Construction method and application of space transcriptomics library

Abstract

The invention provides a construction method and application of a spatial transcriptomics library. The construction method comprises the following steps: S1, carrying out in-situ reverse transcription on a cell sample or a tissue sample by using a photolysis primer to obtain a cDNA: mRNA compound; s2, carrying out ultraviolet irradiation on a specific space region of the cDNA: mRNA compound; s3, carrying out in-situ connection marking on the compound subjected to ultraviolet irradiation by utilizing a marking primer composition; s4, performing library construction on the compound subjected to in-situ connection labeling to obtain a spatial transcriptomics library; wherein the photolysis primer comprises oligonucleotide and a photosensitive substance which is connected with o-nitrobenzyl and is located at the 5'end of the oligonucleotide, the photolysis primer based on photochemical reaction is used for carrying out in-situ reverse transcription, accurate space positioning of target nucleic acid molecules can be achieved, and the accuracy and the resolution ratio of space transcriptomics analysis are greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of gene libraries, and in particular, to a method for constructing a spatial transcriptomics library and its application. Background Art

[0002] Life activities rely on the spatial cooperation of different cells and different bioactive molecules. Decoding the spatial distribution of various molecules or cells can effectively analyze interactions, detect pathological states, and discover drug targets. The detection of ribonucleic acid (RNA) can confirm the state, type, and functions of cells by analyzing the abundance of different transcripts, which is one of the core technologies of contemporary omics; spatial transcriptomics can simultaneously identify the types of RNA and their spatial distribution in tissues or cells, which is a frontier field in omics research in recent years.

[0003] Existing spatial transcriptomics includes in-situ capture, multi-round fluorescence imaging of in-situ hybridization, microdissection, etc. Among them, in-situ capture requires specific chips, and multi-round imaging and microdissection require complex instruments and experimental operations. Although these methods have their own characteristics, they are generally complex to use and the cost of a single sample is high, which hinders the widespread application and commercialization prospects of spatial transcriptomics in biological research. Taking Visium of the US sequencing leader company 10xGenomics as an example, its cost is as high as $3000 - $5000 per single sample, which is expensive and can only be used for frozen section samples, and it is completely unable to be applied to cultured cells or the study of transcript mechanism at subcellular resolution.

[0004] Therefore, how to provide a method for constructing a spatial transcriptomics library with relatively low cost, wide application range, and high accuracy is of great significance for the further research of future omics. Summary of the Invention

[0005] The main object of the present invention is to provide a method for constructing a spatial transcriptomics library and its application to solve the problem of low resolution in constructing a spatial transcriptomics library in the prior art.

[0006] To achieve the above object, according to the first aspect of the present invention, a method for constructing a spatial transcriptomics library is provided. The construction method includes: S1, performing in-situ reverse transcription on a cell sample or a tissue sample using a photocleavable primer to obtain a cDNA:mRNA complex; S2, performing ultraviolet light irradiation on a specific spatial region of the cDNA:mRNA complex; S3, performing in-situ ligation labeling on the complex after ultraviolet light irradiation using a labeled primer composition; S4, constructing a library for the complex after in-situ ligation labeling to obtain a spatial transcriptomics library; wherein, the photocleavable primer includes an oligonucleotide and a photosensitive substance located at the 5' end of the oligonucleotide and linked by o-nitrobenzyl.

[0007] Further, the construction method further includes: steps S2 and S3 can be repeated multiple times. After the completion of the previous step S3, the complex is washed and then the operation of the next step S2 is carried out; wherein, the labeled primer compositions used for each execution of steps S2 and S3 are different; preferably, at 65°C, washing is carried out using a PBS solution.

[0008] Further, the preparation method of the cell sample or the tissue sample includes: successively performing fixation, permeabilization, denaturation, pH adjustment and heating treatment on the cells or tissue sections attached to the carrier to obtain a cell sample or a tissue sample, and storing it at 4°C for standby; wherein, denaturation is carried out using a 0.1N hydrochloric acid solution; preferably, the thickness of the tissue section is 10 μm.

[0009] Further, the photosensitive substance includes: AlexaFluor 488, AlexaFluor 568, AlexaFluor 594, Cy3 or Cy5; preferably, the in-situ reverse transcription includes: pretreating the photocleavable primer and dNTP and then mixing them with the reverse transcription system to obtain an in-situ reverse transcription reaction system; before performing in-situ reverse transcription, preheating the cell sample or the tissue sample at 42°C. When the sample is a cell sample, dropping the in-situ reverse transcription reaction system onto a hydrophilic glass slide, and buckling the carrier with the attached cells onto the in-situ reverse transcription reaction system on the hydrophilic glass slide to form an in-situ reverse transcription reaction chamber, and performing in-situ reverse transcription reaction to obtain a cDNA:mRNA complex; when the sample is a tissue sample, directly dropping the in-situ reverse transcription reaction system onto the tissue sample adsorbed on the hydrophilic glass slide, and completely covering the tissue sample with the carrier to form an in-situ reverse transcription reaction chamber, and performing in-situ reverse transcription reaction to obtain a cDNA:mRNA complex; preferably, the in-situ reverse transcription reaction is carried out in a light-shielded environment.

