Single-nucleus high-resolution multimodal spatial genomics

Abstract

The embodiments disclosed herein provide spatially tagged nuclei that are compatible with any genomic or multi-omic single cell / nucleus assay, enabling the generation of spatially resolved single cell sequencing libraries with single cell resolution.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 434,345, filed December 21, 2022. The entire contents of the above application are incorporated herein by reference in their entirety.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under grant numbers HG010647, CA246632, and NS132135 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Reference to electronic sequence list The contents of the electronic sequence listing (BROD-5740WP_ST26.xml, size 26,852, created December 21, 2023) are incorporated herein by reference in their entirety.

[0004] The subject matter disclosed herein generally relates to spatially resolved single-nucleus sequencing libraries obtained using compositions and methods for tagging nuclei with spatial barcodes. [Background technology]

[0005] Recent technological advances have enabled high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are assembled. However, these measurements lack the ability to routinely and easily spatially identify profiled cells. Approaches for spatial monitoring of genomics data in single cells within tissue samples include traditional histological approaches, such as fixing and staining tissue sections and assessing the presence of individual transcripts across the entire visible area of ​​the fixed tissue section on a microscope slide, as well as more recent in situ techniques for transcriptome monitoring. However, these techniques are laborious to apply, technically challenging, limiting their degree of multiplexing, and suffer from low spatial capture resolution across the array (i.e., only providing a resolution of approximately 100–200 μm).

[0006] Spatially resolved capture of macromolecules from single nuclei is required. Current spatially resolved (multi-)omics tools use thin tissue section inputs, capturing an unclear proportion of each cell, resulting in incomplete cell / nuclei cut during this procedure (e.g., in situ sequencing, in situ capture). Furthermore, some techniques profile macromolecules within voxels that can contain multiple cells and require deconvolution (e.g., 10X Visium, Slide-seq, HOST, LCM), limiting some analyses. Recent techniques (e.g., sci-Space, XYZeq) enable the capture of transcriptomics data from single nuclei while preserving spatial information, but are limited by low capture rates and spatial resolution. Furthermore, they are limited to profiling based on a single combined indexing technique.

[0007] Therefore, there is a need for improved approaches that provide spatial genomics profiling at resolutions approaching single-cell resolution, and more generally, improved approaches that provide spatial macromolecular abundance data (e.g., RNA expression, DNA and / or protein abundance) at resolutions approaching single-cell resolution.

[0008] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention

[0009] In one aspect, the invention provides a method of generating spatially tagged nuclei for use in single-cell genomics, the method comprising: placing a tissue sample on a sequence-verified spatial array, the spatial array comprising nucleic acid sequences comprising spatial barcodes attached to the array via cleavable linkers, the spatial barcodes being the same for each individual location on the array but different for any other location on the spatial array; cleaving the linker to deliver the spatially barcoded nucleic acids to nuclei in the tissue sample; and isolating the tagged nuclei from the tissue sample. In certain embodiments, the method further comprises preparing a single-cell genomics sequencing library using the isolated tagged nuclei, wherein nucleic acid sequences each comprising a cellular barcode sequence identifying a cell of origin and optionally a unique molecular identifier (UMI) are captured from each spatially tagged nucleus to create a nucleic acid sequence comprising a spatial barcode and a cellular barcode, such that genomics data of each single cell can be identified by the cellular barcode, and the spatial location of the same single cell in a tissue can be identified by the same cellular barcode.

[0010] In certain embodiments, the spatial barcode is delivered to the nucleus by diffusion.In certain embodiments, before step (a), the spatial array is sequence verified by in situ sequencing of the nucleic acid sequence comprising the spatial barcode, thereby generating an index of the spatial barcode on the array.In certain embodiments, the in situ sequencing is carried out by sequencing by ligation or sequencing by synthesis.

[0011] In certain embodiments, the spatial array comprises a solid support fixed to each location on the array and linked to a nucleic acid sequence comprising a spatial barcode via a cleavable linker, wherein the spatial barcode is identical for each solid support but different for any other solid support within the spatial array. In certain embodiments, the solid support is covalently fixed to the spatial array. In certain embodiments, the solid support is fixed to the spatial array by a vinyl polymer. In certain embodiments, the solid support is a bead. In certain embodiments, the bead is a polystyrene bead. In certain embodiments, the diameter of the bead is 50 μm or less. In certain embodiments, the bead is 1, 3, 10, 15, or 20 μm.

[0012] In certain embodiments, the linker is a photocleavable, chemically cleavable, or enzymatically cleavable linker. In certain embodiments, the spatial barcode nucleic acid comprises a polyA sequence for capture by a cellular barcode nucleic acid comprising a polyT sequence. In certain embodiments, the tissue sample is treated to permeabilize nuclei. In certain embodiments, the tissue sample is permeabilized in a buffer that increases diffusion of the spatial barcode nucleic acid. In certain embodiments, the spatial barcode nucleic acids are of different lengths. In certain embodiments, the spatial barcode nucleic acid further comprises one or more modifications that promote diffusion into the nucleus. In certain embodiments, the spatial barcode nucleic acid is modified by the addition of one or more lipid or cholesterol groups. In certain embodiments, the spatial barcode nucleic acid further comprises one or more fluorescent labels that can be used to identify tagged nuclei.

[0013] In certain embodiments, the tissue sample is a fresh frozen tissue section. In certain embodiments, the tissue sample is a fresh unfixed tissue section. In certain embodiments, the tissue sample is a fixed tissue section.

[0014] In certain embodiments, the location of each cell in a tissue sample is computationally determined based on sequencing the library. In certain embodiments, the cell barcode nucleic acid comprises a UMI sequence, and the location of each cell in the tissue is determined based on the number of UMIs sequenced for each spatial barcode that has the same cell barcode sequence.

[0015] In certain embodiments, the single-cell genomics sequencing library is a single-nuclear RNA sequencing library (snRNA-seq). In certain embodiments, the single-cell genomics sequencing library is a single-cell DNA accessibility library. In certain embodiments, the single-cell genomics sequencing library is a single-cell ATAC sequencing library (ATAC-seq). In certain embodiments, the single-cell genomics sequencing library is a single-cell chromatin immunoprecipitation (ChIP) sequencing library. In certain embodiments, the single-cell genomics sequencing library is a single-cell genome sequencing library. In certain embodiments, the single-cell genomics sequencing library is a single-cell DNA methylation sequencing library. In certain embodiments, the single-cell genomics sequencing library is a single-cell Hi-C sequencing library. In certain embodiments, the single-cell genomics sequencing library is a single-cell enzyme-linked chromatin profiling sequencing library. In certain embodiments, the single-cell genomics sequencing library is a single-cell genomics and transcriptome sequencing library (G&T-seq). In certain embodiments, the single-cell genomics sequencing library is a single-cell proteome library.

[0016] In another aspect, the present invention provides a kit comprising a plurality of solid supports attached via a cleavable linker to a nucleic acid sequence comprising a spatial barcode and a capture sequence, wherein the spatial barcode is the same for each solid support but different for any other solid support in the spatial array, and the capture sequence is the same across all solid supports. In certain embodiments, the solid support further comprises a chemical linking moiety for covalently immobilizing the solid support to the array. In certain embodiments, the solid support is a bead. In certain embodiments, the bead is a polystyrene bead. In certain embodiments, the bead has a diameter of 50 μm or less. In certain embodiments, the bead is 1, 3, 10, 15, or 20 μm, preferably 10 μm. In certain embodiments, the linker is a photocleavable, chemically cleavable, or enzymatically cleavable linker. In certain embodiments, the capture sequence comprises a polyA sequence. In certain embodiments, the spatial barcode nucleic acids attached to the solid supports vary in length. In certain embodiments, the spatial barcode nucleic acid further comprises one or more modifications that facilitate diffusion into the nucleus. In certain embodiments, the spatial barcode nucleic acid is modified by the addition of one or more lipid or cholesterol groups. In certain embodiments, the spatial barcode nucleic acid further comprises one or more fluorescent labels that can be used to identify the tagged nuclei.

[0017] These and other aspects, objects, features, and advantages of the exemplary embodiments will become apparent to those skilled in the art upon consideration of the following detailed description of the exemplary embodiments. [Brief explanation of the drawings]

[0018] The features and advantages of the present invention will be understood by reference to the following detailed description and accompanying drawings that set forth illustrative embodiments in which the principles of the invention may be utilized.

[0019] [Figure 1] FIG. 1 illustrates a slide tag protocol. [Figure 2A]This is a molecular reaction of slide tagging. Oligonucleotides cleaved from beads (SEQ ID NOs: 1-7) are used to tag nuclei, and the tagged nuclei are encapsulated in droplets with cell barcode beads, where the mRNA and spatial barcode are captured by the cell barcode beads. [Figure 2B] This is the molecular reaction of slide tags (SEQ ID NOs: 6 to 12). It separates spatial barcodes and RNA by size. [Figure 2C] Molecular reaction of slide tags (SEQ ID NOs: 13-21) Generation of sequencing library. [Figure 3] A computational workflow for generating spatially resolved single-nucleus RNA-seq maps. [Figure 4] A comparison of slide tag with other spatial technologies. [Figure 5A] Slide tagging results in the mouse hippocampus. The UMAP plot shows cells identified by snRNA-seq. [Figure 5B] Results of slide tagging in the mouse hippocampus. Expression of marker genes projected onto a UMAP plot. [Figure 5C] 5C shows the results of slide tagging in the mouse hippocampus. 5C shows the mapping of cell types on tissue samples. [Figure 6] Volcano plot showing the increase in the proportion of spatially localizable signals and cells since the initial slide tag experiment. [Figure 7A] Slide tagging results in the mouse hippocampus. Spatial map of slide tagging compared to immunohistochemistry of sections from the same specimen using the same marker gene. [Figure 7B] Slide tag results in the mouse hippocampus. Spatial location of specific cell types. [Figure 8A]Slide tagging results for a mouse embryo. Images of embryo sections and UMAP plots showing clustering of single nuclei based on snRNA-seq data from the sections. The UMAP plots show cell types, and graphs show the percentages of cell types. [Figure 8B] Slide tag results in mouse embryos. Spatial location of nuclei in sections. [Figure 8C] 8C shows the results of slide tagging in mouse embryos. Spatial locations of brain regions identified by slide tagging compared to histology images. [Figure 8D] 1 shows the results of slide tagging in a mouse embryo. Images of slide tagging UMIs in sections and histology images of the location in the sections. [Figure 8E] 1 shows slide tag results in mouse embryos. Images of slide tags showing the spatial location of nuclei expressing the indicated marker genes in sections, and histology images of the section locations. [Figure 8F] Slide tagging results in a mouse embryo. Spatial location of cell types in the section. The spatial location of different neuronal clusters is also shown. [Figure 8G] Slide tagging results in mouse embryos. Spatial location of individual neuron clusters. [Figure 9A] Pseudo-time results of slide tagging. The panels show the dynamic events that occur during neuronal development. [Figure 9B] Pseudotime results of slide tagging: UMAP plot and specific clusters analyzed in pseudotime, spatially resolved pseudotime analysis, and expression of specific markers during pseudotime. [Figure 10] Pseudo-time results for slide tagging. The UMAP plot shows the pseudo-time values ​​and the spatial location of the pseudo-time values. [Figure 11] Slide tag results in a mouse embryo. The spatial locations of cell types in a transverse section are shown. The spatial locations of various neuronal clusters are also shown. [Figure 12]This is a panel that explains three-dimensional (3D) slide tags. [Figure 13A] 1 is a comparison of the thickness of tissue sections, and FIG. 2 is a diagram showing nuclei in tissue sections. [Figure 13B] Comparison of tissue section thickness. UMAP plot of nuclei from 20 μm sections and graph showing percentage of each cell type. [Figure 13C] Comparison of tissue section thickness. UMAP plot of nuclei from 40 μm sections and graph showing percentage of each cell type. [Figure 14A] Plate-based slide tag. (SEQ ID NO: 22) Flow diagram showing single-cell genome and transcriptome analysis. [Figure 14B] 1 is a plate-based slide tag. FIG. 2 is a flow diagram for determining spatially resolved genomic and transcriptomic data using tagged nuclei. [Figure 15A] Capture of spatial barcodes and mRNA in droplets (10X GEMS and beads). 10X beads contain capture sequence 1 oligonucleotides for capturing spatial barcodes and poly(dT) oligonucleotides for capturing mRNA. Template switching oligonucleotides (TSOs) add adapter sequences to the RT products. A spatial barcode sequencing library and a gene expression library are obtained. [Figure 15B] Reverse transcription and extension performed on GEM droplets. [Figure 15C] cDNA amplification of RT products. [Figure 15D] The prepared sample index library. [Figure 16A] Schematic of a slide-tag experiment for profiling the mouse hippocampus. The output from an snRNA-seq experiment is a cell x gene matrix and an accompanying spatial coordinate matrix. [Figure 16B] A dimensionality reduction plot highlighting a population of cell types is shown, each of which has an associated cluster of spatial barcodes. [Figure 17] Slide tagging enables single-nucleus spatial transcriptomics in the mouse hippocampus. Slide tagging experiments show the spatial localization of nuclei in the mouse hippocampus, and cells are color-coded according to cell type annotation. Spatial expression of known marker genes was compared with in situ hybridization data from the Allen Mouse Brain Atlas (scale, normalized mean counts). [Figure 18] Spatial resolution measurements and slide-tag snRNA-seq in the mouse hippocampus allow characterization of deep and superficial sublayers of the mouse hippocampal CA1 region. 10 μm Nissl-stained sections (left) were taken adjacent to slide-tag profiled sections (right). The CA1 nuclei were subset in each case, and a line was fitted to measure the midpoint of this structure. For slide-tag, nuclei were selected based on cell type assignment, and two spatial outliers were removed. For Nissl, nuclei were computationally segmented. The orthogonal distance from this midpoint was then calculated, and points were shaded according to this distance. [Figure 19] Spatial resolution measurements and slide-tagging snRNA-seq in the mouse hippocampus enable characterization of deep and superficial sublayers of the mouse hippocampal CA1 region. PCA plots show cells in the CA1 cluster after subsetting, reprojection, and reclustering. Cells are shaded according to their new subcluster assignment. Cells are plotted according to their spatial location. Violin plots show gene expression differences between each subcluster and the spatial expression of these genes. [Figure 20] The sliding tags provide high molecular quality and spatial resolution. The schematic shows that each nucleus receives multiple spatially barcoded oligos from multiple different beads. The graph shows an estimate of spatial resolution of less than 10 microns. [Figure 21]Comparison metrics plotted for snRNA-seq compared to slide-tagged snRNA-seq performed on serial sections. Cell type percentage and average UMI per cell are plotted by cell type. Normalized average UMI counts were determined per gene across all cells. Normalized average counts were compared. [Figure 22] Slide-tag generates high-quality spatial single-cell data. Violin plot of log10-transformed genes and UMIs per nucleus (Slide-tag) or per 20 μm spatial spot (Slide-seqV2, DBiT-seq, or Xenium) in mouse brain. Elbow plot of the standard deviation of the principal components for Slide-tag snRNA-seq, Slide-seqV2, DBiT-seq, and Xenium in mouse brain. [Figure 23] Slide tags expand the repertoire of spatial genomics at single-cell resolution. Schematic showing how slide-tagged nuclei can be used for a variety of single-cell techniques. [Figure 24A] Spatially resolved snRNA-seq in genetically heterogeneous samples. Slide tagging was performed on human metastatic melanoma samples. Schematic diagram showing experimental design. H&E images of melanoma samples. [Figure 24B] Spatially resolved snRNA-seq in a genetically heterogeneous sample. Slide tagging was performed on a human metastatic melanoma sample. The spatial locations of transcriptome clusters and single cells are shown. [Figure 25A] Spatial separation of distinct tumor subpopulations. 25A Mapping of transcriptionally distinct melanoma subpopulations in space. [Figure 25B] Spatial segregation of different tumor subpopulations. Copy number variations were inferred from transcriptome data using inferCNV. [Figure 26A]Tumor clusters with distinct spatial proximity. Plots show the percentage of cells adjacent to tumor cells. The Y-axis indicates tumor clusters, and the X-axis indicates immune cell types. [Figure 26B] Figure 1 shows tumor clusters with distinct spatial neighborhoods. Figure 2 shows spatial plots of enrichment of CD8 T cell percentages in the neighborhood of each tumor. [Figure 27A] Different immune status and receptors between tumor clones. Differential expression of T cells between tumor lobes. [Figure 27B] Different immune status and receptors between tumor clones. (SEQ ID NOs: 23-24) T cell receptors (TCRs) recovered from slide tags of tumor 1 and tumor 2 are shown. Spatially mapped TCR clones in tumor 1 and tumor 2 are shown. [Figure 28] Downregulation of HLA loci in tumor 1. Copy number variations were inferred from transcriptome data using inferCNV (Chr6 boxed). [Figure 29A] Spatial multi-omics sequencing using slide tags. Clustering of single cells by RNA expression and chromatin accessibility using slide tags. [Figure 29B] Spatial multi-omics sequencing using slide tags. Chromatin accessibility and single gene expression in single cell types are shown in violin plots for each cell type. [Figure 29C] Spatial multi-omics sequencing using slide tags. 29C is a spatial map of a single cell. [Figure 30A] Epigenomic differences between Tumor 1 and Tumor 2 populations. Plot showing comparison of ATAC-seq and RNA expression in single cells (TNC in bold). [Figure 30B] Epigenomic differences between tumor 1 and tumor 2 populations. 30B is chromatin accessibility in each tumor and gene expression in TNC shown in violin plots for each tumor. [Figure 30C]Epigenomic differences between Tumor 1 and Tumor 2 populations. Single-cell spatial maps of TNC chromatin gene scores for Tumor 1 and Tumor 2. [Figure 31A] Figure 1. Tumor 1 cell state driven by spatially clustered TF motifs. Mesenchymal-like cell state gene expression in Tumor 1 and Tumor 2. [Figure 31B] Figure 1 shows the state of tumor 1 cells driven by spatially clustered TF motifs. Figure 2 shows that transcription factor motifs identified by ATAC-seq correlate positively and negatively with mesenchymal score. [Figure 32A] Postmortem human cortical slide tag profiling. Slide tagging was performed on a 5.5x5.5 area of ​​human prefrontal cortex. [Figure 32B] Slide tag profiling of postmortem human cortex. Spatial map showing all cortical cell types. [Figure 32C] Slide tag profiling of postmortem human cortex. Spatial map showing genetic markers for cortical eel species. [Figure 32D] Slide tag profiling of postmortem human cortex. Spatial maps and plots showing all cortical layer types. [Figure 32E] Slide tag profiling of postmortem human cortex. 32E is a spatial map showing CUX2 expression. [Figure 32F] Slide tag profiling of postmortem human cortex. Spatial map showing RORB expression. [Figure 32G] Slide tag profiling of postmortem human cortex. 32G is a spatial map showing FOXP2 expression. [Figure 33A] Slide tag profiling of human tonsils. snRNA-seq data from tonsils. Clustering by cell type is shown. [Figure 33B] Slide tag profiling of human tonsils. Spatial maps of single cells and H and E staining of tissue. [Figure 34A] 1. The use of spatially mutated genes to define dark and light regions in germinal centers. 2. Schematic of a germinal center with light and dark regions. [Figure 34B] Spatially varying genes were used to identify dark and light regions of germinal centers, and germinal center slide tags were used to cluster cell types. [Figure 34C] Using spatially variable genes to identify dark and bright regions of germinal centers. B cell clusters and marker gene expression. Spatial map of germinal center B cells. [Figure 35A] This designates spatially significant receptor-ligand interactions. It is a conventional method for inferring receptor-ligand interactions using single-cell data. [Figure 35B] Designating spatially significant receptor-ligand interactions. Using spatial information to curate receptor-ligand interactions in germinal centers. [Figure 36A] Slide tagging generates high-quality spatial multi-omics data. A schematic diagram showing the experimental design for multimodal analysis of P1 mouse brain. [Figure 36B] Slide tags generate high-quality spatial multi-omics data: single-cell ATAC-seq and snRNA-seq from the same single nucleus. [Figure 37A] Slide tags enable spatial ATAC & RNA-seq within the same single cell, clustering single nuclei by RNA expression, accessible chromatin (ATAC), and weighted nearest neighbor (WNN) analysis (see, e.g., Hao Y, Hao S, Andersen-Nissen E, et al. Integrated analysis of multimodal single-cell data. Cell. 2021;184(13):3573-3587.e29). [Figure 37B]Slide tags enable spatial ATAC & RNA-seq within the same single cell. (A) Plot of clusters in space. [Figure 38A] Transcription and open chromatin can define spatially distinct elements. Spatial map of the outer layers of the developing cortex (isocortex, posterior callosal cortex, and subiculum) using RNA expression. [Figure 38B] Transcription and open chromatin can define spatially distinct elements. Representative top RNA hits. [Figure 38C] This allows us to define spatially distinct elements of transcription and open chromatin. Clustering using ATAC and top chromatin accessibility hits. [Figure 38D] Transcription and open chromatin can define spatially distinct elements. Transcription factor motifs and top hits are mutated along this axis. [Figure 39] These are oligonucleotide variations for slide tags. (SEQ ID NOS: 2-3, 25-28) Capture sequence 1 can be a capture sequence on a spatial barcode oligonucleotide. Capture sequence 1 is complementary to the 10X Genomics chromium 3' v3.1 platform. Oligonucleotide variation 1 indicates the addition of a capture sequence using 10X capture sequence 1 and splint. Oligonucleotide variation 2 indicates a spatial barcode oligonucleotide with capture sequence 1 downstream of the spatial barcode. Oligonucleotide variation 3 indicates a capture sequence as a polyA sequence used to capture a polyT sequence. Oligonucleotide variation 4 indicates a chemical modification that can be present on either of the spatial barcode oligonucleotides to promote nuclear uptake or retention (e.g., cholesterol-TEG, squalene, fatty acid, or α-tocopherol). Oligonucleotide variation 5 indicates a chemical modification that can be present on either of the spatial barcode oligonucleotides to facilitate detection and quantification of the spatial barcode (e.g., a trifunctional linker that can have a fluorescent dye or a combination of a fluorescent dye and any of the above chemical modifications).

