Reagent and application thereof in biological carrier function restoration

Abstract

The invention relates to the technical field of biology, in particular to a reagent and application thereof in biological carrier function restoration. Wherein the reagent comprises at least one of urea, sodium dodecyl sulfate and citicoline sodium. The reagent can be used for removing nucleic acid, protein, cells or tissues on the surface of a biological carrier and releasing occupied probes to realize carrier function recovery.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology. Specifically, the present application relates to a reagent and its application in the functional restoration of biological carriers. More specifically, the present application relates to a reagent and its application, a reagent set and its application, a kit, a biological carrier restoration method, a spatial omics sequencing method and an in situ hybridization method. Background Art

[0002] The emergence of spatial omics technologies has revolutionized researchers' understanding of the spatial organization of molecular processes within complex biological systems, providing unprecedented insights into cell and tissue biology. Among these technologies, spatial transcriptomics is considered a groundbreaking approach, having been named the 2020 Nature Methods Technology of the Year. By combining spatial information with transcriptomic data, this technique enables precise localization of gene expression within tissues, achieving cellular resolution or even higher, facilitating comprehensive investigation of the heterogeneity and spatial dynamics of cell populations within tissues.

[0003] Building on the foundation laid by spatial transcriptomics, scientists and researchers have expanded spatial localization methods to other omics fields, promoting the rapid development of spatial proteomics, spatial immunoomics, and other fields. These multi-omics approaches can detect known target biomolecules through targeted imaging, or capture multi-omics information through random probes and convert it into cDNA for high-throughput sequencing, thereby obtaining spatial localization information.

[0004] Among them, BGI's Stereo-seq technology loads probes with spatial position information onto biochips with nanometer-level resolution through sequencing. These biochips capture target nucleic acids or proteins in biological tissues, and then convert the captured information into corresponding nucleotide sequences through biochemical reactions and recover them. Finally, the captured information and spatial coordinates are matched one-to-one through second-generation sequencing.

[0005] As a biological carrier, chip carriers play an extremely important role in spatiotemporal omics technology and account for a considerable proportion of the cost of spatiotemporal omics experiments. After the chip is prepared, the tissue or cell suspension is attached to the chip surface, which is the first step in obtaining biological information. If there is a problem in this operation (such as: tissue folding, incomplete tissue placement, tissue fragmentation, etc.), the biological information of the tissue or cell cannot be obtained truthfully (such as: incorrect tissue position information, incomplete tissue position information, etc.). In the past, when users encountered patch errors, they often had to continue the experiment or use a new chip patch, which resulted in poor quality and inaccurate final experimental results or increased experimental time and consumables costs.

[0006] Therefore, there is an urgent need for a method for restoring the function of a biological carrier. Summary of the Invention

[0007] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, one purpose of the present application is to provide a reagent that effectively restores the function of the carrier.

[0008] Another object of the present application is to provide a method for restoring vector function.

[0009] Specifically, this application provides the following technical solutions:

[0010] In a first aspect, the present application provides a reagent. According to embodiments of the present application, the reagent includes at least one of urea, sodium dodecyl sulfate (SDS), and citicoline sodium. In some examples of the present application, the reagent can be used to remove nucleic acids, proteins, cells, or tissues from the surface of a biological carrier, releasing the occupied probes and restoring carrier function.

[0011] According to an embodiment of the present application, the above reagent may further include at least one of the following technical features:

[0012] According to an embodiment of the present application, the reagent does not include formamide when used for restoring the function of a biological carrier.

[0013] According to an embodiment of the present application, the reagent does not include protease when used for restoring the function of a biological carrier.

[0014] According to an embodiment of the present application, the concentration of urea in the reagent is 1M to 8M. In some examples of the present application, the concentration of urea in the reagent can optionally be 1M, 2M, 3M, 4M, 5M, 6M, 7M, or 8M. In some preferred examples of the present application, the concentration of urea in the reagent is 2M. Urea at the aforementioned concentrations can effectively remove nucleic acids, proteins, cells, or tissues from the surface of biological carriers.

[0015] According to an embodiment of the present application, the mass volume fraction (g / mL) of the sodium dodecyl sulfate in the reagent is 1% to 20%. In some examples of the present application, the mass volume fraction (g / mL) of sodium dodecyl sulfate in the above-mentioned reagent is optionally 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some preferred examples of the present application, the mass volume fraction (g / mL) of sodium dodecyl sulfate in the above-mentioned reagent is 10%. Based on the above-mentioned mass volume fraction, sodium dodecyl sulfate can effectively remove nucleic acids, proteins, cells or tissues on the surface of biological carriers.

[0016] According to an embodiment of the present application, the mass volume fraction (g / mL) of citicoline sodium in the reagent is 1% to 10%. In some examples of the present application, the mass volume fraction (g / mL) of citicoline sodium in the above reagent is optionally 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In some preferred examples of the present application, the mass volume fraction (g / mL) of citicoline sodium in the above reagent is 3%. Citicoline sodium based on the above mass volume fraction can effectively remove nucleic acids, proteins, cells or tissues on the surface of biological carriers.

