Methods for deep non-targeted profiling of spatial transcriptome and proteome in intact tissues

Abstract

The present invention provides a method for physically expanding a sample and spatially profiling mRNA and optionally protein in the sample. An associated kit is also provided. The methods herein are compatible with different samples, sample staining methods, and sequencing technologies, and are suitable for spatial transcriptomic and proteomic profiling of biological samples.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to international patent application PCT / CN2023 / 121991, filed on September 27, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to space omics, and in particular to the preparation of biomaterials for spatially resolved high-throughput next-generation sequencing and mass spectrometry analysis, methods for producing expanded biomaterials and their uses, and compositions for inflating biomaterials and combining them with transcriptome sequencing and mass spectrometry-based proteomics analysis. Background Technology

[0004] In natural tissue environments, the spatial distribution of transcripts is crucial for understanding cellular tissue structure and state at the organismal level. In recent years, various methods have been developed for in situ gene expression spatial mapping using non-targeted mRNA sequencing. One of the most commonly used methods in spatial transcriptomics is based on ordered arrays of oligonucleotides deposited on glass slides using microarray printing technology. However, these methods have several limitations, such as high cost, limited spatial resolution, and incomplete RNA release. For example, methods such as High Resolution Spatial Transcriptomics (HDST), Slide-seq, and Stereo-seq use microbeads or nanospheres with spatial barcodes immobilized on the surface. The location of each DNA-based barcode must be pre-determined before the experiment begins, which is not only time-consuming but also requires sophisticated fluorescence imaging equipment or in situ sequencing devices.

[0005] On the other hand, expansion sequencing (ExSeq) is an in situ sequencing technique that combines tissue expansion with fluorescence imaging. This technique is cumbersome, time-consuming, and extremely difficult to implement on a large scale. When targeting a large number of molecules, optical crowding, as with any imaging-based spatial transcriptomics (ST) method, can cause problems. While sample expansion helps separate molecules, imaging time must be extended to detect larger regions. Furthermore, when using non-targeted in situ sequencing techniques, ribosomal RNA (rRNA) constitutes a large portion of the sequenced RNA molecules due to its extremely high content. The significant limitations of existing spatial transcriptomics methods make the development of new spatial sequencing methods that can improve ease of use, throughput, and sequencing depth an urgent priority.

[0006] A new method is still needed to enable super-resolution imaging of various sample types, including large-sized samples, and to perform spatial analysis of target biomolecules (such as mRNA) in the samples. Summary of the Invention

[0007] This disclosure includes a method for physically magnifying biological samples, enabling spatial analysis of mRNA and / or proteins in the same sample in an expanded state. This method is referred to herein as "ExiST" or "ExSTP". This method achieves sub-diffraction-limited structures in biological samples through reversible and controllable physical magnification, allowing imaging under a conventional microscope. The method can expand biological samples linearly by approximately 2 to approximately 8–10 times, and is applicable to various types of biological samples without causing significant deformation or structural damage.

[0008] The method presented in this paper is easy to implement in any laboratory and readily applicable. It is compatible with a wide range of sample preparation types, from tissue cultures and large tissue blocks to complete organs. The chemical reagents used in this protocol are chemically stable, inexpensive, and commercially available, and their operation requires no specialized chemical background.

[0009] Furthermore, the method described in this paper is compatible with standard methods for staining all samples (including immunohistochemistry, fluorescent proteins, and chemical dyes) as well as high-throughput sequencing (such as next-generation sequencing).

[0010] On the one hand, this paper provides a method for physically expanding biological samples and spatially analyzing biomolecules (e.g., mRNA and proteins) within the samples.

[0011] In some implementations, the method includes: (a) The sample is incubated with an mRNA probe, wherein the mRNA probe is capable of hybridizing with mRNA in the sample and has been modified to contain hydrogel-reactive chemical groups; (b) Infuse the sample with a hydrogel precursor solution, or embed the sample in a hydrogel precursor solution and polymerize it to form a sample-hydrogel complex; (c) Physically expand the sample-hydrogel composite; (d) Select one or more target regions from the expanded sample-hydrogel composite and cut the regions; and (e) Sequencing of mRNA from the cut region.

[0012] In some alternative implementations, the method includes: (a) The sample is incubated with an mRNA probe and a reverse transcriptase (e.g., in a reverse transcription reaction mixture), wherein the mRNA probe is capable of hybridizing with mRNA in the sample and has been modified to contain hydrogel-reactive chemical groups, and wherein the reverse transcriptase uses the mRNA probe as a primer to reverse transcribe the mRNA in the sample into cDNA. (b) Infuse the sample with a hydrogel precursor solution, or embed the sample in a hydrogel precursor solution and polymerize it to form a sample-hydrogel complex; (c) Physically expand the sample-hydrogel composite; (d) Select one or more target regions from the expanded sample-hydrogel composite and cut the regions; and (e) Sequencing of cDNA from the cut region.

[0013] In some embodiments, before or during step (b), the sample is further incubated with a bifunctional protein anchoring agent comprising protein-reactive chemical groups and hydrogel-reactive chemical groups. In some embodiments, the bifunctional protein anchoring agent is selected from N-succinimide acrylate (NSA), N-(allyloxycarbonyloxy)-succinimide (NAS), and any combination or mixture thereof. Accordingly, the method may also include, after step (d) or after cDNA amplification, extraction of peptides or proteins from the excised region for proteomics identification, for example by mass spectrometry analysis (such as LC-MS / MS and HPLC-MS).

[0014] In some embodiments, during step (a), the sample is incubated in a hybridization buffer together with both the mRNA probe and the bifunctional protein anchoring agent. In some other embodiments, during step (a), the sample is first incubated in the hybridization buffer with the mRNA probe, and then incubated with the bifunctional protein anchoring agent. In some other embodiments, when the sample is incubated with a reverse transcription reaction mixture containing the mRNA probe and reverse transcriptase, the sample is first incubated with the reverse transcription reaction mixture, and then incubated with the bifunctional protein anchoring agent.

[0015] In some implementations, the mRNA probe contains a 10-40 nucleotide sequence comprising dT nucleotides, analogues and / or derivatives of dT nucleotides (such as thymidine nucleotides, dT+), for example, the mRNA probe contains a 15-35 nt nucleotide sequence consisting of alternating dT and dT+.

[0016] In some further embodiments, the mRNA probe is modified with a hydrogel reactive chemical group selected from Acrydite (acrylyl-phosphoramide modification), primary amino, azide, Uni-Link™ amino modifiers, and any combination or mixture thereof. For example, the 5' end of the mRNA probe is modified with Acrydite (i.e., acrylyl-modified oligonucleotide).

[0017] In some embodiments, the method further includes homogenizing the sample-hydrogel complex between steps (b) and (c). Homogenization can be performed by physical, chemical, physicochemical, and / or enzymatic treatment of the sample-hydrogel complex, for example, by treating the sample-hydrogel complex with a protease, an alkaline buffer (such as an alkaline buffer containing detergent), or by heating in a buffer.

[0018] In some specific implementations, homogenization is achieved by treating the sample-hydrogel complex with proteinase K, trypsin, or an SDS-containing buffer.

[0019] In some implementations, the method includes: (1) In step (a), the sample is incubated in hybridization buffer with both the mRNA probe and the protein anchoring agent, and the sample-hydrogel complex is treated with trypsin between steps (b) and (c) to homogenize it; (2) In step (a), the sample is incubated in hybridization buffer with both the mRNA probe and the protein anchoring agent, and the sample-hydrogel complex is treated with SDS for homogenization between steps (b) and (c); (3) First, incubate the sample with the mRNA probe, then with the protein anchoring agent, and between steps (b) and (c), treat the sample-hydrogel complex with trypsin to homogenize it; or (4) First, incubate the sample with the mRNA probe, then incubate it with the protein anchoring agent, and treat the sample-hydrogel complex with SDS between steps (b) and (c) to homogenize it.

[0020] In some embodiments, the hydrogel precursor solution comprises one or more precursors selected from: sodium acrylate (SA), sodium methacrylate (SMA), itaconic acid (IA), trans-aconitic acid (TAA), ethyl-2-(hydroxymethyl)-acrylate (EHA), N,N'-methylenebisacrylamide (Bis-AA), N,N-dimethylacrylamide (DMAA), pentaerythritol tetraacrylate (PT), propoxylated trimethylolpropane triacrylate (TPT), pentaerythritol triacrylate (PA), and dipentaerythritol pentaacrylate. Di(trimethylolpropane) hexaacrylate or dipentaerythritol hexaacrylate (DPHA), trimethylolpropane triacrylate (TTA), di(trimethylolpropane)-tetraacrylate (DiTA), trimethylolpropane trimethacrylate (TTMA), glycerol propoxylated (1PO / OH) triacrylate (GPT), ethoxylated trimethylolpropane triacrylate (TET), pentaerythritol allyl ether (PAE), sodium 4-hydroxy-2-methylenebutyrate (SHMB), N,N-dimethylaminopropylacrylamide (DMPAA), and acrylamide (AA).

[0021] A variety of hydrogel precursors can be used in the methods disclosed herein. In some embodiments, the hydrogel precursor solution comprises any one of the following combinations of precursors: (a) SMA, AA and PAE; (b) SMA, DMAA, and TPT; (c) SMA, AA, and Bis-AA; (d) SA, AA, and Bis-AA; (e) SMA, SA, AA, and Bis-AA; and (f) DMAA, SMA and PAE.

[0022] In some implementations, a polymerization activator or promoter, such as VA-044, V50 (an azo initiator), ammonium persulfate (APS), potassium persulfate, or TEMED, is added to the hydrogel precursor solution immediately before the start of hydrogel polymerization.

[0023] In some embodiments, the methods disclosed herein further include staining the sample with a marker or label before, during, or after steps (a), (b), and (c), or after homogenization. The sample may be stained with DNA, RNA, and / or protein.

[0024] In some implementations, during step (c), the sample-hydrogel complex is incubated with a solvent or aqueous solution (such as pure water or an aqueous buffer) for a sufficient time to allow it to swell.

[0025] In some implementations, the sample is selected from cells (such as cultured cells), biological tissues (such as tissue sections), specimens, biopsies, intact organs and parts thereof, and whole organisms (such as bacteria, fungi, or viruses).

[0026] In some implementations, the sample is a tissue section with a thickness of 50 μm or less, or a tissue sample with a thickness in the range of 30 μm to 400 μm.

[0027] In some embodiments, the method further includes imaging the expanded sample-hydrogel composite using a microscope.

[0028] In some implementations, step (d) involves manually cutting the region using a punch or with the assistance of a robot or mechanical micromanipulation system, for example, by LCM cutting. For instance, the diameter of the target region is 1-1000 μm.

[0029] In some implementations, step (e) includes reverse transcription of the mRNA released from the cleaved region, cDNA amplification (e.g., by RT-PCR), and cDNA library construction. Optionally, the mRNA can be released from the cleaved region by incubating the cleaved gel in SSC buffer at 40–50 °C.

[0030] In some alternative implementations, step (e) includes cDNA amplification (e.g., by PCR) of the cDNA in the cut region and cDNA library construction.

[0031] In one respect, this paper provides a sample-hydrogel composite prepared by the method disclosed herein.

[0032] In one respect, this document provides a kit containing one or more containers: (a) mRNA probes capable of hybridizing with mRNA and crosslinking with hydrogel polymer chains; (b) A hydrogel precursor, or a hydrogel precursor stock solution containing the hydrogel precursor; (c) Optionally, a bifunctional protein anchor, such as NSA, NAS or a combination thereof.

[0033] In some implementations, the mRNA probe contains a 10-40 nucleotide sequence comprising dT nucleotides, analogues of dT nucleotides, and / or derivatives of dT nucleotides (such as thymidine nucleotides, dT+). For example, the mRNA probe contains a 15-35 nt nucleotide sequence consisting of alternating dT and dT+.

[0034] In some embodiments, the mRNA probe is modified with a hydrogel-reactive chemical group selected from Acrydite, primary amino, azide, Uni-Link™ amino modifiers, and any combination or mixture thereof. For example, the 5' end of the mRNA probe is modified with Acrydite.

[0035] In some implementations, the hydrogel precursor is selected from any one of the following groups: (a) SMA, AA and PAE; (b) SMA, DMAA, and TPT; (c) SMA, AA, and Bis-AA; (d) SA, AA, and Bis-AA; (e) SMA, SA, AA, and Bis-AA; and (f) DMAA, SMA and PAE.

[0036] In some implementations, the kit further includes one or more of the following components: A container containing a gelling activator or accelerator, said gelling activator or accelerator being selected, for example, VA-044, TEMED, APS and potassium persulfate; Containers containing hybridization buffers (such as SSC buffer); A container containing a homogenization buffer, optionally containing trypsin or SDS; Containers containing washing buffers (such as SSC buffer or PBS buffer); Containers containing markers; Containers containing fixatives (such as ethanol); A container containing reagents for mRNA reverse transcription; A container containing reagents for cDNA PCR; A container containing reagents for cDNA library preparation; and A container containing reagents for peptide digestion used in mass spectrometry identification.

[0037] In some implementations, the kit further includes one or more of the following components: A gelling chamber configured to hold a sample prior to the gelling reaction, and optionally, a cover for sealing the gelling chamber; The imaging chamber is configured to hold the sample after the gelation reaction. Gel handling device; and Cooling devices, such as ice packs.

[0038] On the one hand, this article provides a system for spatial transcriptomics analysis of biological samples, comprising: Hybridization module, used to anchor mRNA probes to mRNA in biological samples, optionally reverse transcribes mRNA into cDNA; A gelation module for hydrogel polymerization to form sample-hydrogel complexes; The homogenization module is used to homogenize the sample-hydrogel composite. A staining module for staining DNA, RNA, and / or proteins; DNA amplification module for PCR amplification of reverse-transcribed cDNA from regions of interest in a sample-hydrogel complex; and cDNA library preparation module, used to construct cDNA libraries for sequencing; and Optionally, a mass spectrometry analysis module is provided, which is used to preprocess the sample obtained by local sampling prior to mass spectrometry, and the module is connected to the mass spectrometry detection device.

[0039] The above and other features and advantages of this disclosure will become more apparent from the following detailed description of several embodiments with reference to the accompanying drawings. Attached Figure Description

[0040] Figure 1 The illustrated flowchart of the ExiST technology depicts the main steps and procedures, including mRNA and optional protein anchoring, gelation, sample homogenization, staining, imaging, and image analysis. The swollen sample is microdissected; the excised gel blocks are used for mRNA recovery, cDNA amplification, and NGS library preparation. The NGS library is sequenced using standard methods and analyzed according to experimental requirements.

[0041] Figure 2 Images of brain tissue before and after expansion using different homogenization conditions are shown. (a) Brain images before and after expansion using homogenization buffers containing trypsin or SDS. Scale bar, 100 µm. (b) Brain tissue-hydrogel in a test tube for RNA release and purification.

[0042] Figure 3 RNA released from the expanded brain tissue-hydrogel complex is shown. (a) Fragment bands of RNA released from brain tissue-hydrogel under different homogenization conditions, where A1 and B1 are two replicates homogenized with trypsin, and C1 and D1 are two replicates homogenized with SDS; (b) Concentration of RNA released under different homogenization conditions; and (c) Distribution of RNA fragments released from brain tissue-hydrogel under different homogenization conditions.

[0043] Figure 4The library quality of micro-cut samples is shown. (a) Library length distribution of samples cut under trypsin homogenization conditions; (b) Library length distribution of samples cut under SDS homogenization conditions.

[0044] Figure 5 The sequencing quality of microcut samples digested with trypsin was shown. Basic statistics include the quality of each base sequence, the quality of each tile (i.e., chip partition) sequence, the quality fraction of each sequence, the N content of each base, and the adapter content.

