Methods and applications of spatial transcriptomics libraries

Abstract

This invention provides a method for constructing and applying a spatial transcriptomics library. The construction method includes: S1, performing in situ reverse transcription on cell or tissue samples using photolytic primers to obtain a cDNA:mRNA complex; S2, irradiating a specific spatial region of the cDNA:mRNA complex with ultraviolet light; S3, performing in situ ligation labeling on the ultraviolet-irradiated complex using a labeled primer composition; and S4, constructing a library from the in situ ligation-labeled complex to obtain a spatial transcriptomics library. The photolytic primers comprise oligonucleotides and a photosensitive substance located at the 5' end of the oligonucleotide linked by an o-nitrobenzyl group. Utilizing photolytic primers based on photochemical reactions for in situ reverse transcription enables precise spatial localization of target nucleic acid molecules, significantly improving the accuracy and resolution of spatial transcriptomics analysis.

Description

Technical Field

[0001] This invention relates to the field of gene library technology, and more specifically, to a method for constructing and applying a spatial transcriptomics library. Background Technology

[0002] Life activities depend on the spatial coordination of different cells and 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 ribonucleotides (RNA) can confirm the state, type, and function of cells by analyzing the abundance of different transcripts, and is one of the core technologies of contemporary omics. Spatial transcriptomics can simultaneously confirm the types of RNA and their spatial distribution in tissues or cells, and is a cutting-edge field of omics research in recent years.

[0003] Existing spatial transcriptomics methods include in situ capture, multi-round in situ hybridization fluorescence imaging, and microdissection. In situ capture requires specific microarrays, while multi-round imaging and microdissection require complex instruments and experimental procedures. While these methods each have their own characteristics, they are generally complex to use and costly per sample, hindering the widespread application and commercialization of spatial transcriptomics in biological research. For example, Visium from 10xGenomics, a leading US sequencing company, costs $3,000-$5,000 per sample, is expensive, and can only be used on frozen sections, making it completely unsuitable for studying transcript mechanisms in cultured cells or at subcellular resolution.

[0004] Therefore, providing a low-cost, widely applicable, and highly accurate method for constructing spatial transcriptomics libraries is of great significance for future research in omics. Summary of the Invention

[0005] The main objective of this invention is to provide a method for constructing and applying spatial transcriptomics libraries, in order to solve the problem of low resolution in the construction of spatial transcriptomics libraries in the prior art.

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

[0007] Furthermore, the construction method also includes: steps S2 and S3 can be repeated multiple times. After the previous step S3 is completed, the complex is washed before the next step S2 is performed; wherein, the labeled primer composition used in each step S2 and S3 is different; preferably, washing is performed with PBS solution at 65°C.

[0008] Furthermore, the method for preparing cell samples or tissue samples includes: sequentially fixing, permeabilizing, denaturing, pH adjusting and heating the cells or tissue slices attached to the carrier to obtain cell samples or tissue samples, which are then stored at 4°C for later use; wherein denaturation is performed using a 0.1N hydrochloric acid solution; preferably, the thickness of the tissue slices is 10μm.

[0009] Furthermore, the photosensitive materials include: AlexaFluor 488, AlexaFluor 568, and AlexaFluor... 594, Cy3, or Cy5; preferably, in situ reverse transcription includes: pretreating photolysis primers and dNTPs, 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 tissue sample at 42°C; when the sample is a cell sample, adding the in situ reverse transcription reaction system onto a hydrophilic glass slide, and inverting a carrier with attached cells onto the in situ reverse transcription reaction system on the hydrophilic glass slide to form an in situ reverse transcription reaction chamber, performing the in situ reverse transcription reaction to obtain a cDNA:mRNA complex; when the sample is a tissue sample, directly adding the in situ reverse transcription reaction system onto the tissue sample adsorbed on the hydrophilic glass slide, completely covering the tissue sample with a carrier to form an in situ reverse transcription reaction chamber, performing the in situ reverse transcription reaction to obtain a cDNA:mRNA complex; preferably, the in situ reverse transcription reaction is performed in a light-protected environment.

[0010] Furthermore, ultraviolet irradiation of specific spatial regions of the complex includes: placing the cDNA:mRNA complex under a microscope to locate the specific spatial region to be observed; and irradiating the specific spatial region with light of wavelengths of 405 nm or 365 nm.