[0017] According to embodiments of the present application, the reagent includes urea and sodium dodecyl sulfate, with the urea and sodium dodecyl sulfate used in a ratio of (0.1-0.8) mol: (1-20) g, preferably (0.1-0.4) mol: (1-2) g. In some examples of the present application, the urea and sodium dodecyl sulfate at the aforementioned concentration ratio can effectively remove nucleic acids, proteins, cells, or tissues from the surface of biological carriers.

[0018] According to an embodiment of the present application, the reagent includes urea and citicoline sodium, and the ratio of urea to citicoline sodium is (0.1-0.8) mol: (1-10) g, preferably (0.4-0.8) mol: (3-4) g. In some examples of the present application, the urea and citicoline sodium at the aforementioned concentration ratio can effectively remove nucleic acids, proteins, cells, or tissues from the surface of biological carriers.

[0019] According to an embodiment of the present application, the reagent includes urea, sodium dodecyl sulfate, and citicoline sodium in a ratio of (0.1-0.8) mol: (1-10) g: (1-10) g, preferably (0.4-0.8) mol: (1-5) g: (2-6) g, and more preferably (0.4-0.8) mol: (1-2) g: (3-4) g. In some examples of the present application, urea and citicoline sodium in the aforementioned concentration ratio can effectively remove nucleic acids, proteins, cells, or tissues from the surface of a biological carrier.

[0020] In a second aspect of the present application, a reagent set is provided. According to embodiments of the present application, the reagent set includes: a first reagent, the first reagent being as defined in the first aspect of the present application or any embodiment of the first aspect. In some examples of the present application, the reagent set can be used to restore the function of a biological carrier.

[0021] According to an embodiment of the present application, the above reagent set may further include at least one of the following technical features:

[0022] According to an embodiment of the present application, the reagent set further includes: a second reagent, the second reagent including an acid-base buffer. In some examples of the present application, the pH of the acid-base buffer is selected from 6.5 to 7.5, preferably 7. In some preferred examples of the present application, the acid-base buffer includes Tris-HCl. In some examples of the present application, the acid-base buffer is used to neutralize the pH value of the surface of the biological carrier.

[0023] According to an embodiment of the present application, the reagent set further includes: a third reagent, and the third reagent includes nuclease-free water.

[0024] In a third aspect of the present application, a kit is provided. According to embodiments of the present application, the kit includes: the reagents described in the first aspect or any embodiment of the first aspect of the present application, or the reagent set described in the second aspect or any embodiment of the second aspect of the present application. In some examples of the present application, the kit can be used to quickly, easily, and cost-effectively restore the function of a biological vector.

[0025] According to an embodiment of the present application, the above-mentioned kit may further include at least one of the following technical features:

[0026] According to an embodiment of the present application, the method further comprises at least one of anhydrous methanol, sodium citrate buffer and glycerol.

[0027] In the fourth aspect of the present application, the present application proposes the use of the reagent described in the first aspect or any embodiment of the first aspect or the reagent group described in the second aspect or any embodiment of the second aspect of the present application in preparing a kit for restoring the function of a biological carrier.

[0028] According to an embodiment of the present application, the above-mentioned use may further include at least one of the following technical features:

[0029] According to an embodiment of the present application, the biological carrier is selected from a biochip.

[0030] According to an embodiment of the present application, the surface of the biochip has nucleic acid probes and / or protein modifications.

[0031] In a fifth aspect, the present application proposes a method for restoring the function of a biocarrier. According to embodiments of the present application, the method includes: performing a first incubation treatment on a functional biocarrier to be restored in a restoring reagent to restore the function of the biocarrier; wherein the restoring reagent is selected from at least one of urea, sodium dodecyl sulfate, and citicoline sodium. In some examples of the present application, restoring the function of the biocarrier based on the above method is simple to operate, has low experimental safety risks, and reduces the loss of biochip capture capacity after restoration (within 10%).

[0032] According to an embodiment of the present application, the above-mentioned biological carrier recovery method may further include at least one of the following technical features:

[0033] According to an embodiment of the present application, the biological carrier is selected from a biochip.

[0034] According to an embodiment of the present application, the surface of the biochip has nucleic acid probes and / or protein modifications.

[0035] According to an embodiment of the present application, the reconstitution reagent includes urea, and the concentration of urea in the reconstitution reagent is 1M to 8M. In some examples of the present application, the concentration of urea in the reconstitution reagent can optionally be 1M, 2M, 3M, 4M, 5M, 6M, 7M, or 8M. In some preferred examples of the present application, the concentration of urea in the reconstitution reagent is 2M. Urea at the aforementioned concentrations can effectively remove nucleic acids, proteins, cells, or tissues from the surface of biological carriers.

[0036] According to an embodiment of the present application, the reconstitution reagent includes sodium dodecyl sulfate, and the mass volume fraction (g / mL) of the sodium dodecyl sulfate in the reconstitution reagent is 1% to 20%. In some examples of the present application, the mass volume fraction (g / mL) of sodium dodecyl sulfate in the reconstitution reagent is optionally 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some preferred examples of the present application, the mass volume fraction (g / mL) of sodium dodecyl sulfate in the reconstitution reagent is 10%. Based on the above-mentioned mass volume fraction, sodium dodecyl sulfate can effectively remove nucleic acids, proteins, cells or tissues on the surface of biological carriers.

