A spatial transcriptome sequencing chip, and a preparation method and application thereof

Abstract

This invention relates to the field of spatial transcriptomics or spatial multi-omics technology, and particularly to a spatial transcriptome sequencing method based on photolytically cleavable linkers and its applications. In this invention, by utilizing probes with photolytically cleavable linkers bound to a spatial transcriptome chip, single-cell-level resolution transcriptional information analysis with location information is achieved on tissue sections, thereby effectively solving the problems of insufficient probe release efficiency and inadequate localization accuracy in existing technologies.

Description

Technical Field

[0001] This invention relates to the field of spatial transcriptomics or spatial multi-omics technology, and particularly to a spatial transcriptomics sequencing chip, its preparation method and application. Background Technology

[0002] Spatial omics, particularly spatial transcriptomics, is of great value in studying the spatial distribution of gene expression. This technology mainly relies on spatial omics chips with coding probes, which capture or release probes on tissue slices to achieve in-situ detection and spatial localization of RNA molecules.

[0003] Currently, there are two main technical approaches to probe-based chip development, but both have significant limitations: One type is the coupled-release microsphere chip: This type of chip usually couples nucleic acid probes onto a microsphere carrier. The preparation process is relatively complex, and the overall stability of the chip is difficult to control. At the same time, the spatial barcode uses microspheres as carriers. Each microsphere generally carries the same spatial barcode. The diameter of the microspheres (usually on the order of 10 μm) is often close to or larger than the size of a single cell nucleus. They are generally distributed in the form of discrete units with physical gaps. The types or number of spatial barcode units that a single cell nucleus can contact and participate in spatial labeling are limited and the coverage density is insufficient, which causes spatial positioning deviation and affects the accuracy of transcriptional information parsing and subsequent cell nucleus spatial positioning.

[0004] Another type is the enzyme / reagent cleavage microarray: this type of microarray relies on enzyme cleavage or chemical reagents to release probes. Its disadvantages are that the enzyme or chemical cleavage process is limited by osmosis and reaction kinetics, often resulting in limited efficiency and incomplete probe release; furthermore, the introduced cleavage reagent system may interfere with the stability or integrity of endogenous RNA in tissues, thus affecting the accurate acquisition of transcript information.

[0005] It is evident that existing release-type probe chips generally suffer from technical shortcomings such as insufficient spatial barcode coverage density at the cell nuclear scale, low enzyme digestion efficiency, and potential interference with transcriptional information. Therefore, there is an urgent need to provide a new scheme for spatial transcriptomics probe release and spatial information acquisition that can achieve efficient and precise probe release in tissue-attached states without affecting downstream molecular detection, thereby improving the reliability and application scope of spatial omics data. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Addressing the issues of insufficient coverage density, low release efficiency, and limited spatial positioning accuracy of spatial barcodes at the single-cell nucleus scale in existing spatial transcriptomics technologies, this invention proposes a spatial transcriptomics technology scheme based on photolytically cleavable linkers and its application.

[0007] In a first aspect, the present invention provides a spatial transcriptome chip, the spatial transcriptome chip comprising: a substrate and at least one nucleic acid probe loaded on the surface of the substrate.

[0008] In some embodiments of the present invention, at least one of the nucleic acid probes includes a photolytically cleavable linker (PCLinker).

[0009] In some embodiments of the present invention, the photodegradable linker breaks and is released from the substrate surface under specific wavelength illumination conditions.

[0010] In some embodiments of the present invention, the wavelength is in the ultraviolet band, including any wavelength in the range of 280-400 nm.

[0011] In some embodiments of the present invention, the illumination time is 1-10 min.

[0012] In some embodiments of the present invention, the photolytically cleavable linker is a chemical linker group that can break under specific wavelength light conditions.

[0013] In some embodiments of the present invention, the photolytic linker comprises an aromatic nitro compound.

[0014] In some embodiments of the present invention, the photodegradable linker comprises a compound represented by Formula I;

[0015] Formula I.

[0016] In this invention, the material of the substrate is not limited, and any conventional support material and structure used in chip fabrication in the art can be used, including but not limited to: glass, silicon wafers, or polymeric materials (such as polyacrylamide gel, SU8 polymer, polytetrafluoroethylene, etc.). The choice of support material and structure does not affect the realization of spatial information release and analysis in the technical solution of this invention.

[0017] In some embodiments of the present invention, the nucleic acid probe further includes a sequencing adapter sequence.

[0018] In this invention, the sequencing adapter sequence can be a sequencing adapter sequence commonly used in the art, such as P5 from the Illumina platform, and its length is generally 20-30 bp.

[0019] In some embodiments of the present invention, the sequencing adapter sequence is shown as SEQ ID NO: 1.

[0020] In some embodiments of the present invention, the photolyzable linker is attached to one end of the sequencing adapter sequence.

[0021] In some embodiments of the present invention, the photolyzable linker is attached to the 5' end of the sequencing adapter sequence.

[0022] In some embodiments of the present invention, the substrate is further loaded with another nucleic acid probe, the nucleic acid probe containing a sequencing adapter sequence that does not contain a photolytically cleavable linker.

[0023] In this invention, the sequencing adapter sequence that does not contain a photolyzable linker can be another type of sequencing adapter sequence commonly used in the art, including but not limited to: P7 sequencing adapter sequence.

[0024] In some embodiments of the present invention, the sequencing adapter sequence length without photolytically cleavable linkers is generally 20-30 bp.

[0025] In some embodiments of the present invention, the sequencing adapter sequence that does not contain a photolytically cleavable linker is shown in SEQ ID NO: 2.

[0026] In some embodiments of the present invention, the sequencing adapter sequence that does not contain photolytically cleavable linkers contains enzyme cleavage sites.

[0027] In some embodiments of the present invention, the restriction enzyme site is located at one end of the sequencing adapter sequence that does not contain a photolytically cleavable linker.

[0028] In some embodiments of the present invention, the restriction enzyme site is located at the 5' end of the sequencing adapter sequence that does not contain a photolytic linker.

[0029] In some embodiments of the present invention, the enzyme cleavage site may undergo nucleotide modification or chemical linking group breakage under enzyme action conditions.

[0030] In some embodiments of the present invention, the enzyme cleavage site includes, but is not limited to, the uracil (U) base.

[0031] In some embodiments of the present invention, the uracil (U) base is specifically cleaved in the presence of a dissociating enzyme (such as USER enzyme), allowing it to be released after precoding sequencing is completed.

[0032] In some embodiments of the present invention, the structure of the nucleic acid probe containing the photolytically cleavable linker is as follows: From one end of the connector substrate outwards, it includes: a photolyzable linker and a sequencing adapter sequence (P5).

[0033] In some embodiments of the present invention, the structure of the nucleic acid probe without photolytically cleavable linkers is as follows: Starting from one end of the connecting substrate and moving outwards, the sequence includes: restriction site (U) - sequencing adapter sequence (P7).

[0034] In some embodiments of the present invention, the ratio of the number of nucleic acid probes containing photolytic linkers to the number of nucleic acid probes without photolytic linkers is 0.5-2:1.

[0035] In some embodiments of the present invention, the ratio of the number of nucleic acid probes containing photolytic linkers to the number of nucleic acid probes without photolytic linkers is 1:1.

[0036] In some embodiments of the present invention, the average diameter of the nucleic acid probe cluster is distributed between 0.5 μm and 1.5 μm.

[0037] In some embodiments of the present invention, the pitch between the centers of adjacent nucleic acid probe clusters on the surface is between 1 μm and 2 μm.

[0038] In some embodiments of the present invention, the nucleic acid probe containing the photolytically cleavable linker is pre-coded.

[0039] In some embodiments of the present invention, the precoding process includes: obtaining the spatial location information of the nucleic acid probe and anchoring the spatial location information to the corresponding nucleic acid probe.

[0040] In some embodiments of the present invention, the spatial location information corresponding to the nucleic acid probes on the chip surface can be obtained by means including but not limited to sequencing, so as to achieve pre-coding of spatial information. The method of obtaining the spatial location information does not constitute a limitation of the present invention.

[0041] In some embodiments of the present invention, after precoding sequencing is completed, nucleic acid probes or other nucleic acid molecules that do not contain photolytically cleavable linkers can be removed by denaturation or cleavage, so that the nucleic acid probes loaded on the spatial transcriptome chip are in a single-stranded state.

[0042] In some embodiments of the present invention, the spatial location information is set by a spatial barcode probe.

[0043] In some embodiments of the present invention, the spatial barcode probe includes: a nucleic acid molecule complementary to the sequencing adapter sequence described above, a nucleic acid molecule complementary to the sequencing primer, a random sequence, a pre-encoding sequencing primer binding sequence, and a binding site for sequencing primer Read 2.

[0044] In some embodiments of the present invention, the spatial barcode probe refers to a spatial barcode probe composed of a sequencing adapter sequence (P5), a sequencing primer binding sequence, a pre-coding random sequence, a pre-coding sequencing primer binding sequence, a polyA sequence or a target sequence, a primer binding sequence, and a P7 complementary sequence in the nucleic acid probe.

[0045] In some embodiments of the present invention, the spatial barcode probe comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 3.

[0046] In some embodiments of the present invention, the spatial barcode probe is as shown in SEQ ID NO: 3.

[0047] In some embodiments of the present invention, the method for preparing the nucleic acid probe includes: The spatial barcode probe is loaded onto the substrate surface, and a stable optically spliable linker is synthesized on the substrate surface using a solid-phase amplification method, while the spatial barcode probe is connected to the synthesized optically spliable linker.

[0048] In some embodiments of the present invention, the solid-phase amplification method includes bridge amplification.

[0049] In some embodiments of the present invention, the primers for the bridging amplification are the P5 adapter sequence (SEQ ID NO: 1) and the P7 adapter sequence (SEQ ID NO: 2).

[0050] In some embodiments of the present invention, the template for the bridge amplification is the aforementioned spatial barcode probe (SEQ ID NO: 3).

[0051] In some embodiments of the present invention, the amplification system of the bridge amplification is shown in Table 1 of the specification.

[0052] In some embodiments of the present invention, after constructing the nucleic acid probe, the random sequence in the spatial barcode probe contained in the nucleic acid probe cluster can be determined by conventional sequencing methods to obtain the spatial position information of the nucleic acid probe on the chip surface, thereby realizing the pre-coding of spatial information.

[0053] In some embodiments of the present invention, the sequencing includes next-generation sequencing.

[0054] In some embodiments of the present invention, after sequencing is completed, the detection surface of the chip is cleaned to remove sequencing primers, amplification products and other residues introduced during the sequencing reaction.

[0055] In this invention, the term "detection surface" refers to the side surface of the chip that is loaded with nucleic acid probes.

[0056] In some embodiments of the present invention, the cleaning methods include, but are not limited to: alkaline denaturation, enzyme digestion, buffer washing, or combinations thereof.

[0057] In some embodiments of the present invention, the cleaning method is as follows: cleavage of the uracil site in the P7 adapter sequence with enzyme and alkaline denaturation elution.

[0058] In this invention, the chip size is not limited and can be cut according to actual application requirements using mechanical or optical methods to obtain a chip size suitable for tissue attachment. The cutting method and parameters do not affect the subsequent spatial information release process based on photodisintegrable linkers.

[0059] A second aspect of the present invention provides a reagent kit or kit comprising the spatial transcriptome chip described above.