[0011] Furthermore, 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 ortho-nitrobenzyl group, and the splint sequence and the spatial coding sequence have partially complementary pairing regions.

[0012] Further, the in-situ ligation labeling includes: using a labeling primer composition to perform a ligation reaction on the complex after UV irradiation; after the ligation reaction is completed, adding 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 ≥1h.

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

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

[0015] Further applications include: sequencing spatial transcriptome libraries to obtain sequencing results; analyzing gene expression levels and constructing two-dimensional or three-dimensional spatial distribution maps based on the sequencing results.

[0016] By applying the technical solution of this invention, in-situ reverse transcription using photochemical reaction-based photolysis primers can achieve precise spatial localization of target nucleic acid molecules, greatly improving the accuracy and resolution of spatial transcriptomics analysis. Furthermore, the construction method of this application has the advantages of simple operation and low cost (as low as $10 per sample), making it suitable for researchers to conduct spatial transcriptomics analysis on a large number of samples. A single sample only needs to be exposed to spatial structured light to construct a spatial transcriptomics library and perform data analysis, without the need for complex instruments and image analysis procedures. Attached Figure Description

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

[0018] Figure 1 A schematic diagram of the spatial transcriptomics library construction process in this application is shown.

[0019] Figure 2 The image shows the results of observing cell samples from Example 1 of this application under a confocal microscope;

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

[0021] Figure 4 The image shows the results of observing cell samples in Example 2 of this application under a confocal microscope;

[0022] Figure 5 This diagram illustrates the results obtained by transcriptomic species source statistics for sequencing results of HEK293T cell samples and Neuro-2a cell samples in Example 2 of this application.

[0023] Figure 6 The following figures show the results of observation of different subtypes of tissue samples in Example 3 of this application under a confocal microscope;

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

[0025] Figure 8 The results of heatmap analysis of differential gene expression of different subtypes DG, CA1 and CA3 in Example 3 of this application are shown. Detailed Implementation

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0027] As mentioned in the background section, existing technologies for constructing spatial transcriptomics libraries suffer from several drawbacks, including high cost, complex experimental procedures, high skill requirements for operators, easy sample loss, limited applicability, and low resolution, which hinder the development of research in this field. Therefore, this application aims to provide a method for constructing spatial transcriptomics libraries that is low-cost, simple to operate, widely applicable, and has good resolution.

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

[0029] like Figure 1 The flowchart shown illustrates the construction method of this application. In this method, the o-nitrobenzyl group in the photolysis primer is active to short-wavelength light and undergoes cleavage under ultraviolet light irradiation, causing the photosensitive material to separate from the oligonucleotide and releasing the protected phosphate groups of the nucleotides. This exposes only the phosphate groups of the primer nucleotides in specific spatial regions exposed by light, allowing for subsequent labeling to obtain sequencing information with spatial location characteristics. The aforementioned photochemical reaction utilizing the photolysis primer enables relatively simple capture of transcriptome information in specific regions without compromising sample integrity, and it offers good resolution, making it possible to further promote research in this field.

[0030] The construction method described in this application is applicable to samples including: in vitro cultured cell samples; normal tissue sections, including animal or plant tissues; and pathological tissue samples, including tumor tissue, inflammatory tissue, or tissue samples in other pathological states. Pathological tissue samples can be frozen sections, paraffin-embedded sections, or precipitates from liquid biopsies.

[0031] The construction method of this application can also repeatedly label 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 before the next step S2 is performed; wherein, the labeling primer composition used in each step S2 and S3 is different. Any suitable temperature for washing is applicable to this application. Preferably, washing is performed at 65°C using PBS solution. In this application, each UV irradiation is only performed on a specific position. That is, except for the irradiated area, the o-nitrobenzyl groups in the photolysis primers in other areas of the sample do not break and will not be labeled by subsequent primers. Therefore, as long as the sample is washed after one photolabeling, the same sample can be labeled multiple times at different positions, thereby obtaining more comprehensive spatial transcriptome information with minimal sample loss.

[0032] To further ensure the structural integrity and RNA stability of each region in the sample, in a preferred embodiment, the method for preparing cell or tissue samples includes: sequentially fixing, permeabilizing, denaturing, pH adjusting, and heating cells or tissue slices attached to a carrier to obtain cell or tissue samples, which are then stored at 4°C for later use; wherein denaturation is performed using a 0.1N hydrochloric acid solution. Any suitable hydrochloric acid concentration for protein denaturation is applicable to this application, and using hydrochloric acid for protein denaturation can further improve the efficiency of in situ reverse transcription.