[0020] The drawings shown herein are for illustrative purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION OF THE INVENTION

[0021] General definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology can be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M.A. Usubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.); Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.); Antibodies A Laboratory Manual, 2nd edition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers,Inc.,1995(ISBN 9780471185710);Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

[0022] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly indicates otherwise.

[0023] The term "optionally" or "optionally" means that the subsequently described event, circumstance, or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0024] The recitation of numerical ranges by endpoints includes not only the recited endpoints but also all numbers and fractions subsumed within each range.

[0025] As used herein, the term "about" or "approximately," when referring to a measurable value such as a parameter, amount, duration, etc., is meant to encompass variations from the specified value, for example, variations of no more than + / - 10%, no more than + / - 5%, no more than + / - 1%, and no more than + / - 0.1% from the specified value, insofar as such variations are appropriate for practice in the disclosed invention. It is to be understood that the value to which the "about" or "approximately" modifier refers is itself specifically, preferably disclosed.

[0026] As used herein, a "biological sample" can contain whole cells and / or viable cells and / or cell debris. A biological sample can contain (or be derived from) a "body fluid." The present invention encompasses embodiments in which the body fluid is selected from amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, earwax (ear wax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal mucus and phlegm), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof. Biological samples include cell cultures, body fluids, and cell cultures from body fluids. Body fluids can be obtained from a mammal, for example, by paracentesis and other collection or sampling procedures.

[0027] As used herein, the terms "subject," "individual," and "patient" are used interchangeably and refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, murines, apes, humans, farm animals, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0028] Various embodiments are described below. It should be noted that specific embodiments are not intended to be exhaustive or limiting of the broader aspects discussed herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment and may also be practiced in any other embodiment(s). Throughout this specification, references to "one embodiment," "one embodiment," or "exemplary embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment," "in one embodiment," or "exemplary embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, although they may. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, some embodiments described herein include some features but not other features included in other embodiments, meaning that combinations of features from different embodiments are within the scope of the present invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0029] See US Patent Application Publication No. US20210123040A1. Also, “Slide-tags: scalable, single-nucleus barcoding for multi-modal spatial genomics,” Andrew JCRussell, Jackson A. Weir, Naeem M. Nadaf, Matthew Shabet, Vipin Kumar, Sandeep Kambhampati, Ruth Raichur, Giovanni J. Marrero, Sophia Liu, Karol S. Balderrama, Charles R. Vanderburg, Vignesh See also Shanmugam, Luyi Tian, ​​Catherine J. Wu, Charles H. Yoon, Evan Z. Macosko, Fei Chen, bioRxiv 2023.04.01.535228. See also Russell, AJC, Weir, JA, Nadaf, NM et al. Slide-tags enables single-nucleus barcoding for multimodal spatial genomics. Nature (2023). All publications, published patent documents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

[0030] overview The embodiments disclosed herein provide spatially tagged nuclei compatible with any genomic or multi-omic single-cell / nucleus assay, enabling the generation of spatially resolved single-cell sequencing libraries with single-cell resolution. Previous methods have been limited to detecting mRNA expression and the amount of mRNA captured via diffusion from the cell (see, e.g., Stickels RR, Murray E, Kumar P, et al. Highly sensitive spatial transcriptomics at near-cellular resolution with Slide-seq V2. Nat Biotechnol. 2021;39(3):313-319). The present invention overcomes these limitations by tagging nuclei with spatial barcodes, making analyte capture independent of diffusion from the cell. Nuclei can be completely lysed within the reaction volume to release all analytes (e.g., RNA) and capture all RNA. Furthermore, performing multi-omic spatial studies using slide-seq requires each omics measurement to be performed individually. The present invention eliminates the need to perform every spatial experiment twice or multiple times to obtain multi-omic spatial results. The present invention is based on the use of modified slide-seq arrays (polystyrene-supported beads bearing barcoded oligonucleotides) to deliver spatial barcodes to nuclei within fresh-frozen thin tissue sections. The use of slide-seq arrays and the lack of tissue fixation distinguish this invention from XYZeq and sci-Space. Furthermore, the structure of the spatial barcode allows for plate-, microfluidic-, or nanowell-based capture of macromolecules and spatial barcodes from single nuclei, in contrast to XYZeq and sci-Space, which require split-pool index-based profiling.

[0031] Current methods only require the delivery of one or more spatial barcode tags to permeabilized nuclei. The tagged nuclei can be preserved or directly used in single-cell genomics assays. In other words, the present invention integrates spatial profiling and single-cell sequencing. Furthermore, the present invention provides arrays in which the distance between different spatial barcodes is smaller than the size of a cell, enabling single-cell resolution. Furthermore, the use of fluorescently labeled spatial barcode tags allows only tagged nuclei to be used in subsequent single-cell genomics assays. Therefore, because a single tagged nucleus is used as input for single-nucleus / cell genomic assays, the analyte capture efficiency approaches the detection efficiency of non-spatially resolved single-cell genomic sequencing techniques. For example, RNA capture rates approach approximately 100% for single-nucleus RNA sequencing data.

[0032] Spatial barcoding of nuclei within tissue samples In an exemplary embodiment, a tissue sample is placed on an array containing spatial barcodes, and the spatial barcodes are released from the array and tag nuclei within the tissue. The tissue sample is permeabilized to allow the spatial barcodes to tag the nuclei. Tagged single nuclei are then isolated from the tissue sample.

[0033] Spatial Array In an exemplary embodiment, nuclei in a tissue sample are tagged with spatial barcode nucleic acids. A spatial barcode nucleic acid is a nucleic acid that is linked or connected to an array at a specific location and contains a barcode sequence. Thus, the spatial barcode can identify a location within the array. As used herein, the term "array" refers to a collection of features or sites that can be distinguished from one another depending on their relative positions. Different molecules at different sites of an array can be distinguished from one another depending on the site's location within the array. Each site of an array can contain one or more molecules of a particular type. For example, a site can contain a single target nucleic acid molecule with a particular sequence, or a site can contain multiple nucleic acid molecules with the same sequence (and / or its complementary sequence). The sites of an array can be different features arranged on the same substrate. Exemplary features include, but are not limited to, wells within a substrate, beads (or other particles) within or on a substrate, protrusions from a substrate, ridges on a substrate, or channels within a substrate. The sites of an array can be separate substrates, each holding a different molecule. Different molecules attached to individual substrates can be identified according to the substrate's location on the surface to which it is bound, or the substrate's location within a liquid or gel. Exemplary arrays in which distinct substrates are arranged on a surface include, but are not limited to, beads in wells, beads arranged on a flat surface (e.g., a slide), beads optionally captured on a flat surface (e.g., a layer of beads adhered to or otherwise stably associated with a slide (e.g., a layer of beads adsorbed to an elastomeric surface attached to a slide)), and the like. In exemplary embodiments, arrays of the invention comprise over 10,000 individual locations, each with a different spatial barcode. In exemplary embodiments, arrays of the invention comprise between 10,000 and over 1 million individual locations, each with a different spatial barcode.

[0034] As used herein, the term "feature" refers to a location within an array of a particular molecular species. A feature can contain only a single molecule, or it can contain a population of multiple molecules of the same species. Array features are typically discrete. Discrete features can be contiguous and have spaces between them. Feature size and / or spacing can vary to result in high-density, medium-density, or low-density arrays. High-density arrays are characterized by inter-site distances of less than about 15 μm. Medium-density arrays have sites separated by about 15-30 μm, while low-density arrays have sites separated by more than 30 μm. Arrays useful herein can have sites separated by, for example, less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. The disclosed devices or methods can be used to detect arrays with sufficient resolution to distinguish sites at the above densities or density ranges.

[0035] As used herein, the term "attached" refers to the state in which two things are bound, fixed, adhered, connected, or associated with each other. For example, an analyte such as a nucleic acid can be attached to a material such as a gel or solid support by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of electron pairs between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of electron pairs and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0036] Spatially barcoded nucleic acids In an exemplary embodiment, the spatial barcode nucleic acid is a nucleic acid sequence comprising a barcode sequence. In an exemplary embodiment, the spatial barcode nucleic acid is a nucleic acid sequence comprising, from 5' to 3', a cleavable linker, a barcode sequence, and a capture sequence. In an exemplary embodiment, the spatial barcode nucleic acid is blocked at the 3' end to prevent extension. Thus, upon capture by the cellular barcode, the cellular barcode sequence primes extension to the spatial barcode nucleic acid to add the spatial barcode sequence to the cellular barcode sequence, and the spatial barcode nucleic acid is not extended. In an exemplary embodiment, the spatial barcode nucleic acid comprises a barcode sequence that identifies a location on an array. As used herein, "barcode" refers to a short sequence of nucleotides (e.g., DNA or RNA) or a series of nucleotides in a nucleic acid that can be used to identify a nucleic acid, a property of a nucleic acid (e.g., the identity and optionally the location of the nucleic acid), or a manipulation (e.g., perturbation) performed on a nucleic acid that is used as an identifier of a target molecule and / or related molecule, such as a target nucleic acid, or an identifier of the source of the related molecule, such as the cell of origin. As used herein, the term "spatial barcode" or "spatial tag" refers to a series of nucleotides in a nucleic acid that can be used to identify a location on an array where the nucleic acid is immobilized. As used herein, the term "spatial barcode" or "spatial tag" is also intended to refer to a nucleic acid having a sequence that indicates a location. Typically, the nucleic acid is a synthetic molecule having a sequence that is not found in one or more biological specimens with which the nucleic acid is used. However, in some embodiments, the nucleic acid molecule may be naturally occurring, and the sequence of the nucleic acid may be naturally occurring, for example, in the biological specimen with which the nucleic acid is used. The location indicated by the spatial tag may be a location within or on a biological specimen, within or on a solid support, or a combination thereof. A barcode sequence can function as a spatial tag.In exemplary embodiments, the location of tags that function as spatial tags is determined only after a population of beads (each having a different barcode sequence) is arrayed on a solid support (optionally randomly arrayed on the solid support) and the sequencing of such bead-associated barcode sequences is determined in situ on the solid support.