[0045] Figure 6 The sequencing quality of the micro-cut samples after SDS digestion is shown. Basic statistics include the quality of each base sequence, the quality of each tile sequence, the quality score of each sequence, the content of each base sequence, the N content of each base, and the adapter content.

[0046] Figure 7 The evaluation of transcriptome sequencing results is shown. (a) Quality analysis and filtering of raw sequencing data from samples treated with trypsin digestion and SDS homogenization using FastQC; (b) Alignment results of sequencing reads from samples treated with trypsin digestion and SDS homogenization using HISAT2; (c) Alignment regions for genomic analysis of two samples treated with trypsin digestion and SDS homogenization using HISAT2.

[0047] Figure 8 The transcriptome expression results are shown. (a) Gene body coverage analysis was performed on two samples treated with trypsin digestion and SDS homogenization using HISAT2; (b) Transcript expression analysis was performed on two samples treated with trypsin digestion and SDS homogenization using HISAT2.

[0048] Figure 9 The distribution of RNA detected by fragment analysis and the mRNA bands released on BeyoMag™ Oligo (dT) 25 magnetic beads are shown. (a) Fragment distribution of liver tissue-hydrogel mRNA released from the magnetic beads after incubation with the tissue-hydrogel. (b) Regional table of the analyzed mRNAs. (c) Fragment bands of liver tissue-hydrogel RNA released from the magnetic beads after incubation with the tissue-hydrogel.

[0049] Figure 10 Compatibility with fluorescence staining using small molecule dyes is shown. (a) Fluorescence image of mouse brain sections stained with DAPI. (b) Fluorescence image of mouse brain sections stained with Sypro red. LEF = ~3 in (ab), imaged using a 4x objective.

[0050] Figure 11 Super-resolution fluorescence imaging and image-guided microdissection, followed by transcriptomic analysis of selected regions of interest (ROIs), are shown. (a) Fluorescence image of homogenized mouse brain slices treated with trypsin digestion, with the target region in the mouse hippocampus marked. (b) Fluorescence image of homogenized mouse brain slices treated with SDS-containing buffer, with the target region in the hippocampus marked. (c) Fluorescence image of the S3 region shown in Figure b (right: before microdissection, white circles indicate the selected ROI for microdissection; left: after microdissection). (d) The proportion of uniquely aligned reads from the sequencing results of the target region shown in Figures a and b (SDS group corresponds to Figure b, trypsin group corresponds to Figure a). LEF = ~3x, target region diameter = 2 mm, imaging using a 4x objective. (e) Alignment results of the target region shown in Figures a and b, including unaligned reads, uniquely aligned reads, multiple aligned reads, non-splicing reads, and spliced ​​reads.

[0051] Figure 12 This paper presents a genome alignment analysis of transcriptomics results under two conditions: trypsin digestion and homogenization with SDS buffer, using HISAT2 software. (a) Figure 11 Genome alignment analysis of three replicate samples under SDS homogenization conditions shown in (b). Figure 11 Genome alignment analysis of three replicate samples under homogenization conditions for trypsin digestion, as shown in figure a. (S1-S3: Figure 11 SDS group repeated samples 1-3; T1-T3: Figure 11 The trypsinome of repeated samples 1-3.

[0052] Figure 13 Showing Figure 12 Transcript expression level analysis (FPKM: number of exons per million aligned fragments) of two groups of samples under SDS homogenization and trypsin digestion conditions is shown. (a) Bar chart of expression level analysis of 6 samples under the two homogenization conditions. (b) Density distribution plot of expression level analysis of 6 samples under the two homogenization conditions. (S1-S3: SDS group replicates 1-3, T1-T3: trypsin group replicates 1-3).

[0053] Figure 14 The correlation between samples and principal component analysis (PCA) are shown under two conditions: trypsin digestion and SDS homogenization. (a) Pearson correlation between samples under the two homogenization conditions. (b) PCA analysis between samples under the two homogenization conditions.

[0054] Figure 15Genome analysis of samples under two homogenization conditions is shown. (T1-T3: trypsinome replicates 1-3, S1-S3: SDS replicates 1-3).

[0055] Figure 16 Representative images of fluorescent brain sections after incubation with probes labeled with fluorescent groups are shown. (a) Fluorescence images of brain sections after incubation with anchors of different lengths. (b) Calculated fluorescence intensity of anchors of different lengths in the hippocampus.

[0056] Figure 17 The mechanical properties of different gel components and their compatibility with the ExiST workflow are shown. (a) Stress curves of different gel components. (b) Calculated properties of different gel components, including mean linear expansion factor, mean strain, and mean stress. (c) Total number of detected genes for different gel components. (d) Composition of different gel components.

[0057] Figure 18 The workflow of the in situ RT and gel embedding procedure (called neoExiST) is shown, including in situ RT-PCR using an Oligo (dT) probe with an Acrydite-modified 5' end, gelation, sample homogenization, staining, imaging, and image analysis. The swollen sample was microdissected; the excised gel blocks were used for cDNA amplification and NGS library preparation. The NGS library was sequenced using standard methods and analyzed according to experimental requirements.

[0058] Figure 19 Transcriptomic analysis using the in situ RT-based method (neoExiST) compared to ExiST is shown. (a) Total gene counts for two samples based on in situ RT (neoExiST). (b) PCA analysis of samples based on both methods: in situ RT (neoExiST, the new method) and gel-embedded RT (ExiST, the original method). (c) Expression level analysis of samples based on both methods: in situ RT and gel-embedded RT.

[0059] Figure 20 The workflow of the combined spatial transcriptomics and proteomics protocol (ExiSTP) is shown. This workflow includes both RNA anchor incubation and protein anchor incubation.

[0060] Figure 21The distribution of RNA fragments released from liver tissue-hydrogels after treatment with different homogenization conditions (nt, hnt, ns, and hns) is shown. nt: NSA was added to the hybridization buffer, followed by trypsin homogenization; hnt: mRNA probe hybridization was performed first, followed by NSA addition, and then trypsin homogenization; ns: NSA was added to the hybridization buffer, followed by SDS homogenization; hns: mRNA probe hybridization was performed first, followed by NSA addition, and then SDS homogenization.

[0061] Figure 22 The workflow of the combined spatial transcriptomics and proteomics protocol (neoExiSTP) is shown. This protocol includes in situ RT and protein anchoring incubation.

[0062] Figure 23 The total gene count detected by transcriptomics (a) and the total peptide and protein count detected by proteomics (b) are shown.

[0063] Figure 24 Expression analyses from the same samples using proteomics and transcriptomics are shown. (a) PCA analysis of genes detected by spatial transcriptomics and proteomics. (N1-N3: transcriptomics replicates 1-3; N1p-N3p: proteomics replicates 1-3). (b) Volcano plot of differentially expressed genes analysis between transcriptomics and proteomics identification information.

[0064] Figure 25-29 The overexpression analysis of differentially expressed genes between transcriptomics and proteomics under GO enrichment is shown. Figure 25 Bubble chart of the top-ranked enrichment pathways; Figure 26 Heatmaps of the top-ranked enriched pathway groups; Figure 27 Heatmaps of enrichment pathways corresponding to specific molecules; Figure 28 A tree diagram of the top-ranked enriched pathway groups; Figure 29 Upset plot of overexpression analysis. Detailed Implementation

[0065] The disclosures and embodiments set forth herein should be interpreted as exemplary only and not as limiting the scope of the invention. Although specific terms are used herein, they are used in a general and descriptive sense only, and not for limiting purposes, unless otherwise stated. Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure should be understood in the manner commonly understood by one of ordinary skill in the art. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0066] definition

[0067] As used in this article, the singular forms “a,” “an,” and “the” also refer to the plural forms, unless the context clearly indicates otherwise.

[0068] The term “about” used in conjunction with numerical values ​​or ranges is intended to encompass variations in that value or range (i.e., slightly more or less than that value or range within ±20%, ±10%, ±5%, ±1%, ±0.5%, ±0.1%, or ± one standard deviation). While every effort has been made to ensure the accuracy of the figures used (e.g., ranges, amounts, concentrations, etc.), some experimental bias should be taken into account.

[0069] As used herein, the term "sample" encompasses a wide range of biological, chemical, or biochemical materials, including but not limited to cells, cultured cells, biological tissues, specimens, biopsies, intact organs, whole organisms, or, in principle, other target sample types such as bacteria, fungi, and viruses. Typically, samples are intended for microscopic analysis. The term "biological sample" can refer to all or part of a tissue or organ, including but not limited to the brain, spinal cord, heart, lungs, liver, kidneys, stomach, colon, bones, muscles, skin, glands, lymph nodes, genitals, breast, pancreas, prostate, bladder, thyroid, and eyes.

[0070] In this document, the terms “amplification,” “expansion,” and “swelling” are used interchangeably and refer to the physical expansion of the hydrogel or sample-hydrogel composite, which is preferably tunable and reversible.

[0071] The term “precursor” as used in this article includes monomers, comonomers, oligomers, and crosslinking agents that make up the small units that constitute the hydrogel polymer chain.

[0072] The term "swellable material" or "expandable material" generally refers to a material that expands linearly upon contact with a liquid, such as water or another solvent. Preferably, the swelling material expands uniformly in three dimensions, and the material is transparent, allowing light to pass through the sample after expansion. As illustrated herein, a swelling material is a swelling sample-hydrogel composite. In some embodiments, the swelling material is formed from a hydrogel precursor, monomer, or oligomer thereof via in-situ polymerization. For example, monomers comprising water-soluble groups containing polymerizable olefinic unsaturated groups can be used. The monomer or oligomer may comprise one or more substituted or unsubstituted methacrylates, acrylates, acrylamides, methacrylamides, vinyl alcohols, vinylamines, allylamines, allyl alcohols, including their diethylene crosslinking agents (e.g., N,N-alkylenebisacrylamide). The precursor may also be mixed with a polymerization initiator and a crosslinking agent prior to the polymerization reaction. In some embodiments, the swelling polymer is a polyacrylate and its copolymers or crosslinking copolymers. Alternatively or additionally, swellable materials can be formed in situ by chemically crosslinking water-soluble oligomers or polymers. Therefore, the present invention envisions adding a precursor of a swellable material (such as a water-soluble precursor) to a sample and enabling the precursor to swell in situ.

[0073] As used herein, the term "anchoring agent" includes RNA anchoring agents and protein anchoring agents that crosslink RNA and proteins in a sample to hydrogel polymer chains, respectively. The anchoring agent is preferably a bifunctional linker comprising biomolecular reactive chemical groups that bind, link, or hybridize with a target biomolecule (e.g., RNA, protein, DNA) and hydrogel reactive chemical groups that bind, link, or hybridize with the hydrogel polymer chains. In some embodiments, one or more functional chemical anchoring agents attach reactive groups to functional groups (e.g., primary amines or thiol groups) of target biomolecules within a biological sample, such as proteins, nucleic acids, lipids, proteoglycans, lipopolysaccharides, etc. The chemical anchoring agent functionalizes the biomolecules within the sample, thereby reacting with the growing chains of the hydrogel polymer during the sample embedding step, thus covalently anchoring the functionalized biomolecules to the hydrogel mesh.

[0074] The term "thymidine locked nucleic acid" refers to a modified thymidine (T) RNA monomer, which may be abbreviated as "dT+" in this document. The "locked" portion of locked nucleic acid (LNA) originates from a methylene bridge linking the 2' oxygen and 4' carbon of the RNA pentose ring. This bridge anchors the pentose ring in a 3'-endo conformation. LNA has been found to exhibit significant affinity and specificity for native DNA targets. LNA can be synthesized using conventional phosphoramide reagents, which are commercially available.

[0075] The mRNA probe hybridizes with the mRNA under conditions that minimize non-specific hybridization at a set temperature in a physiological buffer (e.g., pH 6-9, 25-150 mM chloride). In some cases, the mRNA probe hybridizes with the mRNA if at least about 6, 8, 10, 12, 14, 16, or 18 consecutive complementary nucleotides to the mRNA sequence are present. In some cases, the mRNA probe hybridizes with a common sequence shared by a set of target mRNAs. The set temperature for specific hybridization can be room temperature or higher. In some embodiments, the defined temperature for specific hybridization is at least about 37°C.

[0076] The term "polymerase chain reaction" or "PCR" refers to a method for exponentially amplifying a specific fragment or subsequence of a target double-stranded DNA. PCR is well known to those skilled in the art; see, for example, U.S. Patent Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, edited by Innis et al., 1990. Exemplary PCR reaction conditions typically consist of two or three cycles. A two-step cycle includes a denaturation step followed by a hybridization / extension step. A three-step cycle includes a denaturation step followed by a hybridization step, and then a separate extension step.

[0077] ExiST

[0078] This disclosure provides a conceptually novel spatial transcriptomics approach, referred to herein as expanded-assisted spatial transcriptomics (PEST). E xpansion Ass i sted S patial T ExiST (Exi-Structured Biological Samples) combines the physical expansion and amplification of biological tissues with RNA sequencing technology. In one aspect, this disclosure provides a method involving the physical expansion of biological samples for spatial analysis of target biomolecules (such as mRNA) within the sample, wherein the mRNA is retained in the expanded sample-hydrogel complex without degradation, allowing for subsequent sequencing of the mRNA in the target region. This method can provide spatial resolution well below the diffraction limit of optical microscopy, thus providing valuable insights into the spatial distribution of biomolecules (such as mRNA) in biological samples. This method can enhance the level of microscopic research on different organs and tissues (e.g., mapping the brain) and facilitate the development of diagnostic, personalized medicine, histopathology, and other medical applications.

[0079] In some embodiments, this disclosure provides a method comprising the following steps: (a) Incubate the biological sample with the mRNA probe for a sufficient time to allow the mRNA probe to hybridize with the mRNA in the sample; (b) The sample is embedded in a hydrogel precursor solution or the sample is perfused with a hydrogel precursor solution and then a polymerization reaction occurs to form a sample-hydrogel complex; (c) Homogenize the sample-hydrogel complex by physical, chemical, physicochemical and / or enzymatic treatment; (d) Optionally, stain the sample-hydrogel complex; (e) Expand the sample-hydrogel composite to the required extent according to the end-user's needs; (f) Selecting a target region from the expanded sample-hydrogel complex and cutting that region, for example, by sample micro-cutting; and (g) Release the mRNA from the cut region and sequence it.

[0080] Furthermore, the method may include washing the sample or sample-hydrogel complex with a washing buffer before, after, or between any of steps (a)-(f) (e.g., before step (a) and after step (c)). The biological samples disclosed herein can be selected from a variety of biological materials, including but not limited to cells (such as cultured cells), biological tissues, specimens, biopsies, intact organs, and whole organisms. Biological samples may be derived from bacteria, fungi, viruses, or mammals, including non-human animals (such as mice and rats) and humans. Samples may be live, fixed, or preserved, such as live cultured cells or fixed tissue sections. The methods described herein are applicable not only to cells and thin samples but also to large or dense samples that can be sufficiently expanded and become optically transparent after hydrogel swelling.

[0081] In some embodiments, the biological sample is cultured cells, such as cultured tumor cells. In some other embodiments, the biological sample is a tissue section or sheet with a thickness of 50 µm or less, i.e., a thin tissue sample. In some further embodiments, the biological sample is a tissue sample with a thickness in the range of 30 μm to 400 μm, for example, in the ranges of 30 μm-100 μm, 100 μm-200 μm, 200 μm-300 μm, 300 μm-400 μm, 50 μm-150 μm, 150 μm-250 μm, and 250 μm-350 μm. In some further embodiments, the biological sample is a tissue sample with a thickness greater than 400 μm.

[0082] In some embodiments, the biological sample is a tissue sample (e.g., a slide) from, but not limited to, the brain, spinal cord, heart, lungs, liver, kidneys, stomach, colon, bones, muscles, skin, lymph nodes, genitals, breast, pancreas, prostate, bladder, thyroid, and eyes. In some embodiments, the biological sample is a slide of liver tissue, heart tissue, kidney tissue, brain tissue, or cancerous tissue.

