WO2025067452A1

WO2025067452A1 - Methods for deep untargeted profiling of spatial transcriptome and proteome in intact tissues

Info

Publication number: WO2025067452A1
Application number: PCT/CN2024/121821
Authority: WO
Inventors: Kiryl PIATKEVICH; Shuchang ZHAO
Original assignee: Westlake Laboratory Of Life Sciences And Biomedicine
Current assignee: Westlake Laboratory Of Life Sciences And Biomedicine
Priority date: 2023-09-27
Filing date: 2024-09-27
Publication date: 2025-04-03
Anticipated expiration: 2026-03-27

Abstract

Provided is a method for physical magnification of a sample and spatially analyzing mRNAs and optionally, the proteins, in the sample. Also provided is a related kit. The methods herein are compatible with different samples, sample staining methods and sequencing technologies, and suitable for spatial transcriptomics and proteomics profiling of biological samples.

Description

Methods for Deep Untargeted Profiling of Spatial Transcriptome and Proteome in Intact Tissues

CROSS-REFERENCING

This application claims the benefit of International application PCT/CN2023/121991, filed on September 27, 2023, which is incorporated by reference in its entirety.

FIELD

The present disclosure relates to spatial omics and, in particular to, preparation of biological materials for spatially resolved high-throughput next-generation sequencing and mass-spectrometry, methods for producing expanded biological materials and uses thereof, and compositions for use in the enlargement of the biological materials to be combined with transcriptomic sequencing and mass-spectrometry based proteomics.

BACKGROUND

The spatial distribution of transcripts in a native tissue environment is crucial for comprehending cellular organization and cell states on the organism level. In recent years, numerous methods have been developed for spatial profiling of gene expression in situ using untargeted mRNA sequencing methods. One of the most popular approaches in spatial transcriptomics is based on an ordered array of oligonucleotides deposited on a glass slide by using microarray printing technologies. However, these methods have several limitations, such as high cost, limited spatial resolution, and incomplete RNA release. For example, beads/nanoballs with spatial barcodes affixed to a surface are used in methods such as high-definition spatial transcriptomics (HDST) , Slide-seq, and Stereo-seq. Prior to the experiment, the location of each DNA-based barcode must be predetermined, which takes time and needs sophisticated fluorescence imaging equipment or in situ sequencing setup.

On another hand, Expansion sequencing (ExSeq) is a kind of in situ sequencing that combines both tissue expansion and fluorescence imaging. It is arduous, time-consuming, and extremely difficult to implement for large-scale sequencing. With a large number of targets, optical crowding can be an issue, as it is with any imaging-based spatial transcriptomics (ST) approach. Although sample expansion can help to separate molecules, longer imaging sessions are necessary to examine bigger regions. Finally, when employing untargeted in situ sequencing techniques, ribosomal RNA can account for many sequenced RNA molecules since it is plentiful. Significant drawbacks of existing ST methods create a great need in novel approaches to spatial sequencing that can improve accessibility, throughput, and sequencing depth.

There is a need for a novel method that can enable super-resolution imaging of various sample types including large samples, and spatially analyzing biomolecules of interest, e.g. mRNAs, in the samples.

SUMMARY

The present disclosure, inter alia, provides a method capable of physical magnification of biological samples while spatially analyzing mRNAs or/and proteins within the same samples in an expanded state. The method is referred to herein as “ExiST” “ExSTP” . The method enables imaging of sub-diffraction limited structure in biological specimens under conventional microscopes via reversible and tunable physical magnification of biological specimens. The method herein can expand the sample by 2 to up to 8-10 times in linear dimensions and can be applied to a wide variety of biological samples without deformation or breakdown of the samples.

The method provided herein is easy to implement in any lab and easily accessible. It is compatible with a large selection of sample preparation ranging from tissue culture, to large tissue block and whole organs. Chemical reagents used in the protocol are chemically stable, cheap and commercially available, their application does not require chemistry background.

In addition, the method herein is compatible with all standard methods for sample staining including immunohistochemistry, fluorescent proteins, and chemical dyes and high-throughout sequencing, such as next generation sequencing.

In one aspect, provided herein is a method for physically expanding a biological sample and spatially analyzing biomolecules, such as mRNAs and proteins, within the sample.

In some embodiments, the method comprises:

(a) incubating the sample with a mRNA probe, wherein the mRNA probe can hybrid to mRNAs in the sample and has been modified to comprise a hydrogel-reactive chemical group;

(b) perfusing the sample with a hydrogel precursor solution or embedding the sample in the hydrogel precursor solution, and polymerize to form a sample-hydrogel composite;

(c) physically expanding the sample-hydrogel composite;

(d) selecting a region (s) of interest from the expanded sample-hydrogel composite and dissecting the region (s) ; and

(e) sequencing the mRNAs from the dissected region (s) .

In some alternative embodiments, the method comprises:

(a) incubating the sample with a mRNA probe and a reverse transcriptase (e.g. in a reverse transcription mix) , wherein the mRNA probe can hybrid to mRNAs in the sample and has been modified to comprise a hydrogel-reactive chemical group, and wherein the mRNAs in the sample are reverse transcribed to cDNAs by the reverse transcriptase, with the mRNA probe acting as a primer;

(c) physically expanding the sample-hydrogel composite;

(e) sequencing the cDNAs from the dissected region (s) .

In some embodiments, prior to or during step (b) , the sample is further incubated with a bifunctional protein anchor, the bifunctional protein anchor comprises a protein-reactive chemical group and a hydrogel-reactive chemical group. In some embodiments, the bifunctional protein anchor is selected from N-Succinimidyl Acrylate (NSA) , N- (Allyloxycarbonyloxy) -succinimide (NAS) , and any combination/mixture thereof. Correspondingly, the method may further comprise extracting peptides or proteins from the dissected region after step (d) or after cDNA amplification for proteomic identification, e.g. by mass spectrometry analysis, such as LC-MS/MS and HPLC-MS.

In some embodiments, during step (a) , the sample is incubated with both the mRNA probe and the bifunctional protein anchor in a hybridization buffer. In some other embodiments, during step (a) , the sample is incubated with the mRNA probe in a hybridization buffer first and then with the bifunctional protein anchor. In some other embodiments, when the sample is incubated with a reverse transcription mix comprising the mRNA probe and a reverse transcriptase, the sample is incubated with the reverse transcription mix first and then with the bifunctional protein anchor.

In some embodiments, the mRNA probe comprises a 10-40 nucleotide sequence comprising dT nucleotide, an analogue and/or derivative of dT nucleotide such as thymidine-locked nucleic acid (dT+) , for example, the mRNA probe comprises a 15-35 nt nucleotide sequence consisting of alternating dT and dT+.

In some further embodiments, the mRNA probe is modified with a hydrogel-reactive chemical group selected from acrydite, a primary amino group, azide, a Uni-Link^TM amino modifier, and any combination/mixture thereof, for example, the mRNA probe is 5’ modified with acrydite.

In some embodiments, the method further comprises subjecting the sample-hydrogel composite to homogenization between step (b) and step (c) . The homogenization may be performed by a physical, chemical, physicochemical, and/or enzymatic treatment of the sample-hydrogel composite, for example, the homogenization is performed by treating the sample-hydrogel composite with a protease, an alkaline buffer (such as a detergent containing alkaline buffer) , or heating in a buffer.

In some specific embodiments, the homogenization is performed by treating the sample-hydrogel composite with Proteinase K, Trypsin or SDS-containing buffer.

In some embodiments, the method comprises:

(1) incubating the sample with both the mRNA probe and the protein anchor in a hybridization buffer in step (a) , and treating the sample sample-hydrogel composite with Trypsin for homogenization between step (b) and step (c) ;

(2) incubating the sample with both the mRNA probe and the protein anchor in a hybridization buffer in step (a) , and treating the sample sample-hydrogel composite with SDS for homogenization between step (b) and step (c) ;

(3) incubating the sample with the mRNA probe first and then with the protein anchor, and treating the sample sample-hydrogel composite with Trypsin for homogenization between step (b) and step (c) ; or

(4) incubating the sample with the mRNA probe first and then with the protein anchor, and treating the sample sample-hydrogel composite with SDS for homogenization between step (b) and step (c) .

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) , Trimethylolpropane Propoxylatetriacrylate (TPT) , Pentaerythritol triacrylate (PA) , Dipentaerythritol penta-/hexa-acrylate (DPHA) , Trimethylolpropane triacrylate (TTA) , Di (trimethylolpropane) -tetraacrylate (DiTA) , Trimethylolpropane Trimethacrylate (TTMA) , Glycerol propoxylate (1PO/OH) triacrylate (GPT) , Trimethylolpropane ethoxylate triacrylate (TET) , Pentaerythritol allyl ether (PAE) , Sodium 4-hydroxy-2-Methylenebutanoate (SHMB) , N, N’-Dimethylaminopropyl acrylamide (DMPAA) , and Acrylamide (AA) .

A variety of hydrogel precursors may be used in the methods as disclosed herein. In some embodiments, the hydrogel precursor solution comprises any of the following groups 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.

In some embodiments, a polymerization activator or accelerator, such as VA-044, V50 (an Azo Initiator) , ammonium persulfate (APS) , potassium persulfate or TEMED, is added to the hydrogel precursor solution immediately before starting polymerization.

In some embodiments, the method as disclosed herein further comprises staining the sample either with a label or tag before, during or after steps (a) , (b) , (c) , or after homogenization. The sample may be subjected to DNA staining, RNA staining and/or protein staining.

In some embodiments, during step (c) , the sample-hydrogel composite is incubated with a solvent or aqueous solution such as pure water or aqueous buffer for a sufficient amount of time for expansion.

In some embodiments, the sample is selected from cell (such as cultured cells) , biological tissue (such as tissue section) , specimen, biopsy, intact organ and parts thereof, and whole organisms (such as bacteria, fungus, or viruses) .

In some embodiments, 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 –400 μm.

In some embodiments, the method further comprises imaging the expanded sample-hydrogel composite via microscopy.

In some embodiments, dissecting the region in step (d) is performed manually via a punch or with assistance of robotic or mechanical micromanipulating system, e.g. with LCM dissection. For example, the region of interest has a diameter of 1-1000 μm.

In some embodiments, step (e) comprises subjecting the mRNAs released from the dissected region to reverse transcription, cDNA amplification (e.g. by RT-PCR) and cDNA library construction. Optionally, the mRNAs are released from the dissected region by incubating the dissected gel in SSC buffer at 40-50℃.

In some alternative embodiments, step (e) comprises subjecting the cDNAs in the dissected region to cDNA amplification (e.g. by PCR) and cDNA library construction.

In one aspect, provided herein is a sample-hydrogel composite produced by the method as disclosed herein.

In one aspect, provided herein is a kit, wherein the kit comprises, in one or more containers:

(a) a mRNA probe capable of hybridizing to mRNAs and crosslinking with the hydrogel polymer chain;

(b) hydrogel precursors or a hydrogel precursor stock solution comprising the hydrogel precursors;

(c) optionally, a bifunctional protein anchor such as NSA, NAS or a combination thereof;

in one or more containers.

In some embodiments, the mRNA probe is modified with a hydrogel-reactive chemical group selected from acrydite, a primary amino group, azide, a Uni-Link^TM amino modifier, and any combination/mixture thereof, for example, the mRNA probe is 5’ modified with acrydite.

In some embodiments, the hydrogel precursors are selected from any 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.

In some embodiments, the kit further comprises one or more of the following components:

a container comprising a gelation activator or accelerator selected from VA-044, TEMED, APS and potassium persulfate;

a container comprising a hybridization buffer such as an SSC buffer;

a container comprising a homogenization buffer, optionally the homogenization buffer comprises trypsin or SDS;

a container comprising a wash buffer such as an SSC buffer or PBS buffer;

a container comprising a labeling agent;

a container comprising a fixative such as ethanol;

a container comprising reagents for reverse-transcription of mRNAs;

a container comprising reagents for cDNA PCR;

a container comprising reagents for cDNA library preparation; and

a container comprising reagents for peptide digestion for mass spectrometry identification.

a gelation chamber configured to accommodate the sample prior to gelation and, optionally a cover to seal the gelation chamber;

an imaging chamber configured to accommodate the sample after gelation;

a gel handling device; and

a cooling device, such as an ice bag.

In one aspect, provided herein is a system for spatial transcriptomics analysis of a biological sample, comprising:

a hybridization module for anchoring an mRNA probe to the mRNAs in the biological sample, optionally the mRNAs are reverse transcribed to cDNAs;

a gelation module for hydrogel polymerization to form a sample-hydrogel composite;

a homogenization module for homogenization of the sample-hydrogel composite;

a staining module for staining DNAs, RNAs and/or proteins;

a DNA amplification module for PCR amplification of the reverse transcribed cDNAs from a region of interest of the sample-hydrogel composite; and

a cDNA library preparation module for constructing a library of the cDNAs for sequencing; and

optionally, mass spectrometry analysis module for pre-processing of the samples obtained by local sampling before mass spectrometry and which is connected with a mass spectrometry detection device.

The foregoing and other features and advantages of the disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

DESCRIPTION OF FIGURES

Figure 1 shows an exemplary workflow of the ExiST technology, depicting major steps and procedures, including mRNA and optional protein anchoring, gelation, sample homogenization, staining, imaging, and image analysis. The expanded sample is microdissected; excised gel pieces are used for mRNA recovery, cDNA amplification, and NGS library preparation. NGS library is sequenced using standard methods and analyzed based on experimental needs.

Figure 2 shows images of brain tissues before and after expansion using different homogenization conditions. (a) Brain images before and after expansion using trypsin or SDS containing homogenization buffer. Scale bars, 100 μm. (b) Brain tissue-hydrogel in tube for RNA release and purification.

Figure 3 shows the RNA release from expanded brain tissue-hydrogel composite. (a) Fragment bands of brain tissue-hydrogel released RNA under different homogenization conditions, in which A1 and B1 are two replicates of trypsin treated homogenization, C1 and D1 are two replicates of SDS treated homogenization; (b) Released RNA concentration of different homogenization conditions; and (c) brain tissue-hydrogel RNA released fragment distribution of different homogenization conditions.

Figure 4 shows the quality of the micro-dissected sample library. (a) Library length distribution of dissected sample under trypsin homogenization condition; (b) Library length distribution of dissected sample under SDS homogenization condition.

Figure 5 shows the sequencing quality of micro-dissected sample with trypsin digestion, basic statistics includes per base sequence quality, per tile sequence quality, per sequence quality scores, per base N content, and adapter content.

Figure 6 shows the sequencing quality of micro-dissected sample with SDS digestion, basic statistics includes per base sequence quality, per tile sequence quality, per sequence quality scores, per base sequence content, per base N content, and adapter content.

Figure 7 shows assessment of transcriptome sequencing results. (a) Sequencing raw data quality analysis and filtering of samples of trypsin digestion and SDS homogenization condition using FastQC; (b) Sequencing alignment reads of samples of trypsin digestion and SDS homogenization condition using HISAT2; (c) Alignment regions of genome analysis of two samples of trypsin digestion and SDS homogenization condition using HISAT2.

Figure 8 shows assessment of transcriptome expression results. (a) Gene body coverage analysis of two samples of trypsin digestion and SDS homogenization condition using HISAT2; (b) Transcripts expression analysis of two samples of trypsin digestion and SDS homogenization condition using HISAT2.

Figure 9 shows the detected RNA distribution and bands of mRNA release on BeyoMag^TMOligo (dT) 25 Magnetic Beads by Fragment Analysis. (a) Fragment distribution of liver tissue-hydrogel mRNA released from beads incubated with tissue-hydrogel. (b) Region table of analyzed mRNA. (c) Fragment bands of liver tissue-hydrogel RNA released from beads incubated with tissue-hydrogel.

Figure 10 shows the compatibility with fluorescence staining with small dyes. (a) fluorescence image of mouse brain slice stained with DAPI. (b) fluorescence image of mouse brain slice stained with Sypro red. LEF = ～3 in (a-b) , imaged with 4x objective.

Figure 11 shows the super-resolution fluorescence imaging and image-guided microdissection followed by transcriptomics of the selected Regions of Interest (ROIs) . (a) fluorescent images of mouse brain slice homogenized using trypsin digestion processing labeled with targeted regions in the mouse hippocampus. (b) fluorescent images of mouse brain slice homogenized using SDS-containing buffer homogenization labeled with targeted regions in the hippocampus. (c) fluorescence images of S3 region shown in b (right, before microdissection, white circle indicated ROI selected for microdissection; left, after microdissection) . (d) Unique mapped reads ratio of sequencing results of targeted regions shown in a and b (SDS group corresponds to panel b, and trypsin group corresponds to panel a) . LEF = ～3x, diameter of targeted region = 2 mm, imaged with 4x objective. (e) Alignment results of targeted regions shown in a and b including unmapped reads, unique mapped reads, multi-mapped reads, non-splice reads and splice reads.

Figure 12 shows genome alignment analysis of transcriptomic results for two conditions using trypsin digestion and SDS-containing buffer homogenization performed with the HISAT2 software. (a) genome alignment analysis of three replicates for SDS homogenization conditions shown in Figure 11b. (b) genome alignment analysis of three replicates for trypsin digestion homogenization condition shown in Figure 11a. (S1-S3: SDS group replicate 1-3, T1-T3: trypsin group replicate 1-3 from Figure 11)

Figure 13 shows transcripts expression level analysis (FPKM: fragments per kilobase of exon per million mapped fragments) of two groups for SDS homogenization and trypsin digestion conditions for samples shown in Figure 12. (a) bar plot of 6 samples expression level analysis under two homogenization conditions. (b) density distribution plot of 6 samples expression level analysis under two homogenization conditions. (S1-S3: SDS group replicate 1-3, T1-T3: trypsin group replicate 1-3) .

Figure 14 shows the correlation between samples and PCA analysis of two conditions of trypsin digestion and SDS homogenization condition. (a) person correlation between samples under two homogenization conditions. (b) PCA analysis between samples under two homogenization conditions.

Figure 15 shows the gene body analysis of samples under two homogenization conditions. (S1-S3: SDS group replicate 1-3, T1-T3: trypsin group replicate 1-3) .

Figure 16 shows representative images of fluorescent brain slices after incubation with a fluorescence group labeled probe. (a) fluorescent images of brain slices after incubation of different length of anchors. (b) The calculated fluorescent intensity of different lengths of anchors in the hippocampus region of the brain.

Figure 17 shows the mechanical properties and the compatibility of the different gel compositions with the ExiST workflow. (a) the stress curve of different gel compositions. (b) the calculated properties including average linear expansion factor, average strain, and average stress of different gel compositions. (c) the total identified gene counts numbers of different gel compositions. (d) the composition of different gels.

Figure 18 shows the workflow of in situ RT and gel embedding procedures (referred to as neoExiST) , including in situ RT-PCR with oligo dT 5’ modified with acrydite probe, gelation, sample homogenization, staining, imaging, and image analysis. The expanded sample is microdissected; excised gel pieces are used for cDNA amplification and NGS library preparation. NGS library is sequenced using standard methods and analyzed based on experimental needs.

Figure 19 shows the transcripts analysis of the method based on in situ RT (neoExiST) compared to ExiST. (a) total gene count of two samples based on in situ RT (neoExiST) . (b) PCA analysis of samples based on two methods: in situ RT (neoExiST, i.e. Newmethod) and RT after gel embedding (ExiST, i.e. Original) . (c) the expression level analysis of samples based on two methods: in situ RT and RT after gel embedding.

Figure 20 shows the workflow of spatial transcriptomic and proteomic combined procedures (ExiSTP) . The procedure includes both RNA anchor incubation and protein anchor incubation.

Figure 21 shows liver tissue-hydrogel RNA released fragment distribution of different homogenization conditions nt, hnt, ns and hns. nt: NSA in hybridization buffer, then Trypsin homogenization; hnt: mRNA probe hybridization, then NSA, then Trypsin homogenization; ns: NSA in hybridization buffer, then SDS homogenization; hns: mRNA probe hybridization, then NSA, then SDS homogenization.

Figure 22 shows the workflow spatial transcriptomic and proteomic combined procedures (neoExiSTP) . The procedures include in situ RT and protein anchor incubation.

Figure 23 shows the total identified gene counts for transcriptomics (a) and total identified peptides and proteins for proteomics (b) .

Figure 24 shows the expression anlysis of proteomics and transcriptomics from same samples. (a) the PCA analysis of spatial transcriptomic and proteomic identified genes. (N1-N3: transcriptomic replicates 1-3; N1p-N3p: proteomic replicates 1-3) . (b) volcanno plot of the analysis of differentially expressed genes between transcriptomic and proteomic identified information.

Figures 25-29 show over-expression analysis of differential expressed genes between transcriptomic and proteomic under GO enrichment. (Fig. 25) dot plot of top enriched pathways; (Fig. 26) heatmap of top enriched pathways group; (Fig. 27) heatmap of enriched pathway corresponding to certain molecules; (Fig. 28) tree plot of top enriched pathways group; (Fig. 29) upset plot of over-expression analysis.

DETAILED DESCRIPTION

The disclosures and embodiments set forth herein are to be construed as exemplary only and not as limiting the scope of the invention. Although specific terms are employed herein, unless otherwise noted, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.

Definitions

As used herein, the singular forms “a, ” “an, ” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The term “about” as used herein in conjunction with a stated numerical value or range of numerical values is meant to encompass variations of the stated numerical value or range of numerical values (i.e., denoting somewhat more or somewhat less than the stated numerical value or range of numerical values, to within a range of ±20%, ±10%, ±5%, ±1%, ±0.5%, ±0.1%, or ±one standard deviation of the stated value or range of numerical values) . Although efforts have been made to ensure accuracy with respect to numbers used (e.g. ranges, amounts, concentrations, etc. ) , some experimental deviations should be accounted for.

The term “sample” as used herein encompass all varieties of biological, chemical or biochemical materials, including but not limited to, a cell, cultured cells, a biological tissue, a specimen, biopsies, an intact organ, a whole organism, or in principle other types of samples of interest, such as bacteria, fungus, and viruses. Generally, the sample is suitable for microscopic analysis. The term “biological sample” may be all or a part of a tissue or organ including, but not limited to brain, spinal cord, heart, lung, liver, kidney, stomach, colon, bones, muscle, skin, glands, lymph nodes, genitals, breasts, pancreas, prostate, bladder, thyroid, and eyes.

The terms “magnification” , “expansion” and “swell” can be used interchangeably herein and refers to physical expansion of the hydrogel or sample-hydrogel composite, which is preferably tunable and reversible.

The term “precursor” as used herein include monomers, comonomers, oligomers and cross-linkers constructing the small units of polymer chain of the hydrogel.

The term “swellable material” or “expandable material” generally refers to a material that linearly expands when contacted with a liquid, such as water or other solvent. Preferably, the swellable material uniformly expands in 3 dimensions, and the material is transparent such that, upon expansion, light can pass through the sample. As exemplified herein, the swellable material is a swellable sample-hydrogel composite. In some embodiments, the swellable material is formed in situ by polymerization of hydrogel from precursors, monomers or oligomers thereof. For example, monomers comprising water soluble groups containing a polymerizable ethylenically unsaturated group can be used. Monomers or oligomers can comprise one or more substituted or unsubstituted methacrylates, acrylates, acrylamides, methacrylamides, vinylalcohols, vinylamines, allylamines, allylalcohols, including divinylic crosslinkers thereof (e.g., N, N-alkylene bisacrylamides) . Precursors can also be mixed with polymerization initiators and crosslinkers prior to polymerization. In some embodiments, the swellable polymer is polyacrylate and copolymers or crosslinked copolymers thereof. Alternatively or additionally, the swellable material can be formed in situ by chemically crosslinking water soluble oligomers or polymers. Thus, the invention envisions adding precursors (such as water soluble precursors) of the swellable material to the sample and rendering the precursors swellable in situ.

The terms “anchor” as used herein include both RNA anchors and protein anchors that crosslink RNAs and proteins in the sample to hydrogel polymer chain, respectively. The anchor is preferably a bifunctional linker comprising a biomolecule-reactive chemical group that binds to, links to or hybridizes to the biomolecule of interest (e.g. RNAs, proteins, DNAs) , and a hydrogel-reactive chemical group that binds to, links to or hybridizes to the hydrogel polymer chain. In certain embodiments, the chemical anchors, containing one or more functionalities, attach reactive groups to functional groups (e.g., primary amines or sulfhydryls) of biomolecules of interest within a biological sample, which may be proteins, nucleic acids, lipids, proteoglycans, lipopolysaccharides etc. The chemical anchors functionalize biomolecules within the sample to react with growing chains of hydrogel polymer during sample embedding step, thus covalently anchoring functionalized biomolecules into hydrogel mesh.

The term “thymidine locked nucleic acid” refers to modified RNA monomers of thymidine (T) and may be abbreviated as “dT+” herein. The “locked” part of locked nucleic acid (LNA) comes from a methylene bridge bond linking the 2′ oxygen to the 4′ carbon of the RNA pentose ring. The bridge bond fixes the pentose ring in the 3′-endo conformation. It has been found that LNAs may show remarkable affinity and specificity against native DNA targets. LNAs can be synthesized using traditional phosphoramidite reagents and are commercially available.

A mRNA probe “hybridizes” to a mRNA under conditions such that non-specific hybridization is minimal at a defined temperature in a physiological buffer (e.g., pH 6-9, 25-150 mM chloride salt) . In some cases, the mRNA probe can hybridize to a mRNA if there are at least about 6, 8, 10, 12, 14, 16, or 18 contiguous complementary nucleotides that are complementary to the mRNA sequence. In some cases, the mRNA probe hybridizes to a common sequence shared among a group of target mRNAs. The defined temperature at which specific hybridization occurs may be room temperature or higher. In some embodiments, the defined temperature at which specific hybridization occurs is at least about 37 ℃.

The term “polymerase chain reaction” or “PCR” refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression. PCR is well known to those of skill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990. Exemplary PCR reaction conditions typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by a hybridization/elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.

ExiST

The present disclosure provides a conceptually novel approach to spatial transcriptomics, designated as Expansion Assisted Spatial Transcriptomics (ExiST) herein. The ExiST approach combines physical magnification of biological tissues and RNA sequencing technology. In one aspect, the disclosure provides a method involving physical expansion of biological samples for spatially analyzing biomolecules of interest (such as mRNAs) in the sample, wherein the mRNAs can be preserved in expanded sample-hydrogel composite without degradation, and subsequently mRNAs in the interested regions can be sequenced. The present method can provide a spatial resolution that is well below the diffraction limit of light microscopy, enabling valuable insights into the spatial distribution of biomolecules such as mRNAs in biological samples. The present method can improve microscopic exploration into different organs and tissues, such as mapping the brain, as well as new diagnostic, personalized medicine, histopathological, and other medical capabilities.