[0033] To further prepare complete and stable sample materials for in situ reverse transcription, in a preferred embodiment, the cultured cell samples or sectioned tissue samples are fixed with formaldehyde solution (any suitable fixation time is applicable to this application, preferably 10 minutes), the fixed samples are then permeabilized with Triton solution (any suitable permeabilization time is applicable to this application, preferably 3 minutes), and then the permeabilized samples are decrosslinked with hydrochloric acid solution (any suitable decrosslinking treatment time is applicable to this application, preferably 5 minutes), and finally the RNA secondary structures in the samples are 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 obtain samples with more easily observable and clearly defined spatial structures, and to acquire spatial transcriptomics information with better resolution, in a preferred embodiment, samples were obtained from cultured cells (approximately 10...). 5 Select 1000, 100, 10 or 1 cells from the tissue sections for subsequent labeling; preferably, the thickness of the tissue section is 10 μm.

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

[0036] To further improve the efficiency of in situ reverse transcription and obtain better data for subsequent ultraviolet light irradiation and library construction, in a preferred embodiment, in situ reverse transcription includes: pretreating photolysis primers and dNTPs, then mixing them with the reverse transcription system to obtain an in situ reverse transcription reaction system; and preheating the cell or tissue sample at 42°C before performing in situ reverse transcription.

[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 on the hydrophilic glass slide to form an in situ reverse transcription reaction chamber, and the in situ reverse transcription reaction is carried out to obtain cDNA:mRNA complex.

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

[0039] To further improve the efficiency of in situ reverse transcription, any pretreatment time and temperature that yields optimal reaction results, as well as the in situ reverse transcription reaction time, temperature, and humidity, are applicable to this application. In a preferred embodiment, the pretreatment time is 5 minutes; preferably, the pretreatment temperature is 65°C; in a preferred embodiment, the in situ reverse transcription reaction time is 1 hour; preferably, the in situ reverse transcription reaction temperature is 42°C; preferably, the in situ reverse transcription reaction humidity is 100%. Preferably, the in situ reverse transcription reaction is carried out in a light-protected environment.

[0040] To accurately locate RNA in specific spatial regions, in a preferred embodiment, ultraviolet (UV) irradiation of the specific spatial region of the complex includes: placing the cDNA:mRNA complex under a microscope to locate the specific spatial region to be observed; and irradiating the specific spatial region with light of a wavelength of 405 nm or 365 nm. The selective cleavage capability of UV irradiation on o-nitrobenzyl groups enables precise spatial labeling of the sample at the subcellular level. Any high-power UV light source is suitable for this application; in a preferred embodiment, a 405 nm laser, a 365 nm laser, or a 365 nm LED is used for irradiation. Higher power density of the irradiated light results in a shorter irradiation time, typically ranging from 5 seconds to 15 minutes.

[0041] To further distinguish gene information in specific spatial regions, exposed phosphate groups are linked with labeled primers. In a preferred embodiment, the labeled primer composition includes a splinter sequence and a barcode sequence; wherein the barcode sequence is linked to the nucleotide at the cleavage site of the ortho-nitrobenzyl group, and the splinter sequence and the barcode sequence have partially complementary pairing regions.

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

[0043] To obtain a spatial transcriptomics library more efficiently, in a preferred embodiment, S4 includes: lysing the in situ ligation-labeled complex to obtain an aqueous cDNA:mRNA solution; using the cDNA in the aqueous cDNA:mRNA solution 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 a second purification; using the second-purified RNA product as a template for reverse transcription to obtain a cDNA product; and using the cDNA product as a template for PCR amplification to obtain a spatial transcriptomics library.

[0044] In a second typical embodiment of this application, an application is provided for a spatial transcriptomics library obtained using the above-described construction method in cell biology, medicine, or developmental biology. Specifically, this can include research on cell-microenvironment interactions; research on disease-related biomarkers; research on gene expression regulation mechanisms during development; and the establishment of molecular databases related to disease diagnosis or treatment.

