Coloring agent and application thereof in sequencing of Stereo-seq space transcriptome

Abstract

The invention relates to the technical field of sequencing of a Stereo-seq space transcriptome, in particular to a coloring agent and application of the coloring agent in sequencing of the Stereo-seq space transcriptome. The staining agent provided by the invention comprises two reagents which are used for staining a cell nucleus and a cell wall respectively, and when the staining agent is applied to tissue chip imaging and / or Stereo-seq space transcriptome sequencing, cellbin data can be quickly provided for plants, manual operation is reduced, the expression data zoning precision is improved, a clustering annotation result is more reliable, and the method has the advantages that the method is simple and convenient to operate, and the staining agent is suitable for large-scale popularization and application in the field of tissue chip imaging and / or Stereo-seq space transcriptome sequencing. A more accurate scientific result is mined; and a basis is established for a plant automation space transcriptome data analysis process.

Description

Technical Field

[0001] The present invention relates to the technical field of Stereo-seq spatial transcriptome sequencing, and in particular to a dye and an application thereof in Stereo-seq spatial transcriptome sequencing. Background Art

[0002] Spatial transcriptome sequencing can combine microscopic imaging and sequencing technology to obtain gene expression data while retaining the spatial location information of the sample to the greatest extent. It provides an important research tool in many fields such as tissue cell function, microenvironment interaction, developmental lineage tracing, disease pathology, etc.

[0003] Stereo-seq spatial transcriptome sequencing technology is a high-throughput spatiotemporal sequencing technology that uses two sequencings to confirm the spatial position and corresponding expression of mRNA sequences. It has now been applied in multiple biological and medical fields and has become one of the mainstream methods of spatial transcription sequencing. It will provide detailed spatial information on the basis of traditional transcriptome sequencing, helping researchers to identify the location of transcription within tissues, accurate to the cellular level or even higher resolution, which will promote researchers' understanding and interpretation of single cells and cell groups within the entire tissue. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a dye and its application in Stereo-seq spatial transcriptome sequencing. Based on the dye and dyeing scheme provided by the present invention, plant cell walls and track line signals can be captured simultaneously in one imaging, providing accurate cell contours for plants while automatically aligning with the expression matrix based on the track line without manual integration.

[0005] The dye provided by the present invention comprises: fluorescent dye A and fluorescent dye B;

[0006] The fluorescent stain A is a cell nucleus stain;

[0007] The fluorescent dye B is a cell wall dye or a cell membrane dye.

[0008] In an embodiment of the present invention, the cell nucleus stain is a fluorescent dye for the cell nucleus, selected from at least one of acridine orange, ethidium bromide, propidium iodide, DAPI, Hoechst dye, EthD III, 7-AAD, RedDot1 or RedDot 2. In some embodiments, the fluorescent stain A is DAPI dye.

[0009] The DAPI is 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, also known as DAPI dihydrochloride, with a molecular formula of C 16 H 15 N5·2HCl, with a molecular weight of 350.25, is a blue fluorescent dye that can penetrate cell membranes and can produce fluorescence that is 20 times stronger than DAPI itself after binding to double-stranded DNA. In the present invention, the DAPI dye is a DAPI staining solution, the solvent of which is water, and the mass fraction of DAPI is 1 / 5000.

[0010] In an embodiment of the present invention, the cell wall stain or cell membrane stain is a fluorescent stain selected from: calcium fluorescent white dye, DiD dye, DiR dye, DiO dye or DiI dye. In some embodiments, the fluorescent stain B is calcium fluorescent white dye.

[0011] Calcofluor White (CW) is a non-specific fluorescent brightening dye that binds to β-linked glucosides, such as cellulose and chitin. Its trade name is Fluorescent Brightener 28 (FB28 for short). In the present invention, the Calcofluor White dye is a dyeing solution of FB28, the solvent of which is anhydrous ethanol, and the mass fraction of FB28 is 2%.

[0012] In the embodiment of the present invention, the volume ratio of the fluorescent dye A to the fluorescent dye B is 50:(0.5-2.5).

[0013] In some embodiments, the volume ratio of the fluorescent dye A to the fluorescent dye B is 50:0.5, or 50:1.0, or 50:1.5, or 50:2.0, or 50:2.5.

[0014] In the embodiment of the present invention, the volume fraction of the fluorescent dye A is 50%; the volume fraction of the fluorescent dye B is 0.5% to 2.5%.

