A reusable spatial indexing template chip, replicable template chip, spatial transcriptome chip and preparation method and application thereof

Abstract

The application discloses a reusable spatial index template chip, a replicable template chip, a spatial transcriptome chip and a preparation method and application thereof. A composite index library compatible with a general sequencing platform is designed, spatial barcode mapping is completed on a commercial chip to obtain a spatial index template chip. Through molecular hybridization and enzymatic extension technology, a known coordinate array is copied to a receptor chip, and multiple generations of copying are supported, so that a batch spatial transcriptome chip preparation system sharing the same spatial coordinate atlas is constructed. The application realizes deep compatibility with a standard sequencing platform through composite index library design, decouples a spatial capture carrier from an original sequencing chip, significantly reduces experimental starting cost, and provides a large-scale solution for high-throughput and low-cost spatial omics large-scale application.

Description

(I) Technical Field

[0001] This invention belongs to the field of spatial omics and high-throughput sequencing technology, specifically relating to a reusable spatial index template chip, a reproducible template chip, and a spatial transcriptome chip, as well as their preparation methods. More specifically, this invention relates to a method for batch preparation of spatial transcriptome chips that share the same spatial coordinate map by copying a nucleic acid array with known spatial coordinates across a medium onto an independent receptor chip after completing spatial barcode mapping using a standard commercial sequencing chip, and by re-copying the template. (II) Background Technology

[0002] Spatial transcriptomics technology can acquire gene expression information while preserving its in situ spatial location information. It is an important tool for studying cellular heterogeneity, tissue functional zoning, and disease microenvironment. With the continuous improvement of resolution, spatial transcriptomics technology has put forward higher requirements for the spatial density of capture vectors, barcode decodability, RNA capture efficiency, and preparation cost.

[0003] Currently, aside from commercial platforms, the technical routes for preparing high-resolution spatial transcriptome capture vectors mainly fall into two categories. The first category is based on commercial sequencing flow cells. First, a correspondence between barcodes and coordinates is established, and then the flow cell is directly modified into a spatial capture vector. For example, Open-ST and Seq-Scope technologies both belong to the route of directly reusing Illumina or NovaSeq flow cells as spatial transcriptome capture regions. Open-ST discloses the sequencing of oligonucleotides with barcodes, adapters, and poly-dTs on a NovaSeq 6000 S4 flow cell, using customized Read1-DraI primers and a specific sequencing program. This type of approach uses the flow cell directly as the final spatial capture vector. During implementation, the flow cell is a disposable consumable, and it is impossible to construct an intermediate template chip system that allows for repeated sequence transfer.

[0004] The second type of approach, represented by Pixel-seq, discloses spatial barcode arrays based on continuous polonygels, reproducible gel-to-gel transfer, peg-based sequencing, and spatial barcode map construction processes based on microscopic images. This approach can achieve array replication, but its key lies in using peg-based gels as templates and completing spatial decoding through additional in-situ sequencing and image analysis. This method does not use commercially available sequencing chips with known coordinates and completed barcode sequencing as donor templates for cross-media replication; therefore, each generation of template or replication product often requires separate and complex decoding operations.

[0005] In summary, developing a system for mass-producing spatial transcriptome chips that can use commercially available barcode-based sequencing chips as the original spatial coordinate source, without using flow cells directly as the final experimental vector or requiring separate clustering or image decoding for each generation of replication templates, and enabling the replication products themselves to continue serving as donor templates, thus forming a system for mass-producing spatial transcriptome chips that can share the same barcode-coordinate map, has significant scientific and industrial value for promoting the large-scale popularization of spatial transcriptomics. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a reusable spatial index template chip, a reproducible template chip, and a spatial transcriptome chip, as well as a method for preparing the same, to overcome the limitations of existing methods that directly utilize flow cells and continuous dot-matrix gels, specifically achieving the following objectives:

[0007] (1) Complete a spatial barcode mapping using a standard commercial sequencing chip;

[0008] (2) After completing the mapping, retain the portability of the chip surface donor array so that it can be used as a reusable spatial index template chip.

[0009] (3) Copy the known coordinate array of the spatial index template chip onto the independent receptor chip to form a reproducible template chip and the final spatial transcriptome chip;

[0010] (4) The replicated chip is used as a donor template for repeated transfer, forming multiple spatial transcriptome chips that share the same barcode-coordinate map.