[0037] The barcode sequence may be a sequence that naturally occurs in the organism from which the barcoded nucleic acid or tagged sample was obtained, or a sequence that does not naturally occur. The barcode sequence may be unique to a single nucleic acid species within a population, or may be shared by multiple different nucleic acid species within a population (e.g., all nucleic acid species attached to a defined location on an array or a single bead may have the same barcode sequence, but different defined locations or beads may each exhibit a different shared barcode sequence that helps identify the different location or bead). As a further example, each nucleic acid probe within a population may contain a barcode sequence that is different from all other nucleic acid probes within the population. Alternatively, each nucleic acid within a population may contain a barcode sequence that is different from some or most of the other nucleic acids within the population. In certain embodiments, one or more barcode sequences used in a biological specimen (e.g., a tissue sample) are not present in the genome, transcriptome, or other nucleic acids of the biological specimen. For example, a barcode sequence may have less than 80%, 70%, 60%, 50%, or 40% sequence identity with a nucleic acid sequence within a particular biological specimen. As used herein, the term "different" when used with respect to nucleic acids means that the nucleic acids have nucleotide sequences that are not identical to each other. Two or more nucleic acids can have different nucleotide sequences over their entire length.Or, two or more nucleic acids can have different nucleotide sequences over the majority of their length.For example, two or more nucleic acids can have different target nucleotide sequence parts for two or more molecules, but at the same time, they can also have the universal sequence part that is the same on two or more molecules.

[0038] As used herein, the term "biological specimen" means one or more cells, tissues, organisms, or portions thereof. Biological specimens can be obtained from a variety of organisms. Exemplary organisms include, but are not limited to, mammals, such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates (i.e., humans or non-human primates), plants, such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, canola, or soybeans, algae, such as Chlamydomonas reinhardtii, nematodes, such as C. elegans, insects, such as Drosophila melanogaster, mosquitoes, fruit flies, honeybees, or spiders, fish, such as zebrafish, reptiles, amphibians, such as frogs or Xenopus laevis, Dictyostelium discoideum, fungi, Pneumocystis carinii, tiger pufferfish, yeast, Saccharomyces cerevisiae or Schizosaccharomyces pombe, or Plasmodium falciparum. The target nucleic acid can also be derived from a prokaryote, such as a bacterium, E. coli, Staphylococcus aureus, or Mycoplasma pneumoniae, an archaea, a virus, such as Hepatitis C virus or human immunodeficiency virus, or a viroid. The specimen can be derived from a homogenous culture or population of the above organisms, or from a collection of multiple different organisms, for example, within a community or ecosystem.

[0039] In exemplary embodiments, the spatial barcode nucleic acid includes a universal sequence used to capture the spatial barcode nucleic acid to another nucleic acid sequence (e.g., a barcode nucleic acid that identifies a cell of origin). In some embodiments, the universal sequence is also referred to as a capture sequence or a handle sequence, e.g., a ligation, PCR, or indexing handle sequence. In preferred embodiments, the capture sequence is at the 3' end of the spatial barcode nucleic acid. In exemplary embodiments, the spatial barcode nucleic acid also includes a universal sequence used as a primer binding sequence. As used herein, the term "universal sequence" refers to a string of nucleotides that is common to two or more nucleic acid molecules, even if the molecules have sequence regions that differ from one another. A universal sequence present in different members of a population of molecules can enable the capture of multiple different nucleic acids using a population of universal capture nucleic acids that are complementary to the universal sequence. Similarly, a universal sequence present in different members of a population of molecules can enable the replication or amplification of multiple different nucleic acids using a population of universal primers that are complementary to the universal sequence. Thus, a universal capture nucleic acid or universal primer includes a sequence that can specifically hybridize to a universal sequence. The target nucleic acid molecule can be modified, for example, by attaching a universal adapter to one or both ends of different target sequences. Non-limiting examples of 3' universal sequences used to capture spatially barcoded nucleic acids include polyA sequences for capture by polyT sequences, or sequences complementary to commercially available capture sequences, such as tagmentation adapter sequences (e.g., the read 1 (read 1N) sequence on the beads of Chromium Next GEM Single Cell ATAC Reagent Kit v1.1 (10x Genomics, Pleasanton, CA, USA)).

[0040] As used herein, the term "poly T or poly A" when used with reference to a nucleic acid sequence refers to a run of two or more thiamine (T) or adenine (A) bases, respectively. The poly T or poly A can contain at least about 2, 5, 8, 10, 12, 15, 18, 20, 25, 30 or more T or A bases, respectively. Alternatively or additionally, the poly T or poly A can contain up to about 30, 25, 20, 18, 15, 12, 10, 8, 5, or 2 T or A bases, respectively.

[0041] In exemplary embodiments, spatial barcode nucleic acids do not require a UMI sequence because the UMI sequence is present on the single-cell / nuclear genomics assay capture sequence, which also includes the barcode sequence of the cell of origin. Thus, when spatial barcodes are captured in a single-cell assay, a UMI unique to each spatial barcode capture event is present in each sequencing read.

[0042] In exemplary embodiments, the spatial barcode nucleic acid is approximately 50-250 nucleotides in length. In one example, the spatial barcode nucleic acid includes a linker, a primer binding sequence common to all spatial barcode nucleic acids, a spatial barcode of approximately 6-50 nucleotides, preferably 6-20 nucleotides, and a capture sequence of approximately 6-50 nucleotides. The spatial barcode nucleic acid can also include additional sequences, for example, to modify the length of the sequence. In exemplary embodiments, the spatial barcode nucleic acid is single-stranded, preferably ssDNA. In some embodiments, single-stranded oligonucleotides diffuse better into the nucleus than double-stranded nucleotides. In exemplary embodiments, single-stranded DNA (ssDNA) specifically stains the nuclei of permeabilized cells but not intact cells.

[0043] In an exemplary embodiment, the spatial barcode nucleic acids on the spatial array have the same spatial barcode at each position, but different lengths at each position. Using spatial barcode nucleic acids with different lengths allows the spatial location of single cells to be identified in three dimensions, since short nucleic acids diffuse further into tissue sections than long nucleic acids. In an exemplary embodiment, the computational method can be used to determine the location of a single cell in a tissue section with multiple cell layers, for example, by quantifying the number of spatial barcodes with different lengths in a single nucleus.

[0044] The length of the barcode sequence can vary. Generally, the longer the sequence, the greater the number and variety of barcodes in the population. Typically, multiple probes all have barcodes of the same length (albeit with different sequences), but different probes can use barcodes of different lengths. The length of the barcode sequence can be at least 2, 4, 6, 8, 10, 12, 15, 20, or more nucleotides. Alternatively or additionally, the length of the barcode sequence can be up to 20, 15, 12, 10, 8, 6, 4, or fewer nucleotides. Examples of usable barcode sequences are described, for example, in U.S. Patent Publication No. 2014 / 0342921A1 and U.S. Patent No. 8,460,865, the contents of each of which are incorporated herein by reference.

[0045] Linker In exemplary embodiments, the spatially barcoded nucleic acid comprises a linker sequence for attachment to an array or solid support (e.g., a bead, as further described herein). In preferred embodiments, the linker is cleavable, such that the spatially barcoded nucleic acid can be released upon contact with or proximity to a tissue specimen. In exemplary embodiments, the cleavable linker is chemically cleavable, photocleavable, or enzymatically cleavable.

[0046] In a preferred embodiment, the linker is photocleavable. Photocleavable linkers that can be released by UV irradiation are available. A PC (photocleavable) spacer can be placed between DNA bases or between the oligo and the 5' modifier group. The spacer arm can be cleaved by exposure to UV light in the 300-350 nm spectral range. Cleavage releases the oligo with a 5'-phosphate group. Exemplary photocleavable linkers are commercially available (Integrated DNA Technologies, Inc., Coralville, Iowa) and are shown below. TIFF2026501229000002.tif38170

[0047] In other exemplary embodiments, the spatial barcode nucleic acid can contain one or more cleavable linkers that are cleaved, for example, upon application of an appropriate stimulus. For example, the cleavable sequence can be a photocleavable linker that can be cleaved by application of light, a chemically cleavable linker that can be cleaved by application of an appropriate chemical, or an enzymatically cleavable linker that can be cleaved by application of an enzyme.

[0048] Oligonucleotides with photosensitive chemical bonds (e.g., photocleavable linkers) have various advantages. They can be efficiently and rapidly cleaved (e.g., in nanoseconds and milliseconds). In some cases, a photomask can be used to ensure that only certain regions of the array are exposed to a cleavable stimulus (e.g., exposure to ultraviolet light, light, or laser-induced heat). When a photocleavable linker is used, the cleavage reaction is triggered by light, making it highly selective for the linker and resulting in biorthogonality. Non-limiting examples of photosensitive chemical bonds that can be used in the cleavage domain include those described in Leriche et al. Bioorg Med Chem. 2012 Jan 15;20(2):571-82, U.S. Publication No. 2017 / 0275669, and WO2020190509A9.

[0049] Spatially barcoded nucleic acid modifications In exemplary embodiments, the spatial barcode nucleic acid comprises a nucleotide modification to enhance diffusion into the nucleus. In exemplary embodiments, the spatial barcode nucleic acid is conjugated to a lipophilic or amphipathic moiety. Lipophilic molecules can associate with and / or insert into lipid membranes, such as cell membranes and nuclear membranes. Non-limiting examples of lipophilic molecules that can be used in the methods provided herein include sterol lipids, such as cholesterol, tocopherol, and their derivatives, lignoceric acid, and palmitic acid. Other lipophilic molecules that can be used in the methods described herein include amphipathic molecules that can vary in head group (e.g., charge, aliphatic content, and / or aromatic content) and / or fatty acid chain length (e.g., C12, C14, C16, or C18). For example, fatty acid side chains (e.g., C12, C14, C16, or C18) can be attached to glycerol or glycerol derivatives (e.g., 3-t-butyldiphenylsilylglycerol), which can also include, for example, cationic head groups. The spatial barcode nucleic acids disclosed herein can bind (directly or indirectly) to these amphipathic molecules, which can associate with and / or insert into membranes (e.g., nuclear membranes).

[0050] The spatial barcode nucleic acid can be attached to a lipophilic moiety (e.g., a cholesterol molecule). The spatial barcode nucleic acid can be attached to the lipophilic moiety via a linker, such as a tetraethylene glycol (TEG) linker. Other exemplary linkers include, but are not limited to, amino linker C6, amino linker C12, spacer C3, spacer C6, spacer C12, spacer 9, and spacer 18. The spatial barcode nucleic acid can be attached to a lipophilic moiety or linker at the 5' end of the spatial barcode nucleic acid. The linker can be a glycol or a derivative thereof. For example, the linker can be tetraethylene glycol (TEG) or polyethylene glycol (PEG). The spatial barcode nucleic acid can be releasably attached to the linker or lipophilic moiety (e.g., as described elsewhere herein for releasable attachment of nucleic acid molecules), allowing the spatial barcode nucleic acid, or a portion thereof, to be released from the lipophilic molecule. In an exemplary embodiment, a lipophilic moiety (eg, cholesterol) is indirectly attached to the oligonucleotide (eg, via hybridization or a ligand-ligand interaction such as biotin-streptavidin).

[0051] In exemplary embodiments, the spatial barcode nucleic acid includes a fluorescent label, such that tagged nuclei can be identified and separated from untagged nuclei. The efficiency of nuclei tagging can also be determined by quantifying the proportion of tagged nuclei. In exemplary embodiments, the lipophilic molecule can include a fluorescent moiety. Several different fluorophores can be readily attached to oligonucleotides, such as fluorescein and its tetra- and hexachlorinated derivatives, TET and HEX.

[0052] Sequence-verified arrays In an exemplary embodiment, the spatial array is sequence-verified. As used herein, "sequence-verified" refers to the sequence of the spatial barcode at each position in the array, or at substantially all positions, preferably more than 50, 60, 70, 80, or 90% of the positions, being known. In an exemplary embodiment, the array is sequence-verified because the spatial barcode attached to each position of the array is specifically positioned at each position. In an exemplary embodiment, the sequence of the spatial barcode at each position is determined by in situ sequencing. In an exemplary embodiment, the array is sequence-verified by in situ sequencing before placing a tissue sample on the array. In an exemplary embodiment, in situ sequencing is performed directly on the array by sequencing by ligation or sequencing by synthesis, and captured by a microscope.

[0053] In certain aspects of the present disclosure, in situ sequencing is performed on a surface-immobilized array, which can be performed by any art-recognized parallel (optionally massively parallel) in situ sequencing mode, examples of which include, inter alia, the aforementioned SOLiD™ method, a sequencing-by-ligation technique that can be performed in situ on a solid support (see, e.g., Voelkerding et al., Clinical Chem., 55-641-658, 2009; U.S. Pat. Nos. 5,912,148 and 6,130,073, the entire contents of which are incorporated herein by reference). In certain embodiments of the present disclosure, such sequencing can be performed on an array present on a standard microscope slide, optionally using a standard microscope with sufficient computing power to track and associate individual sequences with their spatial locations during the detection process. The present disclosure also employs fluidics, incubation times, enzyme mixtures, and imaging setups when performing in situ sequencing (see, e.g., US20210123040A1).

[0054] Sequencing techniques, such as sequencing by synthesis (SBS), are a convenient method for determining barcode sequences. SBS can be performed as follows: To initiate an initial SBS cycle, one or more labeled nucleotides, a DNA polymerase, an SBS primer, etc., can be contacted with one or more features on a bead or other solid support (e.g., the feature(s) where a nucleic acid probe is attached to a bead or other solid support). The feature into which the labeled nucleotide is incorporated by SBS primer extension can be detected. Optionally, the nucleotide can contain a reversible termination moiety that stops further primer extension when a nucleotide is added to the SBS primer. For example, a nucleotide analog with a reversible terminator moiety can be added to the primer to prevent further extension until a deblocking agent is delivered to remove the moiety. Thus, in embodiments using reversible termination, a deblocking reagent can be delivered to the bead or other solid support (before or after detection occurs). Washing can be performed between the various delivery steps. This cycle is repeated n times to extend the primer by n nucleotides, thus detecting a sequence of length n. Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use in the compositions, devices, or methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), PCT Publication Nos. WO91 / 06678, WO04 / 018497, or WO07 / 123744, U.S. Patent Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, or 7,405,281, and U.S. Patent Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0055] Ligation sequencing is a DNA sequencing method that uses an enzyme called DNA ligase to identify the nucleotide at a predetermined position in a DNA sequence.Unlike most currently popular DNA sequencing methods, this method does not use DNA polymerase to create a second strand.Instead, the mismatch sensitivity of DNA ligase enzyme is used to determine the underlying sequence of a target DNA molecule.Ligation sequencing reactions are also useful, including, for example, those described in Shendure et al.Science 309:1728-1732 (2005), or U.S. Patent No. 5,599,675 or U.S. Patent No. 5,750,341, each of which is incorporated herein by reference. Some embodiments can include hybridization sequencing procedures, such as those described in, for example, Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); or PCT Publication No. WO1989 / 10977, each of which is incorporated herein by reference. In both ligation sequencing and hybridization sequencing procedures, target nucleic acids (or amplicons thereof) present at sites on the array are subjected to repeated cycles of oligonucleotide delivery and detection. The compositions, devices, or methods described herein or in the references cited herein can be readily adapted for ligation sequencing or hybridization sequencing procedures. Typically, the oligonucleotides are fluorescently labeled and can be detected using fluorescence detectors similar to those described for SBS procedures herein or in the references cited herein.