In certain embodiments, the disclosure provides a method comprising the following steps:

(a) incubating the biological sample with a mRNA probe for a sufficient time period for the mRNA probe to hybridize to the mRNAs in the sample;

(b) embedding the sample in a hydrogel precursor solution or perfusing the sample with a hydrogel precursor solution, and then polymerization takes place to form a sample-hydrogel composite;

(c) homogenizing the sample-hydrogel composite via physical, chemical, physicochemical, and/or enzymatic treatment;

(d) optionally, staining the sample-hydrogel composite;

(e) expanding the sample-hydrogel composite to a desired degree based on end-user needs;

(f) selecting a region (s) of interest from the expanded sample-hydrogel composite and dissecting the region (s) , e.g. via sample microdissection; and

(g) releasing and sequencing the mRNAs in the dissected region.

Additionally, the method may comprise washing the sample or sample-hydrogel composite with a washing buffer before, after or between any of steps (a) - (f) , e.g. before step (a) , after step (c) . The biological sample as 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, whole organisms. The biological sample may be derived from bacteria, fungus, viruses or mammals, including non-human animals such as mouse, rat and human. The sample may be live, fixed or preserved, such as live culturing cells or fixed tissue sections. The methods herein not only apply to cells and thin samples but are also suitable for large or dense samples which can be adequately expanded and optically clear after hydrogel expansion.

In certain embodiments, the biological sample is cultured cells, such as cultured tumor cells. In some other embodiments, the biological sample is a tissue section or slice with a thickness of 50 μm of 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 –400 μm, such as in the range 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 above 400 μm.

In certain embodiments, the biological sample is a tissue sample (e.g. section) from, including but not limited to, the brain, spinal cord, heart, lung, liver, kidney, stomach, colon, bones, muscle, skin, lymph nodes, genitalia, mammary gland, pancreas, prostate, bladder, thyroid, and eye. In certain embodiments, the biological sample is a liver tissue section, a heart tissue section, a kidney tissue section, a brain section, or a cancer tissue section.

The biological sample, such as a tissue slice, may be fixed with a fixative prior to step (a) . In certain 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, such as SSC buffer. In certain embodiments, the biological sample is incubated with the mRNA probe at 37℃ for 35-45 hours.

The advancements and improvements achieved with ExiST concern several major aspects. First, ExiST overcomes the density limitation of spatially barcoded capture arrays by expanding a sample-hydrogel composite with RNA molecules in the sample reversibly anchored to polymer chains. Physically magnified biological structures facilitate manual microdissection of a region of interest based on anatomical features defined by user at a scalable spatial resolution ranging from 100 μm to 1000 μm. Second, optimization of RNA capture and release (including RNA probe design, RNA release conditions) enables the detection of more transcripts from fewer cells per tissue volume compared to the traditional chip-based spatial transcriptomics method improving the sequencing depth.

Besides, ExiST does not require specialized or sophisticated equipment for tissue expansion, microdissection, and RNA recovery, which are needed in imaging-based in situ sequencing methods. As disclosed herein, the ExiST workflow can utilize only commercially available and extremely accessible reagents, which makes ExiST technology ～1,000 times cheaper than other chip-based and bead-based spatial transcriptomic methods.

In addition, ExiST technology is distinct from other method development efforts in the field, which have predominantly concentrated on reducing the size of the capture spot. Consequently, the ExiST approach holds the potential to be integrated with other sequencing modalities, including chip-based and bead-based sequencing technology, to further improve their overall performance. For example, ExiST complements current high-resolution approaches since sample expansion may be paired with smaller spot sizes to enhance spatial resolution even further, while each method may need to optimize RNA capture efficiency. Combining ExiST with other high-resolution methods may allow for unbiased RNA profiling of tiny subcellular structures such as organelles.

Furthermore, compared to other technology that uses hydrogel-assisted sample magnification, ExiST provides several technological advantages. First and perhaps most crucial, ExiST does not require any chip for sequencing. Second, a new chemistry (including hydrogel composition, mRNA probe composition, homogenization conditions and buffers) that allows for higher spatial resolution due to a higher expansion factor is adopted. Third, to enhance spatial resolution, sample microdissection may be used to excise pieces of gel, followed by RT-PCR performed using pieces of gel under optimized conditions directly in an enzymatic mixture, which was not realized for any previously reported methods. Fourth, in addition to RNA analysis, ExiST is compatible with proteomic analysis as proteins can be anchored into the gel polymer chain by using a protein anchor. Fifth, homogenization conditions were optimized to ensure mRNA remains in the gel in intact form, homogenization conditions used in other Expansion Microscopy (ExM) -based methods for structural imaging do not preserve mRNA in the gel. Sixth, ExiST is compatible with super-resolution imaging under conventional diffraction-limited microscopy prior to sequencing, which allows to investigate and perform image-guide dissection and sequencing.

neoExiST

The present disclosure further provides an alternative approach to ExiST, designated as neoExiST herein. The main difference between neoExiST and ExiST is that neoExiST adopts an in situ reverse transcription of the mRNAs before gelation.

(a) incubating the sample with a mRNA reverse transcription mix comprising the mRNA probe and a reverse transcriptase, wherein the mRNA probe can hybrid to mRNAs in the sample and has been modified to comprise a hydrogel-reactive chemical group, and wherein the mRNAs in the sample are reverse transcribed to cDNAs by the reverse transcriptase, with the mRNA probe acting as a primer;

(d) optionally, staining the sample-hydrogel composite;

(g) sequencing the cDNAs from the dissected region.

The biological sample, such as a tissue slice may be fixed with a fixative prior to step (a) . In certain embodiments, the fixative may be selected from ethanol, methanol or acetone. In step (a) , the biological sample may be incubated with the mRNA reverse transcription mix comprising the mRNA probe and a reverse transcriptase, wherein the mRNA probe acts as a dT primer for reverse transcription. In certain specific embodiments, the reverse transcription reaction includes: 2 ℃ for 3 minutes, then placed in an ice bath for 2 minutes, followed by 42℃ for 90 minutes, 70 ℃ for 15 minutes, and then held at 4 ℃. Preferably, the method may further comprise rinsing the biological sample with SSC buffer prior to reverse transcription. After RT, the transcribed cDNA forms a mRNA-cDNA duplex and during hydrogel formation, the duplex is incorporated into the hydrogel via the hydrogel reactive group in the mRNA probe.

The ExiST and neoExiST methods as disclosed herein further comprises cDNA amplification after step (f) . Methods for mRNA reverse transcription and PCR amplification are well established in the art and a variety of commercial kits are available. Preferably, before the cDNA (synthesized from RNA) can be sequenced, a cDNA library is prepared in which the cDNAs are fragmented, end-repaired, and made into sequencing libraries.

ExiSTP

The present disclosure further provides methods combining the ExiST and neoExiST with protein identification, which are designated as Expansion Assisted Spatial Transcriptomics and Proteomics (ExiSTP) and neoExiSTP herein. The difference between ExiSTP (or neoExiSTP) and ExiST (or neoExiST) is that ExiSTP adopts incubating the biological sample not only with the mRNA probe but also with a bifunctional protein anchor, such that the mRNAs and the proteins from the dissected gel region are subjected to both transcriptomics and proteomics analysis to obtain a comprehensive profiling of the region of interest. The bifunctional protein anchor comprises a protein-reactive chemical group and a hydrogel-reactive chemical group, for example the free amine groups of proteins can interact with the anchor molecule. The biological sample may be incubated with a bifunctional protein anchor during step (a) , between step (a) and step (b) , or during step (b) .

In certain embodiments based on ExiST, the disclosure provides a method comprising the following steps:

(a) incubating the biological sample with a hybridization buffer comprising a mRNA probe and a bifunctional protein anchor for a sufficient time period;

(d) optionally, staining the sample-hydrogel composite;

(f) selecting a region (s) of interest from the expanded sample-hydrogel composite and dissecting the region (s) , e.g. via sample microdissection;

(g) the mRNAs in the dissected region are subjected to RT-PCR and then cDNA sequencing, and the peptides in the dissected region are extracted for proteomics identification.

In some alternative embodiments based on ExiST, steps (a) and (b) comprise:

(a) incubating the biological sample with a hybridization buffer comprising a mRNA probe for a sufficient time period to allow mRNA hybridization;

(b) embedding the sample in a hydrogel precursor solution comprising a bifunctional protein anchor or perfusing the sample with a hydrogel precursor solution comprises a bifunctional protein anchor, and then polymerization takes place to form a sample-hydrogel composite.

In some alternative embodiments based on ExiST, steps (a) and (b) comprise:

(a) incubating the biological sample with a hybridization buffer comprising a mRNA probe for a sufficient time period to allow mRNA hybridization, and subsequenctly incubating the biological sample with a bifunctional protein anchor;

(b) embedding the sample in a hydrogel precursor solution or perfusing the sample with a hydrogel precursor solution, and then polymerization takes place to form a sample-hydrogel composite.

The protein anchor may be selected from NSA and NAS, which modify amines on proteins with an acrylamide or allyloxycarbonyloxyamide functional group, respectively, and also allow for functionalized proteins to be anchored to the hydrogel during polymerization.

Preferably, the dissected gel from step (f) may be incubated with a buffer and subjected to in gel RT-PCR, after that, the supernatant comprising the amplified cDNAs may be removed for cDNA library construction, and the gel may be subjected to peptide extraction. In some embodiments, for peptide extraction from the dissected gel, the gel is re-embedded with a hydrogel precursor solution and re-gelated before performing peptide digestion and extraction for MS analysis.

neoExiSTP

In certain embodiments based on neoExiST, the disclosure provides a method comprising the following steps:

(a) incubating the biological sample with a mRNA reverse transcription mix comprising the mRNA probe and a reverse transcriptase, wherein the mRNA probe can hybrid to mRNAs in the sample and has been modified to comprise a hydrogel-reactive chemical group, and wherein the mRNAs in the sample are reverse transcribed to cDNAs by the reverse transcriptase, with the mRNA probe acting as a primer; then incubating the biological sample with a bifunctional protein anchor;

(d) optionally, staining the sample-hydrogel composite;

(g) the mRNAs in the dissected region are subjected to cDNA amplification (e.g. by PCR) and then cDNA sequencing, and the peptides in the dissected region are extracted for proteomics identification.

In some alternative embodiments based on neoExiST, steps (a) and (b) comprise:

(a) incubating the biological sample with a mRNA reverse transcription mix comprising the mRNA probe and a reverse transcriptase, wherein the mRNA probe can hybrid to mRNAs in the sample and has been modified to comprise a hydrogel-reactive chemical group, and wherein the mRNAs in the sample are reverse transcribed to cDNAs by the reverse transcriptase, with the mRNA probe acting as a primer;

The biological sample may be incubated with the bifunctional protein anchor during step (a) , between step (a) and step (b) , or during step (b) . In some embodiments, the biological sample is incubated with a bifunctional protein anchor after step (a) and before step (b) .

Preferably, the dissected gel from step (f) may be incubated with a buffer and subjected to in gel cDNA PCR, after that, the supernatant comprising the amplified cDNAs may be removed for cDNA library construction, and the gel may be subjected to peptide extraction. In some embodiments, for peptide extraction from the dissected gel, the gel is re-embedded with a hydrogel precursor solution and re-gelated before performing peptide digestion and extraction for MS analysis.

Hybridization Incubation

Most mRNAs comprise a poly (A) tail between 100 and 250 residues long at the 3′ ends. Immediately after a gene in a eukaryotic cell is transcribed, the new RNA molecule undergoes several modifications known as RNA processing. These modifications alter both ends of the primary RNA transcript to produce a mature mRNA molecule. The processing of the 3' end adds a poly (A) tail to the mRNA molecule, which plays essential roles in post-transcriptional regulation, including mRNA export, stability and translation. Oligo (dT) probes that can hybridize to or bind to the poly (A) tail have been developed to isolate mRNAs from samples, without interfering with DNAs or other RNAs.

As disclosed herein, the biological sample is contacted with a mRNA probe that can hybridize to mRNAs in the sample. Preferably, the mRNA probe is a bifunctional probe that comprises both a mRNA-reactive group and a hydrogel-reactive group to allow hybridization to the mRNAs and crosslinking to hydrogel polymer chain. Since mRNAs generally comprise a poly (A) tail, the mRNA probe 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-pairing.

In some embodiments, the mRNA probe comprises a nucleotide sequence consisted of dT nucleotides. In some embodiments, the mRNA probe comprises a nucleotide sequence comprising dT nucleotides, analogues and/or derivatives of dT nucleotides. 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 comprising dT nucleotides and thymidine-locked nucleotides (dT+) . In some embodiments, the mRNA probe comprises a nucleotide sequence of alternating dT and thymidine-locked nucleic acid (dT+) .