[0083] Biological samples (e.g., tissue sections) may be fixed with a fixative prior to step (a). In some embodiments, the fixative may be selected from ethanol, methanol, or acetone. In step (a), the biological sample may be incubated with the mRNA probe in a hybridization buffer (e.g., SSC buffer). In some embodiments, the biological sample and mRNA probe are incubated at 37°C for 35-45 hours.

[0084] The advancements and improvements achieved by ExiST involve several key aspects. First, ExiST overcomes the density limitations of spatial barcode capture arrays by expanding the sample-hydrogel complex, in which RNA molecules in the sample are reversibly anchored to polymer chains. The physically magnified biological structures facilitate manual microdissection of target regions at scalable spatial resolutions from 100µm to 1000µm based on user-defined anatomical features. Second, optimizations to RNA capture and release (including RNA probe design and RNA release conditions) enable the detection of more transcripts per unit tissue volume in fewer cells, thereby increasing sequencing depth compared to traditional chip-based spatial transcriptomics methods.

[0085] Furthermore, ExiST does not require specialized or complex equipment for tissue expansion, microdissection, and RNA recovery, unlike imaging-based in situ sequencing methods. As disclosed herein, the ExiST workflow can utilize only commercially available and readily available reagents, making ExiST technology approximately 1000 times less expensive than other chip-based and magnetic bead-based spatial transcriptomics methods.

[0086] Furthermore, ExiST technology differs from other methodologies in this field, which primarily focus on reducing the size of capture spots. Therefore, the ExiST method has the potential to be integrated with other sequencing modalities, including microarray-based and bead-based sequencing technologies, to further enhance its overall performance. For example, ExiST complements current high-resolution methods because sample expansion can be combined with smaller spot sizes to further improve spatial resolution, and each method may require optimization of RNA capture efficiency. Combining ExiST with other high-resolution methods enables unbiased RNA profiling of minute subcellular structures, such as organelles.

[0087] Furthermore, ExiST offers several technical advantages compared to other techniques that use hydrogel-assisted sample amplification. First, and perhaps most critically, ExiST requires no chips for sequencing. Second, it employs a novel chemical system (including hydrogel components, mRNA probe components, homogenization conditions, and buffers) that enables higher spatial resolution due to a higher expansion factor. Third, to further enhance spatial resolution, sample microdissection can be used to remove gel blocks, followed by RT-PCR directly in the enzyme mixture under optimized conditions—a feat not achieved in any previously reported method. Fourth, in addition to RNA analysis, ExiST is compatible with proteomics analysis because proteins can be anchored to the gel polymer chains using protein anchoring agents. Fifth, optimized homogenization conditions ensure that mRNA remains intact in the gel; homogenization conditions used in other expansion microscopy (ExM)-based methods for structural imaging fail to preserve mRNA in the gel. Sixth, ExiST is compatible with super-resolution imaging using conventional diffraction-limited microscopy prior to sequencing, allowing for image-guided microdissection and sequencing.

[0088] neoExiST

[0089] This disclosure further provides an alternative to ExiST, referred to herein as neoExiST. The main difference between neoExiST and ExiST is that neoExiST performs in situ reverse transcription of mRNA prior to the gelation reaction.

[0090] In some embodiments, this disclosure provides a method comprising the following steps: (a) The sample is incubated with an mRNA reverse transcription reaction mixture containing an mRNA probe and a reverse transcriptase, wherein the mRNA probe is hybridizable to mRNA in the sample and has been modified to contain hydrogel-reactive chemical groups, and wherein the reverse transcriptase uses the mRNA probe as a primer to reverse transcribe the mRNA in the sample into cDNA. (b) The sample is embedded in a hydrogel precursor solution or the sample is perfused with a hydrogel precursor solution and then a polymerization reaction occurs to form a sample-hydrogel complex; (c) Homogenize the sample-hydrogel complex by physical, chemical, physicochemical and / or enzymatic treatment; (d) Optionally, stain the sample-hydrogel complex; (e) Expand the sample-hydrogel composite to the required extent according to the end-user's needs; (f) Selecting a target region from the expanded sample-hydrogel complex and cutting that region, for example, by sample micro-cutting; and (g) Sequencing of cDNA in the cut region.

[0091] Furthermore, the method may include washing the sample or sample-hydrogel complex with a washing buffer before, after, or between any of steps (a)-(f) (e.g., before step (a) and after step (c)). The biological samples disclosed herein may be selected from a variety of biological materials, including but not limited to cells (such as cultured cells), biological tissues, specimens, biopsies, intact organs, and whole organisms. The biological sample may be derived from bacteria, fungi, viruses, or mammals, including non-human animals (such as mice and rats) and humans. The sample may be live, fixed, or preserved, such as live cultured cells or fixed tissue sections. The methods described herein are applicable not only to cells and thin samples but also to large or dense samples that can be sufficiently expanded and become optically transparent after hydrogel expansion.

[0092] Biological samples (e.g., tissue sections) may be fixed with a fixative prior to step (a). In some embodiments, the fixative may be selected from ethanol, methanol, or acetone. In step (a), the biological sample may be incubated with an mRNA reverse transcription reaction mixture containing an mRNA probe and reverse transcriptase, wherein the mRNA probe serves as a dT primer for reverse transcription. In some specific embodiments, the reverse transcription reaction comprises: 3 minutes at 72°C, followed by 2 minutes in an ice bath, then 90 minutes at 42°C, 15 minutes at 70°C, and then held at 4°C. Preferably, the method may further include rinsing the biological sample with SSC buffer prior to reverse transcription. After reverse transcription (RT), the synthesized cDNA forms an mRNA-cDNA duplex, and during hydrogel formation, the duplex is incorporated into the hydrogel via hydrogel-reactive groups in the mRNA probe.

[0093] The ExiST and neoExiST methods disclosed herein also include cDNA amplification after step (f). mRNA reverse transcription and PCR amplification are well-established techniques in the field, and various commercial kits are available. Preferably, a cDNA library is prepared before sequencing of the cDNA (synthesized from RNA), wherein the cDNA is fragmented, end-repaired, and made into a sequencing library.

[0094] ExiSTP

[0095] This disclosure further provides a method for combining ExiST and neoExiST with protein identification, referred to herein as expanded-assisted spatial transcriptomics and proteomics. Ex pansion Ass i sted S patial TExiSTP (or neoExiSTP) and ExiST (or neoExiST) differ from ExiST in that in ExiSTP, the biological sample is incubated not only with the mRNA probe but also with a bifunctional protein anchoring agent, allowing for simultaneous transcriptomic and proteomic analysis of mRNA and protein from the cleaved gel region to obtain a comprehensive profile of the target region. This bifunctional protein anchoring agent contains protein-reactive chemical groups and hydrogel-reactive chemical groups; for example, the free amino groups of the protein can interact with the anchoring agent molecules. The biological sample can be incubated with the bifunctional protein anchoring agent during step (a), between steps (a) and (b), or during step (b).

[0096] In some ExiST-based implementations, this disclosure provides a method comprising the following steps: (a) Incubate the biological sample in a hybridization buffer containing mRNA probes and bifunctional protein anchors for a sufficient duration; (b) The sample is embedded in a hydrogel precursor solution or the sample is perfused with a hydrogel precursor solution and then a polymerization reaction occurs to form a sample-hydrogel complex; (c) Homogenize the sample-hydrogel complex by physical, chemical, physicochemical and / or enzymatic treatment; (d) Optionally, stain the sample-hydrogel complex; (e) Expand the sample-hydrogel composite to the required extent according to the end-user's needs; (f) Select a target region from the expanded sample-hydrogel complex and cut the region, for example by sample micro-cutting; (g) RT-PCR was performed on the mRNA in the cut region, followed by cDNA sequencing, and peptides in the cut region were extracted for proteomics identification.

[0097] In some alternative implementations based on ExiST, steps (a) and (b) include: (a) Incubate the biological sample in a hybridization buffer containing the mRNA probe for a sufficient time to allow the mRNA to hybridize; (b) The sample is embedded in a hydrogel precursor solution containing a bifunctional protein anchoring agent, or the sample is perfused with a hydrogel precursor solution containing a bifunctional protein anchoring agent, and then a polymerization reaction occurs to form a sample-hydrogel complex.

[0098] In some alternative implementations based on ExiST, steps (a) and (b) include: (a) Incubate the biological sample in a hybridization buffer containing the mRNA probe for a sufficient time to allow the mRNA to hybridize, and then incubate the biological sample with a bifunctional protein anchoring agent. (b) The sample is embedded in a hydrogel precursor solution or the sample is perfused with a hydrogel precursor solution and then polymerized to form a sample-hydrogel complex.

[0099] The protein anchoring agent can be selected from NSA and NAS, which modify the amino groups on the protein with acrylamide or allyloxycarbonyloxyamide functional groups, respectively, and also allow the functionalized protein to be anchored to the hydrogel during the polymerization reaction.

[0100] Preferably, the cleaved gel from step (f) can be incubated with buffer and subjected to in-gel RT-PCR. Subsequently, the supernatant containing amplified cDNA can be transferred for cDNA library construction, and peptide extraction can be performed on the gel. In some embodiments, to extract peptides from the cleaved gel, the gel is secondarily embedded with a hydrogel precursor solution and subjected to a second gelation reaction before peptide digestion and extraction for MS analysis.

[0101] neoExiSTP

[0102] In some implementations based on neoExiST, this disclosure provides a method comprising the following steps: (a) The biological sample is incubated with an mRNA reverse transcription reaction mixture containing an mRNA probe and a reverse transcriptase, wherein the mRNA probe is hybridizable to mRNA in the sample and has been modified to contain hydrogel-reactive chemical groups, and wherein the reverse transcriptase uses the mRNA probe as a primer to reverse transcribe the mRNA in the sample into cDNA; then the biological sample is incubated with a bifunctional protein anchoring agent. (b) The sample is embedded in a hydrogel precursor solution or the sample is perfused with a hydrogel precursor solution and then a polymerization reaction occurs to form a sample-hydrogel complex; (c) Homogenize the sample-hydrogel complex by physical, chemical, physicochemical and / or enzymatic treatment; (d) Optionally, stain the sample-hydrogel complex; (e) Expand the sample-hydrogel composite to the required extent according to the end-user's needs; (f) Select a target region from the expanded sample-hydrogel complex and cut the region, for example by sample micro-cutting; (g) The mRNA in the cut region is amplified into cDNA (e.g. by PCR), followed by cDNA sequencing, and peptides in the cut region are extracted for proteomics identification.

[0103] In some alternative implementations based on neoExiST, steps (a) and (b) include: (a) A biological sample is incubated with a mixture of mRNA reverse transcription reaction containing an mRNA probe and a reverse transcriptase, wherein the mRNA probe is hybridizable to mRNA in the sample and has been modified to contain hydrogel-reactive chemical groups, and wherein the reverse transcriptase uses the mRNA probe as a primer to reverse transcribe the mRNA in the sample into cDNA. (b) The sample is embedded in a hydrogel precursor solution containing a bifunctional protein anchoring agent, or the sample is perfused with a hydrogel precursor solution containing a bifunctional protein anchoring agent, and then a polymerization reaction occurs to form a sample-hydrogel complex.

[0104] The biological sample may be incubated with the bifunctional protein anchoring agent during step (a), between steps (a) and (b), or during step (b). In some embodiments, the biological sample is incubated with the bifunctional protein anchoring agent after step (a) and before step (b).

[0105] Preferably, the cleaved gel from step (f) can be incubated with buffer and subjected to in-gel cDNA PCR. Subsequently, the supernatant containing amplified cDNA can be transferred for cDNA library construction, and peptide extraction can be performed on the gel. In some embodiments, to extract peptides from the cleaved gel, the gel is secondarily embedded with a hydrogel precursor solution and subjected to a second gelation reaction before peptide digestion and extraction for MS analysis.

[0106] Hybridization

[0107] Most mRNAs contain a poly(A) tail of 100 to 250 residues at their 3' end. In eukaryotic cells, immediately after gene transcription is complete, the newly generated RNA molecule undergoes a series of modifications called RNA processing. These modifications alter both ends of the primary RNA transcript to produce mature mRNA molecules. The 3' end processing adds a poly(A) tail to the mRNA molecule, which plays a crucial role in post-transcriptional regulation, including mRNA export, stability, and translation. Oligo (dT) probes that hybridize or bind to the poly(A) tail have been developed to isolate mRNA from samples without interfering with DNA or other RNA.

[0108] As disclosed herein, a biological sample is contacted with an mRNA probe capable of hybridizing with mRNA in the sample. Preferably, the mRNA probe is a bifunctional probe comprising both an mRNA-reactive group and a hydrogel-reactive group to allow hybridization with mRNA and crosslinking to the hydrogel polymer chain. Since mRNA typically contains a poly(A) tail, the mRNA probes used herein may comprise a series of T and / or dT nucleotides, modified T and / or dT nucleotides, or analogues or derivatives thereof for base complementarity pairing.

[0109] In some embodiments, the mRNA probe comprises a nucleotide sequence consisting of dT nucleotides. In some embodiments, the mRNA probe comprises a nucleotide sequence containing dT nucleotides, dT nucleotide analogs, and / or derivatives. In some embodiments, the mRNA probe comprises a nucleotide sequence consisting of dT nucleotides and LNA-modified dT nucleotides. In some embodiments, the mRNA probe comprises a nucleotide sequence containing dT nucleotides and thymidine-locked nucleic acids (dT+). In some embodiments, the mRNA probe comprises alternating nucleotide sequences of dT and thymidine-locked nucleic acids (dT+).

[0110] The length of the mRNA probe can vary between 10 and 40 nucleotides, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 35 nucleotides. The number of dT-LNA residues and the length of the mRNA probe can be adjusted as needed to achieve suitable stability and specificity. In some embodiments, the mRNA probe comprises alternating nucleotide sequences of dT and thymidine lock nucleic acid (dT+), said nucleotide sequences being 10-30 nucleotides in length, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the mRNA probe comprises a nucleotide sequence as shown in SEQ ID NO: 1 or 2. Furthermore, the mRNA probes disclosed herein are modified to allow crosslinking to the hydrogel polymer chain. This modification may be located at the 5' or 3' end or at an internal nucleotide. In some embodiments, the mRNA probe is modified at the 5' end with Acrydite, a primary amino group, an azide, a Uni-Link™ amino modifier, or any combination or mixture thereof. In some specific embodiments, the mRNA probe comprises the nucleotide sequence shown in SEQ ID NO: 1 or 2 and has a 5'-Acrydite modification.

[0111] The hybridization buffer can be any buffer commonly used for RNA hybridization. In some embodiments, the hybridization buffer is a saline sodium citrate (SSC) buffer, such as a 2×SSC buffer containing formamide, an RNase inhibitor, dextran sulfate, and yeast transfer RNA. In some embodiments, the biological sample is incubated with the mRNA probe in the hybridization buffer at 4°C–37°C for approximately 24–48 hours, such as approximately 25 hours, approximately 26 hours, approximately 27 hours, approximately 28 hours, approximately 29 hours, approximately 30 hours, approximately 32 hours, approximately 34 hours, approximately 36 hours, approximately 38 hours, approximately 40 hours, approximately 42 hours, approximately 44 hours, approximately 46 hours, approximately 48 hours, or any interval within this range.