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

[0046] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

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

[0048]

[0049] In the following examples, all primers used were synthetic DNA primers customized from Sangon Biotech, with the following specific sequences: The 5' end of the photocleavage primer is linked to an iPClink (Photocleavage linker), and the other end of the iPClink is linked to AF568 (Alexa Fluor 568). Additionally, OPTImap is the optically precise tagging for spatially resolved RNA mapping technology proposed in this application.

[0050]

[0051] Sample preparation before experiment

[0052] 1. Cell sample preparation

[0053] Place 14 mm diameter glass slides in 24-well plates and incubate overnight with 100 μL of Matrigel solution (5% concentration, diluted in DMEM medium). Before cell passage, wash twice with 1×PBS solution and add 1 mL of cell culture medium. Passage cells at a ratio of 1:30 for 6 cm dishes. After 24-48 hours, cell coverage will reach 70%-80%, with approximately 1% of cells in the mitotic phase (mitosis cells).

[0054] Remove the culture medium from the 24-well plate, wash twice with 1×PBS, and then fix with 4% formaldehyde solution at room temperature for 10 minutes. After fixation, remove the formaldehyde solution and wash twice again with 1×PBS. Add permeabilization solution (5% Triton X in 1×PBS) for 3 minutes, wash twice with 1×PBS, add 0.1N hydrochloric acid solution to denature the RNA-bound proteins in the sample, and treat at room temperature for 5 minutes. After removing the hydrochloric acid solution, add 1M Tris-HCl solution (pH 8) to restore the pH of the system, then remove and add 65℃ 1×PBS, place in a 65℃ oven for 5 minutes, remove the hot solution and add 4℃ 1×PBS solution, and store in a 4℃ refrigerator for later use.

[0055] 2. Sample preparation

[0056] After removing the experimental tissue samples from the animal or human body, wash the surface blood with 1×PBS and gently blot away all liquid with fibrous paper. After OCT embedding, rapidly cool the samples in liquid nitrogen or dry ice powder. Cryo-embedded samples can be stored at -80°C. When needed, remove them and section them at -20°C using a cryostat, with an optimal thickness of 10 micrometers. Adsorb the sections onto hydrophilic slides and place them in a dry ice container for later use.

[0057] Wash twice with 1×PBS, then fix with 4% formaldehyde solution at room temperature for 10 minutes. After fixation, remove the formaldehyde solution and wash twice more with 1×PBS. Add permeabilization solution (5% Triton X in 1×PBS) for 3 minutes, wash twice with 1×PBS, add 0.1N hydrochloric acid solution to denature the RNA-bound proteins in the sample, and treat at room temperature for 5 minutes. After removing the hydrochloric acid solution, add 1M Tris-HCl (pH 8) solution to restore the pH of the system, then remove the solution and add 65℃ 1×PBS, place in a 65℃ oven for 5 minutes, remove the hot solution and add 4℃ 1×PBS solution, then store in a 4℃ refrigerator for later use.

[0058] Example 1

[0059] Wild-type HEK293T cells were cultured to a density of 40%, of which approximately 1% were mitotic cells. The prepared cell samples were obtained using the cell sample preparation method described above.

[0060] In situ reverse transcription of 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, 5 U / μL RNase OUT, 25 U / 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 system temperature has decreased, add the reverse transcription system to prepare a 10 μL in situ reverse transcription system. The sequence of the photolysis primer (PC-linker primer) used in this example is shown in SEQ ID NO:1.

[0062] For cell samples, remove the processed cell sample slides using tweezers, and drop the in situ reverse transcription system onto a hydrophilic slide. Gently invert the slide onto the solution to form a chamber for the reverse transcription reaction. Place the reverse transcription sample in a preheated 42°C incubator, ensuring adequate humidity (100%) and protection from light, for one hour to perform the in situ reverse transcription reaction. Terminate the reaction by adding 70°C PBS for 10 minutes, remove the PBS, and add 4°C PBS to protect the cDNA:mRNA complex.

[0063] UV light treatment of cDNA:mRNA complex:

[0064] The obtained cDNA:mRNA complex was stained with SYBR Gold dye at a 1:10000 dilution. The stained complex was then positioned under a confocal microscope to observe specific areas, which were then illuminated only with a 405nm laser. Photolysis of a single cell occurred in just 5 seconds. The observed photolysis area was calculated using Matlab-programmed software. The focused illumination and manipulation of the digital micromirror device to achieve targeted UV illumination of specific areas were implemented using custom software written in LabVIEW. All of the above software can be found at https: / / github.com / pkulichen / DMD-Camera-alignment.