[0010] Further, ultraviolet light illumination of a specific spatial region of the complex includes: placing the cDNA:mRNA complex under a microscope and positioning the specific spatial region to be observed; illuminating the specific spatial region with light having a wavelength of 405 nm or 365 nm.

[0011] Further, the labeled primer composition includes a splint sequence and a spatially encoded sequence; wherein, the spatially encoded sequence is linked to the nucleotide at the cleavage site of o-nitrobenzyl, and there is a partially complementary pairing region between the splint sequence and the spatially encoded sequence.

[0012] Further, in-situ ligation labeling includes: performing a ligation reaction on the complex after ultraviolet light illumination using the labeled primer composition; after the ligation reaction is completed, adding a PBS solution at 65 °C to terminate the ligation reaction; preferably, the temperature of the ligation reaction is 25 °C; preferably, the time of the ligation reaction is ≥1 h.

[0013] Further, S4 includes: lysing the complex subjected to in-situ ligation labeling to obtain an aqueous solution of cDNA:mRNA; using the cDNA in the aqueous solution of cDNA:mRNA as a template for second-strand synthesis, followed by first purification and in vitro transcription to obtain an RNA product; adding DNase to the RNA product and performing second purification; using the RNA product after the second purification as a template for reverse transcription to obtain a cDNA product; performing PCR amplification using the cDNA product as a template to obtain a spatial transcriptomics library.

[0014] To achieve the above object, according to the second aspect of the present invention, there is provided an application of a spatial transcriptomics library obtained by the above construction method in cell biology, medicine or developmental biology.

[0015] Further, the application includes: sequencing the spatial transcriptome library to obtain sequencing results; analyzing the gene expression level of the sequencing results and constructing a two-dimensional or three-dimensional spatial distribution map.

[0016] By applying the technical solution of the present invention, in-situ reverse transcription is carried out using a photocleavable primer based on a photochemical reaction, which can achieve precise spatial positioning of target nucleic acid molecules, greatly improving the accuracy and resolution of spatial transcriptomics analysis. Moreover, the construction method of the present application has the advantages of simple operation and low cost (the cost is as low as $10 per sample), which is suitable for researchers to perform spatial transcriptomics analysis of a large number of samples. Each sample only needs to undergo spatial structured illumination to construct a spatial transcriptomics library and perform data analysis, without complex instruments and image analysis processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings of the specification, which form a part of this application, are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0018] Figure 1 It shows a schematic flow diagram of the construction of a spatial transcriptomics library in this application.

[0019] Figure 2 It shows the result diagram of observing the cell sample in Example 1 of this application under a confocal microscope;

[0020] Figure 3 It shows the result diagram of the PCA dimensionality reduction analysis of the sequencing results of mitotic cells and interphase cells in Example 1 of this application;

[0021] Figure 4 It shows the result diagram of observing the cell sample in Example 2 of this application under a confocal microscope;

[0022] Figure 5 It shows the schematic result diagram obtained by statistically analyzing the species sources of transcripts of the sequencing results of the HEK293T cell sample and the Neuro-2a cell sample in Example 2 of this application;

[0023] Figure 6 It shows the result diagram of observing different subtypes of tissue samples in Example 3 of this application under a confocal microscope;

[0024] Figure 7 It shows the result diagram of the PCA dimensionality reduction analysis of the sequencing results of different subtypes of DG, CA1, and CA3 in Example 3 of this application;

[0025] Figure 8 It shows the result of the heat map analysis of gene differential expression of different subtypes of DG, CA1, and CA3 in Example 3 of this application. Detailed implementation manners

[0026] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0027] As mentioned in the background art, there are many defects in the construction of spatial transcriptomics libraries in the prior art, including high costs, complex experimental operations, high requirements for the skills of operators, easy loss of samples, limited applicable samples, low resolution, etc., which are not conducive to the development of research in this field. Therefore, this application aims to provide a method for constructing a spatial transcriptomics library with lower costs, simpler operations, wider applicability, and better resolution.

[0028] In the first typical embodiment of the present application, a method for constructing a spatial transcriptomics library is provided. The construction method includes: S1, performing in-situ reverse transcription on a cell sample or a tissue sample using a photocleavable primer to obtain a cDNA:mRNA complex; S2, performing ultraviolet light irradiation on a specific spatial region of the cDNA:mRNA complex; S3, performing in-situ ligation labeling on the complex after ultraviolet light irradiation using a labeled primer composition; S4, constructing a library from the complex after in-situ ligation labeling to obtain a spatial transcriptomics library; wherein, the photocleavable primer includes an oligonucleotide and a photosensitive substance located at the 5'-end of the oligonucleotide and linked by o-nitrobenzyl.