[0060] In some embodiments of the present invention, the kit or package further includes at least one reagent or component that can be used for photoactivated release, single-cell nuclear transcription library construction or amplification analysis, such as nucleic acid release buffer, reverse transcription reagent, PCR amplification reagent, library construction reagent, sequencing reagent, enzyme, etc., but is not limited thereto.

[0061] In some embodiments of the present invention, the reagents included in the kit or apparatus are used to capture, amplify, or perform library construction analysis on nucleic acid molecules that have entered the nucleus of a single cell after the nucleic acid probe carrying spatial location information is released from the substrate.

[0062] In some embodiments of the present invention, the reagents may be conventional reagents in the art, and their specific composition and usage conditions do not constitute a limitation of the present invention.

[0063] In some embodiments of the present invention, the sequencing reagents include, but are not limited to, sequencing primers, sequencing buffers, etc.

[0064] In some embodiments of the present invention, the enzyme includes: DNA polymerase, reverse transcriptase, restriction endonuclease, exonuclease, etc.

[0065] In some embodiments of the present invention, the buffer solution includes, but is not limited to, any conventional buffer solution in the art, such as PBS, Tris-HCl, etc.

[0066] In some embodiments of the present invention, the reagent kit or kit may also include, but is not limited to, other reagents used in library construction (including amplification, purification, fragmentation, adapter ligation, A-tailing, etc.), sequencing, nucleic acid extraction, and other operations.

[0067] A third aspect of the invention provides the use of the spatial transcriptome chips and / or kits or packages described above in spatially localized transcriptome sequencing.

[0068] In some embodiments of the present invention, the detection samples for spatially localized transcriptome sequencing include, but are not limited to, cells (layers), tissues, organs (including organoids).

[0069] In some embodiments of the present invention, the test sample further includes: processed cells (layers), tissues, organs (including organoids), etc., such as sections (including but not limited to fresh tissue sections, frozen tissue sections, or fixed tissue sections, etc.).

[0070] In some embodiments of the present invention, the thickness of the test sample is not limited and can be appropriately adjusted according to actual usage requirements and conventional practices in the field.

[0071] A fourth aspect of the present invention provides a spatially localized transcriptome sequencing method, comprising the following steps: (1) The tissue to be tested is attached to the detection surface of the spatial transcriptome chip described above, and light is applied to release the nucleic acid probe. Tissue incubation solution is added to permeate the tissue, so that the nucleic acid probe enters the cell nucleus to label the cell nucleus. (2) The tissue is lysed or dissociated to recover the information complex (including nuclear transcript cDNA) in the cell nucleus, construct a sequencing library, and sequence it to obtain spatially localized transcriptome information.

[0072] In some embodiments of the present invention, the information complex further includes a cell tag.

[0073] In some embodiments of the present invention, other operations (such as drying after attachment) may be further introduced to ensure that the tissue to be tested is in full contact with the nucleic acid probe on the detection surface.

[0074] In some embodiments of the present invention, the definition of the tissue to be tested is the same as that of the test sample described above.

[0075] In some embodiments of the present invention, the conditions for light treatment include: ultraviolet light and a power of 0.2-0.6 mW / mm². 2 Irradiation time: 2.5-4 min.

[0076] In some embodiments of the present invention, the conditions for light treatment include: 365 nm ultraviolet light and a power of 0.4 mW / mm². 2 Irradiation time: 3 minutes.

[0077] In some embodiments of the present invention, the wavelength range, irradiation power, and irradiation time are not considered limitations of the present invention.

[0078] In some embodiments of the present invention, the tissue incubation solution includes: buffer solution, inorganic salts, surfactants, protein stabilizers, and ribonuclease inhibitors.

[0079] In some embodiments of the present invention, the buffer solution includes, but is not limited to, at least one of Tris-HCl, HEPES, or PBS.

[0080] In some embodiments of the present invention, the inorganic salt includes: non-divalent cation salts and divalent cation salts.

[0081] In some embodiments of the present invention, the non-divalent cation salt includes, but is not limited to, at least one of potassium salt, ammonium salt, and sodium salt.

[0082] In some embodiments of the present invention, the non-divalent cation salt includes at least one of NaCl or KCl.

[0083] In some embodiments of the present invention, the divalent cation salt includes at least one of calcium salt, magnesium salt, or zinc salt.

[0084] In some embodiments of the present invention, the divalent cation salt includes MgCl2.

[0085] In some embodiments of the present invention, the surfactant includes nonionic surfactants, including but not limited to: NP40, Triton X-100, etc.

[0086] In some embodiments of the present invention, the protein stabilizer includes, but is not limited to, albumin.

[0087] In some embodiments of the present invention, the protein stabilizer includes, but is not limited to, bovine serum albumin.

[0088] In some embodiments of the present invention, the tissue incubation solution comprises:

[0089] In some embodiments of the present invention, the tissue incubation solution is as shown in Table 3 of the specification.

[0090] In some embodiments of the present invention, tissue lysis reagents are used to lyse or dissociate tissues.

[0091] In some embodiments of the present invention, the pyrolysis or dissociation treatment may employ conventional pyrolysis reagent systems in the art, and their specific composition does not constitute a limitation of the present invention.

[0092] In some embodiments of the present invention, the tissue lysis reagent includes: buffer solution, inorganic salt, surfactant, osmotic pressure regulator, protein stabilizer, and ribonuclease inhibitor.

[0093] In some embodiments of the present invention, the buffer solution includes, but is not limited to, at least one of Tris-HCl and HEPES.

[0094] In some embodiments of the present invention, the inorganic salt includes: non-divalent cation salts and divalent cation salts.

[0095] In some embodiments of the present invention, the non-divalent cation salt includes, but is not limited to, at least one of potassium salt, ammonium salt, and sodium salt.

[0096] In some embodiments of the present invention, the non-divalent cation salt includes at least one of NaCl or KCl.

[0097] In some embodiments of the present invention, the divalent cation salt includes at least one of calcium salt, magnesium salt, or zinc salt.

[0098] In some embodiments of the present invention, the divalent cation salt includes MgCl2 or CaCl2.

[0099] In some embodiments of the present invention, the surfactant includes nonionic surfactants, including but not limited to: NP40, digitoxin, or saponins.

[0100] In some embodiments of the present invention, the osmotic pressure regulator includes, but is not limited to, sucrose.

[0101] In some embodiments of the present invention, the protein stabilizer includes, but is not limited to, albumin.

[0102] In some embodiments of the present invention, the protein stabilizer includes, but is not limited to, bovine serum albumin.

[0103] In some embodiments of the present invention, the tissue lysis reagent comprises:

[0104] In some embodiments of the present invention, the tissue lysis reagent is shown in Table 4 of the specification.

[0105] In some embodiments of the present invention, after adding the tissue lysis reagent, incubation is performed for 3-5 minutes.

[0106] In some embodiments of the present invention, the spatial localization transcriptome sequencing method further includes: adding a cleaning agent to accelerate or promote dissociation during or after dissociation using a tissue lysis reagent.

[0107] In some embodiments of the present invention, the cleaning agent includes: buffer solution, inorganic salt, protein stabilizer, and ribonuclease inhibitor.

[0108] In some embodiments of the present invention, the selection of the buffer solution, inorganic salt, protein stabilizer, and ribonuclease inhibitor is the same as described above.

[0109] In some embodiments of the present invention, the library construction and sequencing steps may employ conventional single-cell nuclear RNA sequencing (snRNA-seq) procedures and corresponding reagents or platforms, and the specific implementation method does not affect the implementation of the technical solution of the present invention.

[0110] In some embodiments of the present invention, the method of integrating and analyzing sequencing data to obtain spatial transcriptome results is not limited, and can be accomplished using conventional data processing algorithms or software tools in the art.

[0111] In some embodiments of the present invention, the spatial localization transcriptome sequencing method further includes: constructing a visualization image based on spatial localization transcriptome information.

[0112] In some embodiments of the present invention, the construction of the visualization image can be achieved based on existing technologies.

[0113] In some embodiments of the present invention, the construction of the visualization image includes: extracting cell tag and spatial barcode (SBC) sequence information from the spatial barcode probe library, using the mapping index obtained by precoding sequencing to restore the SBC sequence to physical spatial coordinates, then mapping the restored physical spatial coordinates to the corresponding cDNA library entries using the cell tag as the association key, and using clustering or density analysis methods to calculate the coordinate points associated with each cell nucleus to determine its final spatial location, further using dimensionality reduction and clustering analysis methods to analyze the cell population, and then visualizing it according to the corresponding spatial coordinate positions.

[0114] In some embodiments of the invention, the clustering or density analysis method includes, but is not limited to, the DBSCAN algorithm.

[0115] In some embodiments of the invention, the dimensionality reduction and clustering analysis method includes, but is not limited to, the UMAP algorithm.

[0116] The beneficial effects of this invention are: 1. This invention achieves efficient and precise release of nucleic acid molecules carrying spatial location information in a tissue-attached state by introducing high-density nucleic acid probes with photolytically cleavable linkers onto the surface of a functionalized substrate. This design enables the method in this invention to obtain higher density and more continuous spatial information labeling at the single-cell nucleus scale, thereby effectively improving spatial resolution and labeling throughput, and providing key technical support for constructing high-resolution spatial omics maps.

[0117] 2. The method in this invention eliminates the need for enzymatic or chemical cleavage reactions during spatial information release, avoiding potential interference with endogenous RNA in tissues and significantly reducing the impact on downstream molecular detection processes. Furthermore, the experimental procedure based on this method is simplified, exhibiting good versatility and scalability, and can be widely applied to spatial transcriptome analysis of various tissue samples, thus enhancing the method's adaptability and practicality.

[0118] 3. Compared to existing spatial labeling methods that rely on microsphere transfer or enzymatic cleavage release, the method in this invention achieves in-situ synchronous release of spatially located probes through a photoactivated cleavage mechanism. This better adapts to high-density probe cluster structures and avoids detection bias caused by insufficient cleavage efficiency or the introduction of exogenous reagents. Furthermore, it significantly improves the utilization rate and labeling accuracy of spatial barcodes while maintaining high spatial resolution, thereby enhancing the overall reliability and analytical efficiency of spatial omics data. Attached Figure Description

[0119] Figure 1 The images show HE staining of the tissue in Example 2 (top image) and the corresponding spatial transcriptome map obtained after spatial transcriptome sequencing (bottom image). Detailed Implementation

[0120] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0121] Example 1 In this embodiment, a method for preparing a functional chip for spatial transcriptome analysis is provided. This functional chip is used for subsequent spatial information release and analysis based on photolytically cleavable linkers.

[0122] The functionalized chip includes a support (the chip body) and nucleic acid probes loaded on the surface of the support. The nucleic acid probes are formed by amplifying sequencing adapter sequences and their associated spatial barcodes.

[0123] In this embodiment, the sequencing adapter includes at least one type of adapter sequence containing a photosplitable linker (PC Linker) and at least one type of adapter sequence without a photosplitable linker.

[0124] The adapter sequence containing the photodissociative linker includes an adapter sequence and a photodissociative linker connected thereto. This adapter sequence can be a sequencing P5 adapter sequence commonly used in the field, typically 20-30 bp in length, and includes, for example, the following sequences: 5'-AATGATACGGCGACCACCGAGATCTACAC-3' (SEQ ID NO: 1).