The length of the mRNA probe may vary between 10-40 nucleotides, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 35 nucleotides. Depending on the needs, the number of dT-LNA residues and lengths of the mRNA probe could be adjusted to achieve suitable stability and specificity. In some embodiments, the mRNA probe comprises a nucleotide sequence of alternating dT and thymidine-locked nucleic acid (dT+) in a length of 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 certain embodiments, the mRNA probe comprises a nucleotide sequence as set forth in SEQ ID NO: 1 or 2. Further, the mRNA probe as disclosed herein is modified to allow cross-linking to the hydrogel polymer chain. The modification may be at the 5’ or 3’ terminal or at an internal nucleotide. In some embodiments, the mRNA probe is 5’ modified with acrydite, a primary amino group, azide, Uni-Link^TM amino modifier, or any combination/mixture thereof. In some specific embodiments, the mRNA probe comprises the nucleotide sequence as set forth in SEQ ID NO: 1 or 2 and has a 5’-acrydite modification.

The hybridization buffer may be any of those conventionally used for RNA hybridization. In some embodiments, the hybridization buffer is a Saline Sodium Citrate (SSC) buffer, e.g. a 2×SSC buffer comprising formamide, RNase inhibitor, dextran sulfate and yeast transfer RNA. In certain embodiments, the biological sample is incubated with the mRNA probe in a hybridization buffer at 4℃-37℃ for about 24-48 hours, e.g. about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours or any interval value within the range.

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

In some embodiments, the biological sample is incubated with a protein anchoring solution comprising a protein anchor after incubated with a hybridization buffer comprising the mRNA probe. The protein anchoring solution may be prepared by dissolving or diluting the protein anchor in an aqueous buffer, such as PBS, MES, or a buffer comprising 50 mM sodium carbonate, 50 mM sodium bicarbonate and 10 mM 10% (v/v) Triton X-100. The protein anchoring solution may further comprise a fixative agent. A person skilled in the art can easily determine the components of the buffer based on the selected anchor (s) .

The protein anchors and RNA probes as disclosed herein enable fast anchoring of biomolecules throughout larger biological samples and provide high fluorescence retention. The bifunctional protein anchor comprises two different functional groups, one for functionalization of proteins and the second for reacting with growing hydrogel polymeric chains. The two different functional groups may be separated by chemical linkers of various length and structures including branching linkers. The bifunctional protein anchor may comprise both a protein-reactive chemical group and a hydrogel-reactive chemical group, to allow crosslinking of the protein with the hydrogel polymer chain. In some embodiments, the protein-reactive chemical group includes, but is not limited to, N-hydroxysuccinimide (NHS) ester, epoxy group, aldehyde group, formamide group, which can be reacted 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, acrylamide group.

In certain embodiments, the protein anchor for crosslinking proteins directly to any hydrogel polymeric chains are one or more selected from N-Succinimidyl Acrylate (NSA) , N-(Allyloxycarbonyloxy) -succinimide (NAS) , Allyl glycidyl ether (AGE) , Glycidyl Acrylate (GAL) , methacrolein, and Glycidyl Methacrylate (GME) . In certain embodiments, the protein anchor is selected from N-Succinimidyl Acrylate (NSA) , N- (Allyloxycarbonyloxy) -succinimide (NAS) , and any combination/mixture thereof. For example, treatment with NSA or NAS modifies amines on proteins with an acrylamide or allyloxycarbonyloxyamide functional group, respectively, which allows for functionalized proteins to be anchored to the hydrogel during polymerization. In some embodiments, the protein anchor is NAS or NSA.

In some embodiments, the biological sample is incubated with a hybridization buffer comprising a mRNA probe at 4℃-37℃ for about 24-48 hours, and then incubated with a buffer comprising NAS, NSA or a combination thereof at 4℃-37℃ for about 2-8 hours, prior to step (b) .

The biological sample may be fresh, frozen, or fixed (i.e. preserved) before step (a) . In certain embodiments, the biological sample has been histologically preserved using a fixative, such as ethanol, formaldehyde or paraformaldehyde. The sample may also be embedded in a firm and generally hard medium such as paraffin, wax, celloidin, or a resin, which makes possible the cutting of thin sections for microscopic examination.

Fixation may be performed by conventional methodology. One of skill in the art will appreciate that the choice of a fixative is determined by the purpose for which the sample is to be histologically stained or otherwise analyzed. One of skill in the art will also appreciate that the length of fixation depends upon the size of the tissue sample and the fixative used. By way of example, neutral buffered formalin, Bouin’s or paraformaldehyde, may be used to fix a sample.

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

Hydrogel Polymerization or Gelation

As disclosed above, the sample is permeated (e.g. perfused, infused, soaked, added or other intermixing) with a hydrogel precursor solution or embedded in a hydrogel precursor solution, wherein mRNAs within the sample, which have been hybridized to the mRNA probe, are covalently bound to hydrogel polymer chains via the crosslinking of the mRNA probe. The hydrogel precursors would crosslink with the sample and polymerize to form the hydrogel-sample composite. The polymerized sample-hydrogel composite comprises a swellable polymer network.

Hydrogel compositions that may be used for expansion are known in the art and can be easily selected according to practical needs. The hydrogel precursor solution generally comprises one or more hydrogel monomers or precursors solubilized in an aqueous solution (e.g. RNase-free water) . In addition to hydrogel precursors, the hydrogel precursor solution may further be added with a polymerization activator (such as APS, potassium persulfate, VA-044, ect. ) or accelerators (such as TEMED) right before use. Water is generally used as the dispersion medium in forming the hydrogel, but other solvents may also be used. Since mRNAs in the sample should be retained in an intact state during the process, the reagents in the hydrogel precursor solution should be RNase-free or the hydrogel precursor solution has undergone RNase free treatment, for example, treated with RNase inhibitors (e.g. DEPC) .

In certain embodiments, the precursors of the hydrogel are selected from but 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) , Trimethylolpropane Propoxylate triacrylate (TPT) , Pentaerythritol triacrylate (PA) , Dipentaerythritol penta-/hexa-acrylate (DPHA) , Trimethylolpropane triacrylate (TTA) , Di (trimethylolpropane) -tetraacrylate (DiTA) , Trimethylolpropane Trimethacrylate (TTMA) , Glycerol propoxylate (1PO/OH) triacrylate (GPT) , Trimethylolpropane ethoxylate triacrylate (TET) , Pentaerythritol allyl ether (PAE) , Sodium 4-hydroxy-2-Methylenebutanoate (SHMB) , N, N-Dimethylaminopropyl acrylamide (DMPAA) , Acrylamide (AA) . All reagents are commercially available, chemically stable and relatively safe (can be used in a regular biology lab) .

The hydrogel is desired to retain a high mechanical stability in expanded state in order to achieve a tunable and reversible swelling. Mechanical stability of the hydrogel can be accessed by method familiar to a person in the art, e.g. visually inspecting expanded hydrogel samples for cracks and breaks after they were manually handled imitating real experiment, i.e., transferred from dish to dish, shaken, rocked, and dissected using a scalpel. A detailed test of the hydrogel test can be found in WO 2022/262311, the entire contents of which are incorporated herein by reference.

A variety of precursors can be adopted for forming the hydrogel as long as the formed hydrogel has good mechanical stability, including those already known for use in expansion microscopy and those to be discovered for expansion microscopy. In some embodiments, the precursors for forming the hydrogel are SMA, AA and PAE. In some embodiments, the precursors for forming the hydrogel are DMAA, SMA and TPT. In some other embodiments, the precursors for forming the hydrogel are DMAA, SA, Bis-AA and AA. In some other embodiments, the precursors for forming the hydrogel are DMAA, SMA and PAE. In some other embodiments, the precursors for forming the hydrogel are SMA, Bis-AA and AA. In some other embodiments, the precursors for forming the hydrogel are SA, Bis-AA and AA. In some other embodiments, the precursors for forming the hydrogel are SMA, SA, Bis-AA and AA. Preferably, the formed hydrogel has the capability for expanding large tissue blocks, whole organs and even entire organisms without deformations and mechanical breakdowns under its own weight. The precursors may be mixed in ratios of a wide range which can form hydrogels capable of tunable and reversible swelling in aqueous buffers.

In some embodiments, the hydrogel precursors are SMA, AA and PAE. Preferably, the molar ratio of SMA to AA in the hydrogel precursor solution may 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, 1: 5 to about 1: 10. Preferably, the molar ratio of AA to PAE in the hydrogel precursor solution may 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.

In some embodiments, the hydrogel precursors are SA, AA and Bis-AA. Preferably, the molar ratio of SA to AA may 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 ratios or subranges therebetween. Preferably, the molar ratio of AA: Bis-AA may 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 ratios or subranges therebetween. 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) .

In some embodiments, the hydrogel precursors are SMA, AA and Bis-AA. Preferably, the molar ratio of SMA to AA may 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 ratios or subranges therebetween. Preferably, the molar ratio of AA: Bis-AA may 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 ratios or subranges therebetween. 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) .

In some embodiments, the hydrogel precursors are DMAA, SMA and TPT. Preferably, the molar ratio of DMAA to SMA may 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 ratios or subranges therebetween. Preferably, the molar ratio of SMA to TPT may 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 ratios or subranges therebetween. For example, the molar ratio of DMAA: SMA: TPT may be in the range of (4-30) : 1: (0.0004-0.4) .

In some embodiments, the hydrogel precursors are DMAA, SMA and PAE. Preferably, the molar ratio of DMAA to SMA may 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 ratios or subranges therebetween. Preferably, the molar ratio of SMA to PAE may 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 ratios or subranges therebetween. 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) .

In some embodiments, the hydrogel precursors are DMAA, SMA and TPT. In some embodiments, the molar ratio of DMAA to SMA in the hydrogel precursor solution may 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) . Further, the molar ratio of SMA to TPT in the hydrogel precursor solution may 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) .

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 may 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) . Further, the molar ratio of AA: Bis-AA in the hydrogel precursor solution may 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) .

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 may 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) . Further, the molar ratio of AA: Bis-AA in the hydrogel precursor solution may 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) .

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 may 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) . Further, the molar ratio of SMA to PAE in the hydrogel precursor solution may 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) .

Specifically, in some embodiments, DMAA, SMA and TPT are mixed in molar ratio of about 30: 1: 0.4 in the hydrogel precursor solution. The hydrogel precursor solution may 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, e.g. about 6.5. The hydrogel precursor solution may be freshly prepared before use or a stock solution is prepared and diluted before use.

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

The hydrogel precursor solution may further be optimized to comprise HCl or NaCl in the buffer system (e.g. PBS buffer) , this may promote an acidic or neutral environment.

Before polymerization, the hydrogel precursor solution is added with a polymerization activator and/or accelerator to induce polymerization or gelation, the activator or accelerator such as but not limited to, ammonium persulfate, potassium persulfate, TEMED, VA-044, or a combination thereof. The polymerization activator may be mixed with the hydrogel precursor solution before use, i.e. before perfusing with the sample.

In some embodiments, the sample is perfused with a hydrogel precursor solution added with the protein anchor and the polymerization activator/accelerator. Alternatively, the sample to be gelated is treated with the protein anchor prior to being incubated with the hydrogel precursor solution.

A stock solution of the hydrogel precursor solution may be prepared and diluted before use. The stock solution can have a high concentration (w/w) of the precursors, such as about 50%or more, about 75%or more, about 80%or more, about 90%or more. The stock solution may be diluted to a concentration of about 30-50%before applied onto the sample. Preferably, the solution comprising the precursors is aqueous.

Homogenization

In certain embodiments, the sample-hydrogel composite is subjected to homogenization before expansion. As used herein, “homogenization” refers to mechanical, physical, chemical, biochemical or enzymatic digestion, disruption or break up of the sample so that it will not resist expansion. The homogenization approach further improves the achievable resolution of fluorescent imaging, which is important for imaging techniques with resolution beyond the size of dyes or labels.

In certain embodiments, an enzymatic homogenization is performed before expansion and comprises treating sample-hydrogel composites with specific or non-specific proteases for an appropriate amount of time depending on the sample type and size. For example, the enzymatic homogenization comprises treating the sample-hydrogel composite with Trypsin in a homogenization buffer. In certain alternative embodiments, a physicochemical homogenization is performed by using an alkaline detergent-rich buffer e.g. a buffer comprising SDS (sodium dodecyl sulfate) .

In some specific embodiments, homogenization was performed by incubating the sample-hydrogel composite with a homogenization solution for a suitable amount of time. The homogenization solution may be prepared by mixing a protease (such as Trypsin) with a homogenization buffer. Alternatively, homogenization may be accomplished by incubating the sample-hydrogel composite with a homogenization buffer comprising alkaline detergent (e.g., sodium dodecyl sulfate at pH 7.0 to 10.0) for a sufficient period of time. A lot of homogenization buffers are established in the art and routinely used. The homogenization buffer may be easily prepared in-house or purchased commercially.

In some embodiments, the homogenization buffer comprises Trypsin in PBS buffer. In some other embodiments, the homogenization buffer comprises SDS in a buffer of NaCl and EDTA.

It is preferable that the homogenization does not impact the structure of the hydrogel and the integrity of the mRNA, but sufficient to compromise the integrity of the mechanical structure of the sample. The homogenization treatment allows the biomolecules such as DNAs, RNAs and/or proteins to be labeled in the molecularly decrowded environment, improving access and thus efficiency of staining with dyes or other molecular labels.