[0112] In some embodiments, in step (a) of the method, the biological sample is incubated with an mRNA probe and optionally a bifunctional protein anchoring agent in a hybridization buffer. In some embodiments, the method further includes incubating the biological sample with the bifunctional protein anchoring agent to allow protein anchoring. The protein anchoring agent may be present in the hybridization buffer together with the mRNA probe, such that RNA anchoring and protein anchoring can be performed simultaneously in step (a). Alternatively, RNA probe hybridization and protein anchoring can be performed separately. In some embodiments, the biological sample is incubated with the mRNA probe in a hybridization buffer before being incubated with the protein anchoring agent. The protein anchoring agent may be provided in an aqueous or organic solvent solution. In some embodiments, the biological sample is incubated with the protein anchoring agent between steps (a) and (b) of the method. In some embodiments, protein anchoring and step (b) are performed simultaneously. Specifically, the sample may be embedded in a mixture containing both the bifunctional protein anchoring agent and a hydrogel precursor solution, such that the anchoring and embedding (and polymerization) steps are performed in one step. In some embodiments, the protein anchoring agent is added to the hydrogel precursor solution before the sample is embedded in the solution or before the sample is perfused with the solution. In some other embodiments, the protein anchoring agent is added to the hydrogel precursor solution after the sample is embedded in the hydrogel precursor solution or after the sample is perfused with the solution.

[0113] In some embodiments, after incubation with a hybridization buffer containing an mRNA probe, the biological sample is incubated with a protein anchoring solution containing a protein anchoring agent. The protein anchoring solution can be prepared by dissolving or diluting the protein anchoring agent in an aqueous buffer (such as PBS, MES, or a buffer containing 50 mM sodium carbonate, 50 mM sodium bicarbonate, and 10 mM 10% (v / v) Triton X-100). The protein anchoring solution may also contain a fixative. Those skilled in the art can readily determine the composition of the buffer based on the selected anchoring agent.

[0114] The protein anchoring agents and RNA probes disclosed herein enable rapid anchoring of biomolecules throughout large biological samples, providing high fluorescence retention. The bifunctional protein anchoring agents contain two distinct functional groups: one for protein functionalization and the second for reaction with growing hydrogel polymer chains. These two distinct functional groups can be separated by chemical linkers (including branched linkers) of various lengths and structures. The bifunctional protein anchoring agents may contain both protein-reactive chemical groups and hydrogel-reactive chemical groups to allow the protein to crosslink with the hydrogel polymer chains. In some embodiments, the protein-reactive chemical groups include, but are not limited to, N-hydroxysuccinimide (NHS) esters, epoxy groups, aldehyde groups, and formamide groups, which can react with amino or carboxylic acid groups on proteins, peptides, nucleic acids, and / or lipids. In some embodiments, the hydrogel-reactive groups include, but are not limited to, vinyl, allyl, acrylate, methacrylate, acrylonitrile, and Acrydite groups.

[0115] In some embodiments, the protein anchoring agent used to directly crosslink the protein to any hydrogel polymer chain is one or more selected from N-succinimidyl acrylate (NSA), N-(allyloxycarbonyloxy)-succinimidyl (NAS), allyl glycidyl ether (AGE), glycidyl acrylate (GAL), methacrolein, and glycidyl methacrylate (GME). In some embodiments, the protein anchoring agent is selected from N-succinimidyl acrylate (NSA), N-(allyloxycarbonyloxy)-succinimidyl (NAS), and any combination or mixture thereof. For example, treating the amino groups on a protein modified with acrylamide or allyloxycarbonyloxyamide functional groups with NSA or NAS allows the functionalized protein to be anchored to the hydrogel during polymerization. In some embodiments, the protein anchoring agent is NAS or NSA.

[0116] In some implementations, prior to step (b), the biological sample is incubated with a hybridization buffer containing an mRNA probe at 4°C–37°C for approximately 24–48 hours, and then with a buffer containing NAS, NSA, or a combination thereof at 4°C–37°C for approximately 2–8 hours.

[0117] Biological samples can be fresh, frozen, or fixed (i.e., preserved) prior to step (a). In some embodiments, biological samples have been histologically preserved using fixatives such as ethanol, formaldehyde, or paraformaldehyde. Samples may also be embedded in a solid and typically hard medium such as paraffin, wax, collodion, or resin, which allows for the cutting of thin sections for microscopic examination.

[0118] Fixation can be performed using conventional methods. Those skilled in the art will understand that the choice of fixative depends on the purpose of histological staining or other analysis of the sample. They will also understand that the fixation duration depends on the size of the tissue sample and the fixative used. For example, neutral buffered formalin, Bouin, or paraformaldehyde can be used to fix the sample.

[0119] In some embodiments, the biological sample has been fixed with ethanol (e.g., 75% ethanol) prior to step (a). In some embodiments, the biological sample is fixed after step (a). In some embodiments, the sample is fixed between steps (a) and (b) (i.e., after hybridization incubation and before being embedded in the hydrogel precursor solution).

[0120] Hydrogel polymerization or gelation reaction

[0121] As disclosed above, the sample is permeated (e.g., perfused, injected, soaked, added, or otherwise mixed) or embedded in a hydrogel precursor solution, wherein mRNA within the sample, already hybridized to an mRNA probe, is covalently bound to the hydrogel polymer chains via cross-linking of the mRNA probe. The hydrogel precursor cross-links with the sample and polymerizes to form a hydrogel-sample complex. The polymerized sample-hydrogel complex comprises a swellable polymer network.

[0122] Hydrogel compositions suitable for expansion are known in the art and can be readily selected according to specific needs. Hydrogel precursor solutions typically contain one or more hydrogel monomers or precursors dissolved in an aqueous solution (e.g., RNase-free water). In addition to the hydrogel precursor, polymerization activators (e.g., APS, potassium persulfate, VA-044, etc.) or promoters (e.g., TEMED) may be added to the hydrogel precursor solution immediately before use. Water is typically used as the dispersion medium for hydrogel formation, but other solvents may also be used. Since the mRNA in the sample should remain intact during this process, the reagents in the hydrogel precursor solution should be RNase-free, or the hydrogel precursor solution should have been treated to be RNase-free, for example, with an RNase inhibitor (e.g., DEPC).

[0123] In some embodiments, the precursor of the hydrogel is selected from, but is not limited to: sodium acrylate (SA), sodium methacrylate (SMA), itaconic acid (IA), trans-aconitic acid (TAA), ethyl-2-(hydroxymethyl)-acrylate (EHA), N,N'-methylenebisacrylamide (Bis-AA), N,N-dimethylacrylamide (DMAA), pentaerythritol tetraacrylate (PT), propoxylated trimethylolpropane triacrylate (TPT), pentaerythritol triacrylate (PA), dipentaerythritol pentaacrylate, or dipentaerythritol pentaacrylate. Pentaerythritol hexaacrylate (DPHA), trimethylolpropane triacrylate (TTA), di(trimethylolpropane)tetraacrylate (DiTA), trimethylolpropane trimethacrylate (TTMA), glycerol propoxylated (1PO / OH) triacrylate (GPT), ethoxylated trimethylolpropane triacrylate (TET), pentaerythritol allyl ether (PAE), sodium 4-hydroxy-2-methylenebutyrate (SHMB), N,N-dimethylaminopropylacrylamide (DMPAA), and acrylamide (AA). All reagents are commercially available, chemically stable, and relatively safe (suitable for routine biological laboratories).

[0124] It is desirable for hydrogels to maintain high mechanical stability in the expanded state in order to achieve adjustable and reversible expansion. The mechanical stability of hydrogels can be assessed using methods familiar to those skilled in the art, such as visually inspecting expanded hydrogel samples for cracks and breakage after manual manipulation mimicking real experiments (i.e., transferring, shaking, and cutting with a scalpel). Detailed testing methods for hydrogels can be found in WO2022 / 262311, the entire contents of which are incorporated herein by reference.

[0125] A variety of precursors can be used to form the hydrogel, provided that the formed hydrogel possesses good mechanical stability. These include precursors known for use in dilatation microscopy and those yet to be discovered. In some embodiments, the precursors used to form the hydrogel are SMA, AA, and PAE. In some embodiments, the precursors used to form the hydrogel are DMAA, SMA, and TPT. In some other embodiments, the precursors used to form the hydrogel are DMAA, SA, Bis-AA, and AA. In some other embodiments, the precursors used to form the hydrogel are DMAA, SMA, and PAE. In some other embodiments, the precursors used to form the hydrogel are SMA, Bis-AA, and AA. In some other embodiments, the precursors used to form the hydrogel are SA, Bis-AA, and AA. In some other embodiments, the precursors used to form the hydrogel are SMA, SA, Bis-AA, and AA. Preferably, the formed hydrogel has the ability to expand large tissue blocks, intact organs, or even entire organisms without deforming or mechanically breaking under its own weight. The precursors can be mixed in a wide range of proportions, and the resulting hydrogel can undergo tunable and reversible swelling in an aqueous buffer solution.

[0126] In some embodiments, the hydrogel precursor is SMA, AA, and PAE. Preferably, the molar ratio of SMA to AA in the hydrogel precursor solution can be in the range of about 1:1.4 to about 1:25, 1:2 to about 1:20, 1:3 to about 1:15, or 1:5 to about 1:10. Preferably, the molar ratio of AA to PAE in the hydrogel precursor solution can be in the range of about 200:1 to 30:1. In some embodiments, the molar ratio of SMA:AA:PAE in the hydrogel precursor solution is in the range of about (8-21):(30-200):1.

[0127] In some embodiments, the hydrogel precursor is SA, AA, and Bis-AA. Preferably, the molar ratio of SA to AA can be in the range of about 0.3:1 to about 6:1, about 0.4:1 to about 5:1, about 0.5:1 to about 4:1, about 0.6:1 to about 3:1, about 0.7:1 to about 2:1, about 0.8:1 to about 1:1, or any ratio or subrange thereof. Preferably, the molar ratio of AA:Bis-AA can be in the range of about 1:0.0001 to about 1:0.4, about 1:0.0002 to about 1:0.3, about 1:0.0003 to about 1:0.2, about 1:0.0004 to about 1:0.1, or any ratio or subrange thereof. In some embodiments, the molar ratio of SA, AA, and Bis-AA in the hydrogel precursor solution is in the range of about (12-48):(9-32):(0.01-1).

[0128] In some embodiments, the hydrogel precursor is SMA, AA, and Bis-AA. Preferably, the molar ratio of SMA to AA can be in the range of about 0.3:1 to about 6:1, about 0.4:1 to about 5:1, about 0.5:1 to about 4:1, about 0.6:1 to about 3:1, about 0.7:1 to about 2:1, about 0.8:1 to about 1:1, or any ratio or subrange thereof. Preferably, the molar ratio of AA:Bis-AA can be in the range of about 1:0.0001 to about 1:0.4, about 1:0.0002 to about 1:0.3, about 1:0.0003 to about 1:0.2, about 1:0.0004 to about 1:0.1, or any ratio or subrange thereof. In some embodiments, the molar ratio of SMA, AA, and Bis-AA in the hydrogel precursor solution is in the range of about (12-48):(9-32):(0.01-1).

[0129] In some embodiments, the hydrogel precursors are DMAA, SMA, and TPT. Preferably, the molar ratio of DMAA to SMA can be in the range of about 30:1 to about 4:1, about 20:1 to about 5:1, about 10:1 to about 6:1, or any ratio or subrange thereof. Preferably, the molar ratio of SMA to TPT can be in the range of about 1:0.0001 to about 1:0.4, about 1:0.0002 to about 1:0.3, about 1:0.0003 to about 1:0.2, about 1:0.0004 to about 1:0.1, or any ratio or subrange thereof. For example, the molar ratio of DMAA:SMA:TPT can be in the range of (4-30):1:(0.0004-0.4).

[0130] In some embodiments, the hydrogel precursor is DMAA, SMA, and PAE. Preferably, the molar ratio of DMAA to SMA can be in the range of about 30:1 to about 4:1, about 20:1 to about 5:1, about 10:1 to about 6:1, about 5:1 to about 2:1, or any ratio or subrange thereof. Preferably, the molar ratio of SMA to PAE can be in the range of about 1:0.0001 to about 1:0.4, about 1:0.0002 to about 1:0.3, about 1:0.0003 to about 1:0.2, about 1:0.0004 to about 1:0.1, or any ratio or subrange thereof. In some embodiments, the molar ratio of DMAA:SMA:PAE in the hydrogel precursor solution is in the range of (30-50):(5-15):(0.0004-2).

[0131] In some embodiments, the hydrogel precursor is DMAA, SMA, and TPT. In some embodiments, the molar ratio of DMAA to SMA in the hydrogel precursor solution can be in the range of about 30:1 to 4:1 (e.g., 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1). Additionally, the molar ratio of SMA to TPT in the hydrogel precursor solution can be in the range of about 1:0.0004 to 1:0.4 (e.g., 1:0.0005, 1:0.001, 1:0.005, 1:0.01, 1:0.05, 1:0.1, 1:0.2, 1:0.3). In some embodiments, the molar ratio of DMAA:SMA:TPT in the hydrogel precursor solution is in the range of about (4-30):1:(0.0004-0.4).

[0132] In some embodiments, the one or more precursors are SMA, AA, and Bis-AA. In some embodiments, the molar ratio of SMA:AA in the hydrogel precursor solution can be in the range of about 0.375:1 to about 5.33:1 (e.g., 0.4:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1). Additionally, the molar ratio of AA:Bis-AA in the hydrogel precursor solution can be in the range of about 1:0.0002 to 1:0.12 (e.g., 1:0.0005, 1:0.001, 1:0.005, 1:0.01, 1:0.05, 1:0.10). In some embodiments, the molar ratio of SMA, AA, and Bis-AA in the hydrogel precursor solution is in the range of about (12-48):(9-32):(0.01-1).

[0133] In some embodiments, the one or more precursors are SA, AA, and Bis-AA. In some embodiments, the molar ratio of SA:AA in the hydrogel precursor solution can be in the range of about 0.375:1 to about 5.33:1 (e.g., 0.4:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1). Additionally, the molar ratio of AA:Bis-AA in the hydrogel precursor solution can be in the range of about 1:0.0002 to 1:0.12 (e.g., 1:0.0005, 1:0.001, 1:0.005, 1:0.01, 1:0.05, 1:0.10). In some embodiments, the molar ratio of SA, AA, and Bis-AA in the hydrogel precursor solution is in the range of about (12-48):(9-32):(0.01-1).

[0134] In some embodiments, the one or more precursors are DMAA:SMA:PAE. In some embodiments, the molar ratio of DMAA to SMA in the hydrogel precursor solution can be in the range of about 30:1 to 4:1 (e.g., 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1). Additionally, the molar ratio of SMA to PAE in the hydrogel precursor solution can be in the range of about 1:0.0002 to 1:0.4 (e.g., 1:0.0005, 1:0.001, 1:0.005, 1:0.01, 1:0.05, 1:0.1, 1:0.2, 1:0.3). In some embodiments, the molar ratio of DMAA:SMA:PAE in the hydrogel precursor solution is in the range of (30-50):(5-15):(0.004-2).

[0135] Specifically, in some embodiments, DMAA, SMA, and TPT are mixed in a molar ratio of approximately 30:1:0.4 in the hydrogel precursor solution. The hydrogel precursor solution can be prepared by mixing DMAA, SMA, and TPT (optionally in THF) into water. The pH range of the hydrogel precursor solution is preferably in the range of 6-7, for example, approximately 6.5. The hydrogel precursor solution can be prepared fresh before use, or a stock solution can be prepared and diluted before use.

[0136] In some other embodiments, SA, AA, and Bis-AA are mixed in the hydrogel precursor solution in a molar ratio of about 2.6:1:0.027. In some embodiments, DMAA, SMA, and PAE are mixed in the hydrogel precursor solution in a molar ratio of about 40:10:0.02.

[0137] The hydrogel precursor solution can be further optimized to include HCl or NaCl in the buffer system (e.g., PBS buffer), which can promote an acidic or neutral environment.

[0138] Prior to the polymerization reaction, a polymerization activator and / or accelerator are added to the hydrogel precursor solution to induce polymerization or gelation. The activator or accelerator may be, for example, but not limited to, ammonium persulfate, potassium persulfate, TEMED, VA-044, or combinations thereof. The polymerization activator may be mixed with the hydrogel precursor solution before use (i.e., before sample infusion).