[0011] (5) Preferably, the T7 promoter region is retained in the final capture probe array to support subsequent linear amplification of in vitro transcription.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] In a first aspect, the present invention provides a reusable spatial index template chip, the preparation method of which includes the following steps: (1) designing and synthesizing a composite index library compatible with a universal sequencing platform, wherein the composite index library includes at least a cluster anchoring sequence, a spatial barcode region, an mRNA capture region, and a preferred T7 promoter region; (2) loading the composite index library onto a standard commercial sequencing chip for clustering and barcode sequencing (using standard sequencing primers of the universal sequencing platform to read the spatial barcode sequence) to establish a correspondence between the spatial barcode and the chip coordinates; (3) after sequencing, skipping the default oxidative terminal cleaning step of the universal sequencing platform to prevent the chip from entering an oxidative degradation treatment that would damage the transferability of surface nucleic acid clusters, and introducing a non-oxidative cleaning medium into the sequencing chip channel to remove free sequencing reagents, thereby preserving the structurally intact double-stranded DNA library clusters in situ on the chip surface; (4) recovering the sequencing chip to obtain a spatial index template chip with known spatial location information that can be reused for sequence transfer.

[0014] Preferably, the standard commercial sequencing chip is a P5 / P7 cluster sequencing chip, including but not limited to Illumina sequencing chips; more preferably, it is an S4 Flow Cell compatible with the Illumina NovaSeq 6000 platform.

[0015] Preferably, the composite index library comprises, from 5′ to 3′, a first adapter (P5 sequence), a T7 promoter sequence, a universal sequencing primer binding site, a spatial barcode region, a poly(dT) capture region, and a second adapter (P7 sequence).

[0016] Preferably, the composite index library adopts the sequence shown in SEQ ID NO.1 or an equivalent sequence with the same functional module arrangement relationship.

[0017] In a second aspect, the present invention provides a reproducible template chip prepared from the spatial index template chip, the preparation method of the reproducible template chip comprising the following steps: (1) providing a recipient chip, the recipient chip comprising a solid support and a primer immobilization layer disposed on its surface; the primer immobilization layer being a chemical layer; (2) immobilizing primer pairs for receiving donor sequences on the primer immobilization layer; (3) denaturing the spatial index template chip (strand separation or exposing its surface array) to convert at least one strand of the double-stranded DNA library cluster into a hybridizable state, serving as a donor template chip; (4) in the presence of polymerase, dNTPs and a buffer system, attaching the donor template chip to the recipient chip, and replicating and transferring the donor array to the surface of the recipient chip according to its original spatial distribution through molecular hybridization and enzymatic extension; (5) subsequently separating the donor template chip and the recipient chip to obtain a reproducible template chip; the nucleic acid array on the surface of the reproducible template chip corresponds one-to-one with the spatial distribution of the DNA library cluster on the spatial index template chip, and retaining the primer binding region required for retransfer.

[0018] Preferably, the receptor chip is a silanized glass slide and a functionalized gel layer on its surface.

[0019] Preferably, the gel layer is a polyacrylamide gel layer capable of covalently immobilizing primers, and the gel contains the functionalized monomer N-(5-(2-bromoacetamide)pentyl)acrylamide (BRAPA), which can undergo a covalent immobilization reaction with primers modified with thiophosphoroate.

[0020] Preferably, the primer pair comprises a primer paired with a universal P5 side sequence and a specific primer paired with a sequence on the other side of the donor template; more preferably, the primer pair is the sequence shown in SEQ ID NO.2 and SEQ ID NO.3 or its equivalent functional sequence.

[0021] Thirdly, the present invention provides a spatial transcriptome chip obtained by replication from a reproducible template chip. The reproducible template chip is used as a donor template to be replicated and transferred to a recipient chip and then amplified in situ, thereby forming a high-density capture probe array on the surface of the final chip, resulting in a spatial transcriptome chip. The chip includes a solid support and a capture probe array located on its surface. The spatial position of each probe in the capture probe array corresponds one-to-one with the spatial barcode coordinates in the original spatial index template chip. The probe includes at least a spatial barcode region and a poly(dT) capture region, and preferably also includes a T7 promoter region located upstream of the spatial barcode region, so as to perform in vitro transcriptional linear amplification after capturing tissue mRNA.