[0065] like Figure 2 As shown, obvious DNA aggregation in mitotic cells can be observed using SYBR Gold dye (e.g. Figure 2 The green fluorescence circled in the upper right corner indicates that the mitotic cells obtained from the localization were illuminated with a 405nm laser. It can be seen that cDNA was clearly visible in the mitotic cells before illumination (e.g., ...). Figure 2 The red fluorescence circled in the lower left corner disappears after ultraviolet irradiation (e.g., the red fluorescence circled in the lower left corner). Figure 2 The dotted circle in the lower right corner indicates the breakage of the o-nitrobenzyl group in the photolysis primer.

[0066] In-situ ligation labeling of the complex after UV irradiation:

[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 LRNaseOut, 1×T4 DNA ligase buffer) to the sample slide after UV irradiation, and react at room temperature (25℃) for 60 minutes. After the ligation reaction, add PBS at 65℃ for 5 minutes to terminate the reaction and wash away any unreacted barcode, ready for library construction. The spatial coding sequence (barcode sequence) used in this embodiment is shown in SEQ ID NO:3, and the splint sequence is shown in SEQ ID NO:2.

[0068] Library construction using complexes with in-situ ligation markers:

[0069] Add 100 μL of protease solution (20 mg / mL Proteinase K, 10% Tween, 1×PBS) to the sample after in situ ligation labeling, and scrape off cells by pipetting with a pipette tip. 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 cDNA:mRNA solution.

[0070] Add 5 μl of the two-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, and 0.08 μl of 2 U / μl E. coli RNaseH) to the purified cDNA:mRNA aqueous solution, incubate at 16 °C for 2 hours, and then purify using Vazyme DNA purification magnetic beads.

[0071] The purified double-stranded DNA was mixed with 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), and incubated overnight at 37°C (16-18 hours) to obtain RNA product. DNase was added to degrade all the DNA, and the RNA was purified using Vazyme RNA purification beads.

[0072] After reverse transcription of the purified RNA solution (using primers as shown in SEQ ID NO:9), PCR amplification was performed using cDNA as a template (using primers as shown in SEQ ID NO:10-11, 11-19 cycles) to obtain spatial transcriptomics libraries of mitotic and interphase cells from the cell samples of this embodiment. The number of PCR amplification cycles was related to the number of starting cells; the more starting cells, the fewer the number of cycles. 11 cycles corresponded to 1000 cells, and 19 cycles corresponded to 1 cell.

[0073] Sequencing analysis was performed on the spatial transcriptomics library obtained above, and gene expression profiles of interphase and mitotic cells were analyzed by PCA dimensionality reduction, such as... Figure 3As shown, the sequencing results reveal a very clear division between mitotic cells (red) and interphase cells (blue).

[0074] Example 2

[0075] The cell samples used in this embodiment are HEK293T cells expressing eGFP and wild-type Neuro-2a cells. The two types of cells were co-cultured on the same slide using cell sample preparation methods.

[0076] In this embodiment, the in situ reverse transcription, UV irradiation (but without dye staining, using eGFP for spatial localization), and in situ ligation labeling of cell samples were performed in the same manner as in Example 1. HEK293T cells were labeled using the barcode1 primer (as shown in SEQ ID NO:6) and the clip sequence (as shown in SEQ ID NO:5). After labeling, the samples were washed with PBS solution to remove unreacted barcode1. A new round of UV irradiation and in situ ligation labeling of Neuro-2a cells was then performed using the barcode2 primer (as shown in SEQ ID NO:7) and the clip sequence (as shown in SEQ ID NO:5) to complete the labeling of Neuro-2a cells. The sequence of the photolysis primer used in this embodiment is shown in SEQ ID NO:4.

[0077] like Figure 4 As shown, the left image shows HEK293T cells and Neuro-2a cells observed under bright field, while the green fluorescence in the right image represents HEK293T cells, and the white frame outlines Neuro-2a cells, enabling precise localization of different cell types.

[0078] The library construction using the complex with in situ ligation markers was performed in the same manner as in Example 1, resulting in spatial transcriptomics libraries of HEK293T cells and Neuro-2a cells from the cell samples of this example.