[0037] According to an embodiment of the present application, the reconstitution reagent includes urea and sodium dodecyl sulfate, and the ratio of urea to sodium dodecyl sulfate is (0.1-0.8) mol: (1-20) g, preferably (0.1-0.4) mol: (1-2) g. In some examples of the present application, the urea and sodium dodecyl sulfate in the aforementioned concentration ratio can effectively remove nucleic acids, proteins, cells, or tissues from the surface of biological carriers.

[0038] According to an embodiment of the present application, the reconstitution reagent includes urea and citicoline sodium, and the ratio of urea to citicoline sodium is (0.1-0.8) mol: (1-10) g, preferably (0.4-0.8) mol: (3-4) g. In some examples of the present application, the urea and citicoline sodium at the aforementioned concentration ratio can effectively remove nucleic acids, proteins, cells, or tissues from the surface of biological carriers.

[0039] According to an embodiment of the present application, the reconstitution reagent includes urea, sodium dodecyl sulfate and citicoline sodium, and the usage ratio of urea sodium dodecyl sulfate and citicoline sodium is (0.1-0.8) mol: (1-10) g: (1-10) g, preferably (0.4-0.8) mol: (1-5) g: (2-6) g, and more preferably (0.4-0.8) mol: (1-2) g: (3-4) g. In some examples of the present application, urea and citicoline sodium in the aforementioned concentration ratio can effectively remove nucleic acids, proteins, cells or tissues from the surface of biological carriers.

[0040] According to an embodiment of the present application, the temperature of the incubation treatment is selected from 20° C. to 65° C., optionally 20° C., 22° C., 24° C., 26° C., 28° C., 30° C., 32° C., 34° C., 36° C., 38° C., 40° C., 42° C., 44° C., 46° C., 48° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 61° C., 62° C., 63° C., 64° C. or 65° C., preferably 50° C. to 60° C., more preferably 55° C. Within this temperature range, the biological carrier can be fully recovered.

[0041] According to an embodiment of the present application, the incubation time is selected from 0.5 hours to 4 hours (h), optionally 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, preferably 1h to 3h, more preferably 1h.

[0042] According to an embodiment of the present application, the method further comprises performing a cleaning process on the incubation product, wherein the cleaning process comprises a first cleaning process and a second cleaning process.

[0043] According to an embodiment of the present application, the first cleaning treatment is performed using a cleaning buffer.

[0044] According to an embodiment of the present application, the washing buffer comprises Tris-HCl. In some examples of the present application, the aforementioned Tris-HCl is used to neutralize the pH value of the surface of the biological carrier.

[0045] According to an embodiment of the present application, the pH of the washing buffer is selected from 6.5 to 7.5, preferably 7.

[0046] According to an embodiment of the present application, the second cleaning treatment is performed using nuclease-free water.

[0047] In a fifth aspect of the present application, the present application proposes the use of the reagents described in the first aspect or any embodiment of the first aspect or the reagent group described in the second aspect or any embodiment of the second aspect of the present application in spatial omics sequencing and / or in situ hybridization. In some examples of the present application, in some spatial omics sequencing or in situ hybridization application scenarios, the above reagents or reagent groups can be used to restore the function of the biocarrier patch when it fails.

[0048] In the sixth aspect of this application, a spatial omics sequencing method is proposed. According to embodiments of this application, the method comprises: restoring the function of a biological carrier using the method described in the fourth aspect or any embodiment of the fourth aspect of this application; performing a second incubation treatment on the nucleic acid sample to be tested and the functionally restored biological carrier; and performing library construction and sequencing on the incubation product to obtain spatial and sequence information of the nucleic acid sample to be tested. In some examples of this application, the functionally restored biological carrier can still be used for spatial omics sequencing.

[0049] The biological carrier after functional restoration by the reagent or reagent set of the present application can still be used for various types of spatial omics sequencing. In some examples of the present application, the aforementioned spatial omics includes but is not limited to spatial transcriptome, spatial proteome, spatial immunome and spatial metabolome.

[0050] In its seventh aspect, this application proposes an in situ hybridization method. According to embodiments of this application, the method comprises: restoring the function of a biological carrier using the method described in the fourth aspect or any embodiment of the fourth aspect; performing a third incubation treatment on the functionally restored biological carrier with a nucleic acid sample to be tested; and hybridizing the product of the third incubation treatment using a nucleic acid probe. In some examples of this application, the restored biological carrier can still be used in an in situ hybridization experiment.

[0051] According to an embodiment of the present application, the above-mentioned in situ hybridization method may further include at least one of the following technical features:

[0052] According to an embodiment of the present application, the in situ hybridization method further comprises: performing a detection process on the hybridization treatment product.

[0053] Beneficial effects

[0054] The present application provides a protease-free recovery carrier reagent, reagent set and kit, which uses the reagent of the present application to remove nucleic acids, proteins, cells or tissues on the surface of the biological carrier, reducing the experimental cost and providing a functional recovery scheme with wider applicability. The recovery reagent of the present application does not need to contain the toxic component formamide, the toxicity of the reagent itself is moderate to low, the user operation is safer, and the old slice removal effect is good, and the capture rate is guaranteed. The biological carrier after functional restoration based on the scheme of the present application can effectively reduce the loss of capture volume (within 10%). In some examples, the whole process of biological carrier functional restoration based on the scheme of the present application takes less time (about 40 minutes), which is more convenient for users to reuse and continue the experiment immediately. The reagents and recovery methods of the present application provide opportunities for reuse of biological carriers with unsatisfactory patches, effectively reducing experimental risks and consumables costs.