[0056] The disclosed methods can include performing a nucleic acid detection reaction on beads or other solid supports and determining the barcode sequences of the nucleic acid probes located on the beads or other solid supports. In some embodiments, the probes are randomly arranged on the beads or other solid supports, and the nucleic acid detection reaction provides information for locating each of the different probes. Exemplary nucleic acid detection methods include, but are not limited to, nucleic acid sequencing of the probe, hybridization of a nucleic acid to the probe, ligation of a nucleic acid hybridized to the probe, extension of a nucleic acid hybridized to the probe, extension of a first nucleic acid hybridized to the probe followed by ligation of the extended nucleic acid to a second nucleic acid hybridized to the probe, or other methods known in the art, such as those described in U.S. Pat. Nos. 8,288,103 or 8,486,625, each of which is incorporated herein by reference.

[0057] solid support In exemplary embodiments, the spatial barcode nucleic acid is attached to the solid support via a cleavable linker. In one embodiment, the spatial barcode nucleic acid is directly attached to the solid support. In one embodiment, the spatial barcode nucleic acid is attached to a plurality of solid supports (e.g., beads), each of which has a unique spatial barcode sequence and is further attached to a solid support (e.g., a slide). As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate may be non-porous or porous. The substrate can optionally absorb liquid (e.g., due to porosity), but is typically sufficiently rigid that the substrate does not substantially expand when absorbing liquid and does not substantially shrink when the liquid is removed by drying. Non-porous solid supports are generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon™, cyclic olefins, polyimides, etc.), nylon, ceramics, resins, zeonol, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glass, fiber optic bundles, and polymers. Particularly useful solid supports for some embodiments are slides and beads (e.g., beads with multiple oligonucleotides attached) that can be sorted / packed onto the surface of a slide.

[0058] Any of a variety of solid supports can be used in the methods, compositions, or devices of the present disclosure. Particularly useful solid supports are those used in nucleic acid arrays. Examples include glass, modified glass, functionalized glass, inorganic glass, microspheres (e.g., inert and / or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, optical fibers or fiber optic bundles, polymers, and multiwell (e.g., microtiter) plates. Exemplary plastics include acrylic, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, and Teflon™. Exemplary silica-based materials include silicon and various forms of modified silicon.

[0059] In certain embodiments, the solid support may be within or part of a container such as a well, tube, channel, cuvette, Petri dish, bottle, etc. Optionally, the container is a flow cell, e.g., as described in WO2014 / 142841A1, U.S. Patent Application Publication No. 2010 / 0111768A1, and U.S. Patent No. 8,951,781, or Bentley et al., Nature 456:53-59 (2008), each of which is incorporated herein by reference. Exemplary flow cells are commercially available from Illumina, Inc. (San Diego, Calif.) for use in sequencing platforms such as the Genome Analyzer®, MiSeq®, NextSeq®, or HiSeq® platforms. Optionally, the container is a well within a multiwell plate or microtiter plate.

[0060] In exemplary embodiments, the solid support can include a gel coating. For example, examples of attaching nucleic acids to a solid support via gel are exemplified by flow cells commercially available from Illumina Inc. (San Diego, Calif.), or flow cells described in U.S. Patent Application Publication Nos. 2011 / 0059865 A1, 2014 / 0079923 A1, or 2015 / 0005447 A1, or PCT Publication No. WO 2008 / 093098, each of which is incorporated herein by reference. Exemplary gels that can be used in the methods and devices described herein include, but are not limited to, gels having a colloidal structure such as agarose, a polymer mesh structure such as gelatin, or a cross-linked polymer structure such as polyacrylamide, SFA (see, e.g., U.S. Patent Application Publication No. 2011 / 0059865 A1, which is incorporated herein by reference) or PAZAM (see, e.g., U.S. Patent Application Publication No. 2014 / 0079923 A1, or 2015 / 0005447 A1, each of which is incorporated herein by reference).

[0061] In some embodiments, the solid support can be configured as an array of features to which beads can be attached. The features can be present in any of a variety of desirable formats. For example, the features can be wells, holes, channels, ridges, raised areas, pegs, posts, etc. Exemplary features include wells present in substrates used in commercial sequencing platforms sold by 454 LifeSciences (a subsidiary of Roche, Basel, Switzerland) or Ion Torrent (a subsidiary of Life Technologies, Carlsbad, California). Other substrates having wells include, for example, etched optical fibers and other substrates described in U.S. Patent Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570, 6,210,891, 6,258,568, 6,274,320, U.S. Patent Application Publication Nos. 2009 / 0026082A1, 2009 / 0127589A1, 2010 / 0137143A1, 2010 / 0282617A1, or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. In some embodiments, the wells of the substrate can contain gel materials (with or without beads) as described in U.S. Patent Application Publication No. 2014 / 0243224A1, which is incorporated herein by reference.

[0062] The features can be displayed on the solid support as a grid of spots or patches. The features can be arranged in a repeating pattern or in an irregular, non-repeating pattern. Optionally, the repeating pattern can include a hexagonal pattern, a rectilinear pattern, a grid pattern, a pattern with reflection symmetry, a pattern with rotational symmetry, etc. Asymmetric patterns can also be useful. The pitch of the array can be the same between different pairs of nearest neighbor features or can vary between different pairs of nearest neighbor features.

[0063] In certain embodiments, each feature on the solid support is about 100 nm 2, 250nm 2 , 500nm 2 , 1 μm 2 , 2.5 μm 2 , 5 μm 2 , 10 μm 2 , or 50 μm 2 Alternatively or additionally, each of the features may have an area greater than about 50 μm 2 , 25 μm 2 , 10 μm 2 , 5 μm 2 , 1 μm 2 , 500nm 2 , or 100 nm 2 The foregoing ranges may represent the apparent area of ​​a bead or other particle on a solid support when viewed or imaged from above.

[0064] In an exemplary embodiment, the present disclosure provides a method for generating and using spatially tagged microbead arrays to tag nuclei on frozen section tissue samples with spatially barcoded nucleic acids at high resolution. The method includes the steps of (a) attaching different nucleic acid probes (spatially barcoded nucleic acids) to beads and capturing them on a solid support to generate probe-containing beads randomly arranged on the solid support, where each different nucleic acid probe contains a barcode sequence (shared by all nucleic acid probes on a single bead), and each randomly arranged bead contains a barcode sequence that is different from other randomly arranged beads on the solid support; (b) performing a nucleic acid detection reaction on the solid support to determine the barcode sequences of the randomly arranged beads on the solid support; (c) contacting the solid support with the randomly arranged beads with a biological specimen; (d) releasing the probes displayed by the randomly arranged beads to tag the biological specimen proximal to the randomly arranged beads; and (e) isolating the tagged nuclei, thereby spatially tagging the nuclei of the biological specimen.

[0065] As used herein, "beads," "microbeads," "microspheres," or "particles," or grammatically equivalent terms, can refer to small, individual particles. Bead composition varies depending on the class of capture probe, the synthesis method, and other factors. In exemplary embodiments of the present disclosure, the size of beads of the present disclosure tends to range from 1 μm to 100 μm in diameter (all subranges within this range are expressly contemplated), depending, for example, on the desired range of image resolution, the nature of the solid support used to construct the spatial bead array, the sequencing process (e.g., flow cell sequencing) used, and other factors. In exemplary embodiments, the smaller the beads used, the narrower the space within which each spatial barcode is placed, improving resolution. In particular, if the distance between different spatial barcodes is smaller than the size of a single cell, using smaller beads allows the same nucleus to be tagged with multiple spatial barcodes. In this case, resolution is not affected because single cells can be placed in overlapping locations on the array. If the beads are larger than a single cell, resolution is reduced because multiple nuclei are tagged with the same spatial barcode. In this case, single cells can only be placed at locations on the array that are larger than a single cell.

[0066] Exemplary embodiments of the present disclosure utilize a collection of beads or other particles with oligonucleotides attached. Bead sizes can range from nanometers (e.g., 100 nm) to millimeters (e.g., 1 mm). Typically, beads between about 0.2 μm and about 200 μm are used, with about 5 μm to about 20 μm being the currently exemplified range, although smaller or larger beads may be used in some embodiments. For example, beads smaller than 50 μm, such as 1 μm, 3 μm, 10 μm, 15 μm, 20 μm, and especially about 10 μm.

[0067] Suitable bead compositions include those used in the synthesis of peptides, nucleic acids, and organic moieties, and include, but are not limited to, plastic, ceramic, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, triazoles, carbon graphite, titanium dioxide, latex or cross-linked dextrans such as Sepharose, cellulose, nylon, cross-linked micelles, and Teflon. The "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. is a useful guide and is incorporated herein by reference in its entirety. Beads need not be spherical; irregular particles can also be used. Additionally, the beads are porous, increasing the surface area of ​​the bead available for attachment of capture probes or tags.

[0068] In exemplary embodiments, the beads include any beads used in the single-cell genomics methods described further herein. Non-limiting examples of beads include hydrogel particles (such as polyacrylamide, agarose, etc.), colloidal particles (such as polystyrene, magnetic particles, or polymeric particles), any beads that can utilize phosphoramidate chemistry such as those used in oligonucleotide synthesis known to those of skill in the art (e.g., methyl acrylate, polyester, polyacrylamide, polyethylene glycol), paramagnetic beads, and magnetic beads.

[0069] In exemplary embodiments, the beads can be hydrogel particles (see, e.g., International Patent Application Publication No. WO 2008 / 109176 for examples of hydrogel particles, including hydrogel particles containing DNA). Examples of hydrogels include, but are not limited to, agarose or acrylamide-based gels, such as polyacrylamide, poly-N-isopropylacrylamide, or poly-N-isopropylpolyacrylamide. For example, an aqueous solution of monomers can be dispersed in the droplets and then polymerized to form, for example, a gel.

[0070] In exemplary embodiments, the beads can comprise one or more polymers. Exemplary polymers include, but are not limited to, polystyrene (PS), polycaprolactone (PCL), polyisoprene (PIP), polylactic acid, polyethylene, polypropylene, polyacrylonitrile, polyimide, polyamide, and / or mixtures and / or copolymers of these and / or other polymers. Additionally, in some cases, the particles can be magnetic, potentially enabling magnetic manipulation of the particles. For example, the particles can comprise iron or other magnetic materials. The particles can also be functionalized to attach other molecules, such as proteins, nucleic acids, and small molecules. In some embodiments, the particles can be fluorescent.

[0071] Beads containing spatial barcode nucleic acids of the present invention can be obtained by any of the methods described above. For example, spatial barcode nucleic acids can be synthesized directly on beads, and barcodes can be generated by random synthesis (see, for example, Macosko et al., 2015, "Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets," Cell 161, 1202-1214; and International Patent Application No. PCT / US2015 / 049178, published as WO2016 / 040476 on March 17, 2016). In an exemplary embodiment, beads are obtained by: 1) performing reverse phosphoramidite synthesis on the surface of the beads to synthesize the 5' end of a spatial barcode nucleic acid from a linker on the bead; 2) performing reverse phosphoramidite synthesis on the surface of the beads in a pool-and-split manner, so that in each cycle of synthesis, the beads are split into four reactions with one of the four canonical nucleotides (T, C, G, or A) or unique oligonucleotides; 3) repeating this process multiple times, at least twice, and optimally 12 or more times, so that the latter results in over 16 million unique spatial barcodes on the surface of each bead in the pool; and 4) synthesizing or attaching (e.g., ligating) the 3' end of a spatial barcode nucleic acid containing a universal sequence for capture by a cellular barcode. For synthesis, the beads must be made of a material that can be maintained during organic synthesis. Non-limiting examples include any bead that can utilize phosphoramidite chemistry, such as that used in oligonucleotide synthesis, known to those skilled in the art. Direct synthesis of oligonucleotides on beads is a preferred embodiment as it allows for more diverse barcodes with more compact sequences.

[0072] In another embodiment, spatial barcode nucleic acids can be synthesized by attaching oligonucleotides to beads, followed by split-pool hybridization and extension to generate a unique cell barcode for each bead (e.g., Klein et al., 2015, "Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells," Cell 161, 1187-1201; International Patent Application No. PCT / US2016 / 027734, published October 20, 2016 as WO2016168584A1). In an exemplary embodiment, nucleic acid barcodes can be constructed combinatorially by combining randomly selected indexes (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 indexes selected from a sequence pool for each index). Each such index is a short sequence of nucleotides (e.g., DNA, RNA, or a combination thereof) with a different sequence. The index may have a length of, for example, about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bp or nt. Thus, in some embodiments, potential barcodes are formed from "pools" of one or more individual barcode elements, which are then combined, such as using split-and-pool, to generate a final barcode. The pools can contain, for example, at least about 300, at least about 500, at least about 1,000, at least about 3,000, at least about 5,000, or at least about 10,000 identifiable barcodes. For example, a first pool can contain x1 elements and a second pool can contain x2 elements, and forming a barcode containing elements from the first pool and elements from the second pool generates, for example, x1x2 usable barcodes. Note that x1 and x2 may or may not be equal.This process can be repeated any number of times; for example, the barcode can contain elements from the first pool, the second pool, and the third pool (e.g., generating x1x2x3 possible barcodes), or elements from the first pool, the second pool, the third pool, and the fourth pool, etc. Thus, because of the large number of potential combinations, a much larger number of identifiable barcodes can be generated using a relatively small number of barcode elements.

[0073] In another embodiment, spatially barcoded nucleic acids can be synthesized by attaching the 5' end of an oligonucleotide containing an adapter sequence to a bead to generate a functionalized bead, followed by emulsion PCR using a primer containing a unique barcode sequence (see, e.g., Zheng, et al., 2016, "Haplotyping germline and cancer genomes with high-throughput linked-read sequencing" Nature Biotechnology 34, 303-311; Zheng, et al., 2017, "Massively parallel digital transcriptional profiling of single cells" Nat. Commun. 8, 14049 doi:10.1038 / ncomms14049; International Patent Application No. WO2014210353A2; and Zilionis, et al., 2017, "Single-cell barcoding and sequencing using droplet microfluidics" Nat Protoc. Jan; 12(1):44-73). In this embodiment, each emulsion PCR contains a single primer that hybridizes to the oligonucleotides on the functionalized beads and contains a barcode sequence. Therefore, after several rounds of amplification, the barcode sequence is transferred to all the oligonucleotides on the functionalized beads. As a result, each bead has its own unique barcode.