[0022] Fourthly, this invention provides a method for multi-generation replication of spatial transcriptome chips. The reproducible template chip obtained from the first replication, after enzyme digestion and strand separation, can continue to serve as a secondary donor template, attaching again to a new recipient chip to complete replication and transfer. This yields a secondary replicated chip that shares the same spatial barcode-coordinate map as the original template chip. Multiple chips obtained from the same original template chip can share the same barcode-coordinate database without requiring separate mapping for each chip.

[0023] Fifthly, the reusable spatial index template chip, reproducible template chip, and spatial transcriptome chip described in this invention, through their unique sequence transfer mechanism and multifunctional library design, can be applied to the following technical fields: (The text abruptly ends here, so the translation stops as well.)

[0024] 1. This invention directly utilizes the high-precision cluster distribution (micrometer level) of commercial sequencing chips as a spatial coordinate source to capture whole transcriptome mRNA in various tissue sections (such as brain tissue, developing embryos, etc.), thereby achieving subcellular resolution in situ localization analysis of gene expression.

[0025] 2. The present invention reserves a T7 promoter element in the capture probe, which enables the chip to have in situ linear amplification capability. Combined with T7 RNA polymerase for in vitro transcription (IVT), a large number of RNA copies are generated in situ in the gel substrate to improve the detection sensitivity of low abundance genes, or to be combined with fluorescence in situ hybridization (FISH) for high-throughput multi-round imaging observation.

[0026] 3. The chip of the present invention is not limited to mRNA capture. By modifying the primers on the surface of the receptor chip, it can be extended to the field of multiple omics. Combined with specific antibodies with DNA tags, it can synergistically capture proteins and DNA or RNA in the same spatial coordinate system, realize the synergistic detection of protein abundance and gene expression, and construct a multidimensional spatial interaction network.

[0027] 4. The multiple replication method proposed in this invention can mass-produce dozens or even hundreds of clone chips sharing the same database by using a single original flow cell for sequencing and mapping once. This ensures that the data between different experimental batches have extremely high alignment consistency, solves the bottleneck of high cost in space omics, and is suitable for large-scale industrial applications.

[0028] Compared with the prior art, the present invention achieves the following beneficial effects:

[0029] 1. This invention separates "one-time mapping of commercial sequencing chips" and "batch preparation of final spatial transcriptome chips" into two stages. The original commercial chips are no longer used as the final experimental carriers, but as reusable spatial index template chips, thus establishing a manufacturing system of "template chip - reproducible template chip - final spatial transcriptome chip".

[0030] 2. This invention replicates multiple chips that share the same spatial barcode-coordinate map. Theoretically, only one barcode mapping is needed for the original template chip to provide a common spatial coordinate reference for subsequent multiple derived chips, reducing the cost and complexity caused by repeated mapping and decoding.

[0031] 3. This invention preferably employs a composite index library design with primers compatible with standard platforms, reducing dependence on dedicated primers and dedicated sequencing procedures, and facilitating compatibility with conventional sequencing procedures.

[0032] 4. This invention transfers a known coordinate array to an independent receptor chip through cross-media replication, allowing the chip material, thickness, size, and processing method used for tissue patching and RNA capture to be decoupled from the original commercial sequencing chip, thus increasing the freedom of subsequent process design.

[0033] 5. Preferably, the present invention retains the T7 promoter region in the capture probe array, so that IVT linear amplification can be further performed after tissue mRNA capture is completed, which is beneficial to improving the detection sensitivity of low-abundance transcripts. (iv) Description of the attached drawings

[0034] Figure 1 This is a photograph of the functionalized gel formed on a surface-treated glass slide. The gel area is 1.0 × 0.8 cm and the thickness is approximately 45 μm.

[0035] Figure 2 The results show the fluorescence detection of the donor template chip, the amplified reproducible template chip, and the spatial transcriptome chip.

[0036] Figure 3 Fluorescence detection results of chips from different replication generations after multiple chips were continuously replicated from the same original donor template chip.