[0079] Since HEK293T is derived from human cells and Neuro-2a from mouse cells, the sequencing results, after transcriptomic species origin analysis, show the following statistical results: Figure 5 As shown, over 95% of the sequencing reads corresponding to barcode1 are human genes, and over 96% of the reads corresponding to barcode2 are mouse genes, confirming that the library information obtained by multiple rounds of labeling using the photolysis primers of this application has extremely high specificity.

[0080] Example 3

[0081] The function of the hippocampus is closely related to the formation of long-term memory. Analyzing the gene expression of different subregions of the hippocampus can help in the study of many neurobiological questions, including the generation and development mechanisms of neurodegenerative diseases.

[0082] This embodiment studies spatial transcriptomics of tissue samples from 6-month-old wild-type mice. Mice were euthanized by cervical dislocation after isoflurane anesthesia, and the brains were dissected, embedded, and frozen sectioned. The tissue samples were prepared from the frozen sections using the methods described above.

[0083] In situ reverse transcription of 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, 5 U / μL RNase OUT, 25 U / 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 system temperature has decreased, add the reverse transcription system to prepare a 10 μL in situ reverse transcription system. The sequence of the photolysis primer (PC-linker primer) used in this example is shown in SEQ ID NO:4.

[0085] For tissue samples, the in situ reverse transcription system was directly dropped onto the tissue sample and covered to form a chamber. The reverse transcription sample was placed in a preheated 42°C incubator, ensuring adequate humidity and protection from light, for one hour of in situ reverse transcription. The reaction was terminated by adding 70°C PBS for 10 minutes, the PBS was removed, and 4°C PBS was added to protect the cDNA:mRNA complex.

[0086] In this embodiment, the tissue samples underwent ultraviolet light treatment (but without dye staining; localization was based on morphological differences under bright field) and in-situ binding labeling, similar to the procedures in Example 1. Different regions were selected based on the morphology of DG, CA1, and CA3 in the hippocampal subregions. After each round of ultraviolet light treatment and in-situ labeling of a specific region, the sample was washed with PBS solution. Figure 6 As shown, the hippocampal subregions DG, CA1, and CA3 were selected based on different morphologies observed under bright field.

[0087] Specifically, the hippocampal subregion DG is labeled with a barcode1 primer (as shown in SEQ ID NO:6) and a splice sequence (as shown in SEQ ID NO:5), the hippocampal subregion CA1 is labeled with a barcode2 primer (as shown in SEQ ID NO:7) and a splice sequence (as shown in SEQ ID NO:5), and the hippocampal subregion CA3 is labeled with a barcode3 primer (as shown in SEQ ID NO:8) and a splice sequence (as shown in SEQ ID NO:5).

[0088] The library construction using the complex with the in situ ligation markers was performed in the same manner as in Example 1, yielding spatial transcriptomics libraries of DG, CA1, and CA3 in the hippocampal subregions of the tissue samples in this example. Figure 7 As shown, PCA dimensionality reduction analysis of the DG, CA1, and CA3 gene expression profiles in the hippocampal subregions reveals that the sequencing results show distinct clustering of the transcriptome in different subregions. Figure 8 As shown, the analysis of marker genes in spatial transcriptomics libraries of different subregions reveals that Prox1 is expressed at approximately twice the level in CA3 and CA1, while Dkk3 and Wfs1 are also significantly highly expressed (more than twice the level) in their respective specific regions. The expression levels of different marker genes in different subtypes are consistent with the patterns reported in existing literature.

[0089] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: using the construction method of this application, the sample obtained by the above preparation method is reverse transcribed in situ using photolysis primers. After spatial localization, it is irradiated with ultraviolet light of a specific wavelength and connected using specific labeled primers to locate the gene information at a specific spatial location in the sample. Moreover, it is possible to repeatedly locate the gene information of different spatial regions on the same sample, and obtain more accurate data information when performing sequencing analysis on the spatial transcriptomics library in the subsequent process.