[0074] In exemplary embodiments, the beads can be suspended in solution or disposed on the surface of a substrate (e.g., arrayed on the surface of a solid support such as a glass slide). Examples of arrays in which beads are disposed on a surface recognized in the art include those in which beads are disposed within wells, such as BeadChip arrays (Illumina Inc., San Diego, Calif.), the substrate used in the sequencing platform of 454 LifeSciences (a subsidiary of Roche, Basel, Switzerland), or the substrate used in the sequencing platform of Ion Torrent (a subsidiary of Life Technologies, Carlsbad, Calif.). Other solid supports with beads disposed on their surfaces are described in U.S. Patent Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570, 6,210,891, 6,258,568, or 6,274,320, U.S. Patent Application Publication Nos. 2009 / 0026082A1, 2009 / 0127589A1, 2010 / 0137143A1, or 2010 / 0282617A1, or PCT Publication No. WO00 / 63437, each of which is incorporated herein by reference. Some of the above references describe methods for attaching nucleic acid probes to beads before loading the beads into or onto a solid support. Thus, a collection of beads can include different beads, each with a unique (or, as described elsewhere herein, sufficiently unique and / or nearly unique) probe attached. However, it will be understood that beads can be made to include universal primers, which can then be loaded onto an array, thereby forming a universal array for use in the methods described herein. Solid supports typically used in bead arrays can also be used without beads. For example, nucleic acids such as probes or primers can be directly attached to the wells or attached to a gel material within the wells. Thus, the above references are illustrative of materials, compositions, or devices that can be modified for use in the methods and compositions described herein.

[0075] Thus, the methods of the present invention can use a bead array, with different nucleic acid probes attached to different beads within the array. In this embodiment, each bead can be bound to a different nucleic acid probe, and the beads can be randomly distributed on the solid support to effectively attach the different nucleic acid probes to the solid support. Optionally, the solid support can include a well sized to accommodate up to one bead. In such a configuration, the bead can be attached to the well by the force generated by the bead fitting into the well. As described elsewhere herein, attachment chemicals or capture materials (e.g., vinyl polymer liquid electrical tape) can also be used to attach or stably bind the beads to the solid support, optionally including retaining the beads in the well regardless of whether they are present on the solid support.

[0076] The nucleic acid probes attached to the beads can include barcode sequences. A bead population can be configured such that each bead has only one type of barcode (e.g., a spatial barcode) attached, with many different beads in the population, each with a different barcode. In this embodiment, randomly distributing the beads on the solid support results in random placement of the beads (and their respective barcode sequences) displaying nucleic acid probes on the solid support. In some cases, multiple beads have the same barcode sequence, resulting in redundancy within the population. However, randomly distributing a population on a solid support, particularly one that includes a redundancy of beads with a capacity greater than the number of unique barcodes in the bead population, tends to result in redundancy of barcodes on the solid support, which tends to reduce image resolution in the context of the present disclosure (i.e., if the exact location of a barcoded bead cannot be resolved due to redundancy in the use of barcodes within the arrayed bead population, it is envisioned that such redundant locations will simply be removed from the final image generated by the methods of the present disclosure, or other adjustment modes (e.g., value normalization and / or averaging) may also be used to address such redundancy.) Alternatively, in preferred embodiments, the number of distinct barcodes in a bead population can exceed the capacity of the solid support to generate an array with no redundancy for the barcode population on the solid support. In some embodiments, the capacity of the solid support is determined by the number of features (e.g., single-bead-occupied wells) to which beads are attached or that house beads.

[0077] The beads or other nucleic acid-presenting solid supports of the present disclosure can contain a plurality of different nucleic acid probes or can be made to have a plurality of different nucleic acid probes attached thereto by the methods described herein. For example, the beads or other nucleic acid-presenting solid supports can have at least 10, 100, 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9Alternatively or additionally, the beads or other nucleic acid presenting solid supports may contain up to 1 x 10 different probes. 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 100, or fewer different probes. For example, if the probes are amplified to form clusters, it will be understood that each different probe can be present in multiple copies. Thus, the above ranges can represent the number of different nucleic acid clusters on a bead or other nucleic acid-presenting solid support of the present disclosure. It will also be understood that the above ranges can describe the number of different barcodes, target capture sequences, or other sequence elements described herein as unique (or sufficiently unique) for a particular nucleic acid probe. Alternatively or additionally, the ranges can represent the number of extension or modified probes created on a bead or other nucleic acid-presenting solid support of the present disclosure using the methods described herein.

[0078] Features can be present on beads or other solid supports of the present disclosure before contacting the beads or other solid supports with nucleic acid probes. For example, in embodiments in which probes are bound to beads or other solid supports via hybridization with primers, the primers can be attached to the features, but the interstitial regions outside any features are substantially free of primers. Nucleic acid probes can be captured in preformed features on beads or other solid supports and, optionally, amplified on the beads or other solid supports using methods such as those described in U.S. Patent Nos. 8,895,249 and 8,778,849 and / or U.S. Patent Publication No. 2014 / 0243224 A1, each of which is incorporated herein by reference. Alternatively, the beads or other solid supports can have a lawn of primers but otherwise lack other features. In this case, features can be formed by attaching nucleic acid probes to the beads or other solid supports. Optionally, the captured nucleic acid probes are amplified on the beads or other solid supports, and the resulting clusters become features. While attachment is exemplified above as capture between a primer and the complementary portion of the probe, it will be understood that capture moieties other than primers can be present as preformed features or lawns. Other exemplary capture moieties include, but are not limited to, chemical moieties that can react with a nucleic acid probe to form a covalent bond, or receptors that can non-covalently bind to a ligand on a nucleic acid probe.

[0079] The step of binding nucleic acid probes to beads or other solid supports can be carried out by providing a fluid containing a mixture of different nucleic acid probes and contacting the fluid mixture with beads or other solid supports. This contact can bring the fluid mixture into contact with a surface to which many different nucleic acid probes from the fluid mixture are attached. Thus, the probes can have random access to the surface (whether the surface has preformed features for attaching the probes or a uniform surface configured for attachment). Thus, the probes can be randomly arranged on the beads or other solid supports.

[0080] The total number and types of different probes attached to a surface can be selected for a particular application or use. For example, in embodiments in which a fluid mixture of different nucleic acid probes is contacted with beads or other solid supports to attach the probes to the support, the number of different probe species can exceed the number of beads or other solid supports occupied by the probes. Thus, the number and types of different probes attached to the beads or other solid supports can be equivalent to the number of probes occupied by the beads or other solid supports.

[0081] Alternatively, the number and type of different probe species on the bead or other solid support may be less than the occupancy number (i.e., there is probe species redundancy, such that the bead or other solid support may contain multiple features with the same probe species.) Such redundancy can be achieved, for example, by contacting the bead or other solid support with a fluid mixture containing a number and type of probe species that is substantially lower than the probe occupancy number of the bead or other solid support.

[0082] Attachment of the nucleic acid probe may be mediated by hybridization of the nucleic acid probe to a complementary primer attached to a bead or other solid support, formation of a chemical bond between a reactive moiety on the nucleic acid probe and the bead or other solid support (examples are described in U.S. Pat. Nos. 8,895,249 and 8,778,849, and U.S. Patent Publication No. 2014 / 0243224 A1, each of which is incorporated herein by reference), affinity interactions between a moiety on the nucleic acid probe and a moiety bound to the bead or other solid support (e.g., between known receptor-ligand pairs such as streptavidin-biotin, antibody-epitope, lectin-carbohydrate, etc.), physical interactions between the nucleic acid probe and the bead or other solid support (e.g., hydrogen bonding, ionic forces, van der Waals forces, etc.), or other interactions known in the art for attaching nucleic acids to surfaces.

[0083] In some embodiments, the attachment of nucleic acid probes is nonspecific with respect to any sequence differences between the nucleic acid probe and other nucleic acid probes that are or will be attached to beads or other solid supports. For example, different probes can have universal sequences that complement primers attached to the surface, or different probes can have a common portion that mediates attachment to the surface. Alternatively, each of the different probes (or a subpopulation of different probes) can have a unique (or sufficiently unique) sequence that complements a unique (or sufficiently unique) primer on the bead or other solid support, or can have a unique (or sufficiently unique) portion that interacts with one or more different reactive moieties on the bead or other solid support. In such cases, the unique (or sufficiently unique) primer or unique (or sufficiently unique) portion can be attached to predetermined locations as needed to selectively capture a particular probe or type of probe at each predetermined location.

[0084] One or more features on a bead or other solid support can each contain a single molecule of a particular probe. In some embodiments, these features can be configured to accommodate only a single nucleic acid probe molecule. However, regardless of whether a feature can accommodate multiple nucleic acid probe molecules, it is possible that the feature contains only one nucleic acid probe molecule. Alternatively, an individual feature can contain multiple nucleic acid probe molecules, e.g., a collection of nucleic acid probe molecules having the same sequence as one another. In certain embodiments, the collection can be generated by amplification from a single nucleic acid probe template to generate amplicons, e.g., as a cluster attached to a surface.

[0085] The methods described herein can use any of a variety of amplification techniques. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), or random primed amplification (RPA). In some embodiments, amplification can be performed in solution, for example, if the array features can accommodate the amplicons within a volume having the desired capacity. In certain embodiments, the amplification techniques used in the disclosed methods are performed on a solid phase. For example, one or more primer species (e.g., universal primers for one or more universal primer binding sites present in the nucleic acid probe) can be attached to beads or other solid supports. In PCR embodiments, one or both of the primers used in amplification can be attached to beads or other solid supports (e.g., via a gel). Formats that use two primers attached to beads or other solid supports are often referred to as bridge amplification, because the double-stranded amplicon forms a bridge-like structure between the two surface-attached primers, flanking the copied template sequence. Exemplary reagents and conditions that can be used for bridge amplification are described, for example, in U.S. Patent Nos. 5,641,658, 7,115,400, and 8,895,249, and / or U.S. Patent Publication Nos. 2002 / 0055100A1, 2004 / 0096853A1, 2004 / 0002090A1, 2007 / 0128624A1, and 2008 / 0009420A1, each of which is incorporated herein by reference. Solid-phase PCR amplification can also be performed by attaching one of the amplification primers to a bead or other solid support and placing the second primer in solution.An exemplary format using a combination of surface-bound and soluble primers is that used in emulsion PCR, as described, for example, in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05 / 010145, or U.S. Patent Publication Nos. 2005 / 0130173 A1 or 2005 / 0064460 A1, each of which is incorporated herein by reference. It should be understood that emulsion PCR is an exemplary format, and that for purposes of the methods described herein, the use of an emulsion is optional, and indeed, in some embodiments, no emulsion is used.

[0086] RCA technology can be modified for use in the methods of the present disclosure. Exemplary components that can be used in RCA reactions and the principles by which RCA generates amplicons are described, for example, in Lizardi et al., Nat. Genet. 19:225-232 (1998) and U.S. Patent Publication No. 2007 / 0099208 A1, each of which is incorporated herein by reference. The primers used in RCA can be in solution or attached to beads or other solid supports. The primers can be one or more of the universal primers described herein.

[0087] MDA technology can be modified for use in the methods of the present disclosure. The basic principles and useful conditions of MDA are described, for example, in Dean et al., Proc Natl. Acad. Acad. Sci. USA 99:5261-66 (2002); Lage et al., Genome Research 13:294-307 (2003); Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995; Walker et al., Nucl. Acids Res. 20:1691-96 (1992); U.S. Patent Nos. 5,455,166, 5,130,238, and 6,214,587, each of which is incorporated herein by reference. Primers used in MDA can be in solution or attached to beads or other solid supports at the amplification site. Primers can also be one or more of the universal primers described herein.

[0088] In certain embodiments, a combination of the amplification techniques exemplified above can also be used. For example, RCA and MDA can be used in combination, where RCA is used to generate concatemeric amplicons in solution (e.g., using solution-phase primers). The amplicons can then be used as templates for MDA using primers (e.g., universal primers) attached to beads or other solid supports. In this example, the amplicons generated after combining the RCA and MDA steps are attached to beads or other solid supports.

[0089] In certain aspects of the present disclosure, a capture material is used to attach a bead array to a solid support (e.g., a glass slide). In some embodiments, the capture material is liquid electrical tape. An exemplary liquid electrical tape of the present disclosure is Permatex™ Liquid Electrical Tape, a weather-resistant protectant for wiring and electrical connections. Liquid capture materials, such as liquid tape, can be applied as a liquid and then dry to a vinyl polymer that is resistant to dirt, dust, chemicals, and moisture. The capture materials of the present disclosure can be applied by any of several methods, including brushing onto the solid support, spraying onto the solid support, or immersing the solid support in the capture material. For certain forms of liquid capture materials, the use of a brush-top applicator allows for tight application and access to tight spaces, which in certain embodiments provides advantages over forms of capture material applied in a non-liquid state (i.e., tape).

[0090] While liquid electrical tape has been exemplified as a capture material for use in the methods and compositions of the present disclosure, other capture materials, including art-recognized adhesives or other reagents, that (a) can be applied and / or deposited onto a solid surface (e.g., a slide, optionally allowing light transmission through the slide, e.g., a microscope slide) and (b) are capable of binding or capturing a population of beads sized between 1 and 100 μm, are also contemplated for such applications. Examples of other capture materials explicitly contemplated include latex and other rubbers, such as cis-1,4-polyisoprene, as well as elastomers (generally defined as polymers that are viscoelastic (i.e., both viscous and elastic), have very weak intermolecular forces, and generally have a low Young's modulus and high strain to failure compared to other materials), artificial elastomers (e.g., neoprene), and / or silicone elastomers. Acrylate polymers (e.g., Scotch tape) are also explicitly contemplated for use as capture materials of the present disclosure, for example. Examples of other capture materials explicitly contemplated include superglue adhesives (e.g., Loctite Super Glue), gels (e.g., agarose, acrylamide), rubberized sealant sprays (e.g., Gorilla Glue, PLASTI DIP Multipurpose Rubber Coating), silicone conformal coatings (e.g., Fine-L-Kote SR), or other conformal coatings (e.g., urethane resins, epoxy resins, acrylic resins, parylene). Additionally, bead oligos can be transferred to agarose or acrylamide gel matrices for subsequent release into tissue.

[0091] In exemplary embodiments, the array is printed on a solid support. In one embodiment, the printed spatially barcoded nucleic acids are amplified on the solid support as described herein (e.g., bridge amplification). In exemplary embodiments, the solid support is a slide or an array on a slide. As used herein, the term "slide" includes an "array," "substrate," or "surface" comprising a plurality of spatially barcoded nucleic acids as described herein. In the case of the spatial array-based analytical methods described herein, the substrate serves as a support for directly or indirectly attaching spatially barcoded nucleic acids to features of the array. Furthermore, in some embodiments, the substrate (e.g., the same substrate or a different substrate) can be used to provide support for biological samples, particularly, for example, thin tissue sections. Thus, a "substrate" is a support that is insoluble in aqueous liquids and allows for the placement of biological samples, analytes, features, and / or spatially barcoded nucleic acids on the substrate.

[0092] Furthermore, as used herein, the term "substrate," unless preceded by the modifier "chemical," refers to an element having at least one surface that generally functions to provide physical support for a biological sample, an analyte, and / or any of the other chemical and / or physical moieties, agents, and structures described herein. Substrates can be formed from a variety of solid materials, gel-based materials, colloidal materials, semi-solid materials (e.g., at least partially cross-linked materials), fully or partially cured materials, and materials that undergo a phase change or transition to provide physical support. Examples of substrates that can be used in the methods and systems described herein include, but are not limited to, slides (e.g., slides formed from various glasses, slides formed from various polymers), hydrogels, layers and / or films, membranes (e.g., porous membranes), flow cells, cuvettes, wafers, plates, or combinations thereof. In some embodiments, substrates can optionally include functional elements such as recesses, protruding structures, microfluidic elements (e.g., channels, reservoirs, electrodes, valves, seals), and various markings. Slides and arrays for spatial profiling have been described (see, e.g., Visium Spatial Capture Technology, 10X Genomics, Pleasanton, CA; WO2020047007A2; WO2020123317A2; WO2020047005A1; WO2020176788A1; and WO2020190509A9). Capture probes containing spatial barcodes can be replaced with spatial barcode nucleic acids containing spatial barcodes described herein.

