Method for spatial transcriptome preparation of high water content samples and applications thereof

Abstract

The application discloses a spatial transcriptome preparation method of a high-water-content sample and application thereof. The preparation method comprises the following steps: sequentially performing fixing of a fixing solution, gelatin replacement, embedding and section processing on a high-water-content biological sample to obtain a section sample. The spatial transcriptome sample preparation method of the high-water-content biological sample optimizes a tissue processing, embedding and section process, ensures section morphological integrity and RNA integrity, meets spatial transcriptome sequencing requirements, and has wide applicability, thereby providing reliable technical support for application of spatial transcriptome sequencing in high-water-content samples.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a spatial transcriptome preparation method for a high-water-content sample and application thereof. BACKGROUND

[0002] Spatial transcriptomics (ST) is an emerging biological technology aiming to combine 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, and are directly related to the accuracy of library construction, the reliability of sequencing results, and the repeatability of gene expression data. 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 greater than or equal to 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 kind of lower marine organisms, belong to the phylum Cnidaria. Among them, the most common Aurelia sp. is widely distributed in the Atlantic, Pacific, Indian Ocean and other sea areas between 70° north latitude and 40° south latitude, and has important research value in the fields of genetics, ecology and evolutionary biology. However, the tissue of Aurelia sp. has the characteristics of high polysaccharide content, RNA easy to degrade, and extremely high water content (about 95%-98%), which brings great challenges to its spatial transcriptomics research. Whether the tissue section with complete morphology and high RNA integrity can be obtained is a key technical problem for the spatial transcriptomics research of jellyfish. However, at present, there is no report on the effective tissue section scheme for high-water-content biological samples such as Aurelia sp. under the premise of ensuring RNA integrity. SUMMARY

[0004] The first aspect of the present application aims to provide a spatial transcriptome sample preparation method for high-water-content organisms.

[0005] The second aspect of the present application aims to provide an application of the spatial transcriptome sample preparation method for high-water-content organisms in spatial transcriptome sequencing.

[0006] The third aspect of the present application aims to provide a spatial transcriptome sequencing method for high-water-content organisms.

[0007] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0008] In a first aspect, the present application provides a method for preparing a spatial transcriptome sample of a high water content organism, the method comprising the following steps: sequentially fixing a high water content organism sample with a fixing solution, replacing with gelatin, embedding, and sectioning to obtain a sectioned sample.

[0009] Preferably, the high water content organism sample refers to a biological sample with a water content of ≥70%, for example, a biological sample with a water content of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%.

[0010] Preferably, the high water content organism sample includes at least one of a jellyfish, a sponge, a comb jelly, a seaweed, a hydra, a sea squirts larva, and a bear bee pupa sample.

[0011] Preferably, the sample is a sectioned sample, for example, a tissue sectioned sample.

[0012] Preferably, the jellyfish includes a Cyanea nozakii, a Turritopsis dohrnii, a Cyanea annasetta, a Cyanea lamarckii, and a Cyanea nobilis.

[0013] Preferably, the high water content organism sample includes an umbrella tissue sample of a jellyfish, a transverse bar sample of a jellyfish at an early developmental stage, and a butterfly bar sample of a jellyfish at an early developmental stage.

[0014] Preferably, the fixing solution includes formaldehyde or paraformaldehyde.

[0015] Preferably, the mass fraction of the paraformaldehyde is 3%-5%, for example, 3%, 4%, or 5%.

[0016] Preferably, the fixing time of the fixing solution is 0.5h-2h, for example, 0.5h, 1h, 1.5h, or 2h.

[0017] Preferably, after the fixing with the fixing solution and before the gelatin replacement, the method further comprises a step of cleaning the high water content organism sample after the fixing with the fixing solution, the step comprising: soaking the high water content organism sample after the fixing with the fixing solution in a buffer solution.

[0018] Preferably, the buffer solution includes a PBS buffer solution.

[0019] Preferably, the soaking time is 4-12min, for example, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 11min, or 12min.

[0020] Preferably, the soaking time is 5-10min, for example, 5min, 6min, 7min, 8min, 9min, or 10min.

[0021] Preferably, the number of times of washing is 1-5 times, for example, 1 time, 2 times, 3 times, 4 times, 5 times.

[0022] Preferably, the number of times of washing is 1-3 times.

[0023] Preferably, the gelatin replacement includes: soaking the high-water-content biological sample fixed by the fixing solution in a gelatin solution with a concentration of 0.08-0.15 g / mL.

[0024] 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.

[0025] 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.