[0029] As Figure 1 The flowchart of the construction method of the present application shown, wherein the o-nitrobenzyl group in the photocleavable primer is active to short-wavelength light and undergoes cleavage under ultraviolet light irradiation, separating the photosensitive substance from the oligonucleotide and releasing the phosphate group protected by the nucleotide. Only the primer nucleotide phosphate groups in the specific spatial region irradiated by light are exposed and can be labeled subsequently to obtain sequencing information with spatial position characteristics. The above-mentioned use of the photoreaction in the photocleavable primer can capture the transcriptome information of a specific region simply without destroying the integrity of the sample, and it has good resolution, providing the possibility for the in-depth promotion of research in this field.

[0030] The samples applicable to the construction method of the present application include: cell samples cultured in vitro; normal tissue sections, including animal or plant tissues; pathological tissue samples, including tumor tissues, inflammatory tissues, or tissue samples in other pathological states. The pathological tissue samples can be frozen sections, paraffin-embedded sections, or precipitate samples in liquid biopsies.

[0031] The construction method of the present application can also perform multiple repeated labelings on different positions of the same sample. In a preferred embodiment, the construction method further includes: steps S2 and S3 can be repeated multiple times. After the previous step S3 is completed, the complex is washed and then the operation of the next step S2 is performed; wherein, the labeled primer compositions used for each performance of step S2 and step S3 are different. Any temperature suitable for washing is applicable to the present application. Preferably, at 65°C, the washing is performed using a PBS solution. Each ultraviolet light irradiation in the present application is only directed at a specific position, that is, except for the irradiated area in the current time, the o-nitrobenzyl in the photocleavable primer in other areas of the sample does not break and will not be labeled by subsequent primers. Therefore, as long as the sample after one-time light irradiation labeling is washed, subsequent multiple different position labelings can be performed on the same sample, thereby obtaining more comprehensive spatial transcriptome information with as little loss of the sample as possible.

[0032] To further ensure the structural integrity and RNA stability of each region in the sample, in a preferred embodiment, the method for preparing a cell sample or a tissue sample includes: successively performing fixation, permeabilization, denaturation, pH adjustment, and heating on the cells or tissue sections attached to the carrier to obtain a cell sample or a tissue sample, and storing it at 4°C for later use; wherein, 0.1N hydrochloric acid solution is used for denaturation. Any hydrochloric acid concentration suitable for protein denaturation is applicable to this application. Using hydrochloric acid for protein denaturation can further improve the efficiency of in-situ reverse transcription.

[0033] To further prepare a complete and stable sample material for in-situ reverse transcription, in a preferred embodiment, the cultured cell sample or the sectioned tissue sample is fixed with a formaldehyde solution (any suitable fixation time is applicable to this application, preferably 10 minutes), and then the fixed sample is permeabilized with a Triton solution (any suitable permeabilization time is applicable to this application, preferably 3 minutes). Then, the permeabilized sample is subjected to a de-crosslinking treatment with a hydrochloric acid solution (any suitable de-crosslinking treatment time is applicable to this application, preferably 5 minutes). Finally, the RNA secondary structure in the sample is eliminated by heating (any suitable temperature is applicable to this application, preferably 65°C) (any suitable treatment time is applicable to this application, preferably 10 minutes).

[0034] To further obtain a sample with a clear spatial structure that is easier to observe and obtain better-resolution spatial transcriptomics information, in a preferred embodiment, 1000, 100, 10, or 1 cell is selected from cultured cells (about 10 5 cells) for subsequent labeling; preferably, the thickness of the tissue section is 10 μm.

[0035] Any photosensitive substance that can be observed under a microscope is applicable to this application. In a preferred embodiment, the photosensitive substances include: AlexaFluor 488, AlexaFluor 568, AlexaFluor 594, Cy3, or Cy5.

[0036] To further perform in-situ reverse transcription efficiently and obtain better data for subsequent ultraviolet light irradiation and library construction, in a preferred embodiment, in-situ reverse transcription includes: pretreating the photocleavable primer and dNTP and then mixing them with the reverse transcription system to obtain an in-situ reverse transcription reaction system; before performing in-situ reverse transcription, the cell sample or the tissue sample is preheated at 42°C.