[0055] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0057] Figure 1 This is a schematic diagram comparing the tissue removal effects of the urea multiplexing scheme described in one embodiment of the present application; wherein the reference genome is selected from mice;

[0058] Figure 2 This is a schematic diagram of chip capture comparison after urea multiplexing treatment according to an embodiment of the present application; wherein the reference genome is selected from mouse;

[0059] Figure 3 This is a schematic diagram comparing the tissue removal effects of the SDS multiplexing scheme described in one embodiment of the present application; wherein the reference genome is selected from mouse;

[0060] Figure 4 This is a schematic diagram of chip capture comparison after SDS multiplexing scheme processing according to one embodiment of the present application; wherein the reference genome is selected from mouse;

[0061] Figure 5 This is a schematic diagram comparing the tissue removal effects of the multiplexing scheme of the composite denaturant according to one embodiment of the present application; wherein the reference genome is selected from mice;

[0062] Figure 6 This is a schematic diagram of chip capture comparison after treatment with the multiplex denaturant scheme described in one embodiment of the present application; wherein the reference genome is selected from mouse;

[0063] Figure 7A comparison diagram of the hybridization nucleic acid removal effect of the sodium choline re-use scheme according to an embodiment of the present application; wherein the reference genome is selected from a mouse;

[0064] Figure 8 A comparison diagram of the chip re-use scheme effect according to an embodiment of the present application; wherein the reference genome is selected from a mouse;

[0065] Figure 9 A comparison diagram of the chip re-use scheme effect according to an embodiment of the present application; wherein the reference genome is selected from a rat;

[0066] Figure 10 A detection distribution map of specific rat eyeball expressed genes according to a chip re-use scheme of an embodiment of the present application; wherein the reference genome is selected from a rat. DETAILED DESCRIPTION

[0067] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not to be construed as limiting the present application.

[0068] In this document, the terms "first", "second", etc. are used only to describe the purpose of the features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0069] In this document, the terms "include" or "comprise" are open expressions, i.e. include the contents indicated by the present application, but do not exclude other aspects.

[0070] In this document, unless otherwise specified, the terms "optionally", "optional" or "optional" generally mean that the event or condition described subsequently can but does not necessarily occur, and the description includes both cases where the event or condition occurs and cases where the event or condition does not occur.

[0071] The patching effect of biocarriers is crucial in spatiotemporal omics processes (such as permeabilization and transcriptome capture). However, due to the high cost of biocarriers (such as biochips) and the complex experimental procedures, both novice and experienced technicians are prone to poor patching. Currently, most existing technologies use protease digestion or formamide to denature and melt DNA to achieve the purpose of restoring the function of biocarriers. However, several problems still exist. First, protease digestion and multiplexing protocols are not suitable for cases where the chip surface is protein-modified. Second, formamide, as a DNA denaturant, has a melting function at high temperatures, but it is extremely toxic, difficult to operate, has high experimental safety risks, and is not effective for tissue removal. In addition, the capture yield is significantly reduced after chip function restoration (for example, the transcriptome capture yield of 10X Genomics' Visium chip multiplexing protocol is reduced by approximately 10-20% after restoration), which affects experimental efficiency. Moreover, the multiplexing process is time-consuming, seriously affecting the progress of the experiment on the same day. Therefore, new methods are needed to address these problems and improve the efficiency and safety of biocarrier function restoration technology.

[0072] To this end, this application proposes a method for restoring the function of a biological carrier, comprising:

[0073] 1. Incubate the biological carrier (taking the biochip as an example) with poor patch effect with the aforementioned recovery reagent;

[0074] In one example of the present application, the reconstitution reagent is selected from urea, sodium dodecyl sulfate, and a mixed system of urea and sodium dodecyl sulfate.

[0075] It should be noted that single reagents or compound reagents with similar functions to urea or sodium dodecyl sulfate can also be used for the recovery of biological carriers.

[0076] In some examples of the present application, other components, such as stabilizers, etc., may be added to the above-mentioned reconstitution reagent.

[0077] In one example of the present application, the incubation process can be selected from room temperature incubation or heated incubation.

[0078] In a specific example of the present application, a heating incubation treatment at 55° C. for 1 hour is selected.

[0079] It should be noted that during the incubation process, the liquid needs to be aspirated and observed, and the next step of the experiment can be carried out only when there is no tissue residue on the surface.

[0080] 2. Performing a first cleaning treatment on the incubation product;

[0081] The pH value of the surface of the biological carrier is neutralized by the first cleaning process. In this application, Tris-HCl with a pH of 7 is used as the cleaning buffer for the first cleaning process.

[0082] 3. Performing a second cleaning process on the biological carrier after the first cleaning process;

[0083] This application uses nuclease-free water for the second cleaning process. The biological carrier obtained after the second cleaning process can continue to be used for related experiments.

[0084] It should be noted that the present application solution is more suitable for restoring the function of biological carriers before tissue fixation.

[0085] Restoration of biocarriers using this method is highly effective in removing nucleic acids, proteins, cells, or tissues from their surfaces. It is simple to perform and offers superior safety compared to other denaturant methods. Furthermore, this method is also applicable to chips with protein-modified surfaces, as restoration does not affect their integrity or ability to capture nucleic acids and proteins. Restoring the functionality of biocarriers significantly reduces the cost of experimental consumables.

[0086] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0087] Example 1: Validation of the urea multiplexing scheme on the BGI Stereo-seq spatiotemporal transcriptome chip Chip T

[0088] Experimental materials: mouse brain and mouse testis (Guangdong Medical Laboratory Animal Center), 100% anhydrous methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5X SSC, 0.1X SSC, coverslips (Shitai), 100% glycerol (Diamond), Stereo-seq Transcriptome Kit T (BGI), STOmics Library Preparation Kit-S1 (BGI), Seq2000 Sequencing Kit PE100 (MGI), RNeasy Micro Kit (Qiagen), urea, wash buffer (Tris-HCl pH 7.0), NF water, Stereo-seq spatiotemporal transcriptome chip Chip T (BGI);

[0089] Experimental instruments: microtome (Leica), compressed air tank (CINE EDC GEAR), Seq2000 sequencer (MGI);

[0090] Experimental conditions setting:

[0091] Experiment 1: Whole-brain frozen sections of a mouse brain were mounted on a chip. After multiplexing (incubation at 55°C for 1 hour), the subsequent Stereo-seq spatiotemporal transcriptome process was directly performed, and sequencing was performed to obtain results. A control group was set up as a positive control without any multiplexing after mounting.

[0092] Experiment 2: All chips were multiplexed (incubated at 55°C for 1 hour). The treated chips were then incubated with mouse testis total free RNA, followed by Stereo-seq spatiotemporal transcriptome sequencing. A control group served as a positive control without multiplexing.

[0093] Experimental process:

[0094] Experiment A process:

[0095] Experimental group: Spatiotemporal transcriptome bare chip attached to whole-brain slices of mouse brain → multiplexing protocol → conventional spatiotemporal transcriptome workflow → sequencing → results;

[0096] Control group: Spatiotemporal transcriptome bare chip attached to whole-brain slices of mice → conventional spatiotemporal transcriptome process → sequencing → results;

[0097] Experiment B process:

[0098] Experimental group: Spatiotemporal transcriptome bare chip after multiplexing protocol → chip incubation of mouse testis Total Free RNA → conventional spatiotemporal transcriptome process → sequencing → results;

[0099] Control group: Total Free RNA from mouse testes incubated on chip → conventional spatiotemporal transcriptome workflow → sequencing → results.

[0100] Result analysis:

[0101] The results of Experiment A (Table 1, Figure 1 ) proved that the use of urea-containing reuse schemes can achieve the effect of removing old slices (mouse brain). The results of Experiment B (Table 2, Figure 2 ) demonstrated that the use of a multiplexing scheme containing urea did not reduce chip capture. The unique reads (M), Bin 20 MID (mean / median), and Bin 20 Gene (mean / median) values ​​for all experimental groups were comparable to those of the control chip. These two experiments demonstrate the feasibility of the urea-containing multiplexing scheme.

[0102] Table 1 Comparison of tissue removal effects of urea reuse schemes

[0103]

[0104] Table 2 Comparison of chip capture after urea multiplexing scheme

[0105]

[0106] Example 2: Verification of the application of the SDS multiplexing scheme to the BGI Stereo-seq spatiotemporal transcriptome chip Chip T

[0107] Experimental materials: mouse brain and mouse testis (Guangdong Medical Experimental Animal Center), 100% anhydrous methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5X SSC, 0.1X SSC, cover glass (Shetai), 100% glycerol (Diamond), Stereo-seq transcriptome kit T (BGI), STOmics library preparation kit-S1 (BGI), Seq2000 sequencing kit PE100 (MGI), RNeasy Micro Kit (Qiagen), SDS, washing buffer (Tris-HCl pH 7.0), NF water, Stereo-seq spatiotemporal transcriptome chip Chip T (BGI).

[0108] Experimental instruments: microtome (Leica), compressed air tank (CINE EDC GEAR), Seq2000 sequencer (MGI)

[0109] Experimental condition setting:

[0110] Experiment 1: Mouse brain whole brain frozen sections were pasted on the chip, and after multiplexing treatment (incubation condition: 55°C, 1 hour), the subsequent Stereo-seq spatiotemporal transcriptome process was directly performed, sequencing was then performed, and results were obtained. The control group set was a positive control that was not subjected to any multiplexing treatment after pasting.

[0111] Experiment 2: All chips were subjected to multiplexing treatment (incubation condition: 55°C, 1 hour). After treatment, the chip was incubated with mouse testis Total Free RNA, and then the Stereo-seq spatiotemporal transcriptome process was performed, sequencing was then performed, and results were obtained. The control group set was a positive control that was not subjected to multiplexing treatment.

[0112] Experimental procedure:

[0113] Experimental procedure A:

[0114] Experimental group: spatiotemporal transcriptome bare chip pasted with mouse brain whole brain sections → use multiplexing scheme → perform conventional spatiotemporal transcriptome process → sequencing → results;

[0115] Control group: spatiotemporal transcriptome bare chip pasted with mouse brain whole brain sections → perform conventional spatiotemporal transcriptome process → sequencing → results;

[0116] Experimental B procedure:

[0117] Experimental group: Reuse solution with SDS → Incubate mouse testis Total Free RNA on chip → Perform regular Stereo-seq procedure → Sequencing → Results;

[0118] Control group: Incubate mouse testis Total Free RNA on chip → Perform regular Stereo-seq procedure → Sequencing → Results.

[0119] Results analysis

[0120] Results of Experiment A (Table 3, Figure 3 ) demonstrate that the reuse solution with SDS can remove the old section (mouse brain) successfully. Results of Experiment B (Table 4, Figure 4 ) demonstrate that the reuse solution with SDS does not reduce the chip capture, all experimental groups have unique reads (M), Bin 20 MID (average / median) and Bin 20 Gene (average / median) not lower than the control group chip. The above two experiments demonstrate the feasibility of the reuse solution with SDS.

[0121] Table 3 Tissue removal effect comparison of SDS reuse solution

[0122] No treatment control 20% SDS solution 10% SDS solution 2% SDS solution Chip number D03174C4 A02484C5 A02484C6 A02484C2 Total Reads(M) 404.45 395.92 369.26 357.73 Unique Reads(M) 113.44 33.37 12.22 5.50 Duplication Rate (%) 25.3 64.0 81.1 81.9 Bin 20MID (mean / median) 235 / 210 34.93 / 24 12.74 / 9 5.86 / 4 Bin 20Gene (mean / median) 195 / 177 21.81 / 16 6.96 / 5 3.71 / 3

[0123] Table 4 Chip capture comparison after SDS reuse solution treatment

[0124]

[0125] Example Three: Verification of composite denaturant reuse solution applied to BGI Stereo-seq spatiotemporal transcriptome chip Chip T

[0126] Experimental materials: Mouse brain and mouse testis (Guangdong Medical Experimental Animal Center), 100% anhydrous methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5X SSC, 0.1X SSC, cover glass (Shetai), 100% glycerol (Diamond), Stereo-seq transcriptome kit T (BGI), STOmics library preparation kit-S1 (BGI), Seq2000 sequencing reagent kit PE100 (MGI), RNeasy Micro Kit (Qiagen), composite denaturant (urea + SDS), washing buffer (Tris-HCl pH 7.0), NF water, Stereo-seq spatiotemporal transcriptome chip Chip T (BGI).

[0127] Experimental instruments: microtome (Leica), compressed air tank (CINE EDC GEAR), Seq2000 sequencer (MGI)

[0128] Experimental conditions setting:

[0129] Experiment 1: Whole-brain frozen sections of a mouse brain were mounted on a chip. After multiplexing (incubation at 55°C for 1 hour), the subsequent Stereo-seq spatiotemporal transcriptome process was directly performed, and sequencing was performed to obtain results. A control group was set up as a positive control without any multiplexing after mounting.

[0130] Experiment 2: All chips were multiplexed (incubated at 55°C for 1 hour). The treated chips were then incubated with mouse testis total free RNA, followed by Stereo-seq spatiotemporal transcriptome sequencing. A control group served as a positive control without multiplexing.

[0131] Experimental process:

[0132] Experiment A process:

[0133] Experimental group: Spatiotemporal transcriptome bare chip attached to whole-brain slices of mouse brain → use multiplexing scheme → perform conventional spatiotemporal transcriptome process → sequencing → results;

[0134] Control group: Spatiotemporal transcriptome bare chip attached to whole-brain slices of mice → conventional spatiotemporal transcriptome process → sequencing → results;

[0135] Experiment B process:

[0136] Experimental group: Spatiotemporal transcriptome bare chip after multiplexing protocol → chip incubation of mouse testis Total Free RNA → conventional spatiotemporal transcriptome process → sequencing → results;

[0137] Control group: Total Free RNA from mouse testes incubated with microarray → conventional spatiotemporal transcriptome workflow → sequencing → results;

[0138] Result Analysis

[0139] The results of Experiment A (Table 5, Figure 5 ) proved that the multiplexing scheme using the composite denaturant can achieve the effect of removing the old slices (mouse brain) well. Figure 6) demonstrated that the multiplexing scheme using a combined denaturant did not reduce chip capture. The unique reads (M), Bin 20 MID (mean / median), and Bin 20 Gene (mean / median) values ​​for all experimental groups were comparable to those of the control chip. These two experiments demonstrate the feasibility of the combined denaturant multiplexing scheme.

[0140] Table 5 Comparison of tissue removal effects of multiplex denaturant schemes

[0141]

[0142]

[0143] Table 6 Comparison of chip capture after multiplexing with multiple denaturants

[0144]

[0145] Example 4: Validation of the Citicoline Sodium Multiplexing Scheme on the BGI Stereo-seq Spatiotemporal Transcriptome Chip T

[0146] Experimental materials: mouse brain and mouse testis (Guangdong Medical Laboratory Animal Center), 100% anhydrous methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5X SSC, 0.1X SSC, coverslips (Shitai), 100% glycerol (Diamond), Stereo-seq Transcriptome Kit T (BGI), STOmics Library Preparation Kit-S1 (BGI), Seq2000 Sequencing Kit PE100 (MGI), RNeasy Micro Kit (Qiagen), citicoline sodium, urea, wash buffer (Tris-HCl pH 7.0), NF water, Stereo-seq spatiotemporal transcriptome chip Chip T (BGI);

[0147] Experimental equipment: microtome (Leica), compressed air tank (CINE EDC GEAR), Seq2000 sequencer (MGI);

[0148] Experimental conditions setting:

[0149] Total-free RNA from mouse liver was incubated on the chip and first treated with a nucleic acid denaturant (8M urea or 3% citicoline sodium) for 1 hour (55°C), followed by treatment with 3% citicoline sodium for another 1 hour (two testing protocols). The treated chip was directly processed for the subsequent Stereo-seq spatiotemporal transcriptomics workflow, followed by sequencing and results. A positive control was set up, in which the chip was incubated with total-free RNA and no further treatment was performed.