[0026] Further, the soaking time is 0.5-2 h, at which time the jellyfish sample can ensure both spatial morphology and nucleic acid quality.

[0027] Further, the soaking time is 0.5-1.5 h.

[0028] Preferably, the soaking temperature is 0-6℃, for example, it can be 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃.

[0029] Preferably, the embedding includes frozen embedding.

[0030] Preferably, the embedding agent used in the frozen embedding includes an OCT embedding agent.

[0031] Preferably, the sectioning is performed by a frozen sectioning machine, and the tissue after frozen embedding is cut into a sectioned sample with a thickness of 8-12 μm (for example, 10 μm).

[0032] Preferably, after the sectioning, the method further includes a step of performing RNA quality inspection on the sectioned sample. By introducing RNA quality inspection, the present application ensures that the sample RNA quality meets the requirements of spatial transcriptome detection through RNA integrity number (RIN value) detection.

[0033] Preferably, the method further includes a step of performing H&E staining and sectioning permeabilization gradient fluorescence imaging test detection on the sectioned sample.

[0034] Preferably, the method further comprises a step of snap-freezing the high-water-content biological sample after the gelatin replacement before the embedding; the snap-freezing comprises placing the high-water-content biological sample after the fixation treatment at -76℃ to -82℃ for 25-35 min. Through the gelatin solution immersion and snap-freezing technology, the tissue shrinkage and fragmentation of the high-water-content sample during the embedding and slicing process are effectively reduced, the morphological integrity of the slice is ensured, the high-quality complete slice is obtained, and the spatial distribution information of the cells in the tissue can be accurately captured. In specific applications, samples with small volume and stable sample morphology, such as the transverse and butterfly bodies of the early stage of the development cycle of the jellyfish Aequorea victoria, do not need to be kept unchanged in sample morphology by means of snap-freezing due to their small volume, and the snap-freezing step can be omitted in specific experiments.

[0035] The second aspect of the present application is to provide an application of the high-water-content biological spatial transcriptome sample prepared by the preparation method of the first aspect of the present application in spatial transcriptome detection.

[0036] Preferably, the method further comprises a step of RNA quality detection of the spatial transcriptome sample before the spatial transcriptome detection.

[0037] Preferably, the sample is a slice sample.

[0038] Preferably, the method further comprises a step of H&E staining and slice permeabilization gradient fluorescence imaging test detection of the spatial transcriptome sample before the spatial transcriptome detection.

[0039] Preferably, the method further comprises a step of H&E staining and slice permeabilization gradient fluorescence imaging test detection of the sample after the RNA quality detection and before the spatial transcriptome detection.

[0040] The third aspect of the present application is to provide a method for spatial transcriptome detection of a high-water-content biological sample, the method comprising the following steps: using the sample prepared by the preparation method of the first aspect of the present application to perform spatial transcriptome detection.

[0041] In the present application, the spatial transcriptome detection refers to the detection of the spatial position of the transcript (i.e., RNA), and the common technical means in the field include: in situ spatial transcriptome detection, including FISH, in situ decoding technology such as the Xenium platform of 10xGenomics, etc.; spatial transcriptome sequencing technology, such as the spatial transcriptome sequencing platform of BGI Stomics, the Visium or Visium HD platform of 10x Genomics. The sample prepared by the present application can be used for high-throughput spatial transcriptome detection, for example, the spatial transcriptome sequencing technology can detect the transcripts of 9303 genes.

[0042] Preferably, the spatial transcriptome sequencing is performed using the Stereo-Seq sequencing technology.

[0043] The present application has the following advantages:

[0044] The spatial transcriptome sample preparation method for high water content organisms provided by the present application ensures the integrity of the slice morphology and RNA by optimizing the tissue processing, embedding and slicing process, to meet the requirements of spatial transcriptome sequencing. The gelatin involved is a protein obtained by partial hydrolysis of collagen, which contains a large number of polar groups such as hydroxyl and carboxyl groups in its molecules. At low temperatures, the hydrogen bonds and other interaction forces between these groups are enhanced, forming a more compact network structure between gelatin molecules, thus forming a gel. After gelatin soaking treatment, the water in the jellyfish can be partially replaced by gelatin. After the tissue is frozen, the solidified gelatin can provide the support required for slicing, ensuring the morphological integrity of the sample during slicing is not damaged. Through gelatin soaking and quick freezing techniques, the tissue shrinkage and fragmentation of high water content samples during embedding and slicing are effectively reduced, ensuring the integrity of the slice morphology and obtaining high-quality intact slices, which ensures accurate capture of the spatial distribution information of cells in the tissue.