[0037] When the sample is a cell sample, the in-situ reverse transcription reaction system is dropped onto a hydrophilic glass slide, and the carrier with attached cells is inverted over the in-situ reverse transcription reaction system on the hydrophilic glass slide to form an in-situ reverse transcription reaction chamber, and an in-situ reverse transcription reaction is carried out to obtain a cDNA:mRNA complex;

[0038] When the sample is a tissue sample, the in-situ reverse transcription reaction system is directly dropped onto the tissue sample adsorbed on a hydrophilic glass slide, and the tissue sample is completely covered with a carrier to form an in-situ reverse transcription reaction chamber, and an in-situ reverse transcription reaction is carried out to obtain a cDNA:mRNA complex.

[0039] To further efficiently carry out the in-situ reverse transcription reaction, the time and temperature of any pretreatment that can obtain better reaction effects, as well as the time, temperature and humidity of the in-situ reverse transcription reaction, are applicable to this application. In a preferred embodiment, the pretreatment time is 5 min; preferably, the pretreatment temperature is 65 °C; in a preferred embodiment, the in-situ reverse transcription reaction time is 1 h; preferably, the in-situ reverse transcription reaction temperature is 42 °C; preferably, the humidity of the in-situ reverse transcription reaction is 100%. Preferably, the in-situ reverse transcription reaction is carried out in a light-shielded environment.

[0040] To more accurately locate the RNA in a specific spatial region, in a preferred embodiment, ultraviolet light irradiation of a specific spatial region of the complex includes: placing the cDNA:mRNA complex under a microscope and locating the specific spatial region to be observed; irradiating the specific spatial region with light having a wavelength of 405 nm or 365 nm. The selective cleavage ability of ultraviolet light irradiation on o-nitrobenzyl can perform precise spatial labeling of the sample at the subcellular level. Any high-power ultraviolet light source is applicable to this application. In a preferred embodiment, 405 nm laser, 365 nm laser, or 365 nm LED is selected for irradiation treatment. Among them, the higher the power density of the irradiation light, the shorter the irradiation time required, and the generally required irradiation time is between 5 seconds and 15 minutes.

[0041] To further distinguish the gene information in a specific spatial region, a labeled primer is ligated to the exposed phosphate group. In a preferred embodiment, the labeled primer composition includes a splinter sequence and a barcode sequence; wherein, the barcode sequence is connected to the nucleotide at the cleavage site of o-nitrobenzyl, and there is a partially complementary pairing region between the splinter sequence and the barcode sequence.

[0042] To further obtain a sample with a better labeling effect in a specific spatial region and improve the efficiency and accuracy of labeling. In a preferred embodiment, in-situ ligation labeling includes: performing a ligation reaction on the complex after ultraviolet light irradiation using a labeling primer composition; after the ligation reaction is completed, adding a PBS solution at 65 °C to terminate the ligation reaction; any reaction temperature and time that can obtain better ligation products are applicable to this application. In a preferred embodiment, the temperature of the ligation reaction is 25 °C; preferably, the time of the ligation reaction is ≥1 h.

[0043] To further efficiently obtain a spatial transcriptomics library. In a preferred embodiment, S4 includes: lysing the complex of in-situ ligation labeling to obtain a cDNA:mRNA aqueous solution; using the cDNA in the cDNA:mRNA aqueous solution as a template for second-strand synthesis, and sequentially performing first purification and in-vitro transcription to obtain an RNA product; adding DNase to the RNA product and performing second purification; using the RNA product after the second purification as a template for reverse transcription to obtain a cDNA product; performing PCR amplification using the cDNA product as a template to obtain a spatial transcriptomics library.

[0044] In the second typical embodiment of this application, an application of a spatial transcriptomics library obtained by using the above construction method in cell biology, medicine, or developmental biology is provided. Specifically, it can be the study of the interaction between cells and the microenvironment; the study of disease-related biomarkers; the study of the gene expression regulation mechanism during development; the establishment of a molecular database related to disease diagnosis or treatment.

[0045] In a preferred embodiment, the application includes: sequencing the spatial transcriptome library to obtain sequencing results; analyzing the gene expression level of the sequencing results and constructing a two-dimensional or three-dimensional spatial distribution map.

[0046] The following further describes this application in detail with specific examples, and these examples should not be construed as limiting the scope claimed in this application.

[0047] In the following examples, the reagents used are all commercially available, including the following suppliers:

[0048]

[0049] In the following examples, all primers used were synthetic DNA primers customized from Sangon Biotech, and the specific sequences are as follows: Among them, the 5' end of the photocleavage primer is linked to an iPClink (Photocleavage linker), and the other end of the iPClink is linked to an AF568 (Alexa Fluor 568). In addition, OPTImap is the Optical Precision TaggIng for spatially resolved RNA mapping technology (OPTImap) proposed in this application.

[0050]

[0051] Sample preparation before experiment

[0052] 1. Cell sample preparation

[0053] Place a glass slide with a diameter of 14 mm in a 24-well plate, add 100 μL of Matrigel solution (5% concentration, diluted with DMEM medium) and incubate overnight. When passaging the cells, wash them twice with 1×PBS solution, add 1 mL of cell culture medium, and passage them at a ratio of 1:30 according to the cell amount in a 6-cm dish. The confluence will reach 70%-80% after 24-48 hours, and approximately 1% of the cells are in the mitosis stage (Mitosis cells).