The methods disclosed herein may include different combinations of hybridization and homogenization processes. In some embodiments, the method comprises incubating the sample with a hybridization buffer comprising both the mRNA probe and the protein anchor for anchoring of mRNAs and proteins, and treating the sample-hydrogel composite with SDS for homogenization. In some embodiments, the method comprises incubating the sample with a hybridization buffer comprising both the mRNA probe and the protein anchor for anchoring of mRNAs and proteins, and treating the sample-hydrogel composite with Trypsin for homogenization. In some embodiments, the method comprises incubating the sample with a hybridization buffer comprising the mRNA probe followed by contacting with the protein anchor for anchoring proteins, and treating the sample-hydrogel composite with Trypsin for homogenization. In some embodiments, the method comprises incubating the sample with a hybridization buffer comprising the mRNA probe followed by contacting with the protein anchor for anchoring proteins, and treating the sample-hydrogel composite with SDS for homogenization.

Staining

In certain embodiments, the sample-hydrogel composite is stained after homogenization for the visualization of biomolecules of interest and tissue and cellular morphology. The staining step may be performed before, during or after expansion of the hydrogel. Typically, one or more dyes or labels will bind chemically (e.g., covalently, hydrogen bonding or ionic bonding) to the biomolecules of interest in the sample. The dye or label can be selective for a specific target (e.g., a biomarker or class of molecule, such as DNA, RNA or protein) . The label preferably comprises a visible component, as is typical of a dye or fluorescent molecule. A fluorescently labeled sample, for example, is labeled through techniques such as, but not limited to, immunofluorescence, immunohistochemical or immunocytochemical staining to assist in microscopic analysis. The label or dye is preferably chemically attached to the targeted biomolecule or component thereof. The sample-hydrogel composite may be stained by one or more dyes or labels. For example, each dye or label can have a particular or distinguishable fluorescent property, e.g., distinguishable excitation and emission wavelengths. Further, each dye or label can have a different target specific binder that is selective for a specific and distinguishable target in, or component of the sample. The present method is compatible with fluorescent proteins as well as standard immunofluorescent methods before or after sample-hydrogel composite expansion, providing up to 50 nm lateral resolution under conventional imaging setups.

In some embodiments, the sample-hydrogel composite is stained with a fluorescent dye for the visualization of nucleic acids. The dye for DNA visualization may be selected from, e.g. Propidium Iodide, DAPI, 7-AAD, Hoechst, and YOYO-1/DiYO-1/TOTO-1/DiTO-1, which are commonly used for staining DNAs. In one embodiment, the sample-hydrogel composite is stained with DAPI for DNA visualization.

In some further embodiments, the sample-hydrogel composite is stained with a dye for the visualization of proteins. The dye for protein visualization may be selected from, e.g. the Sypro series, Flamingo Fluorescent Gel stain, Krypton Protein stain, 5-TAMRA NHS ester, among others. In one embodiment, the sample-hydrogel composite is stained with Sypro dye for staining proteins.

The sample-hydrogel composite may be stained for DNA visualization and protein visualization with corresponding dyes, respectively. In some embodiments, the method further comprises staining the sample-hydrogel composite after homogenization, wherein the sample-hydrogel composite may be stained with a DNA dye (such as DAPI) and/or a protein dye (such as Sypro series dyes) to visualize the nucleic acids or proteins. In some embodiments, the sample-hydrogel composite is stained with DAPI to highlight DNA and then with a Sypro dye (e.g. Sypro Ruby) for staining proteins.

The methods disclosed herein can include different combinations of homogenization and staining processes. In some embodiments, the homogenization step is performed by treating with Trypsin, followed by staining with DAPI for staining DNAs. In some embodiments, the homogenization step is performed by treating with SDS, followed by staining with DAPI for staining DNAs. In some embodiments, the homogenization step is performed by treating with Trypsin, followed by staining with DAPI and a Sypro dye (e.g. Sypro Ruby) for staining DNA and proteins. In some embodiments, the homogenization step is performed by treating with SDS, followed by staining with DAPI and a Sypro dye (e.g. Sypro Ruby) for staining DNA and proteins.

Hydrogel Expansion

After homogenization, a tunable expansion of sample-hydrogel composite is performed to a desired degree based on end-user needs. The composite can be expanded isotropically, preferably with nanoscale precision, in three dimensions.

In some embodiments, after homogenization and staining, a solvent or liquid is added to the sample-hydrogel composite which is then absorbed by the composite and causes swelling. The solvent or liquid used herein is preferably RNase-free for RNA integrity. Where the sample-hydrogel composite is water expandable, an aqueous solution can be used. The aqueous solution may be water or a buffer such as a diluted SSC buffer. In some embodiments, the addition of the aqueous solution allows the embedded sample to expand 8x to 10x or more of its original size in 3-dimensions. Thus, the sample can be increased 100-folds or more in volume. This is because the polymer is embedded throughout the sample, therefore, as the polymer swells (grows) it expands the sample as well. Thus, the tissue sample itself becomes bigger. As the material swells isotropically, the anchored labels or tags maintain their relative spatial relationship.

As for most utilities the goal is often to achieve the highest possible spatial resolution, the subcellular structures were imaged using different microscopy protocols. The highest achievable resolution is defined by expansion factor which, for simplicity, is measured by overall gel expansion. Preferably, the hydrogel can expand by up to 8-10 times in linear dimensions thus bringing the resolution limit to 40 nm under a diffraction limited microscope. In contrast, the maximum expansion factor of currently available protocols is less than 4 in linear dimension thus limiting achievable lateral resolution to 80 nm. Some protocols can expand samples by 10 times in linear dimension, but due to mechanically unstable hydrogel used for expansion, these protocols can be applied only for two-dimensional samples like tissue culture or thin tissue sections.

In certain embodiments, the addition of pure water or other aqueous buffers allows for the sample-hydrogel composite to expand up to 8 times in linear dimension of sample original size maintaining high mechanical stability and elasticity. Both mechanical stability and elasticity allow for handling expanded samples easily without mechanical deformation of the sample-hydrogel composite keeping its integrity. These are crucial properties to ensure absence of artifacts that can be caused by deformation or fracturing of sample-hydrogel composite. As a result, the sample volume can be increased by 512-times in three-dimensions isotopically, meaning equally in all dimensions. Isotropic expansion occurs because of molecular chains of swellable polymer formed throughout the sample expand taking apart biomolecules making the tissue sample itself to become larger. Importantly, relative location of biomolecules remains the same after expansion. After expansion sample-hydrogel composite can be subjected to imaging using optical microscope enabling efficient visualization of features that are smaller than the classical diffraction limit. Since after expansion sample-hydrogel composite is transparent, any conventional microscope capable of large volume imaging can be employed.

Once expanded, the tissue may be probed for the presence and/or location of DNAs, mRNAs and proteins. The swollen material with the embedded sample of interest can be imaged on any optical microscope, allowing effective imaging of features below the classical diffraction limit. Since the resultant specimen is preferably transparent, custom microscopes capable of large volume, wide field of view, 3-D scanning may also be used in conjunction with the expanded sample.

After expansion, a set of methods for biochemical or spectroscopic characterization of biological samples in the expanded state may be used for determining the presence, distribution, identity and/or amount of the biomolecules of interest, such as mRNAs.

Sequencing of mRNAs

In some embodiments, the method as disclosed herein further comprises subjecting the mRNAs released from a region of interest in the hydrogel for high-throughput sequencing or next-generation sequencing (NGS) .

High-throughput sequencing is revolutionizing many fields of biology, including cancer diagnostics, disease monitoring, and environmental analysis. In particular, methods of analyzing mRNA molecules by high-throughput sequencing of reverse transcribed cDNAs can reveal the identity and quantity of transcripts in a biological sample at a given moment in time. By combining with hydrogel expansion microscopy, the ExiST/neoExiST approach as disclosed herein can further reveal the location and spatial distribution of transcripts in a biological sample at higher lateral resolution mapped onto tissue morphology image at high resolution.

In general, the core steps in preparing RNA or DNA for next generation sequencing analysis are: (i) fragmenting and/or sizing the target sequences to a desired length, (ii) converting target to double-stranded DNA, (iii) attaching oligonucleotide adapters to the ends of target fragments, and (iv) quantitating 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 attaching adapters to mRNA fragments followed by some form of amplification. mRNA can be primed by random oligomers or by an anchored oligo-dT to generate first strand cDNA.

In some embodiments, RT-qPCR is used to detect and quantify the mRNAs released from the sample-hydrogel composite. The mRNAs are transcribed into complementary DNA (cDNA) and PCR amplified for library preparation. The cDNA may then be used as the template for the quantitative PCR or real-time PCR reaction (qPCR) . In qPCR, the amount of amplification product is measured in each PCR cycle using fluorescence. RT-qPCR is used in a variety of applications including gene expression analysis, RNAi validation, microarray validation, pathogen detection, genetic testing, and disease research. Alternatively, the cDNA library may be subjected to NGS using a variety of sequencing platforms.

The input material for commonly used high-throughput sequencing platforms, such as platforms provided by Illumina, Roche Sequencing, Pacific Biosciences, and others, consists of libraries of transcriptome-derived DNA fragments flanked by platform-specific adaptors. The standard method for constructing such libraries is entirely in vitro and typically includes one or more, or all, of cDNA synthesis, fragmentation of DNA (mechanical or enzymatic) , end-polishing, ligation of adaptor sequences, gel-based size-selection, and PCR amplification. This core protocol may be preceded by additional steps depending on the specific application.

In some embodiments, the platform used to perform cDNA library sequencing is Illumina sequencing although the application is not limited to the Illumina sequencing platform. A variety of next generation sequencing platforms are known in the art, any of which can be used with the present invention to perform the sequencing step. The technology allows analysis of mRNA expression levels and spatial distribution in their relevant cellular context.

With NGS technology, RNA profiling or whole genome sequencing has become a routine practice now in biological research. On the other hand, due to the high throughput of NGS, multiplexed methods have been developed not just to sequence more regions but also to sequence more samples. Compared to the traditional Sanger sequencing technology, NGS enables the detection of mutation for much more samples in different genes in parallel. Due to its superiorities over traditional sequencing method, NGS sequencers are now replacing Sanger in routine diagnosis. In particular, genomic variations of individuals can now be routinely analyzed for a number of medical applications ranging from genetic disease diagnostic to pharmacogenomics fine-tuning of medication in precision medicine practice. NGS consists in processing multiple fragmented DNA sequence reads, typically short ones (less than 300 nucleotide base pairs) . The resulting reads can then be compared to a reference genome by means of a number of bio informatics methods, to identify small variants such as Single Nucleotide Polymorphisms (SNP) corresponding to a single nucleotide substitution, as well as short insertions and deletions (INDEL) of nucleotides in the DNA sequence compared to its reference.

Mass Spectrometry (MS) Analysis

Approaches for proteomic identification are well-established in the art and applicable to the methods disclosed herein. In some embodiments, protein identification is performed using MS, high performance liquid chromatography-mass spectrometry (HPLC-MS) or LC-MS/MS. Liquid chromatography (LC) can effectively separate the organic components in the samples to be tested, while mass spectrometry (MS) can analyze the separated organics one by one to obtain information on the molecular weight, structure and concentration of the organics. HPLC-MS is a method routinely used in the art to analyze and measure large molecular weight compounds, such as proteins and polymers.

Kits

Also provided herein are kits for use in practicing the subject methods, where the kits typically may include (i) the precursors (including monomers, oligomers and crosslinkers) for forming the hydrogel; (ii) amRNA probe for hybridizing mRNAs in the sample and crosslinking with the hydrogel polymer chain; and/or (iii) one or more of the hybridization buffer, the homogenization buffer, the dyes or labels for staining, as described above. The components of the kits are preferably used under RNase-free conditions, or have been treated with RNase inhibitor (e.g. DEPC) before use or comprise an RNase inhibitor. The kit may be placed in -20℃ for storage at least 6 months, or in 4℃ or ambient temperature to preserve the function for at least 3 months. The components of the kit are stable upon storage and the imaging quality is maintained.

In some embodiments, the kit comprises a container comprising a mRNA probe. The mRNA probe may comprise a nucleotide sequence consisted of dT nucleotides, or a nucleotide sequence comprising dT nucleotides, analogues and/or derivatives of dT nucleotides. 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 comprising dT nucleotides and thymidine-locked nucleotides (dT+) . In some embodiments, the mRNA probe comprises a nucleotide sequence of alternating dT and thymidine-locked nucleic acid (dT+) .

The length of the mRNA probe may vary between 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. Depending on the needs, the number of dT-LNA residues and lengths of the mRNA probe could be adjusted to achieve suitable stability and specificity. In some embodiments, the mRNA probe comprises a nucleotide sequence of alternating dT and thymidine-locked nucleic acid (dT+) in a length of 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 certain embodiments, the mRNA probe comprises a nucleotide sequence as set forth in SEQ ID NO: 1 (15 nt) or 2 (25 nt) . Further, the mRNA probe as disclosed herein is modified to allow cross-linking to the hydrogel polymer chain. The modification may be at the 5’ or 3’ terminal or at an internal nucleotide. In some embodiments, the mRNA probe is 5’ modified with acrydite, a primary amino group, azide, Uni-Link^TM amino modifier, or any combination/mixture thereof. In some specific embodiments, the mRNA probe comprises a nucleotide sequence as set forth in SEQ ID NO: 1 or 2 and has a 5′-acrydite modification.

