight parts of the jellyfish umbrella tissue in PFA fixation, PBS washing, and gelatin solution soaking (the detailed durations are shown in Table 1). At the same time, the cryosections of each sample were subjected to H&E staining (as shown in Figure 12 ).

[0093] Table 1

[0094] The results are as Figures 13 - 14As shown, it can be seen from the figure that the jellyfish Ac8-1 in the control group was not soaked in the gelatin solution and complete sections could not be obtained, directly resulting in poor RNA quality inspection results (RQN was 1); in the experimental group, from jellyfish Ac8-2 to jellyfish Ac8-8, the PFA fixation and gelatin solution soaking time were increased in turn, and the morphological integrity of the sections could be guaranteed. However, there were different results in the RNA integrity detection. Among them, the RNA integrity of jellyfish Ac8-2, Ac8-3, Ac8-4, Ac8-5, and Ac8-6 was very good (RQN was much greater than 7), while for jellyfish Ac8-7 and Ac8-8, the RNA was degraded to a large extent probably due to too long soaking time (RQN was less than 7). Based on this result, the present invention has a reference range for the processing time to obtain RNA-complete jellyfish tissue sections, and the optimal is 4% PFA soaking time + gelatin soaking time < 3.5 h.

[0095] 6. Application of Spatial Transcriptome Sequencing in the Umbrella Tissue Sections of Aurelia aurita Considering that the original sample was too large, only 1 / 8 of the umbrella tissue, namely Aurelia aurita Ac 8-3 in the gradient treatment of the soaking time of the umbrella tissue sections of Aurelia aurita and the RNA integrity detection (the original sample of Aurelia aurita Ac 8-3, the sections obtained by processing Aurelia aurita Ac 8-3 according to the preparation method of the spatio-temporal transcript sample of Aurelia aurita in Example 1, and the results of H&E staining of the sections are as shown in Figures A and B in Figure 15 ), was selected for spatial transcriptome sequencing. The sequencing method used the spatio-temporal omics technology Stereo-Seq sequencing of BGI. Based on the experiment in Example 1 of the present invention, the spatial transcriptome sequencing of Aurelia aurita was completed in the order of "cryo-embedding - RIN quality inspection - spatial group chip patching - H&E staining - ssDNA fluorescence staining - DNA detection - sequencing", and at the same time, spatial transcriptomics analysis was performed on the sequencing data (that is, the data obtained by Stereo-seq sequencing was processed using the analysis process of the supporting SAW-ST-V8 spatio-temporal bioinformatics software suite).

[0096] The results are as shown in Figures 15 - 21 It can be seen from Figure C in Figure 15 that the quality inspection results show that the RIN value is 8.99; the cDNA fragment range of the chip is between 476 and 1500 bp, and the main peak is 1183 bp, and there is no abnormality in library construction (as shown in Figure 18 ); finally, about 4.3G Reads were obtained in the spatial transcriptome sequencing. After alignment with the reference genome, there were 9303 genes, and the sequencing report showed that the saturation tended to 95.5% (as shown in Figure 19 ).

[0097] In spatial transcriptomics analysis, the spatial gene expression distribution is mainly in the marginal part of the umbrella cap, which is also consistent with expectations (as Figure 20 shown), and the morphological distribution of spatial Spots and UMAP clustering are also relatively clear (as Figure 21 shown). The results show that the preliminary data quality of the spatio-temporal transcriptome samples of Aurelia aurita prepared in the embodiments of the present invention is available for subsequent data analysis. Therefore, the method provided in Embodiment 1 of the present invention can be applied to the spatial transcriptomics analysis of high-water-content samples simultaneously.

[0098] 7. Embedding and cryosectioning of tissue samples in the early stage of the Aurelia aurita development cycle One Aurelia aurita strobila (early stage), two Aurelia aurita strobila (late stage), and six Aurelia aurita ephyrae (the samples are shown in Figure A of Figure 22 ) were selected and processed into sections according to the preparation method of spatio-temporal transcriptome samples of Aurelia aurita strobila and ephyrae in Example 2. Among them, the operations of PFA fixation, PBS washing, and gelatin solution immersion are shown in Figures B - D of Figure 22 .

[0099] Embedding, cryosectioning and staining of tissue samples in the early stage of the Aurelia aurita development cycle Taking one strobila (early stage), one strobila (late stage), and one ephyra in 1. Embedding and cryosectioning of tissue samples in the early stage of the Aurelia aurita development cycle as samples respectively for sectioning, with the section thickness of 10 μm. After the sections were stained with H&E, as Figure 23 shown, it can be clearly observed that the microscopic structure of the sections obtained by the method of the present invention is complete and the staining effect is clear.

[0100] The embodiments of the present invention have been described in detail above with reference to the drawings. However, the present invention is not limited to the above embodiments. Various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for preparing a spatial transcriptome sample of a high-water-content organism, characterized in that, The preparation method includes the following steps: sequentially performing fixation with a fixative, gelatin replacement, embedding, and sectioning on a high-water-content biological sample to obtain a section sample.

2. The preparation method according to claim 1, wherein The high-water-content biological sample refers to a biological sample with a water content ≥ 70%.

3. The preparation method according to claim 2, wherein The high-water-content biological sample includes at least one of jellyfish, sponge, ctenophore, seaweed, hydra, ascidian larvae, and bumblebee pupae samples.

4. The preparation method according to claim 1, characterized in that, The fixative includes formaldehyde or paraformaldehyde.

5. The preparation method according to claim 1, characterized in that, The fixation time with the fixative is 0.5 h - 2 h.

6. The preparation method according to claim 1, characterized in that, The gelatin replacement includes: immersing the high-water-content biological sample fixed with the fixative in a gelatin solution with a concentration of 0.08 - 0.15 g / mL for 0.5 - 4 h.

7. Application of the spatial transcriptome sample prepared by the preparation method according to any one of claims 1 - 6 in spatial transcriptome detection.

8. The application according to claim 7, wherein Before spatial transcriptome detection, it further includes the step of performing RNA quality inspection on the spatial transcriptome sample.

9. A method for detecting spatial transcriptome of high-water-content organisms, characterized in that, The method includes the following steps: performing spatial transcriptome detection on the spatial transcriptome sample prepared by the preparation method according to any one of claims 1 - 6.

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