[0054] Remove the culture medium in the 24-well plate, wash it twice with 1×PBS, then add 4% aqueous formaldehyde solution and fix it at room temperature for 10 minutes. After fixation, remove the formaldehyde solution and wash it twice with 1×PBS again. Add a permeabilization solution (5% TritonX in 1×PBS) and permeabilize for 3 minutes, wash it twice with 1×PBS, add 0.1N hydrochloric acid solution to denature the proteins bound to RNA in the sample, and treat it at room temperature for 5 minutes. After removing the hydrochloric acid solution, add 1M Tris-HCl (pH8) solution to restore the acid-base balance of the system, then remove it and add 1×PBS at 65°C, place it in an oven at 65°C for 5 minutes, take it out, remove the hot solution and add 1×PBS solution at 4°C, and place it in a refrigerator at 4°C for later use.

[0055] 2. Tissue sample preparation

[0056] After taking out the experimental tissue sample from an animal or human body, wash the surface blood with 1×PBS, gently blot all the liquid with a fiber-free paper, quickly cool it in liquid nitrogen or dry ice powder after O.C.T embedding. The frozen-embedded sample can be stored in a -80°C refrigerator and taken out when needed for sectioning with a cryostat at -20°C, with a thickness of 10 μm being optimal. Adsorb the section on a hydrophilic glass slide and place it in a dry ice ice box for later use.

[0057] Wash twice with 1×PBS, then add 4% aqueous formaldehyde solution and fix at room temperature for 10 minutes. After fixation, remove the formaldehyde solution and wash twice again with 1×PBS. Add permeabilization solution (5% TritonX in 1×PBS) for 3 minutes of permeabilization, wash twice with 1×PBS, add 0.1N hydrochloric acid solution to denature the proteins bound to RNA in the sample, and treat at room temperature for 5 minutes. After removing the hydrochloric acid solution, add 1M Tris-HCl (pH8) solution to restore the acid-base balance of the system, then remove and add 1×PBS at 65°C, place in a 65°C oven for 5 minutes, take out, remove the hot solution and add 1×PBS solution at 4°C, and place in a 4°C refrigerator for standby.

[0058] Example 1

[0059] Cultivate wild-type HEK293T cells to 40% density, with approximately 1% being mitotic cells, and obtain the prepared cell samples using the method for preparing cell samples described above.

[0060] Perform in situ reverse transcription on the cell samples:

[0061] Prepare a 6 μL primer system (42 ng / μL PC-linker primer, 0.83 mM dNTP mix) and a 4 μL reverse transcription system (2×First Strand Buffer, 0.025 M DTT, 5U / μL RNase OUT, 25U / μL Superscript II Reverse Transcriptase). Heat the primer system to 65°C for 5 minutes, then immediately place it on ice for cooling. After the system temperature drops, add the reverse transcription system to configure a 10 μL in situ reverse transcription system. The sequence of the photocleavable primer (PC-linker primer) used in this example is shown in SEQ ID NO:1.

[0062] For the cell samples, take out the processed cell sample slides, pick them out with forceps, drop the in situ reverse transcription system on a hydrophilic glass slide, gently invert the slide over the solution to form a chamber for the reverse transcription reaction. Place the reverse transcription sample in a preheated 42°C incubator, ensure sufficient humidity (100%) and perform the in situ reverse transcription reaction in the dark for one hour. Add 10 minutes of PBS at 70°C to terminate the reaction, remove the PBS and add 4°C PBS to protect the cDNA:mRNA complex.

[0063] Perform ultraviolet light irradiation treatment on the cDNA:mRNA complex:

[0064] Dilute the SYBR Gold dye at a ratio of 1:10,000 and stain the cDNA:mRNA complex obtained above. After staining the complex, locate the area to be observed under a confocal microscope, and then only focus the light on this area with a 405 nm laser. Photolysis can be completed for a single cell in only 5 s. The area where photolysis occurs can be calculated by software programmed with Matlab. The focusing illumination and the manipulation of the digital micromirror device to achieve ultraviolet light illumination of a specific area are realized by a self-made software written in Labview. The above software can all be found at the website https: / / github.com / pkulichen / DMD-Camera-alignment.

[0065] As Figure 2 shown, DNA aggregation in obvious mitotic cells can be observed with the SYBR Gold dye (such as Figure 2 the green fluorescence circled by the dotted line in the upper right corner). When the mitotic cells obtained by localization are irradiated with a 405 nm laser, it can be seen that the cDNA in the mitotic cells before irradiation can be clearly observed (such as Figure 2 the red fluorescence circled by the dotted line in the lower left corner), but the fluorescence disappears after ultraviolet irradiation (such as Figure 2 the part circled by the dotted line in the lower right corner), that is, the o-nitrobenzyl in the photolysis primer breaks.