[0150] Experimental process:

[0151] Experimental group: Total Free RNA from mouse liver incubated on a spatiotemporal transcriptome bare chip → multiplexing protocol → conventional spatiotemporal transcriptome workflow → sequencing → results;

[0152] Control group: Total Free RNA of mouse liver incubated on a bare spatiotemporal transcriptome chip → conventional spatiotemporal transcriptome workflow → sequencing → results.

[0153] Result Analysis

[0154] The experimental results are shown in Table 7 and Figure 7 As shown, it was shown that the multiplexing scheme containing 3% citicoline sodium was able to remove hybridized nucleic acid molecules. Compared with the untreated control group, the capture amount was significantly reduced, proving the feasibility of the multiplexing scheme containing citicoline sodium.

[0155] Table 7

[0156]

[0157] Example 5: Multiplexing Application of BGI Stereo-seq Spatiotemporal Transcriptome Chip T

[0158] Experimental materials: rat eyeballs (Guangdong Medical Experimental Animal Center), mouse liver (Guangdong Medical Experimental Animal Center), 100% anhydrous methanol (Sigma-Aldrich), Qubit™ ssDNA Reagent (Invitrogen), 5X SSC, 0.1X SSC, coverslips (Shitai), 100% glycerol (Diamond), STOmics permeabilization kit-S1, STOmics library preparation kit-S1 (BGI), Seq2000 sequencing kit PE100 (MGI), RNeasy Micro Kit (Qiagen), denaturants (formamide, urea and SDS), washing buffer (Tris HCl, pH 7.0), NF water, BGI Stereo-seq spatiotemporal transcriptome chip Chip T.

[0159] Experimental instruments: microtome (Leica), compressed air tank (CINE EDC GEAR), Seq2000 sequencer (MGI)

[0160] Experimental conditions setting:

[0161] The chip was first loaded with a rat eyeball, a difficult-to-remove tissue section. After treatment with a multiplexing protocol containing a combined denaturant (4M urea + 1% SDS) (incubation at 55°C for 2 hours), the entire chip was incubated with mouse liver Total Free RNA. The difference between the tissue species used for the initial application and those used after multiplexing facilitated comparison of the residual rate of the old tissue section on the chip. The use of Total Free RNA provided a more comprehensive view of the effect of multiplexing on the overall chip capture rate. A control was a new transcriptome chip that had not been multiplexed. To compare the multiplexing effect, a multiplexing protocol containing formamide as a nucleic acid denaturant and SDS as a protein denaturant was added as a control. The chip was first treated with formamide for 1 hour, followed by 2% SDS for 1 hour.

[0162] Experimental process:

[0163] Experimental group: Spatiotemporal transcriptome bare chip attached to rat eye patch → fixed for 5 minutes → multiplexing protocol → chip incubated with mouse liver Total Free RNA → conventional spatiotemporal transcriptome workflow → sequencing → results;

[0164] Control group: Total Free RNA from mouse liver incubated on a spatiotemporal transcriptome bare chip → conventional spatiotemporal transcriptome workflow → sequencing → results;

[0165] Result Analysis

[0166] The results are shown in Table 8. Figures 8 to 10As shown, the removal effect of the composite denaturant multiplexing scheme provided by the present invention is obvious and the old slices (rat eyeballs) remain less, and the rat eyeball expression gene remains less, which is closer to the control without multiplexing treatment. The multiplexing scheme containing formamide treatment has a poor removal effect and the old slices (rat eyeballs) remain less, and the rat eyeball expression gene remains more. Among the three samples with very similar sequencing data amounts, the Bin 20 average and median MID number (152.02 / 142) and Gene number (94.32 / 89) of the multiplexing scheme group of the present invention are closer to the MID number (152.98 / 144) and Gene number (93.65 / 90) of the untreated control, and the multiplexing scheme containing formamide treatment has more in these two data amounts (MID number: 166.56 / 159, Gene number: 100.81 / 98), suggesting that there are more old slices remaining. As shown in Table 7, the average MID number of Bin 200 (Duplication rate = 50%), compared with the untreated control, decreased by only (5.15-4.75) / 5.15×100%=7.77% in the multiplexing scheme of the present invention, which is less than 10%, demonstrating that the composite denaturant multiplexing scheme of the present invention has little loss on the chip capture rate.

[0167] Table 8 Comparison of chip reuse solutions

[0168]

[0169] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0170] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A reagent, characterized in that include: At least one of urea, sodium lauryl sulfate and citicoline sodium.