[0125] In this embodiment, the connector sequence containing the photo-disintegrable connector is SEQ ID NO: 1.

[0126] The photolytic linker is attached to the 5' end of the linker sequence shown in SEQ ID NO: 1. The photolytic linker is a chemical linker group that can break under specific wavelength light conditions, preferably an aromatic nitro compound, such as the compound shown in Formula I.

[0127]

[0128] Formula I.

[0129] In this embodiment, the photolytically splitable linker is a compound represented by Formula I.

[0130] In this embodiment, the adapter sequence without photosplittable linkers can be another type of sequencing adapter sequence commonly used in the art, such as the sequencing P7 adapter sequence, which is generally 20-30 bp in length, and includes, for example, the following sequences: 5'-UCAAGCAGAAGACGGCATACGAGAT-3' (SEQ ID NO: 2).

[0131] In this embodiment, the connector sequence without photodisintegrable connectors is SEQ ID NO: 2.

[0132] The 5' end of the P7 linker sequence contains a specific enzyme cleavage site, a nucleotide modification or chemical linker group that can be broken under the corresponding enzyme action conditions, preferably a uracil (U) base, and can be specifically cleaved by adding a specific dissociation enzyme (such as USER enzyme), so that it can be released after the precoding sequencing is completed.

[0133] In this embodiment, the 5' end of the P7 adapter sequence contains uracil (U), and the USER enzyme is used in the system to specifically cleave it so that only single-stranded nucleic acid probes (clusters) remain on the surface of the functionalized chip, and the 3' end of the single-stranded nucleic acid probes is in a free state for subsequent spatial information capture.

[0134] In this embodiment, the spatial barcode probe associated with the sequencing adapter is a collection of nucleic acid molecules used for spatial information encoding, which can be obtained through solid-phase synthesis, fragment ligation, or amplification methods. This spatial barcode probe contains a sequencing adapter sequence (P5), a sequencing primer-binding sequence, a pre-coding random sequence, a pre-coding sequencing primer-binding sequence, a polyA sequence or target sequence, a primer-binding sequence, and a P7 complementary sequence. For example, the spatial barcode probe associated with the sequencing adapter may include the sequence shown in SEQ ID NO: 3.

[0135] 5'-AATGATACGGCGACCACCGAGATCTACACgtcctcacagcgtccgtacgtaNNNNNNNNNNNNNNNNNNNNNNNNNNNNtcttgtgactacagcaccctcgactctcgcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAtgcactcagacgatgtcATCTCGTATGCCGTCTTCTGCTTGA-3' (SEQ ID NO: 3).

[0136] In this embodiment, the spatial barcode probe associated with the sequencing adapter is the sequence shown in SEQ ID NO: 3.

[0137] In the fabrication of the functionalized chip, the aforementioned spatial barcode probes related to sequencing adapters are first loaded onto the surface of the functionalized chip. Then, nucleic acid probe clusters with spatial barcode probes are formed on the surface of the functionalized chip through solid-phase amplification. The solid-phase amplification method can be bridge amplification, and the amplification conditions can be adjusted according to actual needs. By adjusting the amplification parameters, the average diameter of the nucleic acid probe clusters can be distributed between 0.5 μm and 1.5 μm, and the center-to-center distance (pitch) between adjacent probe clusters can be between 1 μm and 2 μm, resulting in a high-density distribution on the chip surface to meet the experimental requirements for subsequent spatial information encoding and release. This submicron to micron-level distribution density ensures that multiple probe units with different spatial indices can be covered within a single cell nucleus scale (typically with a diameter of 5-15 μm), thereby achieving redundant labeling and precise localization.

[0138] In this embodiment, the solid-phase amplification method is bridge amplification, and the amplification primers are the P5 adapter sequence (SEQ ID NO: 1) and the P7 adapter sequence (SEQ ID NO: 2).

[0139] The bridge amplification system is shown in the table below: Table 1 Bridge amplification system

[0140] The buffer solution consists of: 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, and 0.1% Triton X-100.

[0141] The PCR reaction procedure is as follows: Table 2 PCR reaction procedure

[0142] After constructing the nucleic acid probe cluster, the random sequences in the spatial barcode probes contained within the nucleic acid probe cluster can be determined using conventional sequencing methods to obtain the spatial location information of the nucleic acid probes on the surface of the functionalized chip, thereby achieving pre-encoding of spatial information. In this embodiment, the sequencing method used is the conventional second-generation sequencing method in the art.

[0143] After sequencing, the surface of the functionalized chip is treated to remove sequencing primers, amplification products, and other residues introduced during the sequencing reaction, thereby obtaining a regenerated functionalized chip for subsequent space experiments. Treatment methods may include alkaline denaturation, enzyme digestion, buffer washing, or a combination thereof.

[0144] In this embodiment, the uracil site in the P7 linker sequence is cleaved using the USER enzyme, followed by alkaline denaturation elution to remove nucleic acid strands without photolyzable linkers and sequencing residues, leaving only single-stranded nucleic acid probes (clusters) on the surface of the functionalized chip. The processing conditions can be adjusted according to actual needs and are not intended to limit the invention.

[0145] In this embodiment, after the functional chip completes the spatial information precoding, it can be cut by mechanical or optical means according to the actual application requirements to obtain a chip size suitable for tissue attachment. The cutting method and parameters do not affect the subsequent spatial information release process based on optically splitter-like connectors.

[0146] Example 2 In this embodiment, real tissue samples are used to verify the feasibility of the spatial transcriptome analysis method based on the above-mentioned functionalized chip.

[0147] (1) Sample preparation: In this embodiment, the test sample was fresh mouse brain tissue, which was frozen and then embedded using OCT. The frozen OCT embedded block was equilibrated in a microtome and then sectioned to a thickness of 20 μm.