[0066] Perform in-situ ligation labeling on the complex that has completed ultraviolet light illumination:

[0067] Add the ligation system corresponding to the coding sequence selected for this illumination (10 μM barcode sequence, 10 μM splint sequence, 50 U / μL T4 DNA ligase, 0.4 U / μL RNaseOut, 1×T4 DNA ligase buffer) to the sample slide after ultraviolet light illumination, and react at room temperature (25 °C) for 60 minutes. After the ligation reaction is completed, add PBS at 65 °C for 5 minutes to terminate the reaction and wash the unreacted barcode, waiting for library construction. In this example, the sequence of the spatial coding sequence (barcode sequence) used is shown in SEQ ID NO:3, and the sequence of the splint sequence is shown in SEQ ID NO:2.

[0068] Use the complex that has completed in-situ ligation labeling to construct a library:

[0069] Add 100 μL of protease solution (20 mg / mL Proteinase K, 10% Tween, 1×PBS) to the sample that has completed in-situ ligation labeling. Use a pipette tip to pipette and scrape off the cells. Heat the solution to 55 °C and react for 30 minutes. Purify the above reaction solution using a commercial DNA column purification system to obtain an aqueous solution of cDNA:mRNA.

[0070] Add 5 μl of the second-strand synthesis system (2 μl of 5 × First-Strand Buffer, 2.31 μl of Second Strand Buffer, 0.23 μl of 10 mM dNTPs, 0.08 μl of 10 U / μl E. coli DNA ligase, 0.3 μl of 10 U / μl E. coli DNA polymerase, 0.08 μl of 2 U / μl E. coli RNaseH) to the purified aqueous solution of cDNA:mRNA above. Incubate at 16 °C for 2 hours. After taking it out, purify it using Vazyme DNA purification magnetic beads.

[0071] Mix the purified double-stranded DNA with the IVT mix for in vitro transcription (1.6 μl each of A / G / C / UTP solution, 1.6 μl of 10 × T7 reaction buffer, and 1.6 μl of T7 enzyme from the MEGAscript T7 Transcription Kit). Incubate at 37 °C overnight (16 - 18 hours) to obtain an RNA product. Add DNase to degrade all the DNA, and purify it using Vazyme RNA purification magnetic beads.

[0072] After reverse transcription of the purified RNA solution (using primers such as those shown in SEQ ID NO:9), perform PCR amplification using the cDNA as a template (using primers such as those shown in SEQ ID NO:10 - 11, 11 - 19 cycles) to obtain the spatial transcriptomics libraries of mitotic cells and interphase cells in the cell sample of this example respectively. Among them, the number of cycles of PCR amplification is related to the starting cell number. The more starting cells, the fewer cycles. 11 cycles correspond to 1000 cells, and 19 cycles correspond to 1 cell.

[0073] Perform sequencing analysis on the spatial transcriptomics libraries obtained above. Analyze the gene expression profiles of interphase cells and mitotic cells through PCA dimensionality reduction analysis, as Figure 3As shown, the sequencing results show a very obvious clustering of mitotic cells (red) and interphase cells (blue).

[0074] Example 2

[0075] The cell samples used in this example are HEK293T cells expressing eGFP and wild-type Neuro-2a cells. These two types of cells are co-cultured on the same glass slide using the method for preparing cell samples.

[0076] In this example, the operations of in situ reverse transcription, ultraviolet light treatment (without staining with dyes and using eGFP for spatial localization), and in situ ligation labeling of the cell samples are the same as those in Example 1. After labeling the HEK293T cells with the barcode1 primer (as shown in SEQ ID NO:6) and the splint sequence (as shown in SEQ ID NO:5), the sample is washed with PBS solution to remove the unreacted barcode1. Then, a new round of ultraviolet light treatment and in situ ligation labeling of the Neuro-2a cells is performed using the barcode2 primer (as shown in SEQ ID NO:7) and the splint sequence (as shown in SEQ ID NO:5) to complete the labeling of the Neuro-2a cells. The sequence of the photocleavable primer used in this example is as shown in SEQ ID NO:4.

[0077] As Figure 4 shown, the left figure shows HEK293T cells and Neuro-2a cells observed under bright field. The green fluorescence in the right figure is HEK293T cells, and the Neuro-2a cells are outlined by the white box, enabling precise localization of different cells.

[0078] The operation of constructing the library using the complex that has completed in situ ligation labeling is the same as that in Example 1, and the spatial transcriptomics libraries of HEK293T cells and Neuro-2a cells in the cell samples of this example are obtained respectively.