[0139] In some embodiments, the sample is perfused with a hydrogel precursor solution containing a protein anchoring agent and a polymerization activator / accelerator. Alternatively, the sample to be gelled is treated with a protein anchoring agent before incubation with the hydrogel precursor solution.

[0140] A stock solution of the hydrogel precursor can be prepared and diluted before use. The stock solution can have a high concentration (w / w) of the precursor, such as about 50% or higher, about 75% or higher, about 80% or higher, or about 90% or higher. The stock solution can be diluted to a concentration of about 30-50% before application to a sample. Preferably, the solution containing the precursor is an aqueous solution.

[0141] Homogenization

[0142] In some implementations, the sample-hydrogel complex is homogenized prior to expansion. As used herein, "homogenization" refers to mechanical, physical, chemical, biochemical, or enzymatic digestion, disruption, or fragmentation of the sample to allow expansion. Homogenization methods further improve the achievable resolution of fluorescence imaging, which is crucial for imaging techniques with resolutions exceeding those of dyes or markers.

[0143] In some embodiments, enzymatic homogenization is performed prior to expansion, which includes treating the sample-hydrogel complex with a specific or non-specific protease for an appropriate time, depending on the sample type and size. For example, enzymatic homogenization includes treating the sample-hydrogel complex with trypsin in a homogenization buffer. In some alternative embodiments, physicochemical homogenization is performed using a buffer rich in alkaline detergents (e.g., a buffer containing SDS (sodium dodecyl sulfate)).

[0144] In some specific embodiments, homogenization is performed by incubating the sample-hydrogel complex with a homogenization solution for an appropriate time. The homogenization solution can be prepared by mixing a protease (such as trypsin) with a homogenization buffer. Alternatively, homogenization can be accomplished by incubating the sample-hydrogel complex with a homogenization buffer containing an alkaline detergent (e.g., sodium dodecyl sulfate at pH 7.0 to 10.0) for a sufficiently long time. Many homogenization buffers are known and routinely used in the art. Homogenization buffers can be readily prepared in-house or purchased commercially.

[0145] In some embodiments, the homogenization buffer is a PBS buffer containing trypsin. In other embodiments, the homogenization buffer is a buffer containing SDS, NaCl, and EDTA.

[0146] Preferably, homogenization does not affect the structure of the hydrogel and the integrity of the mRNA, but is sufficient to disrupt the mechanical structure of the sample. Homogenization allows biomolecules (such as DNA, RNA, and / or proteins) to be labeled in a molecularly decongested environment, thereby improving the accessibility of dyes or other molecular markers and thus increasing staining efficiency.

[0147] The methods disclosed herein may include different combinations of hybridization and homogenization processes. In some embodiments, the method includes incubating a sample with a hybridization buffer containing both an mRNA probe and a protein anchoring agent to anchor mRNA and protein, and treating the sample-hydrogel complex with SDS for homogenization. In some embodiments, the method includes incubating a sample with a hybridization buffer containing both an mRNA probe and a protein anchoring agent to anchor mRNA and protein, and treating the sample-hydrogel complex with trypsin for homogenization. In some embodiments, the method includes incubating a sample with a hybridization buffer containing an mRNA probe, followed by contacting a protein anchoring agent to anchor the protein, and treating the sample-hydrogel complex with trypsin for homogenization. In some embodiments, the method includes incubating a sample with a hybridization buffer containing an mRNA probe, followed by contacting a protein anchoring agent to anchor the protein, and treating the sample-hydrogel complex with SDS for homogenization.

[0148] dyeing

[0149] In some embodiments, the sample-hydrogel complex is stained after homogenization to visualize target biomolecules and tissue and cell morphology. The staining step can be performed before, during, or after hydrogel swelling. Typically, one or more dyes or labels are chemically bound to the target biomolecules in the sample (e.g., covalently, hydrogenally, or ionicly). The dyes or labels may be selective for specific targets (e.g., biomarkers or a class of molecules such as DNA, RNA, or proteins). The labels preferably contain visible components similar to typical dyes or fluorescent molecules. For example, fluorescently labeled samples are labeled using techniques such as, but not limited to, immunofluorescence, immunohistochemistry, or immunocytochemistry staining to aid microscopic analysis. The labels or dyes are preferably chemically linked to the target biomolecules or components thereof. The sample-hydrogel complex can be stained with one or more dyes or labels. For example, each dye or label may have specific or distinguishable fluorescence properties, such as distinguishable excitation and emission wavelengths. Furthermore, each dye or label may have different target-specific binders that are selective for specific and distinguishable targets or components thereof in the sample. This method is compatible with fluorescent proteins and standard immunofluorescence methods before or after sample-hydrogel complex swelling, providing up to 50 nm lateral resolution in conventional imaging settings.

[0150] In some embodiments, the sample-hydrogel complex is stained with a fluorescent dye to visualize nucleic acids. Dyes used for DNA visualization can be selected from, for example, propidium iodide, DAPI, 7-AAD, Hoechst, and YOYO-1 / DiYO-1 / TOTO-1 / DiTO-1, which are commonly used for DNA staining. In one embodiment, the sample-hydrogel complex is stained with DAPI for DNA visualization.

[0151] In some further embodiments, the sample-hydrogel complex is stained with a dye to visualize the protein. Dyes used for protein visualization may be selected from, for example, the Sypro series, Flamingo fluorescent gel staining, Krypton protein staining, 5-TAMRA NHS ester, etc. In one embodiment, the sample-hydrogel complex is stained with Sypro dye to stain the protein.

[0152] The sample-hydrogel complex can be stained separately with appropriate dyes for DNA and protein visualization. In some embodiments, the method further includes staining the sample-hydrogel complex after homogenization, wherein the sample-hydrogel complex can be stained with DNA dyes (such as DAPI) and / or protein dyes (such as Sypro series dyes) to visualize nucleic acids or proteins. In some embodiments, the sample-hydrogel complex is first stained with DAPI to highlight DNA, and then stained with Sypro dyes (e.g., Sypro Ruby) to stain proteins.

[0153] The methods disclosed herein may include different combinations of homogenization and staining processes. In some embodiments, the homogenization step is performed by treatment with trypsin, followed by staining with DAPI to stain the DNA. In some embodiments, the homogenization step is performed by treatment with SDS, followed by staining with DAPI to stain the DNA. In some embodiments, the homogenization step is performed by treatment with trypsin, followed by staining with DAPI and Sypro dye (e.g., Sypro Ruby) to stain both DNA and protein. In some embodiments, the homogenization step is performed by treatment with SDS, followed by staining with DAPI and Sypro dye (e.g., Sypro Ruby) to stain both DNA and protein.

[0154] hydrogel swelling

[0155] After homogenization, the sample-hydrogel composite is tunably expanded to the desired extent according to the end-user's requirements. This composite can expand isotropically in three-dimensional space, preferably with nanometer-level precision.

[0156] In some embodiments, after homogenization and staining, a solvent or liquid is added to the sample-hydrogel complex, which is then absorbed by the complex, causing swelling. The solvent or liquid used herein is preferably RNase-free to maintain RNA integrity. In cases where water swelling of the sample-hydrogel complex is possible, an aqueous solution can be used. The aqueous solution can be water or a buffer, such as diluted SSC buffer. In some embodiments, the addition of the aqueous solution allows the embedded sample to swell in three-dimensional space to 8 to 10 times or more its original size. Therefore, the sample volume can increase by 100 times or more. This is because the polymer is embedded throughout the entire sample, and thus, as the polymer swells (grows), it also causes the sample to swell. Consequently, the tissue sample itself becomes larger. Due to the isotropic swelling of the material, the anchored markers or tags maintain their relative spatial relationships.

[0157] The goal of most applications is typically to achieve the highest possible spatial resolution, thus employing various microscopy protocols to image subcellular structures. The highest achievable resolution is defined by the expansion factor, which, for simplicity, is measured by the overall expansion of the hydrogel. Preferably, the hydrogel can expand up to 8-10 times in the linear dimension, thereby increasing the resolution limit to 40 nm under diffraction-limited microscopy. In contrast, existing protocols have a maximum expansion factor of less than 4 in the linear dimension, thus limiting the achievable lateral resolution to 80 nm. Some protocols can expand the sample 10 times in the linear dimension, but due to the mechanical instability of the hydrogel used for expansion, these protocols are only applicable to two-dimensional samples, such as tissue cultures or thin tissue sections.

[0158] In some implementations, the addition of pure water or other aqueous buffers allows the sample-hydrogel complex to expand linearly to up to eight times its original sample size while maintaining high mechanical stability and elasticity. Both mechanical stability and elasticity allow for easy manipulation of the expanded sample without causing mechanical deformation of the sample-hydrogel complex, thus preserving its integrity. These key properties ensure the absence of artifacts that could be caused by deformation or breakage of the sample-hydrogel complex. Therefore, the sample volume can increase isotropically (i.e., equally in all dimensions) by 512-fold in three-dimensional space. This isotropic expansion occurs because the molecular chains of the swellable polymer formed throughout the sample expand, separating the biomolecules and causing the tissue sample itself to enlarge. Importantly, the relative positions of the biomolecules remain unchanged after expansion. After expansion, the sample-hydrogel complex can be imaged using an optical microscope, enabling effective visualization of features smaller than the classical diffraction limit. Since the expanded sample-hydrogel complex is transparent, any conventional microscope capable of large-volume imaging can be used.

[0159] Once swelling is complete, the presence and / or location of DNA, mRNA, and proteins in the tissue can be observed. The swollen material with the embedded target sample can be imaged on any optical microscope, allowing for effective imaging of features below the classical diffraction limit. Since the resulting specimen is preferably transparent, custom microscopes capable of large-volume, wide-field-of-view, 3D scanning can also be used in conjunction with the swollen sample.

[0160] After expansion, a set of methods for biochemical or spectroscopic characterization of the expanded biological sample can be used to determine the presence, distribution, type, and / or quantity of target biomolecules (such as mRNA).

[0161] mRNA sequencing

[0162] In some implementations, the methods disclosed herein further include high-throughput sequencing or next-generation sequencing (NGS) of mRNA released from a target region in the hydrogel.

[0163] High-throughput sequencing is revolutionizing many areas of biology, including cancer diagnosis, disease surveillance, and environmental analysis. In particular, methods for analyzing mRNA molecules through high-throughput sequencing of reverse-transcribed cDNA can reveal the types and quantities of transcripts in a biological sample at a given time. By combining this with hydrogel expansion microscopy, the ExiST / neoExiST method disclosed in this paper can further reveal the location and spatial distribution of transcripts in biological samples, mapping them to high-resolution tissue morphology images with higher lateral resolution.

[0164] Typically, the core steps in preparing RNA or DNA for next-generation sequencing analysis are: (i) fragmenting and / or screening the target sequence to the target length, (ii) converting the target into double-stranded DNA, (iii) ligating oligonucleotide adapters to the ends of the target fragments, and (iv) quantifying the final library product for sequencing. For mRNA sequencing libraries, methods have been developed based on cDNA synthesis using random primers, Oligo(dT) primers, or by ligating adapters to mRNA fragments followed by some form of amplification. First-strand cDNA can be generated by guiding mRNA with random oligomers or anchored Oligo(dT) primers.

[0165] In some implementations, RT-qPCR is used to detect and quantify mRNA released from the sample-hydrogel complex. The mRNA is transcribed into complementary DNA (cDNA) and amplified by PCR for library preparation. This cDNA can then be used as a template for quantitative PCR or real-time PCR (qPCR). During qPCR, the amount of amplified product is measured using fluorescence at each PCR cycle. RT-qPCR is used in a variety of applications, including gene expression analysis, RNAi validation, microarray validation, pathogen detection, gene assays, and disease research. Alternatively, various sequencing platforms can be used for NGS of the cDNA library.

[0166] Commonly used high-throughput sequencing platforms (such as those provided by Illumina, Roche Sequencing, and Pacific Biosciences) typically use transcriptome-derived DNA fragment libraries flanked by platform-specific adapters. The standard method for constructing such libraries is entirely in vitro and usually includes one or more of the following: cDNA synthesis, DNA fragmentation (mechanical or enzymatic), end trimming, adapter sequence ligation, gel-based size screening, and PCR amplification. Depending on the specific application, additional steps may be performed prior to this core procedure.

[0167] In some embodiments, the platform used for cDNA library sequencing is the Illumina sequencing platform, but this application is not limited to the Illumina sequencing platform. Various next-generation sequencing platforms are known in the art, and any of these platforms can be used in this invention for the sequencing steps. This technology enables the analysis of mRNA expression levels and spatial distribution within a corresponding cellular environment.

[0168] With the help of NGS technology, RNA expression profiling or whole-genome sequencing has become routine practice in biological research. On the other hand, due to the high throughput of NGS, multiplex sequencing methods have been developed for sequencing not only more regions but also more samples. Compared to traditional Sanger sequencing, NGS can detect mutations in different genes in more samples in parallel. Because of its superiority over traditional sequencing methods, NGS sequencers are currently replacing Sanger in routine diagnostics. In particular, it is now possible to routinely analyze individual genetic variations for a variety of medical applications, ranging from genetic disease diagnosis to pharmacogenomics fine-tuning of drugs in precision medicine practices. NGS involves processing multiple fragmented DNA sequence reads, typically short reads (less than 300 nucleotide base pairs). The resulting reads can then be compared to a reference genome using various bioinformatics methods to identify small variations, such as single nucleotide polymorphisms (SNPs) corresponding to single nucleotide substitutions, and short insertions and deletions (INDELs) of nucleotides in the DNA sequence compared to its reference sequence.

[0169] Mass spectrometry (MS) analysis

[0170] Methods for proteomics identification are well known in the art and applicable to those disclosed herein. In some embodiments, MS, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or LC-MS / MS is used for protein identification. Liquid chromatography (LC) can efficiently separate organic components in a sample, while mass spectrometry (MS) can analyze the separated organic compounds one by one to obtain information about their molecular weight, structure, and concentration. HPLC-MS is a conventional method in the art for analyzing and measuring high molecular weight compounds, such as proteins and polymers.

[0171] Reagent test kit

[0172] This document also provides kits for practicing the methods described herein, which typically include: (i) precursors (including monomers, oligomers, and cross-linking agents) for forming the hydrogel; (ii) mRNA probes for hybridizing mRNA in the sample and cross-linking with the hydrogel polymer chains; and / or (iii) one or more hybridization buffers, homogenization buffers, dyes, or markers as described above. The kit components are preferably used under RNase-free conditions, or have been treated with an RNase inhibitor (e.g., DEPC) prior to use, or contain an RNase inhibitor. The kit can be stored at -20°C for at least 6 months, or at 4°C or room temperature to maintain its effectiveness for at least 3 months. The kit components are stable during storage and maintain imaging quality.

[0173] In some embodiments, the kit includes a container containing an mRNA probe. The mRNA probe may contain a nucleotide sequence consisting of dT nucleotides, or a nucleotide sequence containing dT nucleotides, dT nucleotide analogs, and / or derivatives thereof. In some embodiments, the mRNA probe contains a nucleotide sequence consisting of dT nucleotides and LNA-modified dT nucleotides. In some embodiments, the mRNA probe contains a nucleotide sequence containing dT nucleotides and thymidine-intramolecular nucleic acid (dT+). In some embodiments, the mRNA probe contains alternating dT and thymidine-intramolecular nucleic acid (dT+) nucleotide sequences.