[0148] After slicing, the obtained tissue slices are attached to the surface of the functional chip prepared in the above embodiment, and the surface of the functional chip is fixed with nucleic acid probes (clusters) for carrying spatial barcodes.

[0149] The functionalized chip with attached tissue sections was placed in a baking device for a short time to enhance the adhesion stability between the tissue sections and the surface of the functionalized chip. In this embodiment, the baking temperature was 37 ℃ and the time was 1 min.

[0150] Simultaneously, adjacent tissue sections were stained with hematoxylin and eosin (HE) for subsequent tissue morphology comparison analysis.

[0151] (2) Photolysis treatment: Tissue incubation solution is added to the tissue slice area attached to the surface of the functionalized chip to cover the tissue slice. The added tissue incubation solution is used to maintain the tissue structure and provide a suitable environment for the subsequent release of nucleic acid probes with spatial barcodes.

[0152] For example, the tissue incubation solution includes, but is not limited to, one or more of the following components: buffer salts (such as Tris-HCl, HEPES or PBS), inorganic salts (such as NaCl or KCl), divalent cationic salts (such as MgCl2), nonionic surfactants (such as NP40, Triton X-100), protein stabilizers (such as albumin), and ribonuclease inhibitors.

[0153] Specifically, the composition of the incubation solution used in this embodiment is shown in the table below: Table 3 Composition of the incubation solution

[0154] Subsequently, the functionalized chip, which was soaked in tissue incubation solution, was placed under a light source for irradiation to activate the photolytic linkers in the nucleic acid probes (clusters) of the spatial barcode, causing the nucleic acid probes carrying the spatial barcode to break and release from the surface of the functionalized chip.

[0155] In this embodiment, the light source used is 365 nm ultraviolet light with a power of 0.4 mW / mm². 2 , irradiated at room temperature for 3 minutes.

[0156] (3) Release and recovery of the cell nucleus: After photolysis, the liquid on the surface of the functionalized chip is removed, and a tissue lysis reagent is added to the surface of the functionalized chip and incubated for 3-5 min. Exemplary tissue lysis reagents include, but are not limited to, one or more of the following components: buffer salts (such as Tris-HCl or HEPES), inorganic salts (such as NaCl or KCl), divalent cation salts (such as MgCl2 or CaCl2), nonionic surfactants (such as NP40, digitalis saponins, or saponins), osmotic pressure regulators (such as sucrose), protein stabilizers (such as albumin), and ribonuclease inhibitors.

[0157] Specifically, the composition of the tissue lysis reagent used in this embodiment is shown in the table below: Table 4 Composition of tissue lysis reagent

[0158] After incubation, the tissue was lysed 30 times using 1000 μL of washing buffer. The lysate was recovered, filtered through a 40 μm filter, and the filtrate was transferred to centrifuge tubes and centrifuged at 500-1000 × g (800 × g in this example) for 5-10 min (5 min in this example). Exemplarily, the washing buffer may include, but is not limited to, one or more of the following components: buffer salts (such as Tris-HCl, HEPES, or PBS), inorganic salts (such as NaCl or KCl), divalent cation salts (such as MgCl2), protein stabilizers (such as albumin), and ribonuclease inhibitors.

[0159] Specifically, the composition of the washing buffer used in this embodiment is shown in the table below: Table 5 Composition of the washing buffer

[0160] After centrifugation, the supernatant was discarded, and the precipitate was reconstituted with 1×PBS to obtain a cell nucleus suspension.

[0161] (4) Library construction: The nuclear suspension obtained in the above steps is used for subsequent transcriptome library construction and sequencing analysis. Library construction can be achieved using conventional single-cell or single-nuclear transcriptome library construction techniques. In this embodiment, a droplet-based single-nuclear processing method is used to isolate and label the nuclei. Spatial barcoding and nucleic acid reverse transcription reactions are completed within the droplet, ensuring that both the spatial barcode and the nuclear transcript are labeled with the same cell tag (CellBarcode) within the same droplet. The nucleic acid products are then recovered after demulsification.

[0162] The nucleic acid products obtained after demulsification were purified and separated using magnetic beads. Fragment length selection was performed based on the length differences of different nucleic acid products, enabling the separation of different types of nucleic acid molecules under magnetic bead sorting conditions. The specific operation is as follows: (1) Purification of cDNA transcripts carrying cell tags. Specifically, add 60 μL of SPRI magnetic beads to a PCR tube (labeled tube A) containing 100 μL of demulsified solution and mix by pipetting. After incubating at room temperature for 5 minutes, place the PCR tube on a 0.2 mL magnetic rack and let it stand until the solution becomes clear. Transfer the supernatant to a new PCR tube and label it tube B. Add 200 μL of freshly prepared 80% ethanol to tube A (add along the opposite wall where the magnetic beads are located, being careful not to touch the magnetic beads) and let it stand for 30 seconds. Discard the supernatant and repeat the washing step once. Discard the supernatant, centrifuge briefly (with the side containing the magnetic beads facing out), and then place the PCR tube on a magnetic rack and let it stand for 10 seconds. Remove the remaining ethanol in the tube with a 10 μL pipette, repeating this process several times until there is no obvious liquid residue. Then open the PCR tube cap and let the magnetic beads air dry (about 2 min) until there is no water reflection on the surface of the magnetic beads, while avoiding excessive drying that could cause the magnetic beads to crack. Remove the PCR tube from the magnetic rack and immediately add 47 μL of enzyme-free water. Let it stand at room temperature for 5 min. Place it back on the magnetic rack until the solution becomes clear. Transfer 46 μL of the supernatant to a new 0.2 mL PCR tube to obtain purified cell-tagged cDNA transcripts.