[0037] Figure 4 The image shows the microscopic imaging results after capturing tissue mRNA and reverse transcribing it into cDNA using a spatial transcriptome chip prepared based on the present invention. (V) Detailed Implementation Methods

[0038] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0039] Implementation Method 1: Fabrication of Spatial Index Template Chip

[0040] A multifunctional compound index library compatible with universal sequencing platforms was designed and synthesized. The nucleotide sequence of the multifunctional compound index library is SEQ ID NO.1:

[0041] AATGATACGGCGACCACCGAGATCTACACTACGACTCACTATAGGTCTTTCCTACACGACGCTCTTCCGATCTNNVNBVNNVNNVNNVNNVNNVNNNNNCTTTTTTTTTTTTTTTTTTTTTTAAAGATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGCCTTAATCTCGTATGCCGTCTTCTGCTTG.

[0042] Where N represents A, T, C or G; V represents A, C or G; and B represents C, G or T.

[0043] The sequence includes, from the 5' end to the 3' end, the following: First sequencing adapter: a P5 sequence compatible with the Illumina platform; Transcription initiation element: a T7 promoter sequence for in situ signal amplification; Universal sequencing primer binding site; Spatial index region: a spatial barcode sequence composed of highly diverse random base sequences to provide spatial location information; Capture region: an Oligo(dT) tail (25T) for anchoring tissue mRNA, which also serves as a primer pairing sequence for replication; Second sequencing adapter: a P7 sequence compatible with the Illumina platform.

[0044] The library was placed on a standard commercial sequencing chip (in this embodiment, the Illumina novaseq 6000 platform's S4 Flow Cell sequencing chip) for clustering and sequencing. Spatial barcode information was read using the platform's standard Read 1 primers and / or standard Read 2 primers to establish the correspondence between the spatial barcodes and chip coordinates. After sequencing, the chip was protected from oxidative degradation treatment that could impair the transferability of surface nucleic acid clusters, and residual sequencing reagents were removed using a non-oxidizing medium. This process preserved a donor array on the chip surface that could be used for subsequent transfer, resulting in a reusable spatial index template chip.

[0045] Implementation Method 2: Fabrication of Receptor Chips

[0046] In this embodiment, the receptor chip uses a glass slide as a solid support and a functionalized gel layer is disposed on its surface as a primer immobilization layer.

[0047] 1. Slide preparation

[0048] (1) Cleaning the slides: First, rinse the slides (25mm×75mm) with Milli-Q water to remove surface dust; then rinse the slides three times with anhydrous ethanol and let them air dry at room temperature; then soak them in 1M NaOH aqueous solution at room temperature (20-25℃) for 30 minutes, then rinse them three times with Milli-Q water, and then rinse them twice with anhydrous ethanol to remove residual alkali on the surface, and let them air dry at room temperature.

[0049] (2) Silanization modification of glass slides: 150 μL of Bind-silane solution was dropped onto the surface of the glass slide after cleaning in step (1). The solution was spread evenly with lint-free lens paper or cotton swabs and reacted at room temperature for 5-10 min. The Bind-silane solution was composed of propyl 3-(trimethoxysilyl)methacrylate, glacial acetic acid and 96% ethanol by volume, wherein the volume ratio of propyl 3-(trimethoxysilyl)methacrylate to glacial acetic acid was 1:1 and the volume ratio of propyl 3-(trimethoxysilyl)methacrylate to 96% ethanol was 1:18. After the reaction, the slide was rinsed three times with anhydrous ethanol and then rinsed three times with DEPC water. It was then dried naturally at room temperature to obtain silanized glass slides, which were placed in a sealed box for later use.

[0050] (3) Cleaning coverslips: First, rinse the coverslips (24mm×24mm) with Milli-Q water to remove surface dust; then soak them in 1M hydrochloric acid at room temperature for 30 minutes, rinse them three times with Milli-Q water, then rinse them twice with anhydrous ethanol, dry them in an oven at 65℃, and put them in a sealed box for later use.

[0051] 2. Preparation of gel

[0052] (1) Determine the area of ​​the receptor chip: Determine the gel area based on the region to be replicated. Apply a layer of transparent tape to the surface of the silanized glass slide in step 1 and cut off the excess tape around the edges. Use a blade to cut a square of the required gel area on the tape. In this case, the area used is 1.0cm × 0.8cm. After tearing off the square tape, flatten the tape around the edges.