[0093] A slide containing spatially barcoded nucleic acids can be obtained by synthesizing the spatially barcoded nucleic acids and attaching them to a slide or array. In an exemplary embodiment, the spatially barcoded nucleic acids are added to specific locations on an array.

[0094] Arrays can be prepared by depositing features (e.g., droplets, beads) onto a substrate surface to generate spatially barcoded arrays. Methods for depositing features (e.g., droplet manipulation) are known in the art (see U.S. Patent Application Publication No. 2008 / 0132429; Rubina, AY, et al., Biotechniques. 2003 May; 34(5):1008-14, 1016-20, 1022; and Vasiliskov et al. Biotechniques. 1999 September; 27(3):592-4, 596-8, 600 passim). Features can be printed or deposited (e.g., inkjet printing) at specific locations on the substrate. In some embodiments, each feature can have a spatially barcoded nucleic acid. In some embodiments, features can be printed or deposited at specific locations using an electric field. Features can contain photocrosslinkable precursors and oligonucleotides. In some embodiments, photocrosslinkable polymer precursors can be deposited into patterned features (e.g., wells) on a substrate. "Photocrosslinkable polymer precursor" refers to a compound that crosslinks and / or polymerizes upon exposure to light. In some embodiments, one or more photoinitiators can also be included to induce and / or promote polymerization and / or crosslinking (see, e.g., Choi et al. Biotechniques. 2019 Jan;66(1):40-53).

[0095] Arrays can be prepared in a variety of ways. In some embodiments, arrays are prepared by synthesizing oligonucleotides on the array (e.g., in situ synthesis), jet printing, or lithography. In exemplary embodiments, spatially barcoded nucleic acids are deterministically patterned synthetically. For example, light-induced synthesis of high-density DNA oligonucleotides can be achieved by photolithography or solid-phase DNA synthesis. To implement photolithographic synthesis, synthetic linkers modified with photochemical protecting groups can be attached to a substrate, and the photochemical protecting groups can be modified using a photolithographic mask (applied to specific regions of the substrate) and light, thereby generating an array with localized photodeprotection. Many of these methods are known in the art and are described, for example, in Miller et al., "Basic concepts of microarrays and potential applications in clinical microbiology." Clinical Microbiology Reviews 22.4 (2009): 611-633; US201314111482A; US9593365B2; US2019203275; and WO2018091676.

[0096] organization In some embodiments, tissue sections are used. The tissue is derived from a multicellular organism. Exemplary multicellular organisms include, but are not limited to, mammals, plants, algae, nematodes, insects, fish, reptiles, amphibians, fungi, or Plasmodium falciparum. Exemplary species are described previously herein or known in the art. The tissue may be freshly excised from an organism or may have been previously preserved by freezing, embedding in a material such as paraffin (e.g., formalin-fixed paraffin-embedded samples (FFPE)), formalin fixation, infiltration, dehydration, or the like. Optionally, the tissue section can be cryosectioned using techniques and compositions described herein and known in the art. As a further option, the tissue can be permeabilized to allow the spatial barcode nucleic acid to access the nucleus. As used herein, the term "tissue" refers to a collection of cells and, optionally, intercellular substance. Typically, cells within a tissue are not free-floating in solution but are attached to each other to form a multicellular structure. Exemplary tissue types include muscle, nerve, epidermis, and connective tissue. The tissue can also be from a subject with a disease such as an autoimmune disease or cancer (e.g., tissue from irritable bowel disease (IBD) or MS, or tumor tissue).

[0097] The disclosed methods can include contacting a biological specimen (i.e., a frozen section tissue sample) with beads or other solid supports having spatially barcoded nucleic acids attached thereto. In some embodiments, the spatially barcoded nucleic acids are randomly disposed on the beads or other solid supports. The identity and location of the spatially barcoded nucleic acids can be decoded before contacting the biological specimen with the beads or other solid supports. Alternatively, the identity and location of the spatially barcoded nucleic acids can be determined after contacting the beads or other solid supports with the biological specimen.

[0098] As used herein, the term "frozen section" refers to a piece of tissue (e.g., a biopsy) that has been removed from a subject, flash-frozen, embedded in an embedding material of optimal cutting temperature, frozen, and cut into thin sections. In certain embodiments, the thin section can be applied directly to a bead array captured on a solid support (e.g., a slide), or the thin section can be fixed (e.g., in methanol or paraformaldehyde) and applied to a bead-presenting surface (e.g., a slide with a layer / array of microbeads attached).

[0099] In exemplary embodiments, tissues can be obtained from complex multicellular systems (e.g., organoids, tissue explants, or organs on a chip) (e.g., Yin X, Mead BE, Safaee H, Langer R, Karp JM, Levy O. Engineering Stem Cell Organoids. Cell Stem Cell. 2016;18(1):25-38; Clevers, Modeling Development and Disease with Organoids, Cell. 2016 Jun 16;165(7):1586-1597; Porter, RJ, Murray, GI & McLean, MH. Current concepts in tumor-derived organoids. Br J Cancer 123, 1209-1218 (2020). doi.org / 10.1038 / s41416-020-0993-5; Sontheimer-Phelps, A., Hassell, BA & Ingber, DE. Modeling cancer in microfluidic human organs-on-chips.Nat.Rev.Cancer 19,65-81(2019);and Wu,Q.,Liu,J.,Wang,X.et al.Organ-on-a-chip:recent breakthroughs and future prospects.BioMed Eng OnLine 19,9(2020);Ingber,DEDevelopmentally inspired human 'organs on chips'.Development 145,pii:dev156125(2018);Ghosh S,Prasad M,Kundu K,et al.Tumor Tissue Explant Culture of Patient-Derived Xenograft as Potential Prioritization Tool for Targeted Therapy.Front Oncol.2019;9:17;Neil JE,Brown MB,Williams AC.(See Human skin explant model for the investigation of topical therapeutics. Sci Rep. 2020;10(1):21192; and Grivel JC, Margolis L. Use of human tissue explants to study human infectious agents. Nat Protoc. 2009;4(2):256-269.) Tissues or complex multicellular systems include patient-derived organoids (PDOs) or patient-derived xenografts (PDXs).

[0100] Tissues can be prepared for use in the methods, compositions, or devices herein in any convenient or desired manner. Fresh, frozen, fixed, or unfixed tissue can be used. Tissues can be fixed or embedded using methods described herein or known in the art.

[0101] Tissue samples used in the present invention can be fixed by rapid freezing at a temperature suitable to maintain or preserve the integrity of the tissue structure, for example, below -20°C. In another example, tissues can be prepared using formalin-fixed and paraffin-embedded (FFPE) methods known in the art. Other fixatives and / or embedding materials can also be used, if desired. Fixed or embedded tissue samples can be sectioned, i.e., thinly sliced, using known methods. For example, tissue samples can be sectioned using a cooled microtome or cryostat set at a temperature suitable to maintain both the structural integrity of the tissue sample and the chemical properties of the nucleic acids within the sample. Exemplary additional fixatives explicitly contemplated include alcohol fixation (e.g., methanol fixation, ethanol fixation), glutaraldehyde fixation, and paraformaldehyde fixation.

[0102] In some embodiments, the tissue sample is processed to remove embedding material (e.g., removal of paraffin or formalin) from the sample prior to tagging the nuclei. This can be achieved by contacting the sample with a suitable solvent (e.g., xylene and ethanol washes). Processing can be performed before contacting the tissue sample with a spatial array described herein, or can be performed while the tissue sample is on the solid support capture bead array.

[0103] Exemplary methods for engineering tissue for use on solid supports with attached nucleic acids are described in U.S. Patent Application Publication No. 2014 / 0066318A1, which is incorporated herein by reference.

[0104] The thickness of the tissue sample or other biological specimen contacted with the bead array in the methods, compositions, or devices described herein can be any suitable thickness. In representative embodiments, the thickness is at least 0.1 μm, 0.25 μm, 0.5 μm, 0.75 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, or more. Alternatively or additionally, the thickness of the tissue sample contacted with the bead array is 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 0.25 μm, 0.1 μm, or less.

[0105] A particularly suitable source of tissue samples is human. Another source of tissue samples is animal models (e.g., mouse tissue samples). Samples can be derived from organs including, for example, organs of the central nervous system such as the brain, brainstem, cerebellum, spinal cord, cranial nerves, spinal nerves, etc.; organs of the musculoskeletal system such as muscles, bones, tendons, ligaments, organs of the digestive system such as the salivary glands, pharynx, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, organs of the respiratory system such as the larynx, trachea, bronchi, lungs, diaphragm, organs of the urinary system such as the kidneys, ureters, bladder, urethra, reproductive organs such as the ovaries, fallopian tubes, uterus, vagina, placenta, testes, epididymis, vas deferens, seminal vesicles, prostate, penis, scrotum, organs of the endocrine system such as the pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands, organs of the circulatory system such as the heart, arteries, veins, capillaries, organs of the lymphatic system such as lymphatic vessels, lymph nodes, bone marrow, thymus, spleen, sensory organs such as the eye, ear, nose, tongue, or organs of the integumentary system such as the skin, subcutaneous tissue, mammary glands. In some embodiments, tissue samples are obtained from bodily fluids or excretions such as blood, lymph, tears, sweat, saliva, semen, vaginal secretions, earwax, feces, urine, etc.

[0106] The human samples may be considered (or suspected) to be healthy or diseased at the time of use. In some cases, one sample is considered diseased and the second sample is considered healthy (e.g., for use as a healthy control). A variety of conditions may be assessed, including autoimmune diseases, cancer, cystic fibrosis, aneuploidy, pathogenic infections, psychological conditions, hepatitis, diabetes, sexually transmitted diseases, heart disease, stroke, cardiovascular disease, multiple sclerosis (MS), and muscular dystrophy. Certain conditions contemplated include genetic conditions or conditions associated with pathogens that have identifiable genetic signatures.

[0107] In exemplary embodiments, tissues are prepared for nuclear tagging using single-nucleus sequencing (e.g., snRNA-seq) procedures (e.g., Habib et al., 2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons,” Science, Vol. 353, Issue 6302, pp. 925-928; Habib et al., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq,” Nat Methods. 2017 Oct;14(10):955-958; International Patent Application No. PCT / US2016 / 059239, published September 28, 2017 as WO2017164936; International Patent Application No. PCT / US2018 / 060860, published May 16, 2019 as WO / 2019 / 094984; International Patent Application No. PCT / US2019 / 055894, published April 16, 2020 as WO / 2020 / 077236; Drokhlyansky, et al., “The enteric nervous system of the human and mouse colon at a single-cell resolution,” bioRxiv 746743; doi:doi.org / 10.1101 / 746743; Drokhlyansky E, Smillie CS, Van Wittenberghe N, et al. The Human and Mouse Enteric Nervous System at Single-Cell Resolution. Cell. 2020;182(6):1606-1622.e23; and Slyper, M., Porter, CBM, Ashenberg, O. et al. (2020). A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors. Nature Medicine 26(5):792-802).In exemplary embodiments, methods for preparing and isolating nuclei that preserve the nuclear membrane and ribosomes (RAISIN (Ribosomes and Intact Single Nuclei) RNA-seq) or nuclei that preserve both the rough endoplasmic reticulum and ribosomes attached to the outer nuclear membrane (INNER Cell (Nucleus and Endoplasmic Reticulum from Intact Single Cell) RNA-seq) can be applied to tagged nuclei (see WO / 2020 / 077236).

[0108] In an exemplary embodiment, single nuclei are prepared from FFPE tissue sections (see, e.g., WO / 2020 / 077236). For example, FFPE tissue samples are processed by dissolving paraffin in a solvent, preferably xylene or mineral oil, at a temperature between 4°C and 90°C, preferably room temperature (20-25°C) or 90°C. The tissue is then rehydrated using a gradient of 100% to 0% ethanol (EtOH). The rehydrated tissue is then transferred to a volume of a first buffer solution containing a buffer, a detergent, and an ionic strength of 100 mM to 200 mM, optionally containing a protease inhibitor or protease and / or BSA. In an exemplary embodiment, single nuclei are prepared from tissue samples using a buffer containing 10 mM Tris, 0.49% CHAPS, 146 mM NaCl, 1 mM CaCl, 21 mM MgCl, and 0.01% BSA (CST) (see, e.g., Raisin-seq). In an exemplary embodiment, single nuclei are prepared from tissue samples using a buffer containing 10 mM Tris, 0.03% Tween-20, 146 mM NaCl, 1 mM CaCl, 21 mM MgCl, and 0.01% BSA (TST) (see, e.g., INNER Cell-seq). In an exemplary embodiment, a buffer is used to extract nuclei from tissue.

[0109] In some embodiments, biological samples can be permeabilized to facilitate the transfer of spatial barcode nucleic acids into the sample. If the sample is not sufficiently permeabilized, the amount of spatial barcode nucleic acids in the sample may be too low for proper analysis. Conversely, if the tissue sample is too permeabilized, the relative spatial relationships of analytes within the tissue sample may be lost. Therefore, a balance is desired between sufficiently permeabilizing the tissue sample to obtain good signal intensity while maintaining spatial resolution of the distribution of analytes within the sample.

[0110] Generally, biological samples can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, methanol), crosslinkers (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100™, Tween-20™, sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases (e.g., proteinase K)). In some embodiments, the detergent is an anionic detergent (e.g., SDS or N-lauroyl sarcosine sodium salt solution). In some embodiments, the biological sample can be permeabilized using any of the methods described herein (e.g., any of the detergents described herein, e.g., SDS and / or N-lauroyl sarcosine sodium salt solution) before or after enzymatic treatment (e.g., treatment with any of the enzymes described herein, e.g., trypsin, proteases (e.g., pepsin and / or proteinase K)). Further methods of sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entire contents of which are incorporated herein by reference.

[0111] Cleavage of the linker and delivery of the spatial barcode to the nucleus In an exemplary embodiment, spatially barcoded nucleic acids contacted with a permeabilized tissue sample are delivered to the nucleus. In an exemplary embodiment, the tissue sample is incubated in a dissociation buffer described herein (see Dissociation Buffer (DB) in Example 1). In an exemplary embodiment, the incubation is performed at 4-25°C, preferably at about 4°C. The linker is then cleaved (e.g., using light, chemicals, or enzymes), releasing the spatially barcoded nucleic acids from the spatial array. In an exemplary embodiment, the spatially barcoded nucleic acids are delivered to the nucleus by diffusion. In an exemplary embodiment, the spatially barcoded nucleic acids are delivered to the nucleus using electroporation. In electroporation, the spatially barcoded nucleic acids can enter the cell through one or more pores in the cell membrane formed by applied electricity. The membrane pores can be reversible based on the applied electric field strength and pulse duration. In an exemplary embodiment, the spatially barcoded nucleic acids are delivered to the nucleus using sonoporation. Transient permeabilization of the cell membrane using sound waves allows the spatially barcoded nucleic acids to be taken up into the cell. In an exemplary embodiment, the spatially barcoded nucleic acid is delivered to the nucleus using hydroporation. The spatially barcoded nucleic acid can be delivered to the cell by fluid pressure.