[0163] (2) Purify the nucleic acid product carrying spatial barcode information. Specifically, add 60 μL of SPRI magnetic beads to tube B obtained in the above steps, vortex to mix, and incubate at room temperature for 5 min. Then place it on a magnetic rack until the magnetic beads adsorb and the solution becomes clear, and remove the supernatant. Add 200 μL of freshly prepared 80% ethanol (add along the opposite wall where the magnetic beads are located, being careful not to touch the magnetic beads), and let it stand for 30 s. Discard the supernatant and repeat the washing step once. Discard the supernatant, centrifuge briefly (with the side containing the magnetic beads facing out), and then place the PCR tube on a magnetic rack and let it stand for 10 s. Use a 10 μL pipette to remove the remaining ethanol in the tube, repeating this process several times until there is no obvious liquid residue. Open the PCR tube cap and air dry the magnetic beads (about 2 min) until there is no water reflection on the surface of the magnetic beads, while avoiding excessive drying that could cause the magnetic beads to crack. Remove the PCR tube from the magnetic rack, immediately add 47 μL of enzyme-free water to fully resuspend the magnetic beads, and let it stand at room temperature for 5 min. Place it on a magnetic rack until the solution becomes clear, then transfer the supernatant (46 μL) to a new 0.2 mL PCR tube to obtain the purified nucleic acid product with spatial barcode information.

[0164] (3) Library construction: For cDNA transcripts carrying cell tags, after 13 cycles of pre-amplification and purification, appropriate products were used for library construction, and sequencing adapter sequences and index sequences were introduced. The specific steps are as follows: (I) cDNA sample preparation: Take the cell-tagged cDNA transcript (double-stranded cDNA) obtained from the above steps after concentration quantification, and add a cDNA solution containing 150 ng of cDNA to a PCR tube by cDNA mass. If the volume of the added cDNA solution is less than 45 µL, add nuclease-free water to make up to 45 µL, and then place on ice for later use.

[0165] (II) cDNA fragmentation and end repair: The cDNA sample is fragmented by an enzymatic reaction, breaking the long cDNA into fragments suitable for sequencing (300-600 bp in this example), and then performing end repair and adding an A tail to the 3' end.

[0166] The fragmentation and end-of-life repair system is shown in the table below.

[0167] Table 6 Fragmentation and end-treatment reaction systems

[0168] The reagents used in the above reaction system are from Vazyme (catalog number: ND801).

[0169] The reaction procedure was as follows: 30℃ for 9 min; 65℃ for 20 min; and held at 4℃.

[0170] (III) Ligation: The fragmented / end-repair products obtained in the above steps are ligated to universal sequencing adapters. The adapter ligation reaction system is shown in the table below.

[0171] Table 7 Joint Connection Reaction System

[0172] The reagents used in the above reaction system are from Vazyme (catalog number: ND801).

[0173] The reaction procedure was as follows: incubation at 20°C for 15 min to obtain the ligation product.

[0174] (IV) Purification and bilateral sorting of the ligation products: The connection products obtained in the above steps are sorted by length using SPRI magnetic beads. The specific sorting steps include: Primary purification: Add 100 µL of SPRI magnetic beads (1× ratio) to 100 µL of the ligation product obtained in the above steps, mix well and incubate for 5 min. After capture by a magnetic rack, discard the supernatant and wash the magnetic beads twice with 80% ethanol.

[0175] Bilateral sorting: The cleaned magnetic beads were washed with 102 µL of nuclease-free water, and 100 µL of supernatant was recovered.

[0176] Large fragment removal: Add 55 µL of SPRI magnetic beads (0.55× ratio) to 100 µL of recovered supernatant, mix well and incubate for 5 min. At this point, large fragments bind to the magnetic beads. Separate the magnetic beads and retain the supernatant.

[0177] Target fragment recovery: Add 15 µL of SPRI magnetic beads (approximately 0.7×) to the supernatant after removing large fragments and mix well. After the target fragment binds to the magnetic beads, discard the supernatant, wash with 80% ethanol, and then elute with 48 µL of nuclease-free water to obtain the purified ligation product.

[0178] (V) Amplification and sorting of cDNA libraries: The sample index and the P5 / P7 sequencing adapter sequences from the above examples were introduced via PCR amplification. The PCR amplification system is shown in the table below.

[0179] Table 8 PCR amplification system

[0180] The cDNA Index primer sequence is as follows: cDNA primer 1: 5'-AATGATACGGCGACCACCGAGATCTACACTATAGCCTACACTCTTTCCCTACACGACGCTC-3' (SEQ ID NO: 4); cDNA Primer 2: 5'-CAAGCAGAAGACGGCATACGAGATTTAAATCTGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 5).

[0181] The PCR amplification reaction program was as follows: initial denaturation at 95℃ for 3 min; denaturation at 95℃ for 20 s, annealing at 58℃ for 20 s, extension at 72℃ for 30 s, for 13 cycles; final extension at 72℃ for 5 min; and hold at 4℃.

[0182] The obtained PCR amplification products were sorted again using bilateral magnetic beads (sorting steps were the same as above, with a ratio gradient of 0.55× and 0.15×). After eluting the final sorted products with 32 µL of nuclease-free water, 30 µL of supernatant was recovered to obtain a high-quality spatial transcriptome analysis library, which can be directly used for subsequent high-throughput sequencing.

[0183] For nucleic acid products carrying spatial barcode information (hereinafter referred to as barcode), after PCR enrichment and purification, library construction is performed directly, and sequencing adapter sequences and index sequences are introduced.

[0184] Specific methods for building a database include: Prepare the Barcode library amplification reaction system as shown in the table below on ice.