[0015] In some embodiments, the volume fraction of the fluorescent dye B is 0.5%, or 1.0%, or 1.5%, or 2.0%, or 2.5%.

[0016] In the dye of the present invention, the volume fraction of the RNase inhibitor is 1% to 10%.

[0017] In some embodiments, the volume fraction of the RNase inhibitor is 5%.

[0018] In some specific embodiments, every 100 μL of the staining agent includes: 50 μL of DAPI dye, 0.5-2.5 μL of calcium fluorescent white dye and 5 μL of RNase inhibitor.

[0019] In a specific embodiment, every 100 μL of the staining agent includes: 50 μL of DAPI dye, 0.5 μL of calcium fluorescent white dye and 5 μL of RNase inhibitor.

[0020] In another specific embodiment, every 100 μL of the staining agent includes: 50 μL of DAPI dye, 1.0 μL of calcium fluorescent white dye and 5 μL of RNase inhibitor.

[0021] In a specific embodiment, every 100 μL of the staining agent includes: 50 μL of DAPI dye, 1.5 μL of calcium fluorescent white dye and 5 μL of RNase inhibitor.

[0022] In a specific embodiment, every 100 μL of the staining agent includes: 50 μL of DAPI dye, 2.0 μL of calcium fluorescent white dye and 5 μL of RNase inhibitor.

[0023] In a specific embodiment, every 100 μL of the staining agent includes: 50 μL of DAPI dye, 2.5 μL of calcium fluorescent white dye and 5 μL of RNase inhibitor.

[0024] Plant samples can be analyzed at the cellbin level based on ssDNA and FB staining, using the track lines provided by the Stereo-seq technology itself and the cell outlines provided by the cell wall fluorescent staining map. However, this technology requires two staining photos and manual integration of the two images to indirectly achieve alignment with the expression matrix, and it is impossible to quickly and efficiently achieve plant Cellbin analysis. The dye provided by the present invention can capture plant cell wall and track line signals at the same time in one imaging, provide plants with accurate cell outlines, and automatically align with the expression matrix based on the track line without manual integration. The efficiency and accuracy of obtaining plant Cellbin information is greatly improved.

[0025] Experiments show that the best staining effect is achieved when the volume ratio of the fluorescent dye A to the fluorescent dye B is 50:(0.5-2.5), and the degree of interference with the cell nucleus is minimal when the volume ratio of the fluorescent dye A to the fluorescent dye B is 50:2.

[0026] Further, the present invention also provides the use of the aforementioned stain in tissue chip imaging and / or Stereo-seq spatial transcriptome sequencing. In the present invention, the cells in the tissue chip are cells containing cell walls. In some embodiments, the tissue chip is a plant tissue chip.

[0027] Furthermore, the present invention also provides a method for presenting an image of a tissue chip, which comprises: staining the tissue slice with the stain as described above, and then performing fluorescence microscopic imaging.

[0028] The tissue chip imaging method of the present invention is a plant tissue chip imaging method.

[0029] The preparation of tissue sections includes: embedding plant tissues, slicing, pasting, baking at 37° C. for 3 minutes, and fixing in formaldehyde.

[0030] The staining includes: covering the tissue on the chip with the stain as described above, and washing after staining in the dark. The amount of the stain is 100 μL / chip. The staining conditions include: staining at room temperature in the dark for 2 minutes. The washing uses 0.1×SSC buffer containing 5% RNase Inhibitor. The volume of the buffer used for washing is the same as the volume of the stain.

[0031] After the dyeing and before the fluorescent fiber is imaged, glycerol is covered on the fixed chip. The amount of glycerol is 5 μL / chip.

[0032] In the present invention, the fluorescence microscopy imaging adopts an epifluorescence scanning mode, and obtains cell boundary information and track line signals through the DAPI channel.

[0033] DAPI dye emits light after combining with DND on the chip, while the track line on the chip does not emit light. The non-luminescent track line is contrasted by the combination of DAPI and DNB, thereby obtaining the track line signal.

[0034] The cell boundary information is obtained by fluorescence microscopy imaging of the fluorescence emitted after the dye combines with the cell wall or cell membrane of the cell.

[0035] Specifically, the fluorescence microscopy imaging includes: transferring the glass slide carrying the chip to the microscope scanning stage, creating a new folder to save the new image, opening the fluorescence microscope control system, selecting the epifluorescence scanning mode, selecting the DAPI channel, creating a new slice, creating a map with a 4x lens, manually scanning with a 10x lens, quickly adding model points, and adjusting the exposure to scan the entire chip.

[0036] More specifically, the model points are set as follows: 1-2 inside the tissue, based on the clarity of the tissue; 4 points evenly distributed outside the tissue at the four corners of the chip, and the focus is based on the clarity of the track line.

[0037] Furthermore, the present invention also provides a Stereo-seq spatial transcriptome sequencing method, which comprises:

[0038] The tissue microarray was imaged as described above;

[0039] Perform spatiotemporal sequencing to obtain expression data;

[0040] After automatically registering the fluorescent staining image and the expression level image, cell segmentation is performed to form cellbin data.

[0041] In the present invention, the automatic registration includes: performing registration based on track line information shared by the fluorescent staining image and the expression amount, and obtaining cell wall boundary information registered with the expression amount data.

[0042] Specifically, the automatic registration includes: importing the fluorescent staining image and the expression image as described above into the TrakEM2 plug-in in ImageJ, using the track line information shared by the fluorescent staining image and the expression as described above, adjusting the translation, rotation and scaling parameters to achieve accurate registration of the two, and directly obtaining the cell wall boundary information registered with the expression data.

[0043] In the present invention, the expression data include the position information, gene expression level and cell type of each site on the chip carrying the tissue sample.

[0044] In the present invention, the cell segmentation is to obtain the cell outline through the fluorescence microscopy image, and obtain the cellbin data according to the cell outline. In some embodiments, the cell outline is the cell wall outline of the cell.

[0045] Specifically, the acquisition of the cellbin data includes: performing cell segmentation on the fluorescent staining image as described above, obtaining the outline of each cell, and obtaining the corresponding expression amount information based on the outline information to form Cellbin data for subsequent analysis.

[0046] Furthermore, the present invention also provides a Stereo-seq spatial transcriptome sequencing device, comprising:

[0047] Fluorescence imaging module, used for fluorescence imaging of tissue sections to obtain cell wall and track line signals;

[0048] Sequencing module, used for spatiotemporal sequencing to obtain expression data;

[0049] Analysis module, used for automatic registration and acquisition of cellbin data.

[0050] Furthermore, the present invention also provides a storage medium, characterized in that: the storage medium stores computer-readable instructions, and when the computer-readable instructions are executed by one or more processors, the one or more processors execute the steps of the Stereo-seq spatial transcriptome sequencing method as described above.

[0051] Furthermore, the present invention also provides a computer device, characterized in that it includes: one or more processors, and a memory; the memory stores computer-readable instructions, and when the computer-readable instructions are executed by the one or more processors, the steps of the Stereo-seq spatial transcriptome sequencing method as described above are performed.

[0052] The stain provided by the present invention includes two reagents, which stain the cell nucleus and the cell wall respectively. The stain is used in tissue chip imaging and / or Stereo-seq spatial transcriptome sequencing, which can quickly provide cellbin data for plants, reduce manual operations, improve the accuracy of expression data zoning, make clustering annotation results more reliable, and mine more accurate scientific results; and lay the foundation for the automated spatial transcriptome data analysis process of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Shows the imaging effect after staining with 1% FB dye;

[0054] Figure 2 Shows the imaging effect after staining with 5% FB dye;

[0055] Figure 3 Shows the imaging effect after staining with 4% FB dye;

[0056] Figure 4 The track line is shown;

[0057] Figure 5 The analysis results under Bin50 are shown;

[0058] Figure 6 Show the analysis results under cellbin;

[0059] Figure 7 Shows the mapping back to the mask effect;

[0060] Figure 8 14 shows a flow chart of the fluorescent staining scheme of the present invention. DETAILED DESCRIPTION

[0061] The present invention provides a dye and its application in Stereo-seq spatial transcriptome sequencing. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0062] The stain provided by the present invention is mainly used for sample staining based on Stereo-seq spatial transcriptome sequencing technology, wherein fluorescent stain A is a nuclear stain and fluorescent stain B is a cell wall stain. The stain of the present invention can be photographed using the DAPI channel of a microscope, and the plant cell wall and track line signals can be obtained by imaging at one time. In the stain provided by the present invention, the two are stained together to achieve accurate alignment. In the present invention, fluorescent stain A and fluorescent stain B can be mixed or exist independently of each other, and the present invention does not limit this. In order to obtain a better staining effect, in an embodiment of the present invention, fluorescent stain A and fluorescent stain B exist independently of each other.

[0063] The RNaseInhibitor in the stain of the present invention is used to prevent nucleic acid degradation. The present invention does not limit the type of solvent in the stain, and any reagent that can dissolve and / or suspend fluorescent stain A and fluorescent stain B can be used in the cell stain provided by the present invention. In some specific embodiments of the present invention, the solvent is water, more specifically Nuclease free water. In the embodiments of the present invention, Nuclease free water and RNase Inhibitor are used as examples to verify the feasibility of the method of the present invention.

[0064] The staining agent of the present invention is used for staining tissue chips. The reagents for staining tissue chips include the staining agent and chip cleaning solution as described above. The chip cleaning solution is used to keep the chip clean and moist. The present invention does not limit the type of cleaning reagent. In some specific embodiments, 0.1×SSC buffer containing 5% RNase inhibitor is used as the chip cleaning solution.

[0065] The stain of the present invention is used for imaging of tissue chips. The cell imaging reagent of the present invention comprises the stain, chip cleaning solution and imaging solution as described above. The imaging solution in the stain of the present invention is used for applying the tissue to the chip, forming a state in which there is no obvious liquid surface protrusion and the chip is closely attached.

[0066] The cells applicable to the tissue staining reagent and / or the imaging reagent of the tissue chip of the present invention are cells containing cell walls, such as plant cells, bacteria, fungi or algae cells. In the embodiments of the present invention, plant cells are used as an example for verification. The plant cells are from the roots, stems, leaves, flowers, fruits or seeds of plants. The present invention is not limited to this.

[0067] In the present invention, there are track lines on the chip, and during the sequencing process, several fluorescent images can be taken sequentially for parts of a single chip. Several track lines can be distributed in the horizontal and vertical directions of the image of each field of view. The distribution pattern of the track lines on the chip has been specified at the beginning of the chip design. For example, assuming that there are 10 track lines in the vertical direction of the chip, the spacing between them is, for example, 1, 2, 3, 4, 5, 4, 3, 2, 1. Assuming that the position of the first track line is the starting point (the coordinate is 0), the horizontal axis (x-axis) coordinates of the above 10 track lines are 0, 1, 3, 6, 10, 15, 19, 22, 24, 25 respectively.

[0068] The test materials used in the present invention are all common commercial products, which can be purchased in the market. The present invention is further described below in conjunction with the embodiments. It should be noted that in the following embodiments, the somatic cells of Phalaenopsis are used as an example to verify the imaging and spatial transcriptome sequencing. The effects of imaging or transcriptome detection in other plant cells are similar.

[0069] Example

[0070] Stereo-seq spatial transcriptome sequencing was performed on Phalaenopsis somatic embryos. The specific steps are as follows:

[0071] 1. According to the spatiotemporal group technology SOP, plant tissues were embedded, sliced, mounted, baked at 37° for 3 min, and then immersed in formaldehyde.

[0072] 2. Take out the fixed tissue chip from methanol and transfer it to a culture dish with sealing film; after the methanol evaporates and the chip surface becomes dry, stain it in sequence.

[0073] 3. Prepare a mixed fluorescent dye solution (DF fluorescent dye solution) of DAPI and Fluorescent Brightener 28 (FB28) according to Table 1; add DF fluorescent dye solution (100 μL / chip) to cover the tissue on the chip, stain at room temperature in the dark for 2 minutes, and gently wash the chip once with 0.1×SSC (100 μL / chip; containing RNase Inhibitor, 5%).

[0074] Table 1 Formula of each group of dyes

[0075]

[0076] 4. Transfer the chip onto dust-free paper, fix the chip with tweezers in your left hand, and gently blow dry the chip with a compressed air bottle in your right hand; transfer the chip onto a glass slide, slowly drip glycerol in the center of the tissue (5 μL / chip), gently cover with a coverslip (avoid bubbles), and prepare for microscope scanning.

[0077] 5. Transfer the slide with the chip to the microscope scanning stage, create a new folder to save the new image. Open the fluorescence microscope control system, select the epifluorescence scanning mode; select the DAPI channel, create a new slice, create a map with a 4x lens, scan manually with a 10x lens, quickly add model points (1-2 in the tissue, based on clear tissue; 4 evenly distributed outside the tissue at the 4 corners of the chip, focus based on clear track lines), adjust the exposure and scan the entire chip.