[0079] Since HEK293T is from human cells and Neuro-2a is from mouse cells, the results of transcript species origin analysis and statistics of the sequencing results, as Figure 5 shown, it can be seen that more than 95% of the sequencing reads corresponding to barcode1 are human genes, and more than 96% of the reads corresponding to barcode2 are mouse genes, confirming the extremely high specificity of the library information obtained by multiple rounds of labeling using the photocleavable primer of this application.

[0080] Example 3

[0081] The function of the hippocampal region is closely related to the formation of long-term memory. Analyzing the gene expression in different subregions of the hippocampal region contributes to the study of multiple neurobiological problems, including the mechanisms of the occurrence and development of neurodegenerative diseases.

[0082] In this example, spatial transcriptomics research was conducted on tissue samples of 6-month-old wild-type mice. The mice were anesthetized with isoflurane, then decapitated and dissected to obtain the brain. After embedding, cryosections were prepared, and the tissue samples of the mouse cryosections were prepared using the method for preparing tissue samples described above.

[0083] In situ reverse transcription was performed on the tissue samples:

[0084] Prepare a 6 μL primer system (42 ng / μL PC-linker primer, 0.83 mM dNTP mix) and a 4 μL reverse transcription system (2×First Strand Buffer, 0.025 M DTT, 5U / μL RNase OUT, 25U / μL Superscript II Reverse Transcriptase). Heat the primer system to 65°C for 5 minutes, then immediately place it on ice to cool. After the temperature of the system drops, add the reverse transcription system to configure a 10 μL in situ reverse transcription system. The sequence of the photocleavable primer (PC-linker primer) used in this example is shown in SEQ ID NO:4.

[0085] For the tissue samples, directly drop the in situ reverse transcription system on the tissue samples and cover the sections to form a chamber. Place the reverse transcription samples in a preheated incubator at 42°C, ensure sufficient humidity and perform the in situ reverse transcription reaction for one hour in the dark. Add 10 minutes of PBS at 70°C to terminate the reaction, remove the PBS and add PBS at 4°C to protect the cDNA:mRNA complex.

[0086] In this example, the operation of ultraviolet light irradiation treatment (without using dyes for staining and localizing based on different morphologies under bright field) and in situ ligation labeling of the tissue samples was the same as that in Example 1. Different regions were circled according to the morphologies of the hippocampal subregions DG, CA1, and CA3. After each ultraviolet light irradiation and in situ labeling treatment of a specific region, the samples were washed with PBS solution. As Figure 6 shown, the hippocampal subregions DG, CA1, and CA3 were circled respectively according to the different morphologies observed under bright field.

[0087] Among them, the DG sub-region of the hippocampus was labeled with the barcode1 primer (as shown in SEQ ID NO: 6) and the splint sequence (as shown in SEQ ID NO: 5), the CA1 sub-region of the hippocampus was labeled with the barcode2 primer (as shown in SEQ ID NO: 7) and the splint sequence (as shown in SEQ ID NO: 5), and the CA3 sub-region of the hippocampus was labeled with the barcode3 primer (as shown in SEQ ID NO: 8) and the splint sequence (as shown in SEQ ID NO: 5).

[0088] The operation of constructing the library using the complex that has completed in situ ligation labeling is the same as that in Example 1, and the spatial transcriptomic libraries of the DG, CA1, and CA3 sub-regions of the hippocampus in the tissue sample of this example are obtained respectively. As Figure 7 shown, by performing PCA dimensionality reduction analysis on the gene expression profiles of the DG, CA1, and CA3 sub-regions of the hippocampus, it can be seen that the sequencing results show that the transcriptomes of different sub-regions exhibit obvious clustering. As Figure 8 shown, by analyzing the signature genes in the spatial transcriptomic libraries of different sub-regions, it can be seen that the expression level of Prox1 in DG is about twice that in CA3 and CA1, and Dkk3 and Wfs1 are also significantly highly expressed (more than 2 times) in their respective specific regions. The expression levels of different signature genes in different subtypes are consistent with the reported rules in the existing literature.

[0089] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: Using the construction method of the present application, the sample obtained by the above preparation method is subjected to in situ reverse transcription using a photocleavable primer. After spatial localization, it is irradiated with ultraviolet light of a specific wavelength, and a specific labeling primer is used for ligation to localize the gene information at a specific spatial position in the sample, and it is possible to perform repeated localization of gene information in different spatial regions on the same sample. When sequencing and analyzing the spatial transcriptomic library subsequently, more accurate data information can be obtained.