[0185] Table 9 Barcode Library Amplification Reaction System

[0186] The Barcode Index primers are composite primers containing a sequencing platform adapter sequence, a sample index sequence, and a specific binding sequence. The specific binding sequence undergoes complementary hybridization with the fixed sequence at the end of the aforementioned spatial barcode product. The sample index sequence is used to distinguish different library samples, and the sequencing platform adapter sequence is selected from at least one of the aforementioned P5 adapter sequences or P7 adapter sequences. The PCR Master Mix uses a premix containing high-fidelity DNA polymerase.

[0187] Specifically, the Barcode Index primer sequence is: Primer 1: 5'-AATGATACGGCGACCACCGAGATCTACACATAGAGGCACACTCTTTCCCTACACGACGCTC-3' (SEQ ID NO: 6); Primer 2: 5'-CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 7).

[0188] The amplification program was as follows: initial denaturation at 95℃ for 3 min; denaturation at 98℃ for 20 s, annealing at 58℃ for 20 s, extension at 72℃ for 10 s, for 6 cycles; final extension at 72℃ for 5 min; and hold at 4℃. Prior to amplification, a heat-capping treatment was performed at a temperature of 105℃.

[0189] The amplification products were sorted (to remove large fragment impurities and primer dimers) and purified according to the sorting and magnetic bead purification steps described above, and the purified Barcode library was recovered.

[0190] (VI) Sequencing and Data Analysis: In this embodiment, the purified transcript cDNA library and barcode library were subjected to next-generation sequencing. The cDNA library contains cell tags and transcript information, while the barcode library contains cell tags and spatial barcode (SBC) information. Finally, the sequencing data (FASTQ file) was obtained.

[0191] After quality control, filtering, and splitting the sequencing data of the transcript cDNA library, conventional alignment and quantitative analysis methods in the art can be used for processing. For example, in this embodiment, single-cell data analysis software is used to perform reference genome alignment and transcript quantification analysis on the cDNA library.

[0192] Specifically, cell tags and spatial barcode (SBC) sequence information are extracted from the spatial barcode library, and the SBC sequences are restored to physical spatial coordinates using the mapping index obtained from precoding sequencing. Subsequently, using the cell tag as the association key, the restored physical spatial coordinates are mapped to the corresponding cDNA library entries, and clustering or density analysis methods (exemplarily, such as the DBSCAN algorithm used in this embodiment) are used to calculate the coordinate points associated with each cell nucleus to determine its final spatial location.

[0193] After obtaining the cell nuclear transcript expression matrix with matching spatial location information, dimensionality reduction and clustering analysis methods can be further used to analyze the cell population. For example, in this embodiment, a UMAP-based dimensionality reduction algorithm is used to cluster the transcript expression matrix, and different cell groups are visualized according to their respective spatial coordinates.

[0194] HE staining results of adjacent tissue sections used for control and spatial transcriptome data analysis results based on the method of this invention are as follows: Figure 1 As shown, the spatial distribution of different cell populations shows good consistency with the tissue structure revealed by HE staining, indicating that the spatial transcriptome technology provided by this invention can achieve high-precision acquisition of spatial transcriptome information.

[0195] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A spatial transcriptome chip, characterized in that, The spatial transcriptome chip includes: a substrate and at least one nucleic acid probe loaded on the surface of the substrate; In one of the nucleic acid probes, at least one contains a photolytic linker; the photolytic linker breaks and is released from the substrate surface under specific wavelength light conditions.

2. The spatial transcriptome chip according to claim 1, characterized in that, The photolytically degradable linker includes aromatic nitro compounds; Preferably, the photodisintegrable linker comprises a compound represented by Formula I; Formula I.

3. The spatial transcriptome chip according to claim 1, characterized in that, The nucleic acid probe also includes a sequencing adapter sequence.

4. The spatial transcriptome chip according to claim 1, characterized in that, The nucleic acid probe has undergone pre-coding processing; The precoding process includes: obtaining the spatial location information of the nucleic acid probe and anchoring the spatial location information to the corresponding nucleic acid probe; Preferably, the spatial location information is set using a spatial barcode probe.

5. The spatial transcriptome chip according to claim 4, characterized in that, The spatial barcode probe includes: a nucleic acid molecule complementary to the sequencing adapter sequence of claim 3, a nucleic acid molecule complementary to the sequencing primer, a random sequence, a pre-encoding sequencing primer binding sequence, and a binding site for sequencing primer Read 2; Preferably, the spatial barcode probe comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO:

3.

6. A reagent kit or package, characterized in that, The kit or package includes the spatial transcriptome chip according to any one of claims 1-5; Preferably, the kit or assembly further includes: At least one of sequencing reagents, enzymes, and buffer solutions.

7. Use of the spatial transcriptome chip according to any one of claims 1-5 and / or the kit or apparatus according to claim 6 in spatially localized transcriptome sequencing.

8. A spatially localized transcriptome sequencing method, comprising the following steps: (1) The tissue to be tested is attached to the detection surface of the spatial transcriptome chip according to any one of claims 1-5, treated with light, the nucleic acid probe is released, and tissue incubation solution is added to permeate the tissue, so that the nucleic acid probe enters the cell nucleus to label the cell nucleus; (2) The tissue is lysed or dissociated, the information complex in the cell nucleus is recovered, a sequencing library is constructed, and sequencing is performed to obtain spatially localized transcriptome information.

9. The spatial localization transcriptome sequencing method according to claim 8, characterized in that, The tissue incubation solution includes: Buffer solutions, inorganic salts, surfactants, protein stabilizers, and ribonuclease inhibitors; Preferably, the inorganic salt comprises a divalent cation salt; Preferably, the divalent cation salt includes at least one of calcium salt, magnesium salt, or zinc salt.

10. The spatial localization transcriptome sequencing method according to claim 7, characterized in that, The spatial localization transcriptome sequencing method further includes: constructing a visualization image based on spatial localization transcriptome information.

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