[0112] Isolation of labeled nuclei from tissue In an exemplary embodiment, nuclei are extracted from tagged tissue by dissociating the tissue with an extraction buffer (see, e.g., Example 1, Extraction Buffer (ExB)). In an exemplary embodiment, the extraction buffer contains a detergent. In an exemplary embodiment, the detergent is Triton X-100. In an exemplary embodiment, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate). In an exemplary embodiment, the detergent is Tween-20. In one embodiment, the tissue is dissociated by pipetting. In one embodiment, the tissue is dissociated by gentle scraping. In an exemplary embodiment, if the spatial barcode nucleic acid contains a fluorescent dye, the tagged nuclei can be sorted by FACS. Thus, in the next step, only tagged nuclei are used. Once the nuclei are sorted, the same efficiency as using untagged nuclei is obtained.

[0113] Single-cell genomics assays Single-cell omics sequencing of the transcriptome was first achieved in 2009, followed by rapid development of profiling techniques for genomes, DNA methylomes, 3D genome structure, chromatin accessibility, histone modifications, and the like in individual cells (see, e.g., Wen L, Tang F. Recent advances in single-cell sequencing technologies. Precis Clin Med. 2022;5(1):pbac002, published January 31, 2022). In exemplary embodiments, tagged nuclei produced by the present method can be used in any of these single-cell genomics assays to spatially resolve the assay.

[0114] In exemplary embodiments, single-cell genomics assays typically include a barcode (i.e., a cell barcode) used to identify the cell of origin of a genomic analyte. For example, the analyte can be RNA such as polyA-tailed mRNA, a genomic DNA sequence, a bound DNA fragment, immunoprecipitated genomic DNA, or an antibody-specific oligonucleotide. Cell barcodes are also typically associated with a unique molecular identifier (UMI), allowing the number of each analyte in each single cell to be counted (i.e., quantified). As used herein, the term "unique molecular identifier" (UMI) refers to a subtype of sequencing linker or nucleic acid barcode used in methods that use molecular tags to detect and quantify unique amplification products (see, e.g., Islam S. et al., 2014. Nature Methods No: 11, 163-166). In exemplary embodiments, the UMI is a random sequence of 4 to 20 bases. The UMI is used to distinguish effects from a single clone from effects from multiple clones (e.g., amplified products from a single clone contain the same UMI as the original clone). As used herein, the term "clone" can refer to a single mRNA or target nucleic acid to be sequenced. Therefore, the UMI can also be used to determine the number of specific analytes or events that gave rise to an amplification product. In an exemplary embodiment, each cellular barcode in a single nucleus is the same, but each UMI associated with the cellular barcode is randomized, resulting in each capture event having a unique UMI. For example, a spatial barcode nucleic acid can be captured by a cellular barcode nucleic acid containing a UMI. The number of different UMIs aligned with the spatial barcode in a single nucleus identified by the cellular barcode indicates the number of spatial barcodes tagged to the single nucleus (e.g., counting the UMIs of sequence reads with the same cellular barcode but different spatial barcodes).

[0115] In exemplary embodiments, nucleic acid sequences containing cellular barcodes and UMIs can capture multiple spatial barcodes when a single cell is close to or overlaps multiple locations on the array. In exemplary embodiments, computational methods, such as counting UMIs, can be used to resolve the location of a single cell within the spatial array. The computational method can use the distance on the array of various spatial barcodes. If spatial barcodes are close on the array, the location is likely somewhere between them or where their locations overlap. If spatial barcodes are far apart, the spatial barcode nucleic acid is likely diffused from that location, and that location is the spatial barcode with the most UMIs, because the spatial barcode with the highest UMI is the primary spatial barcode diffused in that single cell.

[0116] In single-cell assays, cellular barcodes are typically added in one of three ways: by adding single cells / nuclei to individual wells, each with a unique barcode or combination of barcodes; by pooling and split-indexing intact nuclei / cells; or by separating single cells / nuclei into individual reaction vessels, such as microwells, reaction chambers, or droplets, that contain unique cellular barcodes. In exemplary embodiments, for pooling and split-indexing, the spatial barcode nucleic acid is modified to contain a common universal sequence (e.g., a handle sequence) present in each target analyte, so that both the spatial barcode and the analyte can capture the same index sequence in each pooling and splitting step. In exemplary embodiments, for droplet methods, the spatial barcode nucleic acid is modified to contain a common universal sequence present in each target analyte, so that both can be captured by the cellular barcode nucleic acid present in each droplet (typically on a bead). Single-cell assays typically use capture sequences specific to the analyte, such as analytes with polyA tails or specific adapter sequences (e.g., tagmentation adapters) added by transposases. These sequences can be easily included in spatial barcode nucleic acids.

[0117] The methods of the present invention can be adapted to capture spatial barcodes by cellular barcodes for any single cell assay, specific examples are provided below.

[0118] Spatially resolved single nucleus / cell RNA sequencing In exemplary embodiments, the single-cell genomics sequencing library is a single-cell RNA sequencing library (see, e.g., Trombetta, JJ, Gennert, D., Lu, D., Satija, R., Shalek, A.K. & Regev, A. Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing. Curr Protoc Mol Biol. 107, 4 22 21-24 22 17, doi:10.1002 / 0471142727.mb0422s107 (2014); Qi Z, Barrett T, Parikh AS, Tirosh I, Puram SV. Single-cell sequencing and its applications in head and neck cancer. Oral Oncol. 2019;99:104441; Kalisky, T., Blainey, P. & Quake, SRGenomic Analysis at the Single-Cell Level. Annual review of genetics 45,431-445,(2011);Kalisky,T.& Quake,SRSingle-cell genomics.Nature Methods 8,311-314(2011);Islam,S.et al.Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq.Genome Research,(2011);Tang,F.et al.RNA-Seq analysis to capture the transcriptome landscape of a single cell.Nature Protocols 5,516-535,(2010);Tang,F.et al.mRNA-Seq whole-transcriptome analysis of a single cell.Nature Methods 6,377-382,(2009);Ramskold,D.et al.Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nature Biotechnology 30, 777-782, (2012); and Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification. Cell Reports, Cell Reports, Volume 2, Issue 3, p666-673, 2012). See PMCID: 4338574.

[0119] In an exemplary embodiment, the invention includes plate-based single-cell RNA sequencing (see, e.g., Picelli, S. et al., 2014, "Full-length RNA-seq from single cells using Smart-seq2," Nature protocols 9, 171-181, doi:10.1038 / nprot.2014.006). In an exemplary embodiment, the invention includes high-throughput single-cell RNA-seq. For more information, see Macosko et al., 2015, "Highly Parallel Genome-Wide Expression Profiling of Individual Cells Using Nanoliter Droplets," Cell 161, 1202-1214; International Patent Application No. PCT / US2015 / 049178, published March 17, 2016 as WO2016 / 040476; Klein et al., 2015, "Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells," Cell 161, 1187-1201; International Patent Application No. PCT / US2016 / 027734, published October 20, 2016 as WO2016168584A1; Zheng, et al., 2016, "Haplotyping germline and cancer genomes with high-throughput linked-read sequencing," Nature Biotechnology 34,303-311;Zheng,et al.,2017,“Massively parallel digital transcriptional profiling of single cells”Nat.Commun.8,14049 doi:10.1038 / ncomms14049;International Patent Application No. WO2014210353A2;Zilionis,et al.,2017,“Single-cell barcoding and sequencing using droplet microfluidics”Nat Protoc.Jan;12(1):44-73; Cao et al., 2017, “Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing” bioRxiv preprint first posted online Feb. 2, 2017, doi:dx.doi.org / 10.1101 / 104844; Rosenberg et al., 2017, “Scaling single cell transcriptomics through split pool barcoding” bioRxiv preprint first posted online Feb. 2, 2017, doi:dx.doi.org / 10.1101 / 105163; Rosenberg et al., “Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding” Science 15 Mar 2018; Vitak, et al., “Sequencing thousands of single-cell genomes with combinatorial indexing” Nature Methods, 14(3):302-308, 2017; Cao, et al., Comprehensive single-cell transcriptional profiling of a multicellular organism. Science, 357(6352):661-667, 2017; Gierahn et al., “Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput” Nature Methods 14, 395-398(2017); and Hughes, et al.See, "Highly Efficient, Massively-Parallel Single-Cell RNA-Seq Reveals Cellular States and Molecular Features of Human Skin Pathology," bioRxiv 689273; doi:doi.org / 10.1101 / 689273, the entire disclosures of each of which are incorporated herein by reference.

[0120] In an exemplary embodiment, the present invention includes single-nucleus RNA sequencing. In this regard, see Swiech et al., 2014, "In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9," Nature Biotechnology, Vol. 33, pp. 102-106; Habib et al., 2016, "Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons," Science, Vol. 353, Issue 6302, pp. 925-928; Habib et al., 2017, "Massively parallel single-nucleus RNA-seq with DroNc-seq," Nat Methods, 2017. Oct;14(10):955-958; International Patent Application No. PCT / US2016 / 059239, published September 28, 2017, as WO2017164936; International Patent Application No. PCT / US2018 / 060860, published May 16, 2019, as WO / 2019 / 094984; International Patent Application No. PCT / US2019 / 055894, published April 16, 2020, as WO / 2020 / 077236; Drokhlyansky, et al., “The enteric nervous system of the human and mouse colon at a single-cell resolution,” bioRxiv 746743; doi:doi.org / 10.1101 / 746743; and Drokhlyansky E, Smillie CS, Van Wittenberghe N, et al. The Human and Mouse Enteric Nervous System at Single-Cell Resolution. Cell. 2020;182(6):1606-1622.e23, the entire contents of which are incorporated herein by reference.

[0121] Single-cell / nucleus RNA-seq typically uses a poly-T capture sequence and reverse transcription (RT) to capture mRNAs with poly-A tails. In an exemplary embodiment, the present invention generates tagged nuclei with spatial barcode nucleic acids containing poly-A sequences. The tagged nuclei are then used in any of the RNA-seq methods described above. For example, tagged nuclei can be used in pool-and-split methods or separated into individual wells or droplets. Both the spatial barcode nucleic acid and the mRNA are captured by the cell barcode sequence. In plate- or microwell-based methods, reverse transcription and template-switching primers can be used to tag mRNA with unique barcodes and spatial barcode nucleic acids.

[0122] Spatially resolved single-cell chromatin accessibility In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell assay for transposase-accessible chromatin using sequencing (ATAC-seq). ATAC-seq can be used to identify accessible chromatin within cells (see, e.g., Buenrostro, et al., Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins, and nucleosome position. Nature Methods 2013;10(12):1213-1218). Using plate-, droplet-, or combinatorial indexing-based methods, thousands to hundreds of thousands of individual cells / nuclei can be analyzed in a single sample (e.g., Buenrostro et al., Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-490 (2015); Cusanovich, DA, Daza, R., Adey, A., Pliner, H., Christiansen, L., Gunderson, KL, Steemers, FJ, Trapnell, C. & Shendure, J. Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing. Science. 2015 May 22;348(6237):910-4. doi:10.1126 / science.aab1601. Epub 2015 May 7; Cusanovich DA, Hill AJ, Aghamirzaie D, et al.A Single-Cell atlas of in vivo mammalian chromatin accessibility.Cell.2018;174:1309-24;Lake BB,Chen S,Sos BC,et al.Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain.Nat Biotechnol.2018;36:70-80;Preissl S,Fang R,Huang H,et al.Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.Nat Neurosci.2018;21:432-9;Satpathy AT,Granja JM,Yost KE,et al.Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion.Nat Biotechnol.2019;37:925-36;Xu W,Wen Y,Liang Y,et al.A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility.Nat Protoc. 2021;16:4084-107; US20160208323A1; US20160060691A1; and WO2017156336A1). Single-nucleus ATAC-seq can also be performed by dividing nuclei into droplets and constructing snATAC-Seq libraries using the Chromium Next GEM Single Cell ATAC Reagent Kit v1.1 (10x Genomics, Pleasanton, CA, USA) (see, e.g., Briel N, Ruf VC, Pratsch K, et al.Single-nucleus chromatin accessibility profiling highlights distinct astrocytic signatures in progressive supranuclear palsy and corticobasal degeneration. See Acta Neuropathol. 2022;144(4):615-635.

[0123] In an exemplary embodiment, the present invention generates tagged nuclei using spatially barcoded nucleic acids that contain the same adapter sequences as those inserted into active chromatin by Tn5 transposase. The tagged chromatin fragments and spatially barcoded nucleic acids can then be captured by cellular barcoding using combinatorial indexing with barcoded beads or droplet-based methods.

[0124] Spatially resolved single-cell Hi-C In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell Hi-C sequencing library. In situ Hi-C involves crosslinking cells with formaldehyde, permeabilizing them while leaving the nuclei intact, digesting the DNA with an appropriate 4-cutter restriction enzyme (e.g., MboI), filling in the 5' overhangs while incorporating biotinylated nucleotides, ligating the resulting blunt-ended fragments, cleaving the DNA, capturing the biotinylated ligation junctions with streptavidin beads, and analyzing the resulting fragments by paired-end sequencing (see, e.g., Rao SS, Huntley MH, Durand NC, et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159(7):1665-1680). Hi-C can also be performed on single cells using various barcoding methods, such as combinatorial cell indexing (see, e.g., Ramani, et al., Sci-Hi-C: A single-cell Hi-C method for mapping 3D genome organization in large numbers of single cells. Methods. 2020 Jan 1;170:61-68; Ramani V, Deng X, Qiu R, et al. Massively multiplex single-cell Hi-C. Nat Methods. 2017;14(3):263-266; and Nagano T, Lubling Y, Varnai C, et al. Cell-cycle dynamics of chromosomal organization at single-cell resolution. Nature. 2017;547:61-7).

[0125] In an exemplary embodiment, the present invention generates tagged nuclei using spatial barcode nucleic acids containing double-stranded adapter sequences, crosslinks the tagged nuclei, and digests the DNA with an appropriate restriction enzyme. The nuclei are then distributed into 96-well plates, and a first barcode is introduced through ligation of a barcoded, biotinylated, double-stranded bridge adapter. The intact nuclei are then pooled, subjected to proximity ligation, and then diluted and redistributed into a second 96-well plate. After lysis, a second barcode is introduced through ligation of a barcoded Y adapter. In this example, both the fragmented DNA and the spatial barcode receive the same nucleus-specific barcode.

[0126] In an exemplary embodiment, the present invention involves generating tagged nuclei with spatial barcode nucleic acids containing adapter sequences, crosslinking the tagged nuclei, digesting the DNA with appropriate restriction enzymes, ligating the resulting fragments, and fragmenting the DNA with an adapter-loaded transposase (Tn5) within intact nuclei, after which the nuclei can be used in a droplet-based single-cell sequencing method using beads that capture the adapter sequences and add cellular barcodes.