[0045] At the same time, the preparation method of the present application also optimizes the fixing, washing and gelatin soaking steps, especially controls the operation details and adjusts the processing time, to minimize the degradation of slice RNA, ensuring that the RNA integrity number (RIN or RQN) is ≥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.

[0046] The method of the present application is not only suitable for jellyfish, but also can be extended to other high water content species or tissue samples (such as hydra, sea squirt larvae, bear bee pupa hind legs, etc.), and has wide applicability. Through detailed operation process and parameter setting, the present method ensures the repeatability and scalability of the experiment, and provides reliable technical support for the application of spatial transcriptome sequencing in high water content samples. BRIEF DESCRIPTION OF DRAWINGS

[0047] The present application will be further described below in conjunction with the drawings and examples, in which:

[0048] Figure 1 is the experimental flowchart in Example 1 of the present application;

[0049] Figure 2Figure of slice morphology comparison between other methods in the test example of the present application and the method in embodiment 1 of the present application, wherein A is the embedding slice result figure of traditional paraffin, showing that the tissue shrinks seriously, B is the slice detection result figure of conventional frozen embedding, showing that the sample is easy to break, C is the agar replacement and wax immersion embedding slice result figure, showing that the tissue soft slice is difficult, and D is the slice result figure of embedding by the method in embodiment 1;

[0050] Figure 3 Figure of jellyfish continuous patch slice and H&E staining microscopic image in the test example of the present application, wherein each grid of the ruler represents 1 mm;

[0051] Figure 4 Figure of original sample of jellyfish 5 in the test example of the present application;

[0052] Figure 5 Figure of jellyfish 5 quality inspection result in the test example of the present application, wherein 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;

[0053] Figure 6 Figure of original sample of jellyfish 6 in the test example of the present application;

[0054] Figure 7 Figure of jellyfish 6 quality inspection result in the test example of the present application, wherein 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;

[0055] Figure 8 Figure of original sample of jellyfish 7 in the test example of the present application;

[0056] Figure 9 Figure of jellyfish 7 quality inspection result in the test example of the present application, wherein 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;

[0057] Figure 10 Figure of jellyfish slice permeabilization gradient fluorescence imaging test result in the test example of the present application, wherein A is the sample morphology figure of embedding treatment of jellyfish 7, B is the H&E staining result figure of jellyfish 7, C is the permeabilization gradient fluorescence imaging patch detection result figure of jellyfish 7, and D is the high-definition microscopic image of the permeabilization gradient fluorescence imaging patch detection result figure of jellyfish 7;

[0058] Figure 11Figure 8 is a sample information chart of the jellyfish 8 "one-eighth" in the test example of the present application, wherein the scale is 1 cm;

[0059] Figure 12 Figure 9 is a sample morphology chart and H&E staining result chart of the jellyfish 8 after embedding treatment in the test example of the present application; wherein A is a sample morphology chart of the jellyfish 8 after embedding treatment, B is an H&E staining result chart of the jellyfish 8, and the scale is 1 cm;

[0060] Figure 13 Figure 10 is a jellyfish 8 quality inspection result chart in the test example of the present application;

[0061] Figure 14 Figure 11 is a jellyfish 8 quality inspection peak value result chart in the test example of the present application, wherein A is an Ac8-1 detection result chart; B is an Ac8-2 detection result chart; C is an Ac8-3 detection result chart; D is an Ac8-4 detection result chart; E is an Ac8-5 detection result chart; F is an Ac8-6 detection result chart; G is an Ac8-7 detection result chart; and H is an Ac8-8 detection result chart;

[0062] Figure 15 Figure 12 is a spatial transcriptome sequencing detection result chart in the test example of the present application, wherein A is an original morphology chart of the jellyfish Ac8-3; B is a sample morphology chart and H&E staining result chart of the jellyfish Ac8-3 after embedding treatment, and C is a jellyfish 8-3 quality inspection RIN detection result chart;

[0063] Figure 16 Figure 13 is a jellyfish Ac8-3 spatial group chip patch and H&E staining result chart in the test example of the present application; wherein A is a spatial group chip patch chart of the jellyfish Ac8-3; and B is an H&E staining result chart of the jellyfish Ac8-3;

[0064] Figure 17 Figure 14 is an ssDNA fluorescence staining chart of the jellyfish Ac8-3 in the test example of the present application;

[0065] Figure 18 Figure 15 is a DNA detection result chart of the jellyfish Ac8-3 in the test example of the present application;

[0066] Figure 19 Figure 16 is a spatial transcriptome sequencing saturation statistical chart of the jellyfish Ac8-3 in the test example of the present application;

[0067] Figure 20 Figure 17 is a spatial gene expression distribution chart of the jellyfish Ac8-3 in the test example of the present application;

[0068] Figure 21The spatial Spots morphology distribution and UMAP clustering analysis diagram of jellyfish Ac8-3 in the test example of the present application, wherein A is the spatial Spots morphology distribution diagram; B is the UMAP clustering analysis diagram;

[0069] Figure 22 The process of preparing the sample of the early stage of the cross-split body and the butterfly body of the A. cyanea in Example 2 in the test example of the present application and the H&E staining microscopic diagram of the patch section, wherein A is the sample morphology diagram of the cross-split body and the butterfly body of the A. cyanea, B is the sample morphology diagram during the PFA fixation process, C is the sample morphology diagram during the PBS cleaning process, and D is the sample morphology diagram during the gelatin solution immersion replacement moisture process;

[0070] Figure 23 The actual sample and the corresponding section H&E staining microscopic diagram of the cross-split body (early stage), the cross-split body (late stage), and the butterfly body of the A. cyanea in the test example of the present application. DETAILED DESCRIPTION

[0071] The concept and the technical effects of the present application will be described clearly and completely in combination with the embodiments, so as to fully understand the purposes, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0072] The experimental methods not specified in the following embodiments are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers. The materials, reagents, etc. used in the present embodiments are commercially available reagents and materials, unless otherwise specified.

[0073] Experimental materials:

[0074] Tissue fixative solution (containing 4% paraformaldehyde, brand: meilunbio, catalog number: MA0192-2);

[0075] Nuclease-free water (Nuclease-free Water, brand: TRAN, catalog number: GI101-02);

[0076] 20× buffer solution (20× PBS Buffer, brand: Shenguo, number: B548117-0500);

[0077] Gelatin powder (Gelatin from cold water fish skin, brand: SIGMA, catalog number: G7041-100G).

[0078] Embodiment 1: A method for preparing spatial transcriptome samples of a high water content organism

[0079] The embodiment provides a method for preparing spatial transcriptome samples of a high water content organism (taking Aequorea victoria as an example), and a flow chart is shown in the figure. Figure 1 The specific steps are as follows:

[0080] 1. Preparation of spatial transcriptome samples of Aequorea victoria

[0081] (1) Cutting off the mouth arm of Aequorea victoria

[0082] Select small Aequorea victoria samples with complete body shape and clean umbrella cover with a diameter of about 2 cm (stop feeding 24 hours in advance to ensure sample quality), invert (umbrella top down) in a culture dish, flatten and add seawater just covering the jellyfish, use sterile tools to cut off the mouth arm part of the Aequorea victoria, and reserve the umbrella tissue.

[0083] (2) Fixing the Aequorea victoria sample after cutting off the mouth arm

[0084] Soak the Aequorea victoria sample after cutting off the mouth arm in a fixing solution (4% paraformaldehyde (PFA)) solution, the fixing solution to sample volume ratio is 15:1, and fix on ice for 1 h (0.5 h-2 h are also available), the fixing step is to maintain the tissue morphology and prevent RNA degradation.

[0085] (3) Washing

[0086] Transfer the fixed Aequorea victoria sample to a culture dish, invert and flatten, and add 1x PBS buffer solution for soaking for 5 min for washing (room temperature, the liquid amount is appropriate to cover the sample).

[0087] (4) Gelatin solution soaking

[0088] Soak the washed Aequorea victoria sample in a gelatin solution (10% gelatin solution) with a concentration of 0.1 g / mL.

[0089] The detailed steps for soaking are as follows: according to the size of the jellyfish, an aluminum foil box is prepared, the sample is inverted in the box and flattened and unfolded; pour the 10% gelatin solution cooled to room temperature, and soak in a 4°C refrigerator for 2 h (0.5 h-4 h are also available).

[0090] (5) Quick-freezing the sample

[0091] After soaking in the gelatin solution, the aluminum foil box containing the sample was adjusted to a horizontal flat state, and the gelatin solution was slowly absorbed using a pipette, keeping the sample shape flat during the process. After the solution was completely absorbed, the aluminum foil box edges were flattened to facilitate subsequent removal. The sample was then transferred to a -80°C freezer for rapid freezing for 30 minutes to prevent ice crystal formation.

[0092] (6) Embedding

[0093] Beforehand, pour 1 / 3 of the OCT embedding agent into a transparent embedding mold and place it horizontally on dry ice. When the embedding agent begins to solidify, carefully remove the rapidly frozen sample from the aluminum foil box (operations should be performed in a -25°C environment), quickly transfer it to the transparent embedding mold containing the OCT embedding agent, adjust the sample position to ensure it is placed horizontally, then continue to pour the OCT embedding agent until it is full, and mark the sample position on the embedding box. Place the embedding box on a dry ice block or in a -80°C freezer environment to freeze into a block.

[0094] (7) Frozen sectioning

[0095] Adjust the temperature of the cryostat to -25°C. Use the cryostat to cut the embedded tissue into 10 μm thick sections. Immediately transfer the cut embedding block to a -80°C freezer for storage for subsequent experiments.

[0096] (8) RNA quality inspection

[0097] Take an appropriate amount of tissue sample from the frozen section. Then use the RNeasy FFPE Kit to extract and detect RNA. Skip the deparaffinization step in the kit and directly extract RNA from the lysis operation according to the instructions. Use a bioanalyzer to detect the integrity of the RNA and obtain the RNA Integrity Number (RIN) value to evaluate the quality of the RNA.

[0098] 2. Sample analysis

[0099] (1) H&E staining

[0100] The specific operation steps of H&E staining are performed according to the standard frozen section H&E staining process, which is briefly described as follows: After the frozen section is naturally thawed at room temperature, it is sequentially subjected to methanol fixation, deionized water washing of methanol, hematoxylin staining (staining time is 1-2 min), running water washing, eosin staining (staining time is 1-2 min), dehydration, transparency and mounting.

[0101] (2) Slice permeabilization gradient fluorescence imaging test

[0102] Take 4 consecutive 1 cm x 1 cm sections and sequentially attach them to the permeabilization chip. Observe the tissue morphology by gradient fluorescence imaging.

[0103] Example 2 Spatial transcriptome sample preparation method for high water content samples in early stage of Aurelia aurita development cycle

[0104] This example provides a spatial transcriptome sample preparation method for high water content samples in early stage of Aurelia aurita development cycle, taking Aurelia aurita transverse body and butterfly body samples as examples. The preparation flowchart is similar to that in Example 1 (as shown in Figure 1 The specific steps are as follows:

[0105] 1. Preparation of spatial transcriptome samples of Aurelia aurita transverse body and butterfly body

[0106] (1) Fix the transverse body and butterfly body samples

[0107] Soak the Aurelia aurita transverse body and butterfly body samples in a fixing solution (4% paraformaldehyde (PFA)) solution for 0.5 h. The fixing step is aimed at maintaining the tissue morphology and preventing RNA degradation.

[0108] (2) Washing

[0109] Transfer the fixed Aurelia aurita transverse body and butterfly body samples to a culture dish and add 1x PBS buffer for 5 min for washing.

[0110] (3) Gelatin solution soaking

[0111] Transfer the washed Aurelia aurita transverse body and butterfly body samples to a gelatin solution (10% gelatin solution) with a concentration of 0.1 g / mL and soak them in a 4°C refrigerator for 0.5 h.

[0112] (4) Embedding

[0113] Pour 1 / 3 of the OCT embedding agent into a transparent embedding mold in advance and place it horizontally on dry ice. When the embedding agent gradually solidifies, quickly transfer the sample to the transparent embedding mold containing the OCT embedding agent, adjust the sample position to ensure it is placed horizontally, then continue to fill the OCT embedding agent, and mark the sample position on the embedding box. Place the embedding box on a dry ice block or in a -80°C refrigerator environment to freeze into a block.

[0114] (5) Frozen sectioning

[0115] Adjust the temperature of the cryostat to -25°C. Use the cryostat to cut the embedded tissue into 10 μm thick sections. Immediately transfer the embedded block to a -80°C refrigerator after sectioning for subsequent experimental use.

[0116] 2. Sample analysis

[0117] H&E staining is used for sample analysis:

[0118] The specific operation steps of H&E staining are carried out according to the standard frozen section H&E staining process, and are briefly described as follows: after the frozen section is naturally thawed at room temperature, methanol fixation, deionized water washing methanol, hematoxylin staining (staining time is 1-2 min), water flushing, eosin staining (staining time is 1-2 min), dehydration, transparency and mounting are carried out in turn.

[0119] Test example

[0120] 1. Umbrella tissue of Aequorea coerulescens embedding frozen section processing

[0121] Aequorea coerulescens 1, Aequorea coerulescens 2, Aequorea coerulescens 3 and Aequorea coerulescens 4 were used as research objects, and were respectively treated by traditional paraffin embedding, conventional frozen embedding, agar replacement and embedding in wax and the preparation method of the Aequorea coerulescens sample in Example 1.