[0090] The precise spatial localization of the target nucleic acid molecule is achieved, greatly improving the accuracy and resolution of spatial transcriptomic analysis. The control method using spatially specific ultraviolet light irradiation ensures the controllability of the labeling process. In addition, the spatial transcriptomic method developed based on photochemical reactions has the advantages of simple operation and low cost (as low as $10 per sample), which is suitable for researchers to perform spatial transcriptomic analysis on a large number of samples. Each sample only needs to undergo spatially structured illumination to construct a spatially specific library and perform data analysis, without complex instruments and image analysis processes. And by optimizing the reaction conditions, the efficiency and purity of library construction are further improved, making it possible to perform high-throughput spatial transcriptomic analysis on complex samples, providing a powerful tool for biomedical research, disease diagnosis and treatment.

[0091] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a spatial transcriptomics library, characterized in that, The construction method includes: S1, performing in-situ reverse transcription on a cell sample or a tissue sample using a photocleavable primer to obtain a cDNA:mRNA complex; S2, performing ultraviolet light irradiation on a specific spatial region of the cDNA:mRNA complex; S3, performing in-situ ligation labeling on the complex after the ultraviolet light irradiation using a labeled primer composition; S4, constructing a library for the complex after the in-situ ligation labeling to obtain a spatial transcriptomics library; Wherein, the photocleavable primer includes an oligonucleotide and a photosensitive substance located at the 5' end of the oligonucleotide linked by o-nitrobenzyl.

2. The construction method according to claim 1, characterized in that, The construction method further includes: Steps S2 and S3 can be repeated multiple times. After the previous step S3 is completed, the complex is washed and then the operation of the next step S2 is performed; Wherein, the labeled primer compositions used for each performance of step S2 and step S3 are different.

3. The construction method according to claim 1, characterized in that, The preparation method of the cell sample or the tissue sample includes: Sequentially performing fixation, permeabilization, denaturation, pH adjustment, and heating treatments on the cells or tissue sections attached to the carrier to obtain the cell sample or the tissue sample, and storing it at 4°C for standby; Wherein, the denaturation is performed using a 0.1N hydrochloric acid solution.

4. The construction method according to claim 1, characterized in that, The photosensitive substance includes: AlexaFluor 488, AlexaFluor 568, AlexaFluor 594, Cy3, or Cy5.

5. The construction method according to claim 1, characterized in that, The in-situ reverse transcription includes: Pre-treating the photocleavable primer and dNTP and then mixing them with a reverse transcription system to obtain an in-situ reverse transcription reaction system; Before performing the in-situ reverse transcription, preheating the cell sample or the tissue sample at 42°C. When the sample is the cell sample, dropping the in-situ reverse transcription reaction system onto a hydrophilic glass slide, and buckling the carrier with cells on the in-situ reverse transcription reaction system on the hydrophilic glass slide to form an in-situ reverse transcription reaction chamber, and performing the in-situ reverse transcription reaction to obtain the cDNA:mRNA complex; When the sample is the tissue sample, directly dropping the in-situ reverse transcription reaction system onto the tissue sample adsorbed on the hydrophilic glass slide, and completely covering the tissue sample with the carrier to form the in-situ reverse transcription reaction chamber, and performing the in-situ reverse transcription reaction to obtain the cDNA:mRNA complex; The in-situ reverse transcription reaction is carried out in a light-shielded environment.

6. The construction method according to claim 1, characterized in that, Performing ultraviolet light irradiation on a specific spatial region of the complex includes: Placing the cDNA:mRNA complex under a microscope and positioning the specific spatial region to be observed; Performing light irradiation treatment on the specific spatial region using light with a wavelength of 405 nm or 365 nm.

7. The construction method according to claim 1, characterized in that, The labeled primer composition includes a splint sequence and a spatial coding sequence; Wherein, the spatial coding sequence is linked to the nucleotide at the cleavage site of the o-nitrobenzyl, and the splint sequence and the spatial coding sequence have a partially complementary pairing region.

8. The construction method according to claim 1, characterized in that, Performing the in-situ ligation labeling includes: Perform a ligation reaction on the complex after the ultraviolet light irradiation using the labeled primer composition; After the ligation reaction is completed, add PBS solution at 65 °C to terminate the ligation reaction.

9. The construction method according to claim 1, characterized in that, The S4 includes: Perform cell lysis on the in-situ ligated and labeled complex to obtain a cDNA:mRNA aqueous solution; Use the cDNA in the cDNA:mRNA aqueous solution as a template for second-strand synthesis, and perform first purification and in vitro transcription in sequence to obtain an RNA product; Add DNase to the RNA product and perform second purification; Use the second-purified RNA product as a template for reverse transcription to obtain a cDNA product; Perform PCR amplification using the cDNA product as a template to obtain the spatial transcriptomics library.

10. Use of a spatial transcriptomics library obtained by the construction method according to any one of claims 1-9 in cell biology, medicine or developmental biology.

11. The use according to claim 10, wherein, The application includes: Sequence the spatial transcriptome library to obtain sequencing results; Perform gene expression level analysis and construction of two-dimensional or three-dimensional spatial distribution maps on the sequencing results.

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