[0127] Spatially resolved single-cell DNA methylation sequencing In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell DNA methylation sequencing library. Methods for distinguishing DNA methylation include (i) bisulfite conversion, (ii) Tet-assisted bisulfite conversion, (iii) Tet-assisted conversion with a substituted borane reducing agent, and (iv) protection of hmC followed by Tet-assisted conversion with a substituted borane reducing agent (see, e.g., U.S. Patent Application No. US20210115502A1). Methylation can also be detected using methylation-specific restriction enzymes or methylated DNA immunoprecipitation (MeDIP). In an exemplary embodiment, DNA methylation can be detected when methylated cytosine (mC) and hydroxymethylated cytosine (hmC) are determined by the sequencer itself, independently of one or more agents (e.g., using a PacBio or Nanopore sequencer).Single-cell DNA methylome sequencing techniques have been established using various strategies, such as reduced representation bisulfite sequencing (RRBS) and post-bisulfite adapter tagging (PBAT)-based methods (e.g., Ahn J, Heo S, Lee J, Bang D. Introduction to Single-Cell DNA Methylation Profiling Methods. Biomolecules. 2021;11(7):1013; Guo H, Zhu P, Wu X, et al. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res. 2013;23:2126-35; Luo C, Keown CL, Kurihara L, et al. Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex. Science. 2017;357:600-4; Smallwood SA, Lee HJ, Angermueller C, et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods. 2014;11:817-20; and Shareef SJ, Bevill SM, Raman AT, et al. Extended-representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells. Nat Biotechnol. 2021;39:1086-94).In the sci-MET method, a combinatorial indexing strategy is applied to increase throughput, with the first and second rounds of barcodes being incorporated by Tn5 transposon and random priming, respectively (see Mulqueen RM, Pokholok D, Norberg SJ, et al. Highly scalable generation of DNA methylation profiles in single cells. Nat Biotechnol. 2018;36:428-31).

[0128] Spatially resolved single-cell chromatin immunoprecipitation (ChIP) In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell chromatin immunoprecipitation (ChIP) sequencing library. Chromatin immunoprecipitation (ChIP) is a widely used method for detecting histone modifications within the nucleosomes of chromatin. In single-cell ChIP-seq analysis, cell-specific barcodes are added before cells are aggregated and immunoprecipitated (see Ai S, Xiong H, Li CC, et al. Profiling chromatin states using single-cell itChIP-seq. Nat Cell Biol. 2019;21:1164-72; Grosselin K, Durand A, Marsolier J, et al. High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer. Nat Genet. 2019;51:1060-6; ​​and Rotem A, Ram O, Shoresh N, et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat Biotechnol. 2015;33:1165-72). Of these methods, Drop-ChIP and scChIP-seq add cellular barcodes by MNase digestion and ligation through a droplet microfluidic workflow, whereas itChIP adds cellular barcodes by Tn5 transposase tagmentation through a chromatin-opening step.

[0129] In an exemplary embodiment, the spatial barcode is designed to enable tagmentation or digestion and ligation, such that a barcode sequence comprising a cellular barcode and a spatial barcode is generated. For example, single nuclei can be separated into individual droplets or microwells containing the cellular barcode. The cellular barcode / spatial barcode hybrid is amplified and sequenced from the input chromatin used for immunoprecipitation. This provides the spatial location of each cell. The immunoprecipitated chromatin can be assigned to the same cell based on the cellular barcode.

[0130] Spatially resolved single-cell enzyme-linked chromatin profiling In an exemplary embodiment, the single-cell genomics sequencing library is a single-cell enzyme-linked chromatin profiling sequencing library. Enzyme-linking is a non-immunoprecipitation chromatin profiling method that has become increasingly popular and has been adapted for single-cell analysis (e.g., Carter B, Ku WL, Kang JY, et al. Mapping histone modifications in low cell number and single cells using antibody-guided chromatin tagmentation (ACT-seq). Nat Commun. 2019;10:3747; Harada A, Maehara K, Handa T, et al. A chromatin integration labeling method enables epigenomic profiling with lower input. Nat Cell Biol. 2019;21:287-96; Kaya-Okur HS, Wu SJ, Codomo CA, et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun. 2019;10:1930; Schmid M, Durussel T, Laemmli UK. ChIC and ChEC; genomic mapping of chromatin proteins. Mol Cell. 2004;16:147-57; Skene PJ, Henikoff S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife. 2017;6:e21856; and Wang Q, Xiong H, Ai S, et al. CoBATCH for High-Throughput Single-Cell Epigenomic Profiling. Mol Cell. 2019;76:206-16..e7).In these techniques, Tn5 transposase, MNase, or adenine methyltransferase binds to antibody-binding protein A, directly to the antibody, or to target chromatin proteins, allowing for the marking of genomic regions with specific histone marks. ChIC, CUT&RUN, and scChIC use MNase, while scCUT&Tag, COBATCH, ACT-seq, and ChIL-seq use Tn5 transposase. A critical cation activation step (Ca2+ for MNase and Mg2+ for Tn5 transposase) allows for rapid activation of enzyme activity after washing away nonspecifically bound enzyme.

[0131] In an exemplary embodiment, single tagged nuclei according to the present invention are tagged with a spatial barcode nucleic acid containing the same sequence as a tagmentation or ligation adapter used in enzyme-linked chromatin profiling. In enzyme-linked chromatin profiling, an adapter is added to chromatin bound by an antibody specific for a chromatin modification or protein. These nuclei can then be used in single-cell sequencing (e.g., 10X ATAC-seq kits) with barcode beads containing sequences that hybridize to the adapter sequence and add a bead-specific barcode to the tagged chromatin and spatial barcode. In other words, nuclei are tagged with spatial barcodes, nuclei are isolated, antibodies are added to the nuclei, unbound antibodies are washed away, a secondary antibody containing Tn5 or MNase is added, and Ca is added to tag the antibody-bound DNA. 2+ or Mg 2+ A buffer solution is added and a compatible single-cell sequencing method is performed to add cellular barcodes to the spatially barcoded and marked chromatin.

[0132] Spatially resolved single-cell proteomics In exemplary embodiments, the single-cell genomics sequencing library comprises a single-cell proteomics sequencing library (see, e.g., Yang L, George J, Wang J. Deep Profiling of Cellular Heterogeneity by Emerging Single-Cell Proteomic Technologies. Proteomics. 2020;20(13):e1900226. doi:10.1002 / pmic.201900226; single cell constituents (US20180340939A), single-cell proteomic assay using aptamers (US20180320224A1), and methods of identifying multiple epitopes in cells (US20170321251A1)).

[0133] In exemplary embodiments, tagged nuclei are labeled with an antibody or aptamer conjugated with a barcode oligonucleotide, the nuclei are washed to remove unbound antibody or aptamer, and the barcode oligonucleotide of the bound antibody or aptamer captures the cellular barcode sequence. The antibody or aptamer conjugated oligonucleotide contains a universal sequence complementary to the capture sequence or handle for pool and split indexing or capture by cellular barcode beads. The spatial barcode nucleic acid is modified to contain the same universal sequence, such as to capture the spatial barcode to the cellular barcode sequence.

[0134] Spatially resolved single-cell multi-omics In exemplary embodiments, the single-cell genomics sequencing library comprises a single-cell multiomics sequencing library (e.g., Lee J, Hyeon DY, Hwang D. Single-cell multiomics: technologies and data analysis methods. Exp Mol Med. 2020; 52(9): 1428-1442. doi:10.1038 / s12276-020-0420-2; Wen L, Tang F. Single cell epigenome sequencing technologies. Mol Aspects Med. 2018; 59: 62-69; Cao J, Cusanovich DA, Ramani V, et al. Joint profiling of chromatin accessibility and gene expression in thousands of single cells. Science. 2018; 361: 1380-5; Chen S, Lake BB, Zhang K. High-throughput sequencing of the transcriptome and chromatin accessibility in the same cell.Nat Biotechnol.2019;37:1452-7;Liu L,Liu C,Quintero A,et al.Deconvolution of single-cell multi-omics layers reveals regulatory heterogeneity.Nat Commun.2019;10:470;Zhu C,Yu M,Huang H,et al.An ultra high-throughput method for single-cell joint analysis of open chromatin and transcriptome.Nat Struct Mol Biol.2019;26:1063-70;Ma,S.et al.Chromatin potential identified by shared single cell profiling of RNA and chromatin.bioRxiv 2020.06.17.156943 (2020) doi:10.1101 / 2020.06.17.156943; and Stoeckius, M. et al. Simultaneous epitope and transcriptome measurement in single cells. Nat. Methods 14, 865-868 (2017). In an exemplary embodiment, for a multi-omics assay, the spatial barcode need only be captured by a cellular barcode for one of the omics portions of the multi-omics single-cell assay. For example, if the multi-omics assay includes transcriptome sequencing, the spatial barcode can be configured to be captured by a poly-T sequence.

[0135] Imaging / Image Assembly Using the spatial barcodes of individual nuclei, identified by their cell-of-origin barcodes, and analytes, identified by sequences that also include the cell-of-origin barcodes, high-resolution images identifying analyte expression sites can be readily constructed in silico. In exemplary embodiments, the spatial locations of numerous sites (e.g., beads) within the array can first be assigned to image locations, and expression data for all associated analytes is also assigned to those locations. (Optionally, using spatial barcodes to assign analyte sequence information to array locations effectively separates the spatial barcodes from the array / matrix of analyte sequence information associated with particular sites / beads.) The underlying analyte sequence information can then be used to generate high-resolution images representing the range of individual analytes or grouped analytes across the various spatial locations of the array. The images (i.e., pixel color and / or intensity) can be adjusted and / or normalized using any (or any number) art-recognized techniques deemed appropriate by those skilled in the art.

[0136] In exemplary embodiments, a high resolution image of the present disclosure is an image in which individual features (e.g., pixels) of the image are spaced 50 μm or less apart, hi some embodiments, the spacing between individual features in the image is 40 μm or less, optionally 30 μm or less, optionally 20 μm or less, optionally 15 μm or less, optionally 10 μm or less, optionally 9 μm or less, optionally 8 μm or less, optionally 7 μm or less, optionally 6 μm or less, optionally 5 μm or less, optionally 4 μm or less, optionally 3 μm or less, optionally 2 μm or less, or optionally 1 μm or less.

[0137] Images can be acquired using detection devices known in the art. Examples include microscopes configured for light, bright field, dark field, phase contrast, fluorescence, reflectance, interference, and confocal imaging. Biological specimens can be stained prior to imaging to enhance contrast between different regions or cells. In some embodiments, multiple stains can be used to image different aspects of the specimen (e.g., different regions of tissue, different cells, specific subcellular components, etc.). In other embodiments, biological specimens can be imaged without staining.

[0138] In certain embodiments, a fluorescence microscope (e.g., a confocal fluorescence microscope) can be used to detect biological specimens that fluoresce, for example, due to a fluorescent label. Fluorescent specimens can also be imaged using a nucleic acid sequencing device with optics for fluorescence detection, such as the Genome Analyzer®, MiSeq®, NextSeq®, or HiSeq® platform devices commercialized by Illumina, Inc. (San Diego, Calif.), or the SOLiD™ sequencing platform commercialized by Life Technologies (Carlsbad, Calif.). Other imaging optics that can be used include those found in the detection devices described in Bentley et al., Nature 456:53-59 (2008), PCT Publication Nos. WO 91 / 06678, WO 04 / 018497, or WO 07 / 123744, U.S. Patent Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, or 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0139] For example, images of a biological specimen can be obtained at a desired resolution to distinguish tissues, cells, or subcellular components. Thus, the resolution can be sufficient to distinguish components of the biological specimen that are at least 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 500 μm, 1 mm, or more apart. Alternatively or additionally, the resolution can be set to distinguish components of the biological specimen that are at least 1 mm, 500 μm, 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, or less apart.

[0140] The methods described herein can include correlating the location of a biological specimen within an image with the barcode sequences of nucleic acid probes attached to individual beads with which the biological specimen is in contact, has been in contact, or will be in contact. Thus, identifiable characteristics of the biological specimen within the image can be correlated with the nucleic acids identified to be present in its vicinity. Such correlations can involve any of a variety of morphological characteristics, such as cell shape, cell size, tissue shape, staining patterns, the presence of specific proteins (e.g., as detected by immunohistochemical staining), or other characteristics routinely evaluated in pathology or research applications. Thus, the biological state of a tissue or its components as determined by visual observation can be correlated with molecular biological characteristics as determined by spatially resolved nucleic acid analysis.

[0141] The solid support on which the biological specimen is imaged can include fiducial markers to facilitate orientation of the specimen or its image relative to the probes attached to the solid support. Exemplary fiducials include, but are not limited to, beads (with or without a moiety such as a fluorescent moiety or a nucleic acid to which a labeled probe can bind), fluorescent molecules attached to a known or determinable feature, or structures combining a morphological shape and a fluorescent moiety. Exemplary fiducials are set forth in U.S. Patent Application Publication No. 2002 / 0150909A1 or U.S. Patent Application No. 14 / 530,299, each of which is incorporated herein by reference. Preferably, one or more fiducials are visible when an image of the biological specimen is obtained. Preferably, the solid support includes at least 2, 3, 4, 5, 10, 25, 50, 100, or more fiducial markers. The fiducials can be provided in a pattern, for example, along the outer edge of the solid support or along the perimeter of the location where the biological specimen is located. In one exemplary embodiment, the one or more fiducials are detected using the same imaging conditions used to visualize the biological specimen. However, if desired, separate images can be obtained (eg, one image of the biological specimen and another image of the reference) and the images registered with each other.

[0142] In an exemplary embodiment, spatial filtering can be used to clean up the data. In one example, missing specific beads are removed. The total spatial barcode, nUMI, can be used to remove beads.

[0143] In an exemplary embodiment of slide tagging, spatial barcode oligonucleotides are released into tissue sections, "tagging" nuclei. The nuclei are then isolated from the tissue, and the spatial barcodes are sequenced along with other molecular information. Importantly, each nucleus receives many spatial barcode oligonucleotides from different locations on the bead array. Therefore, nuclei must be computationally spatially positioned based on the distribution of spatial barcodes each nucleus receives. In an exemplary embodiment, density-based spatial clustering of applications with noise (DBSCAN) is used to localize nuclei to spatial locations (see, e.g., Ester, M., Kriegel, H.-P., Sander, J. & Xu, X.A. density-based algorithm for discovering clusters in large spatial databases with noise. In Proc. Second International Conference on Knowledge Discovery and Data Mining (eds Simoudis, E. et al.) 226-231 (AAAI Press, 1996); and Hahsler, M., Piekenbrock, M. & Doran, D. dbscan: fast density-based clustering with RJ Stat. Softw. 91, 1-30 (2019)). DBSCAN is applied to distinguish between "signal" spatial barcodes (those likely to add value to nuclei localization) and background "noise" spatial barcodes (those likely to disrupt nuclei localization). DBSCAN outputs a cluster assignment for each spatial barcode. Clusters = 0 indicate noise barcodes, while clusters > 0 indicate signal barcodes grouped with other signal barcodes that are spatially clustered. Spatial locations where all spatial barcodes are shown as noise are either not assigned to a nucleus or are assigned to a nucleus with multiple signal clusters.From the remaining nuclei with one clear spatial barcode signal cluster, a weighted centroid of the spatial barcode coordinates within the signal cluster is obtained, where the weight is the number of unique molecular identifiers (UMIs) in the sequenced barcodes. Importantly, DBSCAN requires two parameters: minPts and eps (effectively the radius). To determine the optimal parameter set for each slide tagging run, 15 different minPts parameters are iterated, and the parameter set with the highest percentage of nuclei with assigned spatial locations (one DBSCAN signal cluster) is selected.

[0144] While DBSCAN localizes nuclei with relatively high sensitivity and specificity, it is not the only approach to determining location from a set of spatial barcode coordinates. Alternatives include assigning the nucleus the location of the highest UMI spatial barcode or taking the weighted two-dimensional median of the spatial barcode coordinates. Other methods for distinguishing signal from noise barcodes include (1) k-means clustering, (2) affinity propagation, (3) mean shift, (4) spectral clustering, (5) agglomerative clustering, and (6) DBSCAN extensions such as HDBSCAN and OPTICS.

[0145] In exemplary embodiments, the location of nuclei in the Z plane can be measured using, individually or in combination, the number of spