Methods for constructing spatial transcriptome chips and cDNA libraries and methods for transcriptome sequencing analysis

Abstract

The present application provides a spatial transcriptome chip, a method for constructing a cDNA library, and a method for transcriptome sequencing analysis. The chip includes a substrate, on which multiple probe groups are fixed, each of which includes a first probe and a second probe; the first probe includes a sequencing primer segment, a barcode segment, a specific molecular tag, and a first capture structure for capturing the 3' end of mRNA, which are sequentially connected from the substrate upward; the second probe includes a sequencing primer segment, a barcode segment, a specific molecular tag, and a second capture structure for capturing the 5' end of mRNA, which are sequentially connected from the substrate upward; the first probe and the second probe in each probe group have the same barcode segment, and at least two of the multiple probe groups have different barcode segments.

Description

Technical Field

[0001] The present application relates to the field of transcriptome sequencing technology, and in particular to a spatial transcriptome chip, a method for constructing a cDNA library, and a method for transcriptome sequencing analysis. Background Art

[0002] Gene expression is both temporally and spatially specific. This temporal specificity can be analyzed by analyzing samples taken at different time points and combining it with single-cell transcriptome sequencing. Spatial transcriptomics, the latest form of single-cell sequencing, provides both transcriptome and spatial location information. It helps researchers better identify the location of transcripts and further extends this to single-cell resolution, greatly enhancing scientists' understanding and interpretation of individual cells.

[0003] Research related to spatial transcriptomics has shown explosive growth in the past decade, advancing people's understanding of cell functions and having important significance for promoting research in precision medicine, cell mapping, human life and health, and other fields.

[0004] Spatial omics technology dates back to the 1970s, using radioactive in situ hybridization to detect highly amplified ribosomal RNA in cells, enabling quantitative analysis of gene expression levels in tissues or cells. The concept of spatial transcriptomics was first proposed in 2016, and its development has progressed through two phases: bulk transcriptomics and single-cell transcriptomics. Since then, a series of new spatial omics technologies have been continuously developed.

[0005] Currently, the most widely used spatial omics technologies fall into four categories: The first is microdissection, which rapidly and accurately captures target cells from a biopsy sample, visually capturing both spatial and transcriptome information. The second is in situ hybridization, where fluorescent probes hybridize specifically with specific mRNAs in situ within the tissue, revealing the abundance of transcripts within the cell while also providing spatial localization. The third is in situ sequencing, which uses padlock probes to capture mRNA from fixed biological tissue samples and then amplify and sequence them in situ to determine gene expression levels. The fourth category is in situ capture, which combines specific barcode sequences with sequencing data to visually restore gene expression to the original biological tissue sections. It has the advantages of high throughput and high resolution, such as 10×Visium technology, Stereo-Seq technology, slide-seq technology, seq-scope technology, pixel-seq technology, high-definition spatial transcriptomics technology (HDST) and deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq).

[0006] Traditional in situ capture techniques typically use known in situ barcode sequences to mark the spatial location of tissues, then use high-throughput sequencing to read the barcodes and mRNA sequence information, and then restore the measured sequences to the corresponding spatial locations. However, traditional methods cannot obtain full-length mRNA transcriptome information. Summary of the Invention

[0007] Based on this, it is necessary to provide a spatial transcriptome chip, a cDNA library construction method and a transcriptome sequencing analysis method that can obtain the spatial distribution information of mRNA and the full-length transcriptome information of mRNA.

[0008] According to one aspect of the present application, a spatial transcriptome chip is provided, the chip comprising a substrate, a plurality of probe groups fixed on the substrate, each of the probe groups comprising a first probe and a second probe;

[0009] The first probe includes a sequencing primer segment, a barcode segment, a specific molecular tag, and a first capture structure for capturing the 3' end of the mRNA, which are sequentially connected from the substrate upward;

[0010] The second probe includes a sequencing primer segment, a barcode segment, a specific molecular tag, and a second capture structure for capturing the 5' end of the mRNA, which are sequentially connected from the substrate upward;

[0011] The first probe and the second probe in each probe group have the same barcode fragment, and at least two probe groups in the plurality of probe groups have different barcode fragments.

[0012] In one embodiment, the first probe and the second probe are distributed in pairs.

[0013] In one embodiment, the first capture structure is a polyT structure; and / or

[0014] The second trapping structure is a TSO structure.

[0015] In one embodiment, the probes in the probe set are connected to the substrate via covalent bonds.

[0016] In one embodiment, a polymer material is used to connect the substrate and the probe group;

[0017] Optionally, the polymer material includes one or more of alginate gel, agarose gel, polyacrylamide gel, polysaccharide, polylysine and ethylene-acrylamide copolymer.

[0018] In one embodiment, the surface of the substrate is modified with active groups, and the active groups include one or more of amino, hydroxyl, carboxyl, anhydride, acyl, epoxy and thiol groups;

[0019] The probe group is modified with a group that matches the active group on the substrate, and the probe group is modified with one or more of an azide group, a dibenzocyclooctyne group, an amino group, a carboxyl group, an aldehyde group, an acrylamido group, an alkynyl group, a maleimide group, a sulfhydryl group, a dithiol group, a ferrocene group, and a biotin group;

[0020] Optionally, the active groups on the substrate surface are connected to the modified groups on the probe group through an ammonia condensation reaction, a thiol reaction, a cycloaddition reaction or a click chemistry reaction involving a cross-linking agent;

[0021] Further optionally, the cross-linking agent includes one or more of diphenylcyclooctyne polyethylene glycol active ester, azide polyethylene glycol active ester, trans-cyclooctene polyethylene glycol active ester, alkynyl polyethylene glycol active ester, tetrazine polyethylene glycol active ester and cyclopropanecyclooctyne polyethylene glycol active ester.

[0022] According to another aspect of the present application, a method for constructing a cDNA library is provided, comprising the following steps:

[0023] The 3' end of the target mRNA was captured using the above-mentioned spatial transcriptome chip;

[0024] synthesizing cDNA fragments using the target mRNA as a template;

[0025] Connecting the 5' end of the cDNA fragment to the substrate surface of the chip to form a bridge-structured cDNA; and

[0026] The cDNA of the bridge structure is used as a template to perform PCR amplification to construct a cDNA library.

[0027] According to another aspect of the present application, a method for constructing a cDNA library is provided, comprising the following steps:

[0028] A spatial transcriptome chip was used to capture the 3' end of the target mRNA;

[0029] synthesizing cDNA fragments using the target mRNA as a template;

[0030] Connecting a sequencing primer fragment, a barcode fragment, a specific molecular tag, and a second capture structure for capturing the 5' end of mRNA to the 5' end of the cDNA fragment in sequence by microfluidic technology to obtain a double-end labeled cDNA fragment; and

[0031] Using the double-end labeled cDNA fragment as a template, PCR amplification is performed to construct a cDNA library;

[0032] The spatial transcriptome chip comprises a substrate on which a plurality of independently distributed probes are fixed. The probes comprise a sequencing primer fragment, a barcode fragment, a specific molecular tag, and a first capture structure for capturing the 3' end of mRNA, which are sequentially connected upward from the substrate.

[0033] In one embodiment, the first capture structure is a polyT structure; and / or

[0034] The second trapping structure is a TSO structure.

[0035] According to another aspect of the present application, a method for transcriptome sequencing analysis is provided, comprising the following steps:

[0036] Constructing a cDNA library using the above method; and

[0037] The cDNA library was sequenced and analyzed.

[0038] Compared with traditional technologies, this application has the following beneficial effects:

[0039] The present application has been found through analysis that traditional technology cannot obtain mRNA full-length transcriptome information. The reason is that traditional technology only performs sequence decoding on the 3' end of mRNA, and cannot obtain important transcription information on the 5' end of mRNA, such as exon sequence and transcription start sequence. The present application improves the spatial transcriptome sequencing chip captured in situ, so that the 3' end and 5' end of the mRNA sequence are marked with the same barcode sequence, thereby enabling double-end sequencing of mRNA to be achieved, and obtaining the full-length sequence of mRNA. At the same time, using the sequencing chip of the present application, the spatial position information of mRNA can also be obtained.

[0040] In addition, the cDNA library construction method of the present application can label both ends of the mRNA with the same barcode sequence, thereby enabling sequencing analysis of the entire length of the mRNA. The sequencing analysis method of the present application also has the advantages of low cost and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0042] Figure 1 This is a schematic structural diagram of the first probe and the second probe in one embodiment of the present application;

[0043] Figure 2 This is a schematic structural diagram of the first probe and the second probe in one embodiment of the present application;

[0044] Figure 3 This is a schematic diagram of the structure of a chip in one embodiment of the present application;

[0045] Figure 4 This is a schematic diagram of the connection between the first probe and the second probe in one embodiment of the present application;

[0046] Figure 5 Schematic diagram of the construction of a cDNA library in one embodiment of the present application;

[0047] Figure 6 Schematic diagram of the construction of a cDNA library in one embodiment of the present application;

[0048] Figure 7 This is a schematic diagram of the connection between the first probe and the second probe in Example 1;

[0049] Figure 8 Figure a is the histomorphology of the sample to be tested in Example 1 after being stained with cresyl violet. Figure 8 Middle b is the fluorescence imaging image after capturing mRNA reverse transcription;

[0050] Figure 9 In the figure a, a cDNA structure is double-ended with barcode sequences. Figure 9 Figure b is the structure of the cDNA library enriched at the 3' end of mRNA. Figure 9 Middle c is the structure of the cDNA library enriched at the 5' end of mRNA;

[0051] Figure 10 This is the Sanger sequencing peak diagram of the target mRNA in Example 1;

[0052] Figure 11 Schematic diagram of the process of labeling barcode sequences using microfluidic technology in Example 2. DETAILED DESCRIPTION

[0053] In order to make the above-mentioned objects, features and advantages of the present application more clearly understood, the specific embodiments of the present application are described in detail. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotations of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the technical field of this application. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or can be prepared by existing methods.

[0055] According to one aspect of the present application, a spatial transcriptome chip is provided, comprising a substrate, on which a plurality of probe groups are fixed, each probe group comprising a first probe and a second probe;

[0056] The first probe includes a sequencing primer segment, a barcode segment, a specific molecular tag, and a first capture structure for capturing the 3' end of the mRNA, which are sequentially connected from the base upward;

[0057] The second probe includes a sequencing primer fragment, a barcode fragment, a specific molecular tag, and a second capture structure for capturing the 5' end of the mRNA, which are connected sequentially from the base upward;

[0058] The barcode segments of the first probe and the second probe in each probe group are the same, and the barcode segments of at least two probe groups in the plurality of probe groups are different.

[0059] In some specific examples, the first probe and the second probe are distributed in pairs.

[0060] In a specific example, the structures of the first probe and the second probe are as follows Figure 1 shown.

[0061] Understandably, multiple probe sets are distributed in an array on the chip surface. Each probe in the array has a different barcode segment or a different combination of barcode segments. The barcode segment information can be used to determine the specific location of the probe on the chip, thereby obtaining spatial distribution information of the transcriptome. The first and second probes in the probe set have the same barcode information, allowing them to accurately capture the 3' and 5' ends of the same mRNA at the same spatial location.

[0062] In some specific examples, the substrate is selected from a silicon wafer or a silica glass wafer.

[0063] In some specific examples, the first capture structure is a polyT structure, which can complementarily pair with the polyA tail structure at the 3' end of the mRNA, thereby achieving capture of the 3' end of the mRNA.

[0064] In some specific examples, the second capture structure is a TSO structure. "TSO" refers to a template switch oligonucleotide that can hybridize to the non-templated C nucleotide at the 5' end of the mRNA.

[0065] In some specific examples, the sequencing primer fragment is a P7 primer, and its nucleotide sequence is shown in SEQ ID NO: 1 (GCATAGTCGTACGCCGATG).

[0066] A specific molecular identifier (UMI) is a short randomized nucleotide sequence. The inclusion of a specific molecular identifier in a probe can not only perform quantitative analysis of mRNA, but also improve the sensitivity and specificity of detection.

[0067] exist Figure 2 In the example shown, the first probe includes, sequentially from base to base, a sequencing primer fragment, barcode fragment 1, barcode fragment 2, a specific molecular tag, and a first capture structure that captures the 3' end of the mRNA. The second probe includes, sequentially from base to base, a sequencing primer fragment, barcode fragment 1, barcode fragment 2, a specific molecular tag, and a second capture structure that captures the 5' end of the mRNA.

[0068] It is understandable that the barcode sequences in the first probe and the second probe can be one or two, or multiple depending on the needs. Each array point on the chip has unique barcode sequence information, so that its spatial information can be accurately located and identified.

[0069] In some embodiments, the probes in the probe set are linked to the substrate via covalent bonds.

[0070] In some examples, a polymer material is used to connect the substrate and the probe set. Further, the polymer material includes one or more of alginate gel, agarose gel, polyacrylamide gel, polysaccharide, polylysine, and ethylene-acrylamide copolymer.

[0071] In some examples, the surface of the substrate is modified with reactive groups, and the reactive groups include one or more of amino, hydroxyl, carboxyl, anhydride, acyl, epoxy, and thiol.

[0072] Furthermore, a silane coupling agent is used to activate and modify the substrate surface. Furthermore, the silane coupling agent is selected from aminosilane coupling agents. Furthermore, the aminosilane coupling agent includes one or more of 3-aminopropyltriethoxysilane (APTES), 3-aminopropyltrimethoxysilane (APTMS), and 3-aminopropyldimethylethoxysilane (APDMES).

[0073] Furthermore, the probe group is modified with a group that matches the active group on the substrate, and the 5' end of the probe group is modified with one or more of an azide group, dibenzocyclooctyne, amino group, carboxyl group, aldehyde group, acrylamido group, alkynyl group, maleimide, sulfhydryl group, dithiol, ferrocene and biotin.

[0074] In some examples, the active groups on the substrate surface are linked to the modified groups on the probe set via an ammonia condensation reaction, a thiol reaction, a cycloaddition reaction, or a click chemistry reaction involving a crosslinker. Preferably, the active groups on the substrate surface are linked to the modified groups on the probe set via a click chemistry reaction involving a crosslinker.

[0075] Furthermore, the crosslinking agent includes one or more of diphenylcyclooctyne polyethylene glycol active ester (DBCO-PEGn-NHS Ester), azide polyethylene glycol active ester (N3-PEG4-NHS Ester), trans-cyclooctene polyethylene glycol active ester (TCO-PEGn-NHSEster), alkynyl polyethylene glycol active ester (Alkynes-PEGn-NHS Ester), tetrazine polyethylene glycol active ester (Tetrazine-PEGn-NHS Ester), and cyclopropanecyclooctyne polyethylene glycol active ester (BCN-PEGn-NHS Ester). Specifically, n can be any positive integer; preferably, n is any integer between 2 and 10.

[0076] Furthermore, the DBCO-, N3-, TCO-, Alkynes-, Tetrazine-, or BCN- crosslinkers are linked to the 5' end of the probe set. Specifically, the DBCO-, N3-, TCO-, Alkynes-, Tetrazine-, or BCN- crosslinkers are linked to the 5' end of the sequencing primer fragment of the probe set; and the NHS Ester- crosslinker is covalently coupled to the active groups on the substrate surface.

[0077] like Figure 3 As shown, the substrate and probe set can be connected via a bifunctional crosslinker, i.e., the first probe and the second probe are independently connected to the crosslinker. Alternatively, the substrate and probe set can be connected via a trifunctional crosslinker, i.e., the first probe and the second probe are respectively connected to two groups of the crosslinker.

[0078] In some examples, the trifunctional crosslinker is selected from one or more of Bis(DBCO-PEGn)-NHS ester, Bis(N3-PEG4)-NHS Ester, Bis(TCO-PEGn)-NHS Ester, Bis(Alkynes-PEGn)-NHS Ester, Bis(Tetrazine-PEGn)-NHS Ester, and Bis(BCN-PEGn)-NHS Ester. It is understood that Bis(DBCO-PEGn)-NHS ester, for example, refers to a crosslinker containing two DBCO groups and one PEG4-NHS ester; the same applies to the other trifunctional crosslinkers.

[0079] Specifically, the use of a trifunctional cross-linking agent can form a bifurcated structure on the chip surface, allowing each active group to couple with two probes, thereby increasing the probe density and amplifying the recognition signal.

[0080] The chip of the present application can connect the substrate and the probe group via a polymer material; can also connect the substrate and the probe group via a cross-linking agent; or can connect the substrate and the probe group via both a polymer material and a cross-linking agent. It is understood that in other embodiments, the connection method between the substrate and the probe group is not limited to the above, and can also be a connection method commonly used in the art.

[0081] This application improves the in situ captured spatial transcriptome sequencing chip so that the 3' and 5' ends of the mRNA sequence are marked with the same barcode sequence, thereby enabling the use of a sequencing platform to obtain the full-length transcriptome sequence information and spatial location information of the mRNA, and to achieve quantification of the mRNA.

[0082] According to another aspect of the present application, a method for preparing the above-mentioned transcriptome chip is provided, comprising the following steps S1-S2:

[0083] S1: Ligation of substrate and sequencing primer fragments.

[0084] S2: The barcode fragment, specific molecular tag and capture probe fragment are sequentially connected to the end of the sequencing primer fragment to prepare a transcriptome chip.

[0085] In some examples, in step S1, the substrate and the sequencing primer fragment are connected via a cross-linking agent and / or a polymer material. Alternatively, the substrate and the sequencing primer fragment are connected via an amino condensation reaction, a thiol reaction, a cycloaddition reaction, or a click chemistry reaction involving a cross-linking agent.