[0053] (2) Preparation of gel solution:

[0054] The final concentration composition of the gel solution is: BRAPA 16 mg / mL, acrylamide system 8%, ammonium persulfate 0.015%, tetramethylethylenediamine 0.05%, and DEPC water as solvent. The acrylamide system is prepared using acrylamide and N,N'-methylenebisacrylamide in a mass ratio of 19:1.

[0055] (3) Gel preparation: After mixing the gel solution prepared in step (2), add it to the predetermined area in step (1), cover it with a cleaned coverslip, and be careful to remove air bubbles; the gel polymerizes at room temperature for 2 hours.

[0056] (4) Cleaning the gel: After the gel solidifies, carefully remove the coverslip with a blade, cover the gel with DEPC water, let it stand at room temperature for 5 minutes, absorb the liquid, wash twice with DEPC water and dry; then cover the gel with 10 mM phosphate buffer (pH 8.0) to obtain the receptor chip with the functionalized gel layer on the surface. Figure 1 .

[0057] Implementation Method 3: Primer Fixation

[0058] In this embodiment, the first primer and the second primer are fixed to the surface of the functionalized gel layer obtained in Embodiment 2.

[0059] The first primer is the sequence shown in SEQ ID NO.2:

[0060] P1: TTTTTTTTTAATGATACGGCGACCACCGAGATCTACAC.

[0061] The second primer is the sequence shown in SEQ ID NO.3:

[0062] P2: TTTTTTTTTTCGTGTGCTCTTCCGATCTTTAAAAAAAAAAAAAAA.

[0063] Among the above primers This indicates that the terminal nucleotide has been modified by phosphorylation (phosphorothioate).

[0064] The 5' nucleotides of the first and second primers were modified by phosphorylation. The modified primers were dissolved in Milli-Q water and then diluted with 10 mM phosphate buffer (pH 8.0) to a final concentration of 25 μM. The primer solution was added to the gel surface and incubated at 50°C for 1 h. After the reaction, the primer solution was aspirated, and the gel was washed twice with 5×SSC buffer containing 0.05% Tween-20 (containing 0.75 M NaCl and 75 mM sodium citrate). A third wash was performed, retaining the liquid and soaking at room temperature for 30 min to remove unbound primers, resulting in a receptor chip with primers immobilized on its surface.

[0065] Implementation Method 4: First Replication and Transfer of Spatial Index Template Chip to Recipient Chip

[0066] (1) Chip modification process:

[0067] The spatial index template chip obtained by cutting a chip of suitable size according to Implementation Method 1 (cutting area 1cm×1cm, usable area 1cm×0.8cm) was first washed three times with DEPC water and the surface liquid was aspirated. A 0.1M KOH aqueous solution was prepared and added to the chip, and the chip was allowed to stand at room temperature for 5 minutes. The liquid was then aspirated, and the process was repeated once. The chip was then washed three times with 0.1M Tris-HCl buffer (pH 7.5). The chip was then placed in 100% formamide and treated at 60°C for 10 minutes. The chip was then removed and washed twice with 100% formamide, then washed three times with DEPC water and the liquid was aspirated to obtain a template chip that could be used for replication.

[0068] In one detection method, PolyA-QS570 can be used to perform fluorescent hybridization staining on the poly(dT) region of the template chip to observe the signal distribution on the surface of the template chip. 100 μM PolyA-QS570 staining solution is diluted to 0.5 μM with DEPC water. A small piece of the chip is cut off and immersed in the diluted staining solution. After incubation at 98°C for 2 min, the temperature is slowly lowered (0.4°C / s) to 25°C. The chip is then removed, washed twice with DEPC water, and the surface moisture is blotted dry. It is then inverted on a coverslip, and the fluorescence (TRITC channel) is observed and photographed under a microscope. The results are as follows: Figure 2 As shown in Figure A, the results indicate that the signal distribution and brightness on the surface of the template chip are uniform.