[0078] 6. Compare the imaging results of different concentrations of FB dye, such as Figures 1 to 4 As shown in the results, the imaging of tissue microarray treated with 4% FB stain was least disturbed by the cell nucleus, and the staining effects of the other groups were similar. Figure 1 or Figure 2 Therefore, the concentration that was not interfered by the cell nucleus (4% FB) was selected as the concentration used for formal sequencing.

[0079] 7. Repeat the above steps with the selected dye solution in the formal experiment, and perform spatiotemporal sequencing to obtain expression data.

[0080] 8. Import the DF fluorescence staining image and expression level image into the TrakEM2 plug-in in ImageJ, and use the track line information shared by the DF fluorescence staining image and the expression level to adjust the translation, rotation and scaling parameters to achieve accurate alignment between the two, and directly obtain the cell wall boundary information aligned with the expression level data.

[0081] 9. Perform cell segmentation on the DF fluorescent staining image, obtain the outline of each cell, and obtain the corresponding expression information based on the outline information to form Cellbin data for subsequent analysis. The analysis results under Bin50 are as follows: Figure 5 , the analysis results under cellbin are as follows Figure 6 , the mapping back to the mask effect is as follows Figure 7 .

[0082] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Dyes, including: fluorescent stain A and fluorescent stain B; The fluorescent stain A is a cell nucleus stain; The fluorescent dye B is a cell wall dye or a cell membrane dye.

2. The dye according to claim 1, characterized in that The fluorescent dye A is DAPI dye, The fluorescent dye B is calcium fluorescent white dye.

3. The dye according to claim 1 or 2, characterized in that The volume ratio of the fluorescent dye A to the fluorescent dye B is 50:(0.5-2.5).

4. The dye according to claim 3, characterized in that The volume fraction of the fluorescent dye A is 50%; The volume fraction of the fluorescent dye B is 0.5% to 2.5%.

5. The dye according to any one of claims 1 to 4, characterized in that Each 100 μL of the staining agent includes: 50 μL of DAPI dye, 0.5-2.5 μL of calcium fluorescent white dye and 5 μL of RNase inhibitor.

6. Use of the stain according to any one of claims 1 to 5 in tissue chip imaging and / or Stereo-seq spatial transcriptome sequencing.

7. A method for presenting an image of a tissue chip, comprising: After staining the tissue section with the staining agent according to any one of claims 1 to 5, fluorescence microscopic imaging is performed.

8. The imaging method according to claim 7, characterized in that: The fluorescence microscopy imaging adopts an epifluorescence scanning mode, and obtains cell boundary information and track line signals through the DAPI channel.

9. A method for Stereo-seq spatial transcriptome sequencing, comprising: Imaging the tissue chip to obtain a fluorescent staining image, which includes track line information and cell boundary information; Perform spatiotemporal sequencing to obtain expression data; After automatically registering the fluorescent staining image and the expression level image, cell segmentation is performed to form cellbin data.

10. The method according to claim 9, characterized in that The automatic registration includes: performing registration based on track line information shared by the fluorescent staining image and the expression amount, and obtaining cell wall boundary information registered with the expression amount data.

11. The method according to claim 9, characterized in that The cell segmentation is to obtain the cell outline through the fluorescence microscopy image, and obtain cellbin data according to the cell outline.

12. A Stereo-seq spatial transcriptome sequencing device, characterized in that: include: Fluorescence imaging module, used for fluorescence imaging of tissue sections to obtain cell wall and track line signals; Sequencing module, used for spatiotemporal sequencing to obtain expression data; Analysis module, used for automatic registration and acquisition of cellbin data.

13. A storage medium, characterized in that: The storage medium stores computer-readable instructions, and when the computer-readable instructions are executed by one or more processors, the one or more processors execute the steps of the Stereo-seq spatial transcriptome sequencing method as described in any one of claims 9 to 11.

14. A computer device, characterized in that: include: One or more processors, and a memory; the memory stores computer-readable instructions, and when the computer-readable instructions are executed by the one or more processors, the steps of the Stereo-seq spatial transcriptome sequencing method as described in any one of claims 9 to 11 are performed.

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