[0122] The traditional paraffin embedding treatment method is as follows:

[0123] a) Fixation: 4% paraformaldehyde was used to fix the tissue sample at 4℃ overnight to maintain the tissue morphology.

[0124] b) Washing: The sample was washed with 1×PBS buffer for 30 minutes to remove residual fixative.

[0125] c) Gradient dehydration: The sample was sequentially immersed in different concentrations of ethanol solution (30%, 50%, 70%, 85%, 95%), 1 hour per step, slowly shaken on a shaker to gradually remove water from the tissue.

[0126] d) Transparency treatment: The sample was transferred to n-butanol and soaked for 3 times, 1 hour each time, to make the tissue transparent.

[0127] e) Wax immersion and embedding: The sample was placed in 65℃ paraffin for 3 hours, and the paraffin was replaced every 1 hour, and finally paraffin embedding was performed.

[0128] f) Sectioning: The embedded tissue was cut into 10 μm (10-20 μm) thick slices using a microtome.

[0129] The conventional frozen embedding treatment method is as follows:

[0130] a) Fixation: 4% paraformaldehyde was used to fix the tissue sample on ice for 2 hours to maintain the tissue morphology.

[0131] b) Washing: The sample was washed with 1×PBS buffer for 5 minutes to remove residual fixative.

[0132] c) Sucrose sedimentation: The sample was soaked in 30% sucrose solution for 2 hours to enhance the anti-freezing property of the tissue.

[0133] d) Embedding: Embed the tissue using OCT embedding medium.

[0134] e) Sectioning: Section the embedded tissue into 10 pm (10-20 pm) thick slices on a cryostat.

[0135] The agar replacement wax embedding process is as follows:

[0136] a) Gradient dehydration: sequentially immerse the sample in 80%, 85%, 90%, and 95% ethanol for 5 min each time.

[0137] b) Agarose replacement: immerse the dehydrated tissue sample in a 3% agarose solution, and after embedding into a block, place it in a 4°C cooling coagulation to replace the traditional agarose protective layer.

[0138] c) Clearing treatment: transfer the sample to xylene and immerse it 2-3 times for 5 min each time to make the tissue transparent and replace ethanol.

[0139] d) Wax immersion and embedding: place the sample in 65°C paraffin for 3 hours, replace the paraffin every 1 hour, and finally perform paraffin embedding.

[0140] e) Sectioning: use a microtome to section the embedded tissue into 10 pm (10-20 pm) thick slices.

[0141] The sectioning effect comparison is shown in Figure 2 As can be seen from the figure, in the traditional paraffin embedding process (as shown in Figure 2 A), the high-temperature environment in the wax immersion operation can cause the tissue to shrink and the morphological structure to be damaged. This shrinkage not only destroys the integrity of the sample, but also causes severe deformation of the tissue structure, resulting in distortion or even loss of spatial position information. For spatial transcriptome sequencing, the morphological integrity of the sample and the accuracy of the spatial position information are crucial, and the paraffin embedding method is difficult to meet these requirements when processing high-water content samples; conventional frozen embedding (as shown in Figure 2 B) is prone to sample breakage during sectioning, which cannot meet the strict requirements of spatial transcriptome sequencing for sample integrity. In addition, the formation of ice crystals can also damage the cell structure, further affecting the integrity of RNA, which is not conducive to spatial transcriptome sequencing, and therefore it is difficult to obtain complete and high-quality sections; using agar treatment (as shown in Figure 2Although the jellyfish in FIG. C shows no obvious change in morphology after overnight wax immersion, the tissue exhibits a soft state during the subsequent paraffin embedding process, resulting in the inability to obtain complete and continuous sections during sectioning. This is mainly because the agarose fails to effectively support the tissue structure after high-temperature paraffin immersion, and the jellyfish tissue itself has a high water content and soft texture, making the tissue prone to local deformation and softening during the penetration and solidification of the wax. The softened tissue is prone to breakage and shrinkage during sectioning with a microtome blade, which cannot form complete sections, affecting subsequent spatial transcriptome analysis. The method of the present application innovatively uses gelatin solution immersion optimization technology (as shown in FIG. D of Figure 2 based on conventional frozen embedding, effectively ensuring the morphological integrity of the sections and the clarity of the tissue structure.

[0142] 2. Continuous and complete sectioning and staining of umbrella tissue of Aurelia sp. 1

[0143] Using Aurelia sp. 4 as a sample in the embedding and frozen sectioning process of umbrella tissue of Aurelia sp. 1, continuous sections were taken from the umbrella top (1 section) to the endoderm (4 sections) and then to the gonad (3 sections), with a section thickness of 10 μm and a patching interval of 300 μm.