[0069] (2) Preparation for replication:

[0070] Prepare a mixture of 1× isothermal amplification buffer and Bst 3.0 DNA polymerase. The total volume of the Bst 3.0 DNA polymerase mixture is 40 μL, which includes 0.4 μL of Bst 3.0 DNA polymerase (8000 U / mL), 4 μL of 10× Isothermal Amplification Buffer II, 0.8 μL of 10 mM dNTPs, and the remainder is DEPC water.

[0071] Preheat the heating table to 60℃;

[0072] The receptor chip with immobilized primers prepared in Implementation Method 3 was washed three times with DEPC water, 1× isothermal amplification buffer was added to the surface, and it was soaked at room temperature for 2 min. The surface liquid was then aspirated, and Bst3.0 DNA polymerase mixture was added and soaked at 4°C for 30 s. 1× isothermal amplification buffer was added to the surface of the spatial index template chip after denaturation treatment in step (1) above, and it was soaked at room temperature for 2 min. The surface liquid was then aspirated and it was ready for use.

[0073] (3) Sequence replication: The template chip from step (2) was then attached to the recipient chip and kept at 60°C for 5 min under a pressure of 10-30 kPa to allow the template strand to replicate to the surface of the recipient chip through molecular hybridization and enzymatic extension. After replication, the pressure was removed, and 100% formamide was added between the gel and the chip. The mixture was then treated at 60°C for 10 min to untie the double strands, and the two chips were carefully separated. The original template chip was washed three times with DEPC water, dried, and stored in 100% formamide at 4°C for reuse. The recipient chip then yielded a first nucleic acid array that spatially corresponds one-to-one with the original template chip, constituting the first-generation reproducible template chip.

[0074] Implementation Method 5: Replication of the reproducible template chip to a second receptor chip and preparation of a spatial transcriptome chip

[0075] Using the first-generation reproducible template chip obtained in Embodiment 4 as a donor template, it was processed in the same or similar manner as in Embodiment 4, and then bound to a new receptor chip for replication. The nucleic acid array on the surface of the first-generation reproducible template chip was then transferred to the surface of the second receptor chip. After replication was completed, the second receptor chip was amplified in situ to form a high-density capture probe array.

[0076] In this embodiment, in situ amplification is performed for 30 cycles at 60°C, and each cycle includes the following steps:

[0077] Denaturation: Add 40 μL of 100% formamide and let stand for 1 min;

[0078] Annealing: Add 40 μL of 1×buffer II and let stand for 2 seconds;

[0079] Extension: Add 40 μL of Bst 3.0 DNA polymerase mixture and incubate for 1 min.

[0080] After amplification, the sample was washed three times with DEPC water and then covered with TE buffer (pH 8.0) for storage. The resulting chip is a spatial transcriptome chip, in which the spatial positions of each probe in the surface capture probe array correspond one-to-one with the spatial positions of the corresponding DNA library clusters on the original sequenced commercial sequencing chip, and the spatial barcode region, poly(dT) capture region, and T7 promoter region located upstream of the spatial barcode region are retained.

[0081] In one detection method, EvaGreen (a green fluorescent nuclear dye) can be used to stain the amplified first-generation reproducible chip and spatial transcriptome chip to observe the distribution of double-stranded DNA signals on the surface. A 20× EvaGreen aqueous solution is diluted to 1× with DEPC water. The diluted dye solution is dropped onto the chip surface and stained at room temperature in the dark for 10 min. The dye solution is then removed, and the chip is washed three times with DEPC water. DEPC water is then dropped onto the chip surface, and the chip is inverted on a coverslip. Fluorescence (FITC channel) is observed under a microscope and photographed. The results are as follows: Figure 2 As shown in B and C, the results indicate that the spatial index sequence was successfully copied from the donor template chip to the primary reproducible recipient template chip, and from the reproducible template chip to the spatial transcriptome chip. Both chips had a high-density distribution of DNA capture probes, and the fluorescence signal was uniform with low background, demonstrating that the spatial coordinate information maintained a high degree of integrity and consistency during the replication process.