In some embodiments, the kit further comprises a container comprising a bifunctional protein anchor. The protein anchor may be added into a hybridization buffer comprising the mRNA probe before use. The protein anchor may also be added into the hydrogel precursor solution, and the sample is then embedded into the mixture solution or perfused with the mixture solution. The protein anchor may be selected from NSA, NAS, methacrolein, AGE, GME, GAL, BDE, GDE and a combination thereof. In some embodiments, the protein anchor is NSA or NAS.

In certain embodiments, the kit further comprises one or more containers comprising one or more hydrogel precursors. The precursors of the hydrogel may be selected from but 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) , Trimethylolpropane Propoxylate triacrylate (TPT) , Pentaerythritol triacrylate (PA) , Dipentaerythritol penta-/hexa-acrylate (DPHA) , Trimethylolpropane triacrylate (TTA) , Di (trimethylolpropane) -tetraacrylate (DiTA) , Trimethylolpropane Trimethacrylate (TTMA) , Glycerol propoxylate (1PO/OH) triacrylate (GPT) , Trimethylolpropane ethoxylate triacrylate (TET) , Pentaerythritol allyl ether (PAE) , Sodium 4-hydroxy-2-Methylenebutanoate (SHMB) , N, N-Dimethylaminopropyl acrylamide (DMPAA) , Acrylamide (AA) .

In certain embodiments, the precursors comprised in the kit are SMA, AA and PAE. In certain embodiments, the precursors comprised in the kit are DMAA, SMA and TPT. In some other embodiments, the precursors comprised in the kit are DMAA, SA, Bis-AA and AA. In some other embodiments, the precursors comprised in the kit are DMAA, SMA and PAE. In some other embodiments, the precursors comprised in the kit are SMA, Bis-AA and AA. In some other embodiments, the precursors comprised in the kit are SA, Bis-AA and AA.

The precursors may be present in separate containers in the kit, e.g., DMAA is present in a first container, SMA is present in a second container and TPT is present in a third container; or SMA is present in a first container, Bis-AA is present in a second container and AA is present in a third container; and to be mixed before use. Alternatively, any of the following form is also suitable: DMAA and SMA are mixed together (in a certain ratio range) in a first container, TPT is present in a second container; DMAA and TPT are mixed together (in a certain ratio range) in a first container, SMA is present in a second container, or; SMA and TPT are mixed together (in a certain ratio range) in a first container, DMAA is present in a second container; SMA and AA are mixed together (in a certain ratio range) in a first container, Bis-AA is present in a second container; SMA and Bis-AA are mixed together (in a certain ratio range) in a first container, AA is present in a second container, or; AA and Bis-AA are mixed together (in a certain ratio range) in a first container, SMA is present in a second container. The containers may or may not be present in a combined configuration.

The hydrogel precursors may be solubilized in an aqueous buffer such as a PBS buffer. In some embodiments, the kit comprises a hydrogel precursor solution comprising DMAA, SMA and TPT. In some other embodiments, the kit comprises a hydrogel precursor solution comprising DMAA, SA, Bis-AA and AA. In some other embodiments, the kit comprises a hydrogel precursor solution comprising DMAA, SMA and PAE. In some other embodiments, the kit comprises a hydrogel precursor solution comprising SMA, Bis-AA and AA.

The kit may further comprise a container comprising a hybridization buffer, e.g. a SSC buffer. In some specification embodiments, the hybridization buffer comprises 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, the mRNA probe has been added into the hybridization buffer.

The kit may further comprise a container comprising a wash buffer, e.g. an SSC buffer or PBS buffer. In some specification embodiments, the wash buffer comprises 2× SSC or 2× SSC and 30% [v/v] formamide.

The kit may further comprise a container comprising a fixative such as ethanol, e.g. 75%ethanol.

The kit may further comprise a container comprising a staining agent or a labeling agent such as a fluorescent dye for staining DNA, RNA and/or proteins. In some further embodiments, the kit comprises more than one staining agent, including one of DNA staining (e.g. DAPI) and one for protein staining (e.g. Sypro series) . The dyes, fixatives, probes, anchors and hydrogel precursor solution are stable under storage conditions and can be shipped without effecting the performance.

The kit may further comprise a container containing a polymerization activator or accelerator which is to be added before polymerization. The polymerization activator or accelerator may be selected from, but not limited to VA-044, TEMED, potassium persulfate and APS. Preferably, the polymerization activator (e.g., APS) is added into the hydrogel precursor solution at last, on ice and right before perfusing the sample with the hydrogel precursor solution.

The kit may further include a container comprising a homogenization buffer. Specifically, the homogenization buffer may comprise proteinases (e.g. Trypsin) or alkaline detergents (e.g. SDS) , or supplemented with proteinases (e.g. Trypsin) or alkaline (e.g. SDS) before use. In some embodiments, the kit comprises a container comprising Trypsin. In some embodiments, the kit comprises a container comprising commonly used buffer, such as PBS or NaCl buffer, to facilitate the preparation of solutions.

Containers are understood to refer to any structure that may hold or surround the liquid or solid components (e.g. of the hydrogel formulation) ; exemplary containers include bottles, syringes, vials, pouches, capsules, ampules, cartridges, and the like. The containers may be shielded from visible, ultraviolet, or infrared radiation through the use of additional components (e.g. a foil pouch surrounding a vial) or through selection of the material properties of the container itself (e.g. an amber glass vial or opaque syringe) .

The kits may also include a mixing device, for mixing the precursors together to produce the formulation of the invention. The kits may also include a gel handling device, such as a soft brush, a tweezer, and/or a delivery device (which may or may not include a mixing element) for injecting the hydrogel precursor solution onto the sample, and the like.

In some embodiments, the kit comprises one or more of the following components:

a gelation chamber configured to accommodate the sample prior to gelation and optionally a cover to seal the gelation chamber; an imaging chamber configured to accommodate the sample after gelation; a gel handling device; and a cooling device (such as an ice bag) . Additionally, the kit can contain different forms of a mold (e.g. silicone molds) or gelation chamber suitable for a variety of sample gelation including but not limited to cell culture, whole organ, tissue or tissue section, whole organism.

The kit may further include other components, e.g., desiccants or other means of maintaining control over water content in the kit, indicators to convey the maximum temperature experienced by the kit, and the like, that are required to maintain the product in good condition during transport and storage.

In some embodiments, the kit comprises a gelation chamber for forming the sample-hydrogel composite as described above. The chamber is configured to comprise a cavity, well or space for accommodating the sample prior to gelation, such as in the form of a dish, such as MatTek dish. The gelation chamber may also comprise a cover (such as a cover slide) to cover the cavity, well or hole.

In some embodiments, the kit further comprises an expansion chamber configured to accommodate the sample after gelation. The kit may comprise a stock of such chambers, such as a dozen dishes packaged together for convenient usage.

The kits as described herein can be stored for at least 6 months and shipped with some reagents on ice or RT, can include various combinations of reagents packed into suitable container for distribution.

In addition to above-mentioned components, the kits may further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc., or are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded.

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention.

EXAMPLES

Exemplary reagents, materials and storage conditions used in the experiments below were shown in Table 1. The volume or mass of the reagents can be scaled up or down depending on user’s needs.

Table 1

Example 1: Spatially transcriptomic analysis of mouse brain and liver tissues (ExisT)

1.1 Tissue preparation

Mouse were humanely euthanized using 1%sodium pentobarbital. Brain tissue (or other organ of interest) was rapidly dissected. The tissue block was promptly frozen using liquid nitrogen vapor until the tissue block solidified, and subsequently stored at -80 ℃ for future use.

1.2 Cryosection

The tissues were then cryo-sectioned to a thickness of 16 μm using a Leica cryostat and carefully placed onto custom-designed glass slides.

1.3 Ethanol fixation

The slices at first are fixed with 75%ethanol or 100%Methanol or 100%Acetone for 3 mins.

1.4 Hybridization incubation

The samples are incubated with the 1 μM RNA anchor probe (a15 nucleotide sequence of alternating dT and thymidine-locked nucleic acid (dT+) with 5’ modified with acrydite group) and at first for 40h at 37℃. Hybridization buffer comprises 1 μM anchor probe, 2× SSC, 30% [v/v] formamide, 1% [v/v] RNase inhibitor, 10% [w/v] dextran sulfate, 0.1% [w/v] yeast transfer RNA.

RNA anchor probe sequence: T+TT+TT+TT+TT+TT+TT+TT, with 5’ acrydite modification.

1.5 Monomer incubation and gelation

Samples were incubated in the monomer solution comprising 1× PBS, 2 M 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 at 4 ℃. Gelation was initiated by adding 0.3%APS solution and 0.2%TEMED solution. The gelation step was performed at 37℃ for 2 h.

1.6 Homogenization

SDS buffer (1%SDS, 50 mM NaCl, 5 mM EDTA) or trypsin buffer (6.25 ug/mL trypsin in 5X SSC buffer) is applied to the gel for 22℃ overnight.

1.7 Staining and expansion

The homogenized gels are washed with 5x SSC buffer 3 times, 10 min each. DAPI and Sypro series dyes are used to stain gel for 30 min to 3 hours. After staining, the gel is expanded with 1x PBS buffer for 3-5 times, each 15 min.

1.8 Imaging and microdissection

Expanded samples are imaged with the stereomicroscope or fluorescent microscope. Images of brain tissues before and after expansion using different homogenization conditions are shown in Figure 2. The regions of interest identified based on super-resolution imaging or morphological features are micro-dissected using a certain size punch (1.5 mm punch for 16 μm at LEF=3.0 from cortex) from 0.35 mm-3 mm.

1.9 In gel reverse transcription-polymerase chain reaction

At first, after expansion, the whole sample was used for mRNA release and analysis to verify mRNA concentration and integrity (Figure 3 for brain tissue and Figure 4 for liver tissue) . RT-PCR and NGS library preparation were performed according to the standard protocols using commercially available reagents and kits.

After verifying the doability of whole workflow, the in gel reverse transcription-polymerase chain reaction are performed for the microdissected sample. 3.5 μl 0.1x SSC buffer is added to cover the gel particle. Then the RNA release reaction is performed at 40-50℃ (30min) . Then the oligo dT primer is firstly added to the dissected samples then reacts at 72 ℃ for 3 min. The sample is put on the ice immediately after reaction. Then 5’ oligo primer and the reverse transcriptase are added into the reaction system. The reverse transcription reaction is performed at 40-45℃ (30-180 min) followed by 70 ℃ (15 min) . The reaction product is hold at 4 ℃. Then amplification mix and primers are added to the system for cDNA amplification. The cycle set depends on the size of the dissected sample, ranging from 8-18.

1.10 cDNA products purification and detection

Products from section 1.9 are purified with DNA beads and incubated 10 min to bind DNA. The samples are put on the magnetic shelf to separate the beads and liquid. After about 5 min, the supernatant is removed. Use freshly made 80%ethanol to wash the beads 30s, repeat. Open the lid and make the beads dry to remove the ethanol. Get the tubes away from the magnetic shelf and add about 17 ul elution buffer to incubate the beads 2-5 min. Put the tubes on the magnetic shelf to separate the liquid and beads. Gently soak 15 ul supernatant and store at -20℃. The purified cDNA products are detected using bioanalyzer.

1.11 Library preparation

At first cDNA tagmentation is performed with transposase at 55℃ for 10 min. Then reaction is stopped. The fragments are amplified with index. The cycle set depends on the size of the dissected sample from 5-15 range. The product is purified with DNA beads or DNA purification column. The quality of DNA library is detected with bioanalyzer or Qubit. The high-quality library is sending to next generation sequencing using commercially available services.

Result:

For ExiST, the extracted RNA quality (RNA band/size distribution) as shown in Figure 3 proves that both trypsin or SDS treated condition could work well for the ExiST method. For both homogenization conditions (Trypsin or SDS) , the obtained NGS libraries were of sufficient quality for downstream sequencing and analysis (Figure 4 and Table 2) .

Table 2. Properties of micro-dissected samples including length range, average size, concentration, region molarity and percent of total fragments.

FastQC analysis of NGS results demonstrated raw data looks good and there are no problems or biases in the data both with trypsin homogenization (Figure 5) and SDS-containing buffer homogenization (Figure 6) . Specifically, Figure 5 shows the parameters such as per base sequence quality, per tile sequence quality, per sequence quality scores, adapter content, and per base N content were good, and Figure 6 shows FastQC showed a high per base sequence quality, and per tile sequence quality, per sequence quality scores, adapter content, per base N content, and per base sequence content were good.

For the 2.0 mm punch sample homogenized with trypsin, 17, 980 aligned expressed genes were identified. As shown in panel a of Figure 7, the evaluation of sequencing raw data using FastQC indicates both conditions (S-zsc: SDS treated homogenization; T-zsc: Trypsin treated homogenization) showed good quality. Further analysis use HISAT2 to map RNA-seq data to the genome as well as identify splice junctions for both homogenization conditions (trypsin and SDS) (Figure 7. b and c) . The alignment results of panel b of Figure 7 showed the quality of alignment including no bias against sense strain and antisense strand. The alignment region result of panel c of Figure 7 showed the trypsin treated group has more exons alignment regions compared to the SDS treated group.

Analysis of gene body coverage and gene expression like FPKM also showed the identified diversity and region of sequencing results of two homogenization conditions (trypsin or SDS) (Figure 8) . As shown in panel a of Figure 8, the SDS treated group showed more average gene body coverage against the trypsin treated group. As shown in panel b of Figure 8, the SDS treated group showed more expression density and higher expression level against the trypsin treated group.

Figure 9 shows the detected RNA distribution and bands of mRNA release on BeyoMag^TMOligo (dT) 25 Magnetic Beads by Fragment Analysis using step 1.9. The microdissected sample is incubated in buffer at 40-45℃ (30-180 min) followed by mRNA concentration using commercially available dT -beads or RNA purification kit. Compared to results in Figure 21, it shows both RNA directly release in solution and RNA release on magnetic poly dT beads are workable.

Figure 10-11 shows the compatibility of the ExiST workflow with fluorescence super-resolution imaging guided microdissection. Based on this imaging guided microdissection method, the hippocampus regions of brain samples are microdissected and perform ExiST workflow. The mapped reads statistic, genome alignment distribution analysis and expression level show no huge difference between SDS and Trypsin homogenization conditions in Figures 11-13. However, the correlation analysis and PCA analysis show SDS group is lower reproducible than Trypsin digestion group in Figure 14. Gene body shows both two groups have 3’ identification preference in Figure 15, which is caused by poly dT mRNA capture method.

Example 2: Spatially transcriptomic analysis of mouse brain and liver tissues (neoExiST) In this Example, the performance of a condition in which RNA probe hybridization and in-situ reverse transcription were performed simultaneously was tested. Briefly, a procedure same as Example 1 was performed except that the following steps 1.4’ and 1.9’ were adopted instead of steps 1.4 and 1.9, respectively.

1.4’ Alternative step to 1.4

Immediately before reverse transcription, samples were rinsed once in 5X SSC buffer. After aspiration, reverse transcription mixes comprising RNA anchor probe, dNTP, 5 oligo primer, 1^st strand buffer, DTT, RNA inhibitor, and reverse transcriptase according to the Single Cell Full Length mRNA-Amplification Kit (Vazyme, cat. no. N712-01) were added to the chamber. These reactions were then incubated at 72 ℃ for 3 minutes, then placed in an ice bath for 2 minutes, followed by 42℃ for 90 minutes, 70 ℃ for 15 minutes, and then held at 4 ℃. single-stranded cDNA forms RNA-DNA duplex with the template mRNA and then during gelation cDNA gets incorporated to gel via acridate group. The same RNA anchor probe was used as in Example 1: T+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT, with 5’ acrydite modification.

Correspondingly, the following cDNA amplification step 1.9’ was performed instead of the RT-PCR of step 1.9.

1.9’ Alternative step to 1.9

The microdissected sample is incubated in buffer at 40-45℃ (30-180 min) followed by mRNA concentration using commercially available dT-beads or RNA purification kit.

The in-gel cDNA amplification reaction is performed for the microdissected sample. 3.5 μl 0.1x SSC buffer is added to cover the gel particle. Then amplification mix and primers are added to the system for cDNA amplification. The cycle set depends on the size of the dissected sample, ranging from 8-18.

Result:

Figures 18-19 show the neoExiST based on in situ RT reaction on sample before gel embedding. As shown in Figure 19, the identified gene counts validate this workflow has the ability to achieve spatial with less mRNA degradation. The expression level and PCA analysis against the original ExiST shows higher stability of neoExiST. In summary, neoEXiST utilizes in situ reverse transcription (RT) in its initial step, effectively preserving the quality of mRNA due to the formation of stable RNA: DNA complex thus preventing degradation. This approach not only enhances the integrity of the RNA samples but also allows for more comprehensive data collection in conjugation with proteomics. In comparison to the EXiST method, neoEXiST offers greater stability and a richer dataset regarding transcript information, enabling more reliable analysis and interpretation of gene expression. This advancement highlights the potential of neoEXiST in providing detailed insights into cellular processes.

Example 3: Spatially transcriptomic and proteomics analysis of mouse brain and liver tissues (ExiSTP and neoExiSTP)

In this Example, the performance of spatial transcriptomics combined with downstream proteomics analysis was tested. The procedure of Example 1 or Example 2 was modified in step 1.4 or 1.4’ to have the sample incubated with both the RNA anchor probe and a protein anchor, and the peptides extracted from the gels were subjected to proteomics analysis.

1.1 Tissue preparation

1.2 Cryosection

1.3 Ethanol fixation

1.4 Hybridization incubation

The samples are incubated with the 1 μM RNA anchor probe and protein anchor (0.2 mg/ml NSA) at first for 40h at 37℃. Hybridization buffer comprises 1 μM anchor probe, 2× SSC, 30% [v/v] formamide, 1% [v/v] RNase inhibitor, 10% [w/v] dextran sulfate, 0.1% [w/v] yeast transfer RNA.

Together with the homogenization step 1.6, four different conditions were tested. ns: NSA is present in the hybridization buffer and then using SDS for homogenization; nt: NSA is present in the hybridization buffer and then using Trypsin for homogenization; hns: RNA probe hybridization and then NSA incubation for protein anchoring, followed by using SDS for homogenization; hnt: RNA probe hybridization and then NSA incubation for protein anchoring, followed by using Trypsin digestion at 37℃ overnight for homogenization.

RNA anchor probe sequence: T+TT+TT+TT+TT+TT+TT+TT (SEQ ID NO: 1) , with 5’ acrydite modification

1.4’ Alternative procedure to 1.4

Immediately before reverse transcription, samples were rinsed once in 5X SSC buffer. After aspiration, reverse transcription mixes comprising RNA anchor probe, dNTP, 5 oligo primer, 1^st strand buffer, DTT, RNA inhibitor, and reverse transcriptase according to the Single Cell Full Length mRNA-Amplification Kit (Vazyme, cat. no. N712-01) added to the chamber. These reactions are then incubated 72 ℃ for the 3 minutes, then placed on ice bath for 2 minutes, followed by 42℃ for 90 minutes, 70 ℃ for 15 minutes and then be held at 4 ℃.

RNA anchor probe sequence: T+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT, with 5’ acrydite modification

Products are washed by 5X SSC buffer three times for 5 minutes each time, then incubated by protein anchor buffer (0.2 mg/ml NSA diluted by 5X SSC buffer) at 22-25 ℃ for 3 hours.

1.5 Monomer incubation and gelation

Samples are incubated in the monomer solution comprising 1× PBS, 2 M 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 at 4 ℃. Gelation was initiated by adding 0.3%APS solution and 0.2%TEMED solution. The gelation step was performed at 37 ℃ for 2 h.

1.6 Homogenization

SDS (1%SDS, 50 mM NaCl, 5 mM EDTA) homogenization buffer is applied to the gel for 12 hours at 25-95℃ or trypsin buffer (6.25 ug/mL trypsin in 5X SSC buffer) is applied to the gel for 22-37℃ overnight.

1.7 Staining and expansion

The homogenized gels are washed with 5x SSC buffer 3 times, 10 min each. DAPI and Sypro series dyes are used to stain gel for 30 min to 3 hours. After staining, the gel is expanded with 0.1x SSC buffer for 3-5 times, each 15 min.

1.8 Imaging and microdissection

1.9 In gel reverse transcription-polymerase chain reaction

At first, after expansion, the whole sample was used for mRNA release and analysis to verify mRNA concentration and integrity. RT-PCR and NGS library preparation were performed according to the standard protocols using commercially available reagents and kits.

The in gel reverse transcription-polymerase chain reaction are performed for the microdissected sample. 3-6 μl 0.1x SSC buffer is added to cover the gel particle. Then the RNA release reaction is performed at 40-50℃ (30min) . Then the oligo dT primer is firstly added to the dissected samples then reacts at 72 ℃ for 3 min. The sample is put on the ice immediately after reaction. Then 5’ oligo primer and the reverse transcriptase are added into the reaction system. The reverse transcription reaction is performed at 40-45℃ (30-180 min) followed by 70 ℃ (15 min) . The reaction product is hold at 4 ℃. Then amplification mix and primers are added to the system for cDNA amplification. The cycle set depends on the size of the dissected sample, ranging from 8-18.

1.9’ Alternative procedure to 1.9

The microdissected sample is incubated in buffer at 40-45℃ (30-180 min) followed by mRNA concentration using commercially available dT -beads or RNA purification kit.

If alternative produce to 1.9 were performed, the followed step should replace the original RT-PCR step mentioned in 1.9 section: the in-gel cDNA amplification reaction is performed for the microdissected sample. 3.5 μl 0.1x SSC buffer is added to cover the gel particle. Then amplification mix and primers are added to the system for cDNA amplification. The cycle set depends on the size of the dissected sample, ranging from 8-18.

1.10 cDNA products purification and detection

The supernatant of products from section 1.9 or 1.9’ are transferred into new PCR tubes to separate gels and solutions. Solutions are purified with DNA beads and incubated 10 min to bind DNA. The samples are put on the magnetic shelf to separate the beads and liquid. After about 5 min, the supernatant is removed. Use freshly made 80%ethanol to wash the beads 30s, repeat. Open the lid and make the beads dry to remove the ethanol. Get the tubes away from the magnetic shelf and add about 17 ul elution buffer to incubate the beads 2 min. Put the tubes on the magnetic shelf to separate the liquid and beads. Gently soak 15 ul supernatant and store at -20℃.

The purified cDNA products are detected using bioanalyzer.

1.11 Library preparation

1.12 Protein samples preparation

Gels from section 1.8 are incubated by re-embedded monomer solution comprising 0.09% [w/w] acrylamide, 0.003% [w/w] N, N′-methylenebisacrylamide, 0.00129%APS solution, 0.00129%TEMED solution and ddH₂0. After gels re-embedded, transfer it into Lobind tube. Add 100ul 10mM DL-dithiothreitol diluted in 100mM ammonium bicarbonate solution to incubate gels for 30 minutes, followed with disposing supernatant and adding 100ul 55mM iodoacetamide diluted in 100mM ammonium bicarbonate solution to incubate gels for 30 minutes in dark environment. Wash the gels with 100ul 100mM ammonium bicarbonate solution for 5 minutes, repeat. Remove the supernatant and add 100ul acetonitrile wash gels for 10 minutes, repeat. Digest gels with 5ng/ul trypsin dissolved in 100mM ammonium bicarbonate solution on ice bath for 2 hours. After that, add 8ul 100mM ammonium bicarbonate solution into the tube. Invert the tube and place it in an incubator at 37 ℃ overnight.

Digested peptide solutions are collected in the following steps and combined: 1) collect 30–40 μL of the supernatant; 2) add 100 μL 100 mM ABB, shake for 20 min at 37 ℃ and collect supernatant; 3) add 100 μL 10%ACN solution and shake for 20 min at 37 ℃, collect supernatant; 4) add 100 μL 50%ACN solution and shake for 20 min at 37 ℃, collect supernatant; 5) add 100 μL 70%ACN solution and shake for 20 min at 37 ℃, collect supernatant; 6) add 100%ACN and shake at 37 ℃, collect supernatant until the gel pieces turning white and sticky. Peptide samples were placed under vacuum to reduce the volume to 20–30 μL. The peptides were then desalted using C18 spin columns (Pierce^TM C18 Spin Tips, Thermo Fisher Scientific, US) and dried in a SpeedVac. The cleaned peptide samples were subjected to LC-MS/MS for subsequent analysis.

Result:

For ExiSTP, combination of ExiST (adopting steps 1.4 and 1.9) with proteomics workflow are shown in Figure 20. Combining how the protein anchor is incubated with the sample and how homogenization is performed, we tested four different conditions to see which one was better up to the results of extracted RNA. ns: NSA is present in the hybridization buffer and then using SDS for homogenization; nt: NSA is present in the hybridization buffer and then using Trypsin for homogenization; hns: RNA probe hybridization and then NSA incubation for protein anchoring, followed by using SDS for homogenization; hnt: RNA probe hybridization and then NSA incubation for protein anchoring, followed by using Trypsin for homogenization.

The results shown in Figure 21a shows the nt condition (incubating the sample with the RNA probe and the protein anchor (NSA) at the same time and then using Trypsin for homogenization) has a more even fragment distribution from 500bp to 5000bp, which proves the nt condition is best condition among the four conditions.

For neoExiSTP, which combined neoExiST (adopting steps 1.4’ and 1.9’) with proteomics workflow, the protocol is shown in Figure 22. Multi-omics achieve through the workflow neoExSTP and the total identifications of 3 replicate brain samples are shown in Figure 23. For transcriptomics, about 7500-10,000 gene counts > 100 is identified for each sample. For proteomics, about 27,000 peptides and about 3,600 proteins are identified for each sample.

The down-stream analysis shows the difference between transcriptomic and proteomic expression level of one sample in Figures 24-29. Differential expressed proteins analysis between proteomics and transcriptomics is shown in Figure 24. Downstream enrichment analysis shows some pathways including synapse organization are differentially expressed in transcriptomic and proteomic expression level. Figure 25 shows the significant enrichment pathway in differential expressed proteins including the gene counts and p value. Figure 26 shows significant group of enrichment analysis of each sample in heatmap format based on expression level. Figure 27 shows the significant enrichment analysis pathway corresponding to molecules in heatmap format based on fold change. Figure 28 shows the tree architecture of different enrichment pathway based on the number of genes and p adjusted value. Figure 29 shows the relationship between different enrichment analyzed pathway and gene numbers of each group.

Example 4: Spatially transcriptomic analysis with different lengths of poly dT probes

In this Example, different lengths of poly dT probes were tested. Briefly, RNA anchor probes with 15 nt (a15 nucleotide sequence of alternating dT and thymidine-locked nucleic acid (dT+) with 5’ modified with acrydite group, as used in Example 1) , 25 nt (a25 nucleotide sequence of alternating dT and thymidine-locked nucleic acid (dT+) as shown in SEQ ID NO: 2, with 5’ modified with acrydite group) or 35 nt (a35 nucleotide sequence of alternating dT and thymidine-locked nucleic acid (dT+) with 5’ modified with acrydite group) in length were adopted in step 1.4, and the resultant fluorescence intensities were compared.

As shown in Figure 16, the anchor with FAM group labeled reflect the anchoring fluorescence intensity and the intensity are measured using confocal microscope. The intensity comparison shows the 25 dT is somewhat better than other lengths.

Example 5: Spatially transcriptomic analysis using different gel compositions

In this Example, to optimize gel stability and expansion factor, we further explored four different gel compositions and performed the 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 and 0.8%PAE and 0.8M NaCl; Group 4 (SMA group) : 8.625%SMA, 3.8%AA, 0.2%Bis and 0.8M HCl.

From the mechanical perspective, all the gels show acceptable stability and expansion, although HCL and NaCl group show better stability. The transcriptomics results through different gel composition shows HCl group is more compatible with the protocol.

Mechanical stability measurements were performed using a uniaxial tensile machine (Univert, CELLSCALE) with a 2.5-N-load cell at a deformation rate of 53.33%of height/min. For four different hydrogels, the test was carried out on round column-shaped samples with the size 1mm (radius) and 1-5mm (height) . The height of the sample between the two clamps was subject to the actual test.

Figure 17a shows representative stress-strain curves for expanded Bis (LEF=2.8) , SA (LEF=N/A) , NaCl (LEF=5.33) , and HCl (LEF=5.6) hydrogels (n=3, 3, 3, and 3 technical replicates, respectively) . The stability curve shows HCL and NaCl groups have better stability in Figure 17a. Figure 17b shows the average parameters of each group (3 replicates) including LEF (linear expansion factor) , stress (assessed by the force divided by the initial cross-sectional area of the hydrogel sample) , and the strain (assessed by displacement divided by height) . In Figure 17c, the transcriptomics results through different gel composition show HCl group is more compatible with the protocol. The gel composition of Figure 17c is shown in Figure 17d, “. ST/. V” stands for different brand of kits used.

In conclusion, the ExiST and neoExiST workflows could achieve the identification of spatial transcriptomics in micro-region tissue at scalable, user-defined resolution without a need for special equipment or supplies using standard NGS protocols and procedures. The RNA-seq results validate the high quality and coverage of transcriptomics data without any artifacts or biases compared to benchmark standards.

There are several technological advancements and solutions implemented in ExiSTP and neoExiSTP, which were not realized previously for any of the other existing spatial transcriptomic methods. First, we showed that we can perform reversible anchoring of mRNA and proteins in one step, combining reagents for both biomolecules. Second, we found conditions that allow sample homogenization without impact on mRNA integrity and localization in expanded samples, followed by microdissection of regions of interest. Third, we demonstrated and implemented mRNA release from small microdissected gel pieces can be performed in RT-PCR enzymatic mixture for direct generation of cDNA. By doing so, we proved that spatially resolved transcriptomics can be carried out without needing RNA-binding DNA-based chips or spatial barcoding. We also do mRNA release for samples that retain proteins, potentially enabling downstream proteomic analysis. The microdissection step for spatial sampling provides a unique opportunity to define the spatial resolution of analysis by flexibly choosing microdissection dimensions. None of the any other method allows for scalable lateral resolution, they all operate at a predefined and fixed lateral resolution. Overall, ExiST is a technologically novel and unique approach to performing spatially resolved transcriptomics, which does not require any specialized or sophisticated equipment and is simultaneously compatible with any regular DNA sequencing platform including all NGS methods.

These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is intended that such variations are included within the scope of the invention.

Claims

A method for physically expanding a biological sample and spatially analyzing biomolecules within the sample, comprising:

(a) incubating the sample with a mRNA probe, wherein the mRNA probe can hybrid to mRNAs in the sample and has been modified to comprise a hydrogel-reactive chemical group;

(b) perfusing the sample with a hydrogel precursor solution or embedding the sample in the hydrogel precursor solution, and polymerize to form a sample-hydrogel composite;

(c) physically expanding the sample-hydrogel composite;

(d) selecting a region (s) of interest from the expanded sample-hydrogel composite and dissecting the region (s) ; and

(e) sequencing the mRNAs from the dissected region (s) .
A method for physically expanding a biological sample and spatially analyzing biomolecules within the sample, comprising:

(a) incubating the sample with a mRNA probe and a reverse transcriptase, wherein the mRNA probe can hybrid to mRNAs in the sample and has been modified to comprise a hydrogel-reactive chemical group, and wherein the mRNAs in the sample are reverse transcribed to cDNAs by the reverse transcriptase, with the mRNA probe acting as a primer;

(b) perfusing the sample with a hydrogel precursor solution or embedding the sample in the hydrogel precursor solution, and polymerize to form a sample-hydrogel composite;

(c) physically expanding the sample-hydrogel composite;

(d) selecting a region (s) of interest from the expanded sample-hydrogel composite and dissecting the region (s) ; and

(e) sequencing the cDNAs from the dissected region (s) .
The method of claim 1 or 2, wherein prior to or during step (b) , the sample is further incubated with a bifunctional protein anchor, the bifunctional protein anchor comprises a protein-reactive chemical group and a hydrogel-reactive chemical group,

for example, during step (a) , the sample is incubated with both the mRNA probe and the bifunctional protein anchor in a hybridization buffer; or the sample is incubated with the mRNA probe first, and then incubated with the bifunctional protein anchor.
The method of claim 3, wherein the method further comprises extracting peptides or proteins from the dissected region for proteomic identification, e.g. by mass spectrometry analysis, such as LC-MS/MS and HPLC-MS.
The method of any of the preceding claims, wherein the mRNA probe comprises a 10-40 nucleotide sequence comprising dT nucleotide, an analogue and/or derivative of dT nucleotide such as thymidine-locked nucleic acid (dT+) ,

for example, the mRNA probe comprises a 15-35 nt nucleotide sequence consisting of alternating dT and dT+.
The method of claim 5, wherein the mRNA probe is modified with a hydrogel-reactive chemical group selected from acrydite, a primary amino group, azide, a Uni-Link^TM amino modifier, and any combination/mixture thereof,

for example, the mRNA probe is 5’ modified with acrydite.
The method of any of the preceding claims, wherein the method further comprises subjecting the sample-hydrogel composite to homogenization between step (b) and step (c) .
The method of claim 7, wherein the homogenization is performed by a physical, chemical, physicochemical, and/or enzymatic treatment of the sample-hydrogel composite,

for example, the homogenization is performed by treating the sample-hydrogel composite with a protease, an alkaline buffer (such as a detergent containing alkaline buffer) , or heating in a buffer.
The method of claim 7 or 8, wherein the homogenization is performed by treating the sample-hydrogel composite with Trypsin, SDS or Proteinase K.
The method of any of claims 3-9, wherein the bifunctional protein anchor is selected from N-Succinimidyl Acrylate (NSA) , N- (Allyloxycarbonyloxy) -succinimide (NAS) , and any combination/mixture thereof.
The method of claim 3 or 4, wherein the method comprises:

(1) incubating the sample with both the mRNA probe and the protein anchor in a hybridization buffer in step (a) , and treating the sample sample-hydrogel composite with Trypsin for homogenization between step (b) and step (c) ;

(2) incubating the sample with both the mRNA probe and the protein anchor in a hybridization buffer in step (a) , and treating the sample sample-hydrogel composite with SDS for homogenization between step (b) and step (c) ;

(3) incubating the sample with the mRNA probe first and then with the protein anchor, and treating the sample sample-hydrogel composite with Trypsin for homogenization between step (b) and step (c) ; or

(4) incubating the sample with the mRNA probe first and then with the protein anchor, and treating the sample sample-hydrogel composite with SDS for homogenization between step (b) and step (c) .
The method of any of the preceding claims, 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) , Trimethylolpropane Propoxylatetriacrylate (TPT) , Pentaerythritol triacrylate (PA) , Dipentaerythritol penta-/hexa-acrylate (DPHA) , Trimethylolpropane triacrylate (TTA) , Di(trimethylolpropane) -tetraacrylate (DiTA) , Trimethylolpropane Trimethacrylate (TTMA) , Glycerol propoxylate (1PO/OH) triacrylate (GPT) , Trimethylolpropane ethoxylate triacrylate (TET) , Pentaerythritol allyl ether (PAE) , Sodium 4-hydroxy-2-Methylenebutanoate (SHMB) , N, N’-Dimethylaminopropyl acrylamide (DMPAA) , and Acrylamide (AA) .
The method of claim 12, wherein the hydrogel precursor solution comprises any of the following groups 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.
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 before polymerization is started.
The method of any of the preceding claims, further comprising staining the sample either with a label or tag before, during or after steps (a) , (b) , (c) , or after homogenization.
The method of claim 15, wherein the sample is subjected to DNA staining, RNA staining and/or protein staining.
The method of any of the preceding claims, wherein during step (c) , the sample-hydrogel composite is incubated with a solvent or aqueous solution such as pure water or aqueous buffer for a sufficient amount of time for expansion.
The method of any of the preceding claims, wherein the sample is selected from cell (such as cultured cells) , biological tissue (such as tissue section) , specimen, biopsy, intact organ and parts thereof, and whole organisms (such as bacteria, fungus, or viruses) .
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 –400 μm.
The method of any of the preceding claims, further comprising imaging the expanded sample-hydrogel composite via microscopy to acquire super-resolution imaging.
The method of any of the preceding claims, wherein dissecting the region is performed manually via a punch or with assistance of robotic or mechanical micromanipulating system, e.g. with LCM dissection.
The method of claim 21, wherein the region of interest has a diameter of 1-1000 μm.
The method of claim 1, wherein step (e) comprises subjecting the mRNAs released from the dissected region to reverse transcription, cDNA amplification (e.g. by RT-PCR) and cDNA library construction,

optionally, the mRNAs are released from the dissected region by incubating the dissected gel in SSC buffer at 40-50℃.
The method of claim 2, wherein step (e) comprises subjecting the cDNAs in the dissected region to cDNA amplification (e.g. by PCR) and cDNA library construction.
A sample-hydrogel composite produced by the method of any of the preceding claims.
A kit, wherein the kit comprises:

(a) a mRNA probe capable of hybridizing to mRNAs and crosslinking with the hydrogel polymer chain;

(b) hydrogel precursors or a hydrogel precursor stock solution comprising the hydrogel precursors;

(c) optionally, a bifunctional protein anchor such as NSA, NAS or a combination thereof; in one or more containers.
The kit of claim 26, wherein the mRNA probe comprises a 10-40 nucleotide sequence comprising dT nucleotide, an analogue and/or derivative of dT nucleotide such as thymidine-locked nucleic acid (dT+) ,

for example, the mRNA probe comprises a 15-35 nt nucleotide sequence consisting of alternating dT and dT+.
The kit of claim 27, wherein the mRNA probe is modified with a hydrogel-reactive chemical group selected from acrydite, a primary amino group, azide, a Uni-Link^TM amino modifier, and any combination/mixture thereof,

for example, the mRNA probe is 5’ modified with acrydite.
The kit of any of claims 26-28, wherein hydrogel precursors are selected from any 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.
The kit of any of claims 26-29, wherein the kit further comprises one or more of the following components:

a container comprising a gelation activator or accelerator selected from VA-044, TEMED, APS and potassium persulfate;

a container comprising a hybridization buffer such as an SSC buffer;

a container comprising a homogenization buffer, optionally the homogenization buffer comprises trypsin or SDS;

a container comprising a wash buffer such as an SSC buffer or PBS buffer;

a container comprising a labeling agent;

a container comprising a fixative such as ethanol;

a container comprising reagents for reverse-transcription of mRNAs;

a container comprising reagents for cDNA PCR;

a container comprising reagents for cDNA library preparation; and

a container comprising reagents for peptide digestion for mass spectrometry identification.
The kit of any of claims 26-30, wherein the kit further comprises one or more of the following components:

a gelation chamber configured to accommodate the sample prior to gelation and optionally a cover to seal the gelation chamber;

an imaging chamber configured to accommodate the sample after gelation;

a gel handling device; and

a cooling device, such as an ice bag.
A system for spatial transcriptomics analysis of a biological sample, comprising:

a hybridization module for anchoring an mRNA probe to the mRNAs in the biological sample, optionally the mRNAs are reverse transcribed to cDNAs;

a gelation module for hydrogel polymerization to form a sample-hydrogel composite;

a homogenization module for homogenization of the sample-hydrogel composite;

a staining module for staining DNAs, RNAs and/or proteins;

a DNA amplification module for PCR amplification of the reverse transcribed cDNAs from a region of interest of the sample-hydrogel composite; and

a cDNA library preparation module for constructing a library of the cDNAs for sequencing; and optionally, mass spectrometry analysis module for pre-processing of the samples obtained by local sampling before mass spectrometry and which is connected with a mass spectrometry detection device.