[0174] The length of the mRNA probe can vary between 10 and 30 nucleotides, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The number of dT-LNA residues and the length of the mRNA probe can be adjusted as needed to obtain appropriate stability and specificity. In some embodiments, the mRNA probe comprises alternating nucleotide sequences of dT and thymidine lock nucleic acids (dT+) of length 10-30 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). In some embodiments, the mRNA probe comprises nucleotide sequences as shown in SEQ ID NO: 1 (15 nt) or 2 (25 nt). Furthermore, the mRNA probes disclosed herein are modified to enable them to crosslink with hydrogel polymer chains. This modification can be located at the 5' or 3' end, or at an internal nucleotide. In some embodiments, the 5' end of the mRNA probe is modified with Acrydite, a primary amino group, an azide group, a Uni-Link™ amino modifier, or any combination or mixture thereof. In some specific embodiments, the mRNA probe comprises the nucleotide sequence shown in SEQ ID NO: 1 or 2 and has a 5'-Acrydite modification.

[0175] In some embodiments, the kit further includes a container containing a bifunctional protein anchoring agent. The protein anchoring agent can be added to a hybridization buffer containing an mRNA probe prior to use. Alternatively, the protein anchoring agent can be added to a hydrogel precursor solution, and then the sample is embedded in or perfused using the mixture. The protein anchoring agent can be selected from NSA, NAS, methacrolein, AGE, GME, GAL, BDE, GDE, and combinations thereof. In some embodiments, the protein anchoring agent is NSA or NAS.

[0176] In some embodiments, the kit further includes one or more containers containing one or more hydrogel precursors. The hydrogel precursor may be selected from, but is not limited to: sodium acrylate (SA), sodium methacrylate (SMA), itaconic acid (IA), trans-aconitine (TAA), ethyl-2-(hydroxymethyl)-acrylate (EHA), N,N'-methylenebisacrylamide (Bis-AA), N,N-dimethylacrylamide (DMAA), pentaerythritol tetraacrylate (PT), propoxylated trimethylolpropane triacrylate (TPT), pentaerythritol triacrylate (PA), dipentaerythritol pentaacrylate, or dipentaerythritol hexaacrylate. Acrylates (DPHA), trimethylolpropane triacrylate (TTA), di(trimethylolpropane)tetraacrylate (DiTA), trimethylolpropane trimethacrylate (TTMA), glycerol propoxylated (1PO / OH) triacrylate (GPT), ethoxylated trimethylolpropane triacrylate (TET), pentaerythritol allyl ether (PAE), sodium 4-hydroxy-2-methylenebutyrate (SHMB), N,N-dimethylaminopropylacrylamide (DMPAA), and acrylamide (AA).

[0177] In some embodiments, the kit contains precursors SMA, AA, and PAE. In some embodiments, the kit contains precursors DMAA, SMA, and TPT. In some other embodiments, the kit contains precursors DMAA, SA, Bis-AA, and AA. In some other embodiments, the kit contains precursors DMAA, SMA, and PAE. In some other embodiments, the kit contains precursors SMA, Bis-AA, and AA. In some other embodiments, the kit contains precursors SA, Bis-AA, and AA.

[0178] The precursors can be present in separate containers within the kit, for example, DMAA in a first container, SMA in a second container, and TPT in a third container; or SMA in a first container, Bis-AA in a second container, and AA in a third container, for mixing before use. Alternatively, any of the following configurations are also suitable: DMAA and SMA mixed (within a certain ratio) in a first container, with TPT in a second container; DMAA and TPT mixed (within a certain ratio) in a first container, with SMA in a second container; or SMA and TPT mixed (within a certain ratio) in a first container, with DMAA in a second container; SMA and AA mixed (within a certain ratio) in a first container, with Bis-AA in a second container; SMA and Bis-AA mixed (within a certain ratio) in a first container, with AA in a second container; or AA and Bis-AA mixed (within a certain ratio) in a first container, with SMA in a second container. These containers may or may not employ a combined configuration.

[0179] The hydrogel precursor can be dissolved in an aqueous buffer (such as PBS buffer). In some embodiments, the kit includes a hydrogel precursor solution containing DMAA, SMA, and TPT. In some other embodiments, the kit includes a hydrogel precursor solution containing DMAA, SA, Bis-AA, and AA. In some other embodiments, the kit includes a hydrogel precursor solution containing DMAA, SMA, and PAE. In some other embodiments, the kit includes a hydrogel precursor solution containing SMA, Bis-AA, and AA.

[0180] The kit may further include a container containing a hybridization buffer (e.g., SSC buffer). In some specific embodiments, the hybridization buffer contains 2×SSC, 30% [v / v] formamide, 1% [v / v] RNase inhibitor, 10% [w / v] dextran sulfate, and 0.1% [w / v] yeast transfer RNA. Optionally, an mRNA probe has been added to the hybridization buffer.

[0181] The kit may further include a container containing a wash buffer (e.g., SSC buffer or PBS buffer). In some specific embodiments, the wash buffer contains 2×SSC, or 2×SSC and 30% [v / v] formamide.

[0182] The kit may further include a container containing a fixative (such as ethanol, e.g., 75% ethanol).

[0183] The kit may further include a container containing a staining agent or labeling agent (e.g., a fluorescent dye for staining DNA, RNA, and / or proteins). In some further embodiments, the kit contains more than one staining agent, including a staining agent for DNA staining (e.g., DAPI) and a staining agent for protein staining (e.g., the Sypro series). The dye, fixative, probe, anchoring agent, and hydrogel precursor solution are stable under storage conditions and can be transported without affecting performance.

[0184] The kit may further include a container containing a polymerization activator or accelerator, which is added prior to polymerization. The polymerization activator or accelerator may be selected from, but is not limited to, VA-044, TEMED, potassium persulfate, and APS. Preferably, the polymerization activator (e.g., APS) is added to the hydrogel precursor solution on ice, and should be added immediately before the sample is infused with the hydrogel precursor solution.

[0185] The kit may further include a container containing a homogenization buffer. Specifically, the homogenization buffer may contain a protease (e.g., trypsin) or an alkaline detergent (e.g., SDS), or may be replenished with a protease (e.g., trypsin) or alkaline detergent (e.g., SDS) prior to use. In some embodiments, the kit includes a container containing trypsin. In some embodiments, the kit includes a container containing a commonly used buffer (e.g., PBS or NaCl buffer) for easy solution preparation.

[0186] The term "container" should be understood to refer to any structure that can contain or enclose liquid or solid components (such as components in a hydrogel formulation); exemplary containers include bottles, syringes, sample vials, sealed bags, capsules, ampoules, reservoirs, etc. Containers can be made to block visible light, ultraviolet, or infrared radiation by using additional components (such as aluminum foil bags covering sample vials) or by selecting the material properties of the container itself (such as amber glass sample vials or opaque syringes).

[0187] The kit may also include a mixing device for mixing the precursors to formulate the formulation of the present invention. The kit may also include gel handling devices, such as soft brushes, tweezers, and / or delivery devices (which may or may not include mixing elements), for injecting the hydrogel precursor solution onto a sample.

[0188] In some embodiments, the kit includes one or more of the following components: a gelation chamber configured to contain a sample prior to the gelation reaction, and optionally a cover for sealing the gelation chamber; an imaging chamber configured to contain a sample after the gelation reaction; a gel manipulation device; and a cooling device (e.g., an ice pack). Furthermore, the kit may contain different forms of molds (e.g., silicone molds) or gelation chambers suitable for gelation reactions of various samples (including but not limited to cell cultures, intact organs, tissues or tissue sections, and whole organisms).

[0189] The kit may further include other components, such as desiccants or other tools to adjust the moisture content within the kit, indicators for recording the highest temperature experienced by the kit, etc., all of which are necessary to keep the product in good condition during transportation and storage.

[0190] In some embodiments, the kit includes a gelation chamber for forming the sample-hydrogel complex as described above. The gelation chamber is configured to contain cavities, pores, or spaces for containing the sample prior to the gelation reaction, for example, in the form of a petri dish (such as a MatTek petri dish). The gelation chamber may also include a cover plate (e.g., a coverslip) for covering the cavity, pore, or opening.

[0191] In some embodiments, the kit further includes an expansion chamber configured to contain the sample after the gelation reaction. The kit may contain a stock kit of such chambers, such as a dozen petri dishes packaged together for easy access.

[0192] The kits described herein can be stored for at least 6 months, some reagents can be transported on ice or at room temperature (RT), and multiple reagent combinations can be packaged in suitable containers for distribution.

[0193] In addition to the components described above, the kit may also include instructions for using the kit components to implement the methods of the present invention. The instructions for implementing the methods of the present invention may be present within the kit as a packaging insert, in a label on the container of the kit or its components (i.e., associated with the packaging or inner packaging), or may not be included in the kit but may provide access to the instructions from a remote channel (e.g., via the Internet). One example of the embodiment is a kit containing a URL through which the instructions can be viewed and / or downloaded.

[0194] The following embodiments are provided to better illustrate the claimed technical solutions and should not be construed as limiting the scope of protection of this invention. All specific compositions, materials, and methods described below, whether in whole or in part, fall within the protection scope of this invention.

[0195] Example

[0196] The exemplary reagents, materials, and storage conditions used in the experiments described below are shown in Table 1. The volume or mass of reagents can be increased or decreased proportionally according to user needs.

[0197] Table 1

[0198] Example 1: Spatial transcriptomics analysis of mouse brain and liver tissues (ExisT)

[0199] 1.1 Tissue Preparation

[0200] Mice were euthanized using 1% sodium pentobarbital. Brain tissue (or other target organs) was rapidly dissected. The tissue blocks were rapidly frozen in liquid nitrogen vapor until solidified, and then stored at -80°C for later use.

[0201] 1.2 Frozen Sections

[0202] The tissue was then frozen and sectioned to a thickness of 16µm using a Leica cryostat and carefully placed on custom-made slides.

[0203] 1.3 Ethanol fixation

[0204] First, fix the sections with 75% ethanol, 100% methanol, or 100% acetone for 3 minutes.

[0205] 1.4 Hybridization and Incubation

[0206] The sample was first incubated with 1 µM RNA anchoring probe (a 15-nucleotide sequence of alternating dT and thymidine-modified dT+ nucleic acid, 5' end modified with an Acrydite group) at 37 °C for 40 h. The hybridization buffer contained 1 µM anchoring probe, 2×SSC, 30% [v / v] formamide, 1% [v / v] RNase inhibitor, 10% [w / v] dextran sulfate, and 0.1% [w / v] yeast transfer RNA.

[0207] RNA anchoring probe sequence: T+TT+TT+TT+TT+TT+TT+TT, with 5'Acrydite modification.

[0208] 1.5 Monomer incubation and gelation reaction

[0209] The sample was incubated in a monomer solution containing 1×PBS, 2M NaCl, 8.625% [w / w] sodium acrylate or sodium methacrylate, 2.5% [w / w] acrylamide, and 0.15% [w / w] N,N'-methylenebisacrylamide for 2 hours. 0.3% APS solution and 0.2% TEMED solution were added to initiate the gelation reaction. The gelation step was carried out at 37°C for 2 hours.

[0210] 1.6 Homogenization

[0211] Apply SDS buffer (1% SDS, 50mM NaCl, 5mM EDTA) or trypsin buffer (6.25ug / mL trypsin in 5×SSC buffer) to the gel and treat overnight at 22°C.

[0212] 1.7 Staining and Swelling

[0213] Wash the homogenized gel three times with 5×SSC buffer, 10 minutes each time. Stain the gel with DAPI and Sypro series dyes for 30 minutes to 3 hours. After staining, swell the gel 3-5 times with 1×PBS buffer, 15 minutes each time.

[0214] 1.8 Imaging and Micro-cutting

[0215] The expanded sample was imaged using a stereomicroscope or a fluorescence microscope. Figure 2 Images of brain tissue before and after expansion using different homogenization conditions are presented. Target regions were identified based on super-resolution imaging or morphological features, and microdissection was performed using punches of specific sizes (0.35 mm to 3 mm) (a 1.5 mm punch was used for 16 µm cortical tissue with LEF=3.0).

[0216] 1.9 In-gel reverse transcription-polymerase chain reaction

[0217] First, after expansion, mRNA was released and analyzed from the entire sample to verify the concentration and integrity of the mRNA (see brain tissue). Figure 3 For liver tissue, see [link / reference]. Figure 4 RT-PCR and NGS library preparation were performed using commercially available reagents and kits according to standard experimental protocols.

[0218] After validating the feasibility of the entire workflow, in-gel reverse transcription-polymerase chain reaction (RT-PCR) was performed on the microcut samples. 3.5 µl of 0.1×SSC buffer was added to cover the gel particles. RNA release was then performed at 40–50 °C for 30 min. Oligo(dT) primers were then added to the cut samples, followed by incubation at 72 °C for 3 min. The samples were immediately placed on ice after the reaction. 5' oligo primers and reverse transcriptase were then added to the reaction mixture. Reverse transcription was performed at 40–45 °C for 30–180 min, followed by incubation at 70 °C for 15 min. The reaction products were maintained at 4 °C. The amplification mixture and primers were then added to the system for cDNA amplification. The number of cycles ranged from 8 to 18 cycles, depending on the size of the cut samples.

[0219] 1.10 cDNA Product Purification and Detection

[0220] The product from Section 1.9 was purified using DNA magnetic beads, incubated for 10 minutes to bind DNA. The sample was placed on a magnetic rack to separate the beads from the liquid. After approximately 5 minutes, the supernatant was discarded. The beads were washed with freshly prepared 80% ethanol for 30 seconds, and this step was repeated. The tube caps were opened and the beads were allowed to dry to remove the ethanol. The centrifuge tube was removed from the magnetic rack, approximately 17 µl of elution buffer was added, and the beads were incubated for 2–5 minutes. The centrifuge tube was returned to the magnetic rack to separate the liquid from the beads. 15 µl of the supernatant was gently aspirated and stored at -20°C. The purified cDNA product was analyzed using a bioanalyzer.

[0221] 1.11 Library Preparation

[0222] First, cDNA fragmentation was performed using transposase at 55°C for 10 minutes. The reaction was then terminated. The fragments were amplified using index sequences. The number of cycles was set according to the size of the cleaved sample, ranging from 5 to 15 cycles. The product was purified using DNA magnetic beads or a DNA purification column. The quality of the DNA library was assessed using a bioanalyzer or Qubit. The high-quality library was then sent to a commercial sequencing facility for next-generation sequencing (NGS).

[0223] result: For ExiST, such as Figure 3 The extracted RNA quality (RNA band / size distribution) shown demonstrates that both trypsin and SDS treatment conditions are well-suited for the ExiST method. For both homogenization conditions (trypsin or SDS), the resulting NGS libraries possess sufficient quality for downstream sequencing and analysis. Figure 4 (and Table 2).

[0224] Table 2. Properties of micro-dissected samples, including length range, average size, concentration, regional molar concentration, and percentage of total fragments.

[0225]

[0226] FastQC analysis of the NGS results indicated good quality of the raw data, and that the data were homogenized with trypsin ( Figure 5 ) and homogenization with SDS-containing buffer ( Figure 6 Under these conditions, there are no problems or biases in the data. Specifically, Figure 5 The parameters, including the quality of each base sequence, the quality of each tile sequence, the quality fraction of each sequence, the linker content, and the N content of each base, all showed good performance. Figure 6The FastQC results shown have high quality per base sequence, and good performance in terms of quality per tile sequence, quality score per sequence, adapter content, N content per base, and content per base sequence.

[0227] A total of 17,980 aligned expressed genes were identified in 2.0 mm punched samples homogenized with trypsin. Figure 7 As shown in Figure a, evaluation of the raw sequencing data using FastQC indicated that both conditions (S-zsc: homogenization with SDS treatment; T-zsc: homogenization with trypsin treatment) demonstrated good quality. Further analysis used HISAT2 to align the RNA-seq data to the genome and identify splice junctions under both homogenization conditions (trypsin and SDS). Figure 7 (b and c). Figure 7 The alignment results in Figure b demonstrate the alignment quality, showing no bias in both the sense and antisense chains. Figure 7 The alignment results in Figure c show that the trypsin-treated group has more exon alignment regions compared to the SDS-treated group.

[0228] Analysis of gene body coverage and gene expression such as FPKM also demonstrated the detection diversity and regional differences in sequencing results under two homogenization conditions (trypsin or SDS). Figure 8 ).like Figure 8 As shown in Figure a, the SDS-treated group exhibited a higher mean gene body coverage compared to the trypsin-treated group. Figure 8 As shown in Figure b, the SDS-treated group exhibited higher expression density and expression level compared to the trypsin-treated group.

[0229] Figure 9 This demonstrates the RNA distribution and banding of mRNA released from BeyoMag™ Oligo(dT)25 magnetic beads, detected by fragment analysis in step 1.9. Microdissected samples were incubated in buffer at 40–45°C (30–180 min), followed by mRNA concentration using commercially available dT magnetic beads or an RNA purification kit. Figure 21 Compared to the results, this indicates that it is feasible to release RNA directly into the solution as well as to release RNA onto Oligo(dT) magnetic beads.

[0230] Figure 10-11 This demonstrates the compatibility of the ExiST workflow with fluorescence super-resolution imaging-guided microdissection. Based on this imaging-guided microdissection method, the hippocampus of a brain sample was microdissected and the ExiST workflow was executed. Figure 11-13Read statistics, genome alignment distribution analysis, and expression levels showed no significant difference between SDS and trypsin homogenization conditions. However, Figure 14 Correlation analysis and PCA analysis showed that the repeatability of the SDS group was lower than that of the trypsin digestion group. Figure 15 Genome analysis showed that both groups exhibited 3' end bias, which was attributed to the poly(dT)mRNA capture method.

[0231] Example 2: Spatial transcriptomic analysis of mouse brain and liver tissues (neoExiST)

[0232] In this embodiment, the experimental effect of simultaneously performing RNA probe hybridization and in situ reverse transcription was tested. In short, the same experimental procedure as in Example 1 was performed, except that steps 1.4' and 1.9' were used instead of the original steps 1.4 and 1.9, respectively.

[0233] 1.4' Alternative steps to step 1.4

[0234] Before reverse transcription, the sample was washed once with 5×SSC buffer. After aspirating the supernatant, a reverse transcription reaction mixture containing RNA anchoring probes, dNTPs, 5' oligo primers, first-strand buffer, DTT, RNase inhibitors, and reverse transcriptase (according to the Single Cell Full Length mRNA-Amplification Kit, Vazyme, catalog number N712-01) was added to the reaction chamber. The mixture was then incubated at 72°C for 3 minutes, followed by 2 minutes on ice, then at 42°C for 90 minutes, 70°C for 15 minutes, and finally at 4°C. The single-stranded cDNA formed an RNA-DNA duplex with the template mRNA, and subsequently, during the gelation reaction, the cDNA was incorporated into the gel via the Acrydite group. The same RNA anchoring probe as in Example 1 was used: T+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT, modified with 5' Acrydite.

[0235] Accordingly, perform the following cDNA amplification step 1.9' instead of the original RT-PCR step 1.9.

[0236] 1.9' Alternative steps to step 1.9

[0237] Microcut samples were incubated in buffer at 40-45°C for 30-180 minutes, followed by mRNA concentration using commercially available dT magnetic beads or an RNA purification kit.

[0238] Perform in-gel cDNA amplification on the microdissected samples. Add 3.5 µl of 0.1×SSC buffer to cover the gel particles. Then add the amplification mixture and primers to the system for cDNA amplification. The number of cycles is set according to the size of the dissected sample, ranging from 8 to 18 cycles.

[0239] result: Figure 18-19 NeoExiST is demonstrated based on in-situ RT reactions on the sample prior to gel embedding. Figure 19 As shown, the number of genes detected validates the workflow's ability to perform spatial analysis with less mRNA degradation. Expression levels and PCA analysis compared to the original ExiST demonstrate the higher stability of neoExiST. In summary, neoExiST utilizes in situ reverse transcription (RT) in its initial step, effectively preserving mRNA quality by preventing degradation through the formation of a stable RNA:DNA complex. This approach not only improves the integrity of RNA samples but also allows for more comprehensive data collection when combined with proteomics. Compared to the ExiST method, neoExiST offers greater stability and a richer dataset of transcript information, enabling more reliable analysis and interpretation of gene expression. This technological advantage highlights the application potential of neoExiST in the in-depth analysis of cellular physiological processes.

[0240] Example 3: Spatial transcriptomics and proteomics analysis of mouse brain and liver tissues (ExiSTP and neoExiSTP)

[0241] In this embodiment, the experimental effect of combining spatial transcriptomics with downstream proteomics analysis was tested. Step 1.4 or 1.4' of the experimental protocol of Example 1 or Example 2 was modified so that the sample was co-incubated with both the RNA anchoring probe and the protein anchoring agent, and the peptides extracted from the gel were used for proteomics analysis.

[0242] 1.1 Organizational Preparation

[0243] Mice were euthanized using 1% sodium pentobarbital. Brain tissue (or other target organs) was rapidly dissected. The tissue blocks were rapidly frozen in liquid nitrogen vapor until solidified, and then stored at -80°C for later use.

[0244] 1.2 Frozen Sections

[0245] The tissue was then frozen and sectioned to a thickness of 16µm using a Leica cryostat and carefully placed on custom-made slides.

[0246] 1.3 Ethanol fixation

[0247] First, fix the sections with 75% ethanol, 100% methanol, or 100% acetone for 3 minutes.

[0248] 1.4 Hybridization and Incubation

[0249] The sample was first incubated with 1 µM RNA anchoring probe and protein anchoring agent (0.2 mg / ml NSA) at 37 °C for 40 hours. The hybridization buffer contained 1 µM anchoring probe, 2×SSC, 30% [v / v] formamide, 1% [v / v] RNase inhibitor, 10% [w / v] dextran sulfate, and 0.1% [w / v] yeast transfer RNA.

[0250] Combined with homogenization step 1.6, four different conditions were tested: ns: NSA was present in the hybridization buffer, followed by homogenization with SDS; nt: NSA was present in the hybridization buffer, followed by homogenization with trypsin; hns: RNA probe hybridization was performed first, followed by NSA incubation to achieve protein anchoring, and then homogenization with SDS; hnt: RNA probe hybridization was performed first, followed by NSA incubation to achieve protein anchoring, and then homogenization was performed by overnight trypsin digestion at 37°C.

[0251] RNA anchoring probe sequence: T+TT+TT+TT+TT+TT+TT+TT (SEQ ID NO:1), with 5' Acrydite modification.

[0252] 1.4' Alternative to step 1.4

[0253] Before reverse transcription, wash the sample once in 5×SSC buffer. After aspirating the supernatant, add the reverse transcription reaction mixture to the reaction chamber. This mixture contains RNA anchoring probes, dNTPs, 5' oligo primers, first-strand buffer, DTT, RNA inhibitor, and reverse transcriptase (according to the Single Cell Full Length mRNA-Amplification Kit, Vazyme, catalog number N712-01). Incubate the reaction mixture at 72°C for 3 minutes, then on ice for 2 minutes, followed by 42°C for 90 minutes, 70°C for 15 minutes, and then maintain at 4°C.

[0254] RNA anchoring probe sequence: T+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT, with Acrydite modification at the 5' end.

[0255] The product was washed three times with 5×SSC buffer for 5 minutes each time, and then incubated at 22-25°C in protein anchoring buffer (0.2 mg / ml NSA diluted with 5×SSC buffer) for 3 hours.

[0256] 1.5 Monomer incubation and gelation reaction

[0257] The sample was incubated in a monomer solution at 4°C for 2 hours. The monomer solution contained 1×PBS, 2M NaCl, 8.625% [w / w] sodium acrylate or sodium methacrylate, 2.5% [w / w] acrylamide, and 0.15% [w / w] N,N'-methylenebisacrylamide. 0.3% APS solution and 0.2% TEMED solution were added to initiate the gelation reaction. The gelation step was carried out at 37°C for 2 hours.

[0258] 1.6 Homogenization

[0259] Apply SDS (1% SDS, 50mM NaCl, 5mM EDTA) homogenization buffer to the gel and treat at 25-95°C for 12 hours, or apply trypsin buffer (6.25µg / mL trypsin in 5×SSC buffer) to the gel and treat overnight at 22-37°C.

[0260] 1.7 Staining and swelling

[0261] After homogenization, the gel was washed three times with 5×SSC buffer for 10 minutes each time. The gel was then stained with DAPI and Sypro dyes for 30 minutes to 3 hours. After staining, the gel was swollen 3-5 times with 0.1×SSC buffer for 15 minutes each time.

[0262] 1.8 Imaging and Microdissection

[0263] The expanded sample was imaged using a stereomicroscope or a fluorescence microscope. Figure 2 Images of brain tissue before and after expansion using different homogenization conditions are presented. Target regions were identified based on super-resolution imaging or morphological features, and microdissection was performed using punches of specific sizes (0.35 mm to 3 mm) (a 1.5 mm punch was used for 16 µm sections from the cortex with LEF=3.0).

[0264] 1.9 In-gel reverse transcription-polymerase chain reaction

[0265] First, after expansion, the entire sample was subjected to mRNA release and analysis to verify mRNA concentration and integrity. RT-PCR and NGS library preparation were performed using commercially available reagents and kits according to standard experimental protocols.

[0266] Microsurgical reverse transcription-polymerase chain reaction (RT-PCR) was performed on the microcuttered samples. 3–6 µl of 0.1×SSC buffer was added to cover the gel particles. RNA release was then performed at 40–50 °C for 30 min. Oligo dT primers were then added to the cut samples, and the reaction was carried out at 72 °C for 3 min. The samples were immediately placed on ice after the reaction. 5' oligo primers and reverse transcriptase were then added to the reaction mixture. Reverse transcription was performed at 40–45 °C for 30–180 min, followed by 70 °C for 15 min. The reaction products were maintained at 4 °C. The amplification mixture and primers were then added to the mixture for cDNA amplification. The number of cycles ranged from 8 to 18 cycles, depending on the size of the cut samples.

[0267] 1.9' Alternative to step 1.9

[0268] Microcut samples were incubated in buffer at 40-45°C for 30-180 minutes, followed by mRNA concentration using commercially available dT magnetic beads or an RNA purification kit.

[0269] If the alternative to step 1.9 was performed, the original RT-PCR steps mentioned in section 1.9 should be replaced with the following steps: Perform in-gel cDNA amplification on the microdissected sample. Add 3.5 µl of 0.1×SSC buffer to cover the gel particles. Then add the amplification mixture and primers to the system for cDNA amplification. The number of cycles is set according to the size of the dissected sample, ranging from 8 to 18 cycles.

[0270] 1.10 cDNA product purification and detection

[0271] Transfer the product supernatant from section 1.9 or 1.9' to a new PCR tube to separate the gel and solution. Purify the solution with DNA magnetic beads and incubate for 10 minutes to bind DNA. Place the sample on a magnetic rack to separate the beads and liquid. After approximately 5 minutes, discard the supernatant. Wash the beads with freshly prepared 80% ethanol for 30 seconds, repeating once. Open the tube cap and allow the beads to dry to remove ethanol. Remove the centrifuge tube from the magnetic rack, add approximately 17 µl of elution buffer, and incubate the beads for 2 minutes. Place the centrifuge tube on a magnetic rack to separate the liquid from the beads. Gently aspirate 15 µl of the supernatant and store at -20°C.

[0272] The purified cDNA product was detected using a bioanalyzer.

[0273] 1.11 Library Preparation

[0274] First, cDNA fragmentation was performed using transposase at 55°C for 10 minutes. The reaction was then terminated. The fragments were amplified using an index sequence. The number of cycles was set according to the size of the cut sample, ranging from 5 to 15 cycles. The product was purified using DNA magnetic beads or a DNA purification column. The quality of the DNA library was assessed using a bioanalyzer or Qubit. The high-quality library was then sent to a commercial sequencing facility for next-generation sequencing.

[0275] 1.12 Protein Sample Preparation

[0276] The gel from Section 1.8 was incubated in a secondary embedding monomer solution containing 0.09% [w / w] acrylamide, 0.003% [w / w] N,N'-methylenebisacrylamide, 0.00129% APS solution, 0.00129% TEMED solution, and ddH2O. After secondary embedding, the gel was transferred to a lobind centrifuge tube. 100 µl of 10 mM DL-dithiothreitol diluted in 100 mM ammonium bicarbonate solution was added, and the gel was incubated for 30 minutes. The supernatant was then discarded, and 100 µl of 55 mM iodoacetamide diluted in 100 mM ammonium bicarbonate solution was added, and the gel was incubated for 30 minutes in the dark. The gel was washed with 100 µl of 100 mM ammonium bicarbonate solution for 5 minutes, and this was repeated once. The supernatant was discarded, and the gel was washed with 100 µl of acetonitrile for 10 minutes, and this was repeated once. Digest the gel with 5 ng / µl trypsin dissolved in 100 mM ammonium bicarbonate solution for 2 hours on ice. Afterward, add 8 µl of 100 mM ammonium bicarbonate solution to the tube. Invert the tube and incubate overnight at 37°C.

[0277] Collect and combine the digested peptide solutions in the following steps: 1) Collect 30-40 µL of supernatant; 2) Add 100 µL of 100 mM ABB, shake at 37 °C for 20 minutes, and collect the supernatant; 3) Add 100 µL of 10% ACN solution, shake at 37 °C for 20 minutes, and collect the supernatant; 4) Add 100 µL of 50% ACN and 5% formic acid solution, shake at 37 °C for 20 minutes, and collect the supernatant; 5) Add 100 µL of 70% ACN and 5% formic acid solution, shake at 37 °C for 20 minutes, and collect the supernatant; 6) Add 100% ACN and shake at 37 °C, collecting the supernatant until the gel block turns white and sticky. Place the peptide sample under vacuum to reduce the volume to 20-30 µL. The peptides were then desalted using a C18 column (Pierce™ C18 Spin Tips, Thermo Fisher Scientific, US) and dried in a SpeedVac vacuum centrifuge. The purified peptide samples were then analyzed using LC-MS / MS.

[0278] result: For ExiSTP, the integration of ExiST (using steps 1.4 and 1.9) with the proteomics workflow is as follows: Figure 20 As shown. Combining the incubation method of the protein anchoring agent with the sample and the homogenization reaction method, we tested four different conditions to evaluate which condition was optimal based on the extracted RNA results. ns: NSA was present in the hybridization buffer, followed by homogenization with SDS; nt: NSA was present in the hybridization buffer, followed by homogenization with trypsin; hns: RNA probe hybridization was performed first, followed by NSA incubation to achieve protein anchoring, and then homogenization with SDS; hnt: RNA probe hybridization was performed first, followed by NSA incubation to achieve protein anchoring, and then homogenization with trypsin.

[0279] Figure 21 The results shown in a indicate that under nt conditions (simultaneous incubation of the sample with RNA probes and protein anchoring agents (NSA), followed by homogenization with trypsin), there is a more uniform fragment distribution between 500 bp and 5000 bp, demonstrating that nt conditions are the optimal of the four conditions.

[0280] For neoExiSTP, it combines neoExiST (using steps 1.4' and 1.9') with a proteomics workflow, and the experimental protocol is as follows: Figure 22 As shown. Multi-omics analysis was performed using the neoExiSTP workflow, and the overall detection results for three duplicate brain tissue samples are as follows. Figure 23 As shown. For transcriptomics, approximately 7,500–10,000 genes with expression counts greater than 100 were identified per sample. For proteomics, approximately 27,000 peptides and approximately 3,600 proteins were identified per sample.

[0281] Figure 24-29 Downstream analyses revealed differences in transcriptomic and proteomic expression levels within the same sample. Differential protein expression analysis between proteomics and transcriptomics is shown below. Figure 24 As shown, downstream enrichment analysis revealed that some pathways, including synaptic structural organization, exhibited differential expression at both transcriptomic and proteomic levels. Figure 25 Significant enrichment pathways in differentially expressed proteins were shown, including gene number and p-value. Figure 26 The significant groups of each sample based on expression level enrichment analysis are presented in the form of a heatmap. Figure 27 The significant enrichment analysis pathways based on fold changes corresponding to each molecule are presented in the form of heatmaps. Figure 28 A tree structure of different enrichment pathways based on gene number and corrected p-value is presented. Figure 29The relationship between different enrichment analysis pathways and the number of genes in each group was shown.

[0282] Example 4: Spatial Transcriptomics Analysis Using PolydT Probes of Different Lengths

[0283] In this embodiment, polydT probes of different lengths were tested. In short, in step 1.4, RNA anchoring probes of lengths of 15 nt (as used in Example 1, consisting of alternating 15 nucleotide sequences of dT and thymidine (dT+) with an Acrydite modification at the 5' end), 25 nt (as shown in SEQ ID NO:2, consisting of alternating 25 nucleotide sequences of dT and thymidine (dT+) with an Acrydite modification at the 5' end), or 35 nt (as shown in SEQ ID NO:2, consisting of alternating 35 nucleotide sequences of dT and thymidine (dT+) with an Acrydite modification at the 5' end) were used, and the resulting fluorescence intensities were compared.

[0284] like Figure 16 As shown, the anchoring probe labeled with the FAM group reflects the anchoring fluorescence intensity, which was measured using confocal microscopy. Intensity comparisons indicate that the 25 dT probe is slightly superior to probes of other lengths.

[0285] Example 5: Spatial transcriptomics analysis using different gel compositions

[0286] In this embodiment, to optimize gel stability and expansion coefficient, we further explored four different gel components and performed mechanical stability measurements. Group 1 (Bis group (SA:AA:Bis=8.625%:2.5%:0.15%)); Group 2 (HCl group): 8.625% SMA, 30% AA, 0.8% PAE and 10% HCl; Group 3 (NaCl group): 8.625% SMA, 30% AA, 0.8% PAE and 0.8M NaCl; Group 4 (SMA group): 8.625% SMA, 3.8% AA, 0.2% Bis and 0.8M HCl.

[0287] From a mechanical properties perspective, all gels exhibited acceptable stability and swelling, although the HCl and NaCl groups showed superior stability. Transcriptomic results from different gel components indicated that the HCl group was more compatible with the experimental protocol.

[0288] Mechanical stability was measured using a uniaxial tensile tester (Univert, CELLSCALE) equipped with a 2.5 N force sensor at a deformation rate of 53.33% height / min. Tests were conducted on cylindrical samples with dimensions of 1 mm (radius) and 1–5 mm (height) for four different hydrogels. The height of the sample between the two clamps was determined based on actual testing.

[0289] Figure 17 a shows representative stress-strain curves (n=3, 3, 3 and 3 technical replicates) of expanded Bis (LEF=2.8), SA (LEF=N / A), NaCl (LEF=5.33) and HCl (LEF=5.6) hydrogels. Figure 17 The stability curves in group a show that the HCl and NaCl groups have better stability. Figure 17 b shows the average parameters for each group (3 replicates), including LEF (linear expansion factor), stress (assessed by force divided by the initial cross-sectional area of ​​the hydrogel sample), and strain (assessed by dislocation in height). Figure 17 In c, transcriptomic results from different gel components showed that the HCl group was more compatible with the experimental protocol. Figure 17 c, the gel component, such as Figure 17 As shown in d, ".ST / .V" represents different brands of reagent kits used.

[0290] In summary, the ExiST and neoExiST workflows enable the identification of spatial transcriptomics in micro-regions at scalable, user-defined resolution using standard NGS experimental protocols and procedures, without the need for special equipment or consumables. RNA-seq results validated the high quality and coverage of the transcriptomics data, and showed no human interference or bias compared to benchmark standards.

[0291] ExiSTP and neoExiSTP incorporate several technological advancements and solutions not achieved by any other existing spatial transcriptomics approach. First, we demonstrate the ability to combine reagents targeting two biomolecules for reversible anchoring of mRNA and protein in a single step. Second, we discover experimental conditions that enable sample homogenization without compromising mRNA integrity and localization in swollen samples, followed by microdissection of target regions. Third, we demonstrate and implement the release of mRNA from tiny microdissected gel blocks in an RT-PCR enzyme mixture, directly generating cDNA. Thus, we demonstrate that spatially resolved transcriptomics analysis can be performed without the need for RNA-binding DNA-based chips or spatial barcodes. We also enable mRNA release from protein-preserving samples, facilitating downstream proteomics analysis. The microdissection step for spatial sampling allows for flexible selection of the microdissection size to set the spatial resolution of the analysis. No other method allows for scalable lateral resolution; they all operate only at a pre-defined and fixed lateral resolution. Overall, ExiST is a technically novel and unique approach for spatially resolved transcriptomics analysis that requires no specialized or complex equipment and is compatible with any conventional DNA sequencing platform, including all NGS methods.

[0292] These specific compositions, materials, and methods are not intended to limit the invention, but are merely illustrative of specific embodiments falling within the scope of this invention. Those skilled in the art can develop equivalent compositions, materials, and methods without inventive effort and without departing from the scope of this invention. It should be understood that various modifications can be made to the experimental procedures described herein, while still remaining within the scope of this invention, and all such modifications are covered within the scope of this invention.

Claims

1. A method for physically expanding a biological sample and spatially analyzing biomolecules within the sample, the method comprising: (a) The sample is incubated with an mRNA probe, wherein the mRNA probe is capable of hybridizing with mRNA in the sample and has been modified to contain hydrogel-reactive chemical groups; (b) The sample is perfused with a hydrogel precursor solution or the sample is embedded in a hydrogel precursor solution and polymerized to form a sample-hydrogel complex; (c) Physically expand the sample-hydrogel composite; (d) Select a target region from the expanded sample-hydrogel composite and cut the region; and (e) Sequencing of mRNA from the cut region.

2. A method for physically expanding a biological sample and spatially analyzing biomolecules within the sample, the method comprising: (a) The sample is incubated with an mRNA probe and a reverse transcriptase, wherein the mRNA probe is capable of hybridizing with the mRNA in the sample and has been modified to contain hydrogel-reactive chemical groups, and wherein the reverse transcriptase uses the mRNA probe as a primer to reverse transcribe the mRNA in the sample into cDNA. (b) The sample is perfused with a hydrogel precursor solution or the sample is embedded in a hydrogel precursor solution and polymerized to form a sample-hydrogel complex; (c) Physically expand the sample-hydrogel composite; (d) Select a target region from the expanded sample-hydrogel composite and cut the region; and (e) Sequencing of cDNA from the cut region.

3. The method of claim 1 or 2, wherein before or during step (b), the sample is further incubated with a bifunctional protein anchoring agent comprising protein-reactive chemical groups and hydrogel-reactive chemical groups. For example, during step (a), the sample is incubated in a hybridization buffer together with both the mRNA probe and the bifunctional protein anchoring agent; or the sample is incubated first with the mRNA probe and then with the bifunctional protein anchoring agent.

4. The method of claim 3, wherein the method further comprises extracting peptides or proteins from the cleaved region for proteomics identification, for example by mass spectrometry analysis, such as LC-MS / MS and HPLC-MS.

5. The method of any preceding claim, wherein the mRNA probe comprises a sequence of 10-40 nucleotides, said sequence comprising dT nucleotides, analogues and / or derivatives of dT nucleotides, such as thymidine lock nucleic acid (dT+). For example, the mRNA probe contains a 15-35 nt nucleotide sequence consisting of alternating dT and dT+.

6. The method of claim 5, wherein the mRNA probe is modified with a hydrogel reactive chemical group selected from Acrydite, primary amino groups, azides, Uni-Link™ amino modifiers, and any combination or mixture thereof. For example, the 5' end of the mRNA probe is modified with Acrydite.

7. The method of any of the preceding claims, wherein the method further comprises homogenizing the sample-hydrogel composite between step (b) and step (c).

8. The method of claim 7, wherein the homogenization is performed by physical, chemical, physicochemical, and / or enzymatic treatment of the sample-hydrogel composite. For example, the homogenization is performed by treating the sample-hydrogel complex with a protease, an alkaline buffer (e.g., an alkaline buffer containing detergent), or by heating in a buffer solution.

9. The method of claim 7 or 8, wherein the homogenization is performed by treating the sample-hydrogel complex with trypsin, SDS, or proteinase K.

10. The method of any one of claims 3-9, wherein the bifunctional protein anchoring agent is selected from N-succinimide acrylate (NSA), N-(allyloxycarbonyloxy)-succinimide (NAS), and any combination or mixture thereof.

11. The method of claim 3 or 4, wherein the method comprises: (1) In step (a), the sample is incubated in hybridization buffer with both the mRNA probe and the protein anchoring agent, and the sample-hydrogel complex is treated with trypsin between steps (b) and (c) to homogenize it; (2) In step (a), the sample is incubated in hybridization buffer with both the mRNA probe and the protein anchoring agent, and the sample-hydrogel complex is treated with SDS between steps (b) and (c) to homogenize it; (3) First, incubate the sample with the mRNA probe, then incubate with the protein anchoring agent, and between steps (b) and (c), treat the sample-hydrogel complex with trypsin to homogenize it; or (4) First, incubate the sample with the mRNA probe, then incubate it with the protein anchoring agent, and treat the sample-hydrogel complex with SDS between steps (b) and (c) to homogenize it.

12. The method of any preceding claim, wherein the hydrogel precursor solution comprises one or more precursors selected from: sodium acrylate (SA), sodium methacrylate (SMA), itaconic acid (IA), trans-aconitic acid (TAA), ethyl-2-(hydroxymethyl)-acrylate (EHA), N,N'-methylenebisacrylamide (Bis-AA), N,N-dimethylacrylamide (DMAA), pentaerythritol tetraacrylate (PT), propoxylated trimethylolpropane triacrylate (TPT), pentaerythritol triacrylate (PA), dimethylolpropane tetraacrylate, etc. Pentylenetetroxide pentaacrylate or dipentaerythritol hexaacrylate (DPHA), trimethylolpropane triacrylate (TTA), di(trimethylolpropane)tetraacrylate (DiTA), trimethylolpropane trimethacrylate (TTMA), glycerol propoxylated (1PO / OH) triacrylate (GPT), ethoxylated trimethylolpropane triacrylate (TET), pentaerythritol allyl ether (PAE), sodium 4-hydroxy-2-methylenebutyrate (SHMB), N,N-dimethylaminopropylacrylamide (DMPAA), and acrylamide (AA).

13. The method of claim 12, wherein the hydrogel precursor solution comprises any one of the following precursors: (a) SMA, AA and PAE; (b) SMA, DMAA, and TPT; (c) SMA, AA and Bis-AA; (d)SA, AA, and Bis-AA; (e) SMA, SA, AA, and Bis-AA; and (f) DMAA, SMA and PAE.

14. The method of any of the preceding claims, wherein a polymerization activator or accelerator, such as VA-044, V50, APS, potassium persulfate, or TEMED, is added to the hydrogel precursor solution immediately prior to the start of polymerization.

15. The method of any of the preceding claims further comprises staining the sample with a marker or label before, during, or after steps (a), (b), and (c), or after homogenization.

16. The method of claim 15, wherein the sample is stained with DNA, RNA, and / or protein.

17. The method of any of the preceding claims, wherein during step (c), the sample-hydrogel complex is incubated with a solvent or aqueous solution (e.g., pure water or an aqueous buffer) for a sufficient period of time to allow for expansion.

18. The method of any of the preceding claims, wherein the sample is selected from cells (e.g., cultured cells), biological tissues (e.g., tissue sections), specimens, biopsies, complete organs and parts thereof, and whole organisms (e.g., bacteria, fungi, or viruses).

19. The method of claim 18, wherein the sample is a preserved tissue section with a thickness of 50 µm or less, or a tissue sample with a thickness in the range of 30 µm to 400 µm.

20. The method of any of the preceding claims, further comprising imaging the expanded sample-hydrogel composite with a microscope to obtain a super-resolution image.

21. The method of any of the preceding claims, wherein the cutting of the region is performed manually by a punch or with the assistance of a robot or mechanical micromanipulation system (e.g., by LCM cutting).

22. The method of claim 21, wherein the diameter of the target region is 1-1000 μm.

23. The method of claim 1, wherein step (e) comprises reverse transcription of the mRNA released from the cleaved region, cDNA amplification (e.g., by RT-PCR), and cDNA library construction. Optionally, the mRNA can be released from the cleaved region by incubating the cleaved gel in SSC buffer at 40-50°C.

24. The method of claim 2, wherein step (e) comprises cDNA amplification (e.g., by PCR) of the cDNA in the cut region and cDNA library construction.

25. A sample-hydrogel composite prepared by the method as described in any one of the preceding claims.

26. A kit, wherein the kit comprises in one or more containers: (a) mRNA probes capable of hybridizing with mRNA and crosslinking with hydrogel polymer chains; (b) A hydrogel precursor or a hydrogel precursor stock solution containing the hydrogel precursor; (c) Optionally, a bifunctional protein anchor, such as NSA, NAS or a combination thereof.

27. The kit of claim 26, wherein the mRNA probe comprises a sequence of 10-40 nucleotides, said sequence comprising dT nucleotides, analogues and / or derivatives of dT nucleotides, such as thymidine lock nucleic acid (dT+). For example, the mRNA probe contains a 15-35 nt nucleotide sequence consisting of alternating dT and dT+.

28. The kit of claim 27, wherein the mRNA probe is modified with a hydrogel-reactive chemical group selected from Acrydite, primary amino groups, azides, Uni-Link™ amino modifiers, and any combination or mixture thereof. For example, the 5' end of the mRNA probe is modified with Acrydite.

29. The kit according to any one of claims 26-28, wherein the hydrogel precursor is selected from any one of the following: (a) SMA, AA and PAE; (b) SMA, DMAA, and TPT; (c) SMA, AA and Bis-AA; (d)SA, AA, and Bis-AA; (e) SMA, SA, AA, and Bis-AA; and (f) DMAA, SMA and PAE.

30. The kit according to any one of claims 26-29, wherein the kit further comprises one or more of the following components: A container containing a gelling activator or accelerator, said gelling activator or accelerator being selected from VA-044, TEMED, APS and potassium persulfate; Containers containing hybridization buffers (such as SSC buffer); A container containing a homogenization buffer, wherein the homogenization buffer optionally contains trypsin or SDS; Containers containing washing buffers (such as SSC buffer or PBS buffer); Containers containing markers; Containers containing fixatives (such as ethanol); A container containing reagents for mRNA reverse transcription; A container containing reagents for cDNA PCR; A container containing reagents for cDNA library preparation; and A container containing reagents for peptide digestion used in mass spectrometry identification.

31. The kit according to any one of claims 26-30, wherein the kit further comprises one or more of the following components: A gelling chamber configured to contain the sample prior to the gelling reaction, and optionally a cover for sealing the gelling chamber; The imaging chamber is configured to hold the sample after the gelation reaction. Gel handling device; and Cooling devices, such as ice packs.

32. A system for spatial transcriptomics analysis of biological samples, comprising: A hybridization module for anchoring an mRNA probe to mRNA in the biological sample, optionally reverse transcribing the mRNA into cDNA; A gelation module for hydrogel polymerization to form sample-hydrogel complexes; A homogenization module is used to homogenize the sample-hydrogel composite. A staining module for staining DNA, RNA, and / or proteins; DNA amplification module, which is used to perform PCR amplification of reverse transcribed cDNA from the target region in the sample-hydrogel complex; and cDNA library preparation module, which is used to construct a library of the cDNA for sequencing; as well as Optionally, a mass spectrometry analysis module is provided for preprocessing the sample obtained by local sampling prior to mass spectrometry, and the module is connected to the mass spectrometry detection device.

Images