[0082] Implementation Method 6: Verification of the Reusability of Spatial Index Template Chips

[0083] Twenty receptor chips with primers immobilized on their surfaces were prepared according to Embodiments 2 and 3, respectively. The same denatured spatial index template chip was continuously replicated onto the 20 receptor chips using the method described in Embodiment 4. After each replication, 100% formamide was added to the interface to dissociate the template chip and receptor chip, and the template chip was cleaned and reused for the next replication. After all 20 receptor chips had completed replication, chips 5, 10, 15, and 20 were selected for in situ amplification using the method described in Embodiment 5, and observed using EvaGreen staining. The results are as follows: Figure 3 As shown in A, B, C, and D, the chips obtained from different replication rounds all show clear cluster distribution and detectable fluorescence signals, indicating that the spatial index template chip can be reused for sequence transfer, and multiple chips obtained from the same template chip share the same spatial barcode coordinate map.

[0084] Implementation Method 7: Verification of mRNA capture using spatial transcriptome chips

[0085] The spatial transcriptome chip prepared in Implementation Method 5 was used to verify the capture of mRNA from tissue sections.

[0086] In one specific embodiment, hydra were selected as the sample. After anesthetizing the hydra with a 2% urethane solution, the sample was transferred to an embedding mold and OCT embedding agent was added. The sample was then rapidly frozen and embedded in isopentane in a liquid nitrogen bath. The 2% urethane solution was prepared by mixing 450 μL of hydra culture medium with 50 μL of a 20% urethane aqueous solution. The hydra culture medium was obtained by adding 1 mL each of stock solution A, stock solution B, and stock solution C to 1 L of deionized water. Stock solution A was obtained by dissolving 14.61 g NaCl, 1.864 g KCl, and 27.745 g CaCl2 in 500 mL of deionized water; stock solution B was obtained by dissolving 30.285 g Tris in 500 mL of deionized water; and stock solution C was obtained by dissolving 3 g MgSO4 in 500 mL of deionized water.

[0087] The spatial transcriptome chip was washed three times with DEPC water, then washed with a washing solution containing 0.1×SSC buffer and 0.4×Maxima reverse transcription buffer, and dried under ventilation conditions for later use. Frozen tissue blocks were cut into 10 μm thick slices and attached to the chip surface. Tissue hybridization buffer (6×SSC and 2 U / L RNaseOUT) was added to immerse the slices, and they were incubated at room temperature for 15 min.

[0088] Subsequently, a reverse transcription mixture was added, and the mixture was incubated at 42°C for 1 h to complete the reverse transcription reaction. The reverse transcription mixture consisted of 5 μL Maxima H-reverse transcriptase (200 U / μL), 20 μL 5×Maxima reverse transcription buffer, 20 μL 20% Ficoll PM-400, 10 μL dNTPs, 2.5 μL RNase inhibitor (40 U / μL), and 42.5 μL DEPC water; the dNTP system included 500 μM dATP / dGTP / dTTP, 12.5 μM dCTP, and 25 μM Cy3-dCTP. After reverse transcription, tissue digestion solution was added, and the mixture was incubated at 37°C for 40 min to digest the tissue. The tissue digestion solution included 100 mM Tris-HCl (pH 8.0), 200 mM NaCl, 2% SDS, 5 mM EDTA, and 16 mU / μL proteinase K. After incubation, the sample was washed three times with DEPC water, and the fluorescence signal was observed under a TRITC microscope. The results are as follows: Figure 4 As shown, the prepared spatial transcriptome chip can capture tissue mRNA and form cDNA products with spatial information.

[0089] Implementation Method 8: Application of In Vitro Transcription Linear Amplification

[0090] In a preferred embodiment, the spatial transcriptome chip prepared in Embodiment 5, after capturing tissue mRNA and forming corresponding cDNA, can be linearly amplified in vitro using the T7 promoter region retained in the capture probe array, thereby improving the detection sensitivity of low-abundance transcripts. The RNA amplification products obtained through in vitro transcription can be used for subsequent sequencing, fluorescence in situ detection, or coupled with other molecular detection procedures.

[0091] Implementation Method 9: Spatial Multi-Omics Detection Application

[0092] In one embodiment, the spatial transcriptome chip of the present invention can also be used as a nucleic acid capture carrier for spatial multi-omics detection, and can be used in conjunction with protein, DNA or other RNA analysis processes to construct spatial associations of multidimensional molecular information in tissue samples.

[0093] Those skilled in the art will understand that any substitutions, modifications, and adjustments made to the above embodiments without departing from the concept of the present invention should fall within the protection scope of the present invention.