[0090] This method achieves precise spatial localization of target nucleic acid molecules, significantly improving the accuracy and resolution of spatial transcriptomics analysis. The use of space-specific ultraviolet light control ensures the controllability of the labeling process. Furthermore, the photochemical reaction-based spatial transcriptomics method offers advantages such as simplicity and low cost (as low as $10 per sample), making it suitable for researchers to conduct spatial transcriptomics analysis on large sample volumes. A single sample only requires spatial structured illumination for space-specific library construction and data analysis, eliminating the need for complex instruments and image analysis procedures. Moreover, by optimizing reaction conditions, the efficiency and purity of library construction are further improved, enabling high-throughput spatial transcriptomics analysis on complex samples and providing a powerful tool for biomedical research, disease diagnosis, and treatment.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a spatial transcriptomics library, characterized in that, The construction method includes: S1, using photolysis primers to perform in situ reverse transcription on cell or tissue samples to obtain cDNA:mRNA complexes. S2, irradiate a specific spatial region of the cDNA:mRNA complex with ultraviolet light; S3, In situ ligation labeling of the complex after UV irradiation is performed using a labeled primer composition; S4, construct a library from the complex with the in situ ligation marker to obtain a spatial transcriptomics library; The photolysis primer comprises an oligonucleotide and a photosensitive substance located at the 5' end of the oligonucleotide linked by an ortho-nitrobenzyl group; The construction method also includes: Steps S2 and S3 can be repeated multiple times. After the previous step S3 is completed, the complex is cleaned before proceeding to the next step S2. The labeled primer composition used in each of steps S2 and S3 is different; The marker primer composition includes a splice sequence and a spatial coding sequence; The spatial coding sequence is linked to the nucleotide at the cleavage site of the ortho-nitrobenzyl group, and the clamp sequence has a partially complementary pairing region with the spatial coding sequence.

2. The construction method according to claim 1, characterized in that, The method for preparing the cell sample or tissue sample includes: Cells or tissue slices attached to the carrier are sequentially fixed, permeabilized, denatured, pH adjusted, and heated to obtain the cell samples or tissue samples, which are then stored at 4°C for later use. The denaturation is carried out using a 0.1N hydrochloric acid solution.

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

4. The construction method according to claim 1, characterized in that, The in situ reverse transcription includes: After pretreatment of the photolysis primers and dNTPs, they were mixed with the reverse transcription system to obtain an in situ reverse transcription reaction system. Prior to performing the in situ reverse transcription, the cell or tissue sample was preheated at 42°C. When the sample is the cell sample, the in situ reverse transcription reaction system is dropped onto a hydrophilic glass slide, and the carrier with attached cells is inverted on the hydrophilic glass slide onto the in situ reverse transcription reaction system to form an in situ reverse transcription reaction chamber, and the in situ reverse transcription reaction is carried out to obtain the cDNA:mRNA complex. When the sample is the tissue sample, the in situ reverse transcription reaction system is directly added to the tissue sample adsorbed on the hydrophilic glass slide, and the tissue sample is completely covered by the carrier to form the in situ reverse transcription reaction chamber. The in situ reverse transcription reaction is carried out to obtain the cDNA:mRNA complex. The in situ reverse transcription reaction was carried out in a light-protected environment.

5. The construction method according to claim 1, characterized in that, Irradiating specific spatial regions of the complex with ultraviolet light includes: The cDNA:mRNA complex was placed under a microscope to locate the specific spatial region to be observed; The specific spatial region is illuminated using light with a wavelength of 405nm or 365nm.

6. The construction method according to claim 1, characterized in that, Performing the in-situ connection marking includes: The complex after UV irradiation was ligated using a labeled primer composition. After the ligation reaction is completed, PBS solution at 65°C is added to terminate the ligation reaction.

7. The construction method according to claim 1, characterized in that, S4 includes: Cells were lysed to obtain an aqueous cDNA:mRNA solution from the in situ ligated complex. Using the cDNA in the cDNA:mRNA aqueous solution as a template, a second-strand synthesis was performed, followed by a first purification and in vitro transcription to obtain the RNA product. DNase was added to the RNA product and a second purification was performed; Using the second purified RNA product as a template, reverse transcription was performed to obtain the cDNA product; The cDNA product was used as a template for PCR amplification to obtain the spatial transcriptomics library.

8. An application of a spatial transcriptomics library obtained using the construction method according to any one of claims 1-7, the application comprising: The spatial transcriptome library was sequenced to obtain sequencing results; Gene expression level analysis and construction of two-dimensional or three-dimensional spatial distribution maps were performed on the sequencing results.

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