[0144] After H&E staining of the sections, as shown in FIG. 2, the microstructure of the sections obtained by the method of the present application was complete and the staining effect was clear. Figure 3

[0145] 3. RNA integrity detection of umbrella tissue of Aurelia sp.

[0146] Aurelia sp. 5, 6, and 7 were treated according to the preparation and processing method of the spatiotemporal transcriptome sample of Aurelia sp. 1 in Example 1. Due to the difference in size of Aurelia sp., the corresponding combinations of different time lengths were set for PFA immersion and gelatin solution immersion.

[0147] Aurelia sp. 5, with a diameter of about 1 cm, was treated with PFA for 0.5 h, PBS for 5 min, and gelatin solution for 0.5 h.

[0148] Aurelia sp. 6, with a diameter of about 1.8 cm, was treated with PFA for 1.5 h, PBS for 5 min, and gelatin solution for 1 h.

[0149] Aurelia sp. 7, with a diameter of about 2 cm, was treated with PFA for 1.5 h, PBS for 5 min, and gelatin solution for 4 h.

[0150] After embedding, Aurelia sp. 5, 6, and 7 were frozen sectioned, and the integrity of the sections was observed. The sections were also subjected to RNA integrity detection.

[0151] The results are shown in FIG. 3. Figures 4-9 ​As shown in the figure, it can be seen from the figure that the embedded jellyfish of different time combinations can obtain complete slices when slicing, and the RNA integrity of the slices is detected, and the RIN value is obtained. Among them, the RIN value of jellyfish 5 is 8.2, the RIN value of jellyfish 6 is 7.7, and the RIN value of jellyfish 7 is 8.2. The results show that the RNA integrity number (RIN value) of the sample treated by the method of the present application is high, the RIN value is all ≥7, which meets the requirements of spatial transcriptome sequencing, ensures that the sample RNA quality meets the spatial transcriptome sequencing standard, and improves the reliability and repeatability of experimental data.

[0152] 4. Gradient fluorescence imaging test of umbrella tissue section of Aurelia aurita

[0153] Select jellyfish 7 samples (RIN value is 8.20 when RNA integrity is detected, and the slice morphology is complete) for sectioning and gradient fluorescence imaging test, and perform H&E staining on the frozen section of jellyfish 7 samples.

[0154] The results are shown in Figure 10 , four 1cmx1cm sections of jellyfish 7 are sequentially attached to the permeation chip (as shown in Figure 10 C figure), and the jellyfish 7 sample can be observed to have complete and clear tissue morphology at 18 minutes by gradient fluorescence imaging, Figure 10 D high-definition micrograph and Figure 10 B figure shows the same texture characteristics as the H&E staining result.

[0155] 5. Gradient processing of jellyfish umbrella tissue section soaking time and RNA integrity detection

[0156] Select jellyfish 8 (diameter about 4cm) samples and divide the umbrella tissue into eight equal parts (as shown in Figure 11 ), and process them according to the preparation and processing method of jellyfish spatiotemporal transcript samples in embodiment 1. In order to explore the good processing time for protecting the RNA integrity of the section, the eight jellyfish umbrella tissues are gradient combined in terms of PFA fixation, PBS cleaning and gelatin solution soaking time (the detailed time is shown in table 1), and the frozen section of each sample is subjected to H&E staining (as shown in Figure 12 ).

[0157] Table 1

[0158]

[0159] The results are shown in Figures 13-14As shown in the figure, the control group jellyfish Ac8-1 was not soaked with gelatin solution and could not obtain complete sections, which directly led to poor RNA quality inspection results (RQN was 1); the experimental group increased the PFA fixation and gelatin solution soaking time from jellyfish Ac8-2 to jellyfish Ac8-8, and the morphological integrity of the sections could be guaranteed, but the RNA integrity quality inspection test had different results, among which the RNA integrity of jellyfish Ac8-2, Ac8-3, Ac8-4, Ac8-5, Ac8-6 was very good (RQN was much greater than 7), and the RNA of jellyfish Ac8-7 and Ac8-8 was degraded to a greater extent (RQN was less than 7) due to too long soaking time. Based on the results, the present application has a processing time range for obtaining RNA-intact jellyfish tissue sections, 4% PFA immersion time + gelatin immersion time < 3.5 h is optimal.