2. The reagent according to claim 1, characterized in that The concentration of urea in the reagent is 1M to 8M; Optionally, the mass volume fraction (g / mL) of the sodium dodecyl sulfate in the reagent is 1% to 20%; Optionally, the mass volume fraction (g / mL) of the citicoline sodium in the reagent is 1% to 10%.

3. The reagent according to claim 1, characterized in that The reagent comprises urea and sodium dodecyl sulfonate, wherein the usage ratio of the urea and sodium dodecyl sulfonate is (0.1-0.8) mol: (1-20) g, preferably (0.1-0.4) mol: (1-2) g; Optionally, the reagent comprises urea and citicoline sodium, and the usage ratio of urea to citicoline sodium is (0.1-0.8) mol: (1-10) g, preferably (0.4-0.8) mol: (3-4) g; Optionally, the reagent includes urea, sodium dodecyl sulfate and citicoline sodium, and the usage ratio of urea, sodium dodecyl sulfate and citicoline sodium is (0.1-0.8) mol: (1-10) g: (1-10) g, preferably (0.4-0.8) mol: (1-5) g: (2-6) g, and more preferably (0.4-0.8) mol: (1-2) g: (3-4) g.

4. A reagent set, characterized in that: include: A first reagent, wherein the first reagent is as defined in any one of claims 1 to 4; Optionally, further comprising: a second reagent, the second reagent comprising an acid-base buffer; Optionally, the pH of the acid-base buffer solution is selected from 6.5 to 7.5, preferably 7; Preferably, the acid-base buffer comprises Tris-HCl; Optionally, the method further comprises: a third reagent, wherein the third reagent comprises nuclease-free water.

5. A kit, characterized in that include: The reagent according to any one of claims 1 to 3 or the reagent set according to claim 4; Optionally, at least one of anhydrous methanol, sodium citrate buffer and glycerol is further included.

6. Use of the reagent according to any one of claims 1 to 3 or the reagent set according to claim 4 in preparing a kit for restoring the function of a biological carrier; Optionally, the biological carrier is selected from a biochip; Optionally, the surface of the biochip is modified with nucleic acid probes and / or proteins.

7. A method for restoring the function of a biological carrier, characterized in that: include: performing a first incubation treatment on the functional biological carrier to be restored in a restoration agent to restore the function of the biological carrier; Wherein, the reconstitution reagent is selected from at least one of urea, sodium lauryl sulfate and citicoline sodium.

8. The method according to claim 7, characterized in that The biological carrier is selected from a biochip; Optionally, the surface of the biochip is modified with nucleic acid probes and / or proteins.

9. The method according to claim 7, characterized in that The reconstitution reagent includes urea, and the concentration of urea in the reconstitution reagent is 1M to 8M; Optionally, the reconstitution reagent comprises sodium dodecyl sulfate, and the mass volume fraction (g / mL) of the sodium dodecyl sulfate in the reconstitution reagent is 1% to 20%; Optionally, the reconstitution reagent comprises urea and sodium dodecyl sulfonate, and the usage ratio of the urea to the sodium dodecyl sulfonate is (0.1-0.8) mol: (1-20) g, preferably (0.1-0.4) mol: (1-2) g; Optionally, the reconstitution reagent comprises urea and citicoline sodium, and the usage ratio of urea to citicoline sodium is (0.1-0.8) mol: (1-10) g, preferably (0.4-0.8) mol: (3-4) g; Optionally, the reconstitution reagent comprises urea, sodium dodecyl sulfate and citicoline sodium, and the usage ratio of urea sodium dodecyl sulfate and citicoline sodium is (0.1-0.8) mol: (1-10) g: (1-10) g, preferably (0.4-0.8) mol: (1-5) g: (2-6) g, and more preferably (0.4-0.8) mol: (1-2) g: (3-4) g.

10. The method according to claim 7, characterized in that The incubation temperature is selected from 20°C to 65°C, preferably 50°C to 60°C; Optionally, the incubation time is selected from 0.5 hours to 4 hours, preferably 1 hour to 3 hours.

11. The method according to claim 7, characterized in that The method further comprises cleaning the incubation product; Optionally, the cleaning process includes a first cleaning process and a second cleaning process; Optionally, the first washing treatment is performed using a washing buffer; Optionally, the wash buffer comprises Tris-HCl; Optionally, the pH of the washing buffer is selected from 6.5 to 7.5, preferably 7; Optionally, the second washing treatment is performed using nuclease-free water.

12. Use of the reagent according to any one of claims 1 to 3 or the reagent set according to claim 4 in spatial omics sequencing and / or in situ hybridization.

13. A spatial omics sequencing method, characterized in that: include: Restoring the function of the biological carrier using the method according to any one of claims 7 to 11; performing a second incubation treatment on the nucleic acid sample to be tested and the functionally restored biological carrier; The incubation products are subjected to library construction and sequencing to obtain spatial information and sequence information of the nucleic acid sample to be tested.

14. An in situ hybridization method, characterized in that: include: Restoring the function of the biological carrier using the method according to any one of claims 7 to 11; performing a third incubation treatment on the nucleic acid sample to be tested and the functionally restored biological carrier; performing hybridization treatment on the third incubation treatment product using a nucleic acid probe; Optionally, the method further comprises: performing detection processing on the hybridization processing product.

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