[0086] In some examples, in step S1, the 5' end of the sequencing primer fragment is attached to the substrate surface.

[0087] In some examples, in step S2, the barcode fragment, the specific molecular tag, and the capture probe fragment are sequentially connected to the end of the sequencing primer fragment by extension or ligation. Specifically, the barcode fragment, the specific molecular tag, and the capture probe fragment are sequentially connected to the 3' end of the sequencing primer fragment.

[0088] In one example, the first probe and the second probe obtained by the extension method are connected as follows: Figure 4 shown.

[0089] According to another aspect of the present application, a method for constructing a cDNA library is provided.

[0090] In some embodiments, the method for constructing a cDNA library includes the following steps S3 to S6.

[0091] S3: The 3' end of the target mRNA is captured using the above-mentioned spatial transcriptome chip.

[0092] S4: Synthesize cDNA fragments using the target mRNA as a template.

[0093] S5: Connect the 5' end of the cDNA fragment to the substrate surface of the chip to form a bridge-structured cDNA.

[0094] S6: Using the bridge structure cDNA as a template, bridge amplification and PCR amplification are performed sequentially to construct a cDNA library.

[0095] In some examples, in step S5, the first capture structure of the spatial transcriptome chip is ligated to the 3' end of the target mRNA.

[0096] In some examples, in step S5, the 5' end of the cDNA fragment is linked to the substrate surface of the chip by hybridization. Further, the 5' end of the cDNA fragment is linked to the second capture structure on the substrate surface.

[0097] Specifically, if Figure 5 As shown, the cDNA library construction method includes the following steps: The 3' end of the target mRNA is captured using a transcriptome chip connected with a bifunctional or trifunctional crosslinker. Reverse transcription amplification is performed using the captured target mRNA as a template to generate a cDNA-mRNA double-strand. The mRNA strand is then removed by denaturation of the hybridized strands to generate a single-stranded cDNA. The 5' end of the cDNA fragment is then linked to the substrate surface of the chip via hybridization to form a bridge structure. Using the bridge structure as a template, bridge amplification and PCR amplification are sequentially performed to construct the cDNA library.

[0098] In some embodiments, the method for constructing a cDNA library includes the following steps S7 to S10:

[0099] S7: A spatial transcriptome chip is used to capture the 3' end of the target mRNA; the spatial transcriptome chip includes a substrate, on which a plurality of independently distributed probes are fixed, the probes including sequencing primer fragments, barcode fragments, specific molecular tags, and a first capture structure for capturing the 3' end of the mRNA, which are sequentially connected from the substrate upward.

[0100] S8: Synthesize cDNA fragments using the target mRNA as a template.

[0101] S9: Using microfluidics technology, a sequencing primer fragment, a barcode fragment, a specific molecular tag, and a second capture structure for capturing the 5' end of mRNA are sequentially connected to the 5' end of the cDNA fragment to obtain a double-end labeled cDNA fragment.

[0102] S10: Using the double-end labeled cDNA fragments as templates, bridge amplification is performed to construct a cDNA library.

[0103] Specifically, the barcode segments of the first capture structure and the second capture structure are the same.

[0104] In some specific examples, the first capture structure is a polyT structure.

[0105] In some specific examples, the second trapping structure is a TSO structure.

[0106] Specifically, if Figure 6As shown, the cDNA library construction method includes the following steps: The 3' end of the target mRNA is captured using a transcriptome chip connected to a bifunctional crosslinker. Reverse transcription amplification is performed using the captured target mRNA as a template to generate a cDNA-mRNA duplex. The mRNA strand is then removed by denaturation of the hybrid strand to generate a single cDNA strand. Microfluidics is used to deliver a sequencing primer fragment, a barcode fragment, a specific molecular tag, and a second capture structure that captures the 5' end of the mRNA to a fixed location and ligate to the 5' end of the cDNA strand, generating double-end captured cDNA fragments. Using the double-end labeled cDNA fragments as templates, bridge amplification is performed to construct the cDNA library.

[0107] The cDNA library construction method of the present application uses bridge amplification or microfluidics technology to simultaneously label the two ends of mRNA with the same barcode, and can achieve full-length sequence analysis of mRNA on a second-generation sequencing platform.

[0108] According to another aspect of the present application, a method for transcriptome sequencing analysis is provided, comprising the following steps:

[0109] The above method was used to construct a cDNA library, and the cDNA library was sequenced and analyzed.

[0110] The transcriptome sequencing analysis method of this application can obtain the full-length sequence information and spatial location information of mRNA. Compared with third-generation sequencing, it has the advantages of low cost and high accuracy.

[0111] The present application will be further described below with reference to specific embodiments and comparative examples, but they should not be construed as limiting the scope of protection of the present application.

[0112] Example 1:

[0113] (1) Chip preparation

[0114] A silica glass sheet was selected as the substrate of the chip, and the surface of the substrate was amino-modified using an aminosilane coupling agent.

[0115] A P7 primer fragment was provided, and its 5' end was modified with an azide group to form the nucleotide sequence shown in SEQ ID NO: 1. In this example, using one spot on a chip as an example, a sequence fragment with the nucleotide sequence shown in Table 1 was provided, where Barcode1 represents barcode fragment 1, Barcode2-polyT represents the linker of barcode fragment 2, a specific molecular tag, and a polyT structure, and Barcode2-TSO represents the linker of barcode fragment 2, a specific molecular tag, and a TSO structure. The primer sequences were synthesized by Shanghai Bioengineering.

[0116] Table 1

[0117]

[0118] Dilute DBCO-PEG4-NHS Ester crosslinker (Ruixi Bio) to 100 μM in 1× PBS and dropwise apply to the chip substrate. Allow to react at room temperature for 30 min–2 h. This allows the -NHS group of the crosslinker to covalently couple with the amino groups on the substrate surface, and the DBCO end of the crosslinker to covalently link with the azide group on the 5' end of the primer fragment. After the reaction, wash with 1× PBS and remove any remaining reagents.

[0119] Use 1×PBS to dilute the azide-modified P7 primer to 2.5 μM, continue to add it dropwise to the substrate surface, and react at room temperature for more than 4 hours; after the reaction, wash it with 1×PBS.

[0120] Mix 50 μM Barcode1 and 50 μM Splint1 to form Barcode1 mix. Prepare the ligation reaction system as shown in Table 2 and continue dripping onto the substrate surface. Ligase T4 activates the phosphate group at the 5' end of Barcode1 to form a phosphodiester bond with the hydroxyl group at the 3' end of the P7 primer.

[0121] Mix 25 μM Barcode2-polyT, 25 μM Barcode2-TSO, and 50 μM Splint2 to form Barcode2 mix. Prepare the ligation reaction system as shown in Table 2 and continue to drip onto the substrate surface. Under the action of ligase T4, the phosphate groups at the 5' ends of Barcode2-polyT and Barcode2-TSO respectively form phosphodiester bonds with the hydroxyl group at the 3' end of Barcode1, and the ligation is as follows: Figure 7 The first probe and the second probe are shown.

[0122] The T4 ligase used in this example was purchased from New England Biolabs.

[0123] Table 2

[0124]

[0125] (2) Capturing mRNA

[0126] The slices of frozen mouse brain tissue (embryo E13.5) were attached to the surface of the chip and stained with cresyl violet. The morphology of the stained tissue slices was as follows: Figure 8 As shown in a.

[0127] The tissue was then permeabilized with 0.1% pepsin at 37°C for 10 minutes. A 5× sodium citrate buffer (SSC) solution was added and incubated at room temperature for 20 minutes to allow for sufficient hybridization between the polyA at the 3' end of the mRNA and the polyT structure on the chip surface.

[0128] Prepare the reverse transcription reaction system according to Table 3 and react in a 42°C incubator at 40 rpm for 3 hours. After the reverse transcription reaction is completed, add PK enzyme to a Tris buffer system with a pH of 9.0, remove the biological tissue, and wash it with 2×SSC, 0.2×SSC, and 0.1×SSC in sequence, and then take pictures. Figure 8 Figure b shows the fluorescence imaging of mRNA captured in situ after reverse transcription. Figure 8 It can be seen that the chip prepared in step (1) successfully captured the mRNA in the mouse brain tissue.

[0129] The dCTP-CY3 used in this example was purchased from Shenzhen Tanmi Technology Co., Ltd.

[0130] Table 3

[0131]

[0132] (3) 5' end barcode labeling

[0133] The DNA-RNA hybrid strand formed after in situ reverse transcription in step (2) was denatured using a 0.08M KOH solution, removing the mRNA strand and restoring the cDNA on the chip surface to a single-stranded state. The sequence at the 5' end of the cDNA was then hybridized with the TSO structure on the chip surface in a 5×SSC solution to form a bridge structure. Under the action of a high-fidelity polymerase, the reaction system was as shown in Table 4 and the reaction was carried out in a 30°C incubator for 30 minutes, allowing the 3' end of the bridge structure to extend using each as a template to form a DNA double strand.

[0134] Table 4

[0135]

[0136] (4) cDNA amplification, library construction, and sequencing

[0137] Collect the cDNA with barcode sequences on both ends in step (3). The structure of the cDNA with barcode sequences on both ends is as follows: Figure 9 As shown in a, the structure of the cDNA library enriched at the 3' end of mRNA is as follows Figure 9 As shown in b, the structure of the cDNA library enriched at the 5' end of mRNA is as follows Figure 9 As shown in c.

[0138] PCR amplification was performed with the P7 primer according to the system shown in Table 5. The reaction procedure was: 98°C for 3 min; 98°C for 20 s, 60°C for 15 s, 72°C for 1 min, 15 cycles; 72°C for 5 min, and storage at 4°C.

[0139] Table 5

[0140]

[0141] The PCR products were purified using 0.8× magnetic beads and directly subjected to Sanger sequencing. The sequencing results were as follows: Figure 10 As shown in the figure, the polyT end indicates the 3' end of the mRNA, and the TSO sequence indicates the 5' end of the mRNA. The Barcode2-polyT probe on the chip surface is successfully labeled at the 3' end of the mRNA, while the Barcode2-TSO probe is labeled at the 5' end of the mRNA. Both the 3' and 5' ends share the same barcode label.

[0142] The above sequencing results show that the two ends of mRNA can be simultaneously labeled with the same barcode through the bridge extension method, and spatial transcriptome sequencing analysis of the full-length mRNA sequence can be directly realized on the second-generation sequencing platform.

[0143] Example 2:

[0144] (1) Chip preparation

[0145] Silica glass sheet was selected as the substrate, and the surface of the substrate was amino-modified using an aminosilane coupling agent.

[0146] A P7 primer fragment is provided, and its 5' end is modified with an azide group to form a nucleotide sequence as shown in SEQ ID NO: 1. In this example, a specific mRNA is used as an example, and the Barcode1 fragment and the Barcode2-polyT fragment are provided as shown in Table 1.

[0147] Dilute DBCO-PEG4-NHS Ester crosslinker (Ruixi Bio) to 100 μM in 1× PBS and dropwise apply to the chip substrate. Allow to react at room temperature for 30 min–2 h. This allows the -NHS group of the crosslinker to covalently couple with the amino groups on the substrate surface, and the DBCO end of the crosslinker to covalently link with the azide group on the 5' end of the primer fragment. After the reaction, wash with 1× PBS and remove any remaining reagents.

[0148] Use 1×PBS to dilute the azide-modified P7 primer to 2.5 μM, continue to add it dropwise to the substrate surface, and react at room temperature for more than 4 hours; after the reaction, wash it with 1×PBS.

[0149] Mix 50 μM Barcode1 and 50 μM Splint1 to form Barcode1 mix. Prepare the ligation reaction system as shown in Table 2 and continue dripping onto the substrate surface. Incubate with ligase T4 at 30°C and 40 rpm for 1 hour to form a phosphodiester bond between the phosphate group at the 5' end of Barcode1 and the hydroxyl group at the 3' end of the P7 primer.

[0150] (2) The steps of capturing mRNA are the same as those in Example 1.

[0151] (3) 5' end barcode labeling

[0152] Use 0.08M KOH solution to denature the DNA-RNA hybrid chain after in situ reverse transcription in step (2) to remove the mRNA chain, so that the cDNA on the chip surface is restored to a single-stranded state.

[0153] according to Figure 11 The method shown utilizes microfluidics technology to deliver a known barcode sequence to a fixed position for hybridization with the 5' end of the cDNA, so that both the 3' and 5' ends of the cDNA are simultaneously labeled with barcode sequences having spatial position information.

[0154] Specifically, microfluidic technology includes the following steps:

[0155] Cover the chip with the captured mRNA 3' end with a softer PDMS chip with a horizontal stripe structure. Prepare the reaction system containing Barcode1 mix according to Table 2. Add the mixture to the PDMS chip in the order of well positions. Use negative or positive pressure to draw the reagents onto the surface of the carrier chip to modify the carrier chip surface with Barcode1.

[0156] Use 1× PBS to wash away the residual reagents in the PDMS channel and remove the PDMS chip with horizontal stripes.

[0157] Stack a PDMS chip with vertical stripes on top of the chip. Prepare the reaction system containing Barcode2 mix according to Table 2. Add it to the PDMS chip in the order of well locations. Use negative or positive pressure to draw the reagents onto the carrier chip surface, modifying the carrier chip surface with Barcode2.

[0158] Because Barcode 1 and Barcode 2 are crisscrossed, each array point on the chip surface is labeled AiAj, where Ai is the horizontal Barcode 1 and Aj is the vertical Barcode 2. The same Barcode 1 is added to the same row and the same Barcode 2 is added to the same column; different Barcode 1s are added to different rows and different Barcode 2s to different columns. This gives each array point a unique barcode sequence, allowing accurate location and spatial identification.

[0159] (4) The steps of cDNA amplification, library construction and sequencing are the same as those in Example 1.

[0160] The sequencing results obtained using the method of this embodiment are the same as those in Example 1, indicating that microfluidics can achieve the same barcode labeling at both ends of mRNA, and directly realize spatial transcriptome sequencing analysis of the full-length mRNA sequence on the second-generation sequencing platform.

[0161] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0162] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present invention patent shall be determined by the appended claims.

Claims

1. A spatial transcriptome chip, characterized in that The chip includes a substrate, a plurality of probe groups are fixed on the substrate, and each probe group includes a first probe and a second probe; The first probe comprises a sequencing primer segment, a barcode segment, a specific molecular tag, and a first capture structure for capturing the 3' end of mRNA, which are sequentially connected from the substrate upward; the first capture structure is a polyT structure; The second probe comprises a sequencing primer segment, a barcode segment, a specific molecular tag, and a second capture structure for capturing the 5' end of the mRNA, which are sequentially connected from the substrate upward; the second capture structure is a TSO structure; The first probe and the second probe in each probe group have the same barcode fragment, and at least two probe groups in the plurality of probe groups have different barcode fragments; the first probe and the second probe are distributed in pairs.

2. The spatial transcriptome chip according to claim 1, characterized in that The probes in the probe group are connected to the substrate via covalent bonds.

3. The spatial transcriptome chip according to claim 2, characterized in that The substrate and the probe group are connected by using a polymer material.

4. The spatial transcriptome chip according to claim 3, characterized in that The polymer material includes one or more of alginate gel, agarose gel, polyacrylamide gel, polysaccharide, polylysine and ethylene-acrylamide copolymer.

5. The spatial transcriptome chip according to claim 2, characterized in that The surface of the substrate is modified with active groups, and the active groups include one or more of amino, hydroxyl, carboxyl, anhydride, acyl, epoxy and thiol groups; The probe group is modified with a group that matches the active group on the substrate, and the probe group is modified with one or more of an azide group, a dibenzocyclooctyne group, an amino group, a carboxyl group, an aldehyde group, an acrylamide group, an alkynyl group, a maleimide group, a sulfhydryl group, a dithiol group, a ferrocene group, and a biotin group.

6. The spatial transcriptome chip according to claim 5, characterized in that The active groups on the surface of the substrate are connected to the modified groups on the probe group through an ammonia condensation reaction, a thiol reaction, a cycloaddition reaction or a click chemistry reaction involving a cross-linking agent.

7. The spatial transcriptome chip according to claim 6, characterized in that The cross-linking agent includes one or more of diphenylcyclooctyne polyethylene glycol active ester, azide polyethylene glycol active ester, trans-cyclooctene polyethylene glycol active ester, alkynyl polyethylene glycol active ester, tetrazine polyethylene glycol active ester and cyclopropanecyclooctyne polyethylene glycol active ester.

8. The spatial transcriptome chip according to claim 6, characterized in that The cross-linking agent is selected from one or more of Bis(DBCO-PEGn)-NHS ester, Bis(N3-PEG4)-NHS Ester, Bis(TCO-PEGn)-NHS Ester, Bis(Alkynes-PEGn)-NHS Ester, Bis(Tetrazine-PEGn)-NHS Ester, and Bis(BCN-PEGn)-NHS Ester.

9. A method for constructing a cDNA library, characterized in that: The steps include: Using the spatial transcriptome chip according to any one of claims 1 to 8 to capture the 3' end of the target mRNA; synthesizing cDNA fragments using the target mRNA as a template; connecting the 5' end of the cDNA fragment to the substrate surface of the chip to form a cDNA with a bridge structure; as well as The cDNA of the bridge structure is used as a template to perform PCR amplification to construct a cDNA library.

10. A method for transcriptome sequencing analysis, characterized in that: The steps include: Constructing a cDNA library using the method according to claim 9; and The cDNA library was sequenced and analyzed.

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