Claims

1. A reusable spatial index template chip, characterized in that, The method for preparing the spatial index template chip includes the following steps: (1) Design and synthesize a composite index library compatible with a universal sequencing platform, wherein the composite index library includes at least a cluster anchor sequence, a spatial barcode region, an mRNA capture region and a T7 promoter region; (2) The composite index library is loaded onto a standard commercial sequencing chip for clustering and barcode sequencing to establish the correspondence between spatial barcodes and chip coordinates; (3) After sequencing is completed, skip the default oxidative terminal cleaning step of the general sequencing platform and introduce non-oxidative cleaning medium into the sequencing chip channel to remove free sequencing reagents and retain the structurally intact double-stranded DNA library clusters on the chip surface in situ, retaining the hybridization region and / or primer binding region required for further replication to another recipient chip, which can be used as a donor array for subsequent transfer. (4) The sequencing chip is recovered to obtain a spatial index template chip with known spatial location information that can be reused for sequence transfer.

2. The spatial index template chip according to claim 1, characterized in that, The standard commercial sequencing chip is a P5 / P7 type sequencing chip, including but not limited to Illumina sequencing chips.

3. The spatial index template chip according to claim 1, characterized in that, The composite index library includes, from the 5' end to the 3' end, a first adapter, a T7 promoter sequence, a universal sequencing primer binding site, a spatial barcode sequence, a poly(dT) capture sequence, and a second adapter.

4. The spatial index template chip according to claim 1, characterized in that, The composite index library uses the sequence shown in SEQ ID NO.1 or an equivalent sequence with the same functional module arrangement relationship.

5. A reproducible template chip prepared from the spatial index template chip of claim 1, characterized in that, The method for preparing the reproducible template chip includes the following steps: (1) Provide a receptor chip, the receptor chip comprising a solid support and a primer immobilization layer disposed on its surface; (2) Primer pairs for receiving donor sequences are fixed on the primer immobilization layer; (3) The spatial index template chip of claim 1 is denatured to convert at least one strand of the double-stranded DNA library cluster into a hybridizable template strand, which serves as a donor template chip; (4) In the presence of polymerase, deoxyribonucleoside triphosphate and buffer system, the donor template chip is attached to the recipient chip, and the nucleic acid sequence on the template strand is copied and transferred to the recipient chip according to its original spatial distribution through molecular hybridization and enzymatic extension; (5) Separate the spatial index template chip from the receptor chip to obtain a reproducible template chip; The nucleic acid array on the surface of the reproducible template chip corresponds one-to-one with the spatial distribution of the DNA library cluster on the spatial index template chip, and retains the primer binding region required for retransfer.

6. The reproducible template chip according to claim 5, characterized in that, The primer pair includes a primer that pairs with the universal P5 side sequence and a specific primer that pairs with the other side sequence of the donor template; the primer pair is the sequence shown in SEQ ID NO.2 and SEQ ID NO.3 or its equivalent functional sequence.

7. A spatial transcriptome chip capable of batch replication using the reproducible template chip of claim 5, characterized in that, The reproducible template chip described in claim 5 is used as a donor template to be copied and transferred to the recipient chip and then amplified in situ, thereby forming a high-density capture probe array on the final chip surface to obtain a spatial transcriptome chip. The spatial position of each probe in the capture probe array corresponds one-to-one with the spatial barcode coordinates in the original spatial index template chip, and the probe includes at least a spatial barcode area and a poly(dT) capture area.

8. The spatial transcriptome chip according to claim 7, characterized in that, The capture probe array retains the T7 promoter region to enable in vitro transcriptional linear amplification after capturing tissue mRNA.

9. A method for multi-generation replication of a spatial transcriptome chip as described in claim 1, characterized in that, The reproducible template chip obtained by the method of claim 5, after being digested with enzymes and separated from the strand, continues to serve as a secondary donor template. It is then attached to a new recipient chip and the replication and transfer are completed, thereby obtaining a secondary replicated chip that shares the same spatial barcode-coordinate map as the original template chip.

10. The application of the spatial transcriptome chip of claim 1 in tissue section mRNA capture, in vitro transcriptional linear amplification of low-abundance transcripts and / or spatial multi-omics detection.

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