[0160] 6. Application of Aurelia aurita umbrella tissue section spatial transcriptome sequencing

[0161] Considering that the original sample is too large, the experiment only selected 1 / 8 umbrella tissue, i.e., 5, Aurelia aurita umbrella tissue section soaking time gradient processing and RNA integrity detection of Aurelia aurita Ac8-3 (Aurelia aurita Ac8-3 original sample, and sections obtained by processing Aurelia aurita Ac8-3 according to the preparation method of Aurelia aurita spatiotemporal transcript sample in embodiment 1, and the results of H&E staining of the sections are shown in Figure 15 A and B of the figure) for spatial transcriptome sequencing. The sequencing method adopts the spatiotemporal transcriptome technology Stereo-Seq sequencing of Huada, and the spatial transcriptome sequencing of Aurelia aurita is completely carried out according to the process of “freezing embedding-RIN quality inspection-spatial transcriptome chip patch-H&E staining-ssDNA fluorescent staining-DNA detection-sequencing” on the basis of the experiment in embodiment 1 of the present application, and spatial transcriptome analysis is carried out on the sequencing data (i.e., the data obtained by Stereo-seq sequencing is processed by using the matching SAW-ST-V8 spatiotemporal bioinformatics software suite analysis process).

[0162] The results are shown in Figures 15-21 From Figure 15 C in the figure, the quality inspection result shows that the RIN value is 8.99; the cDNA fragment range of the chip is 476-1500 bp, the main peak is 1183 bp, and the library construction is normal (as shown in Figure 18 ); finally, about 4.3G Reads were obtained by spatial transcriptome sequencing, and 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 ).

[0163] In the spatial transcriptomic analysis, the spatial gene expression distribution is mainly in the edge of the umbrella, which is also consistent with the expectation (as shown in Figure 20 FIG. 2B), and the spatial Spots morphology distribution is also clear (as shown in Figure 21 FIG. 2C), which shows that the preliminary data quality of the spatial transcriptome sample of Aurelia coerulea prepared by the method of the present embodiment can be used for subsequent data analysis. Therefore, the method provided in embodiment 1 can be applied to spatial transcriptomic analysis of high water content samples.

[0164] 7. Sample tissue embedding and frozen section processing in early stage of Aurelia coerulea development cycle

[0165] One Aurelia coerulea transverse body (early stage), two Aurelia coerulea transverse bodies (late stage), and six Aurelia coerulea butterfly bodies (as shown in Figure 22 FIG. 1A) were selected and processed according to the method for preparing spatial transcriptome samples of Aurelia coerulea transverse body and butterfly body in embodiment 2 to obtain sections, wherein the PFA fixation, PBS washing, and gelatin solution immersion operations are shown in Figure 22 FIG. 2B-D.

[0166] Sample tissue embedding and frozen section processing in early stage of Aurelia coerulea development cycle

[0167] One Aurelia coerulea transverse body (early stage), one Aurelia coerulea transverse body (late stage), and one Aurelia coerulea butterfly body in 1, sample tissue embedding and frozen section processing in early stage of Aurelia coerulea development cycle were sectioned, with a section thickness of 10 μm. After H&E staining, the section obtained by the method of the present embodiment was clear in microstructure and clear in staining effect, as shown in Figure 23 FIG. 3.

[0168] The embodiments of the present application are described in detail above in combination with the drawings, but the present application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method of preparing a spatial transcriptome sample of a high water content organism, characterized in that, The preparation method comprises the following steps: sequentially performing fixing liquid fixing, gelatin replacement, embedding and section processing on a high water content biological sample to obtain a section sample; the high water content biological sample refers to a biological sample with a water content of ≥ 90%; The gelatin replacement comprises: placing the high water content biological sample fixed by the fixing liquid in a gelatin solution with a concentration of 0.08-0.15 g / mL for soaking treatment for 0.5-4 h; The RNA integrity number of the spatial transcriptome sample is ≥ 7.

2. The production method according to claim 1, characterized by, The high water content biological sample comprises at least one of the following: jellyfish, sponge, comb jelly, seaweed, hydra, tunicate larva and bear bee pupa sample.

3. The preparation method according to claim 1, characterized in that, The fixing liquid comprises formaldehyde or paraformaldehyde.

4. The production method according to claim 1, characterized by, The fixing liquid is fixed for 0.5-2 h.

5. Application of the spatial transcriptome sample prepared by the preparation method in spatial transcriptome detection.

6. Use according to claim 5, characterized in that, Before the spatial transcriptome detection, the spatial transcriptome sample is further subjected to RNA quality detection.

7. A method for spatial transcriptome detection of a high water content organism, characterized by, The method comprises the following steps: using the spatial transcriptome sample prepared by the preparation method in spatial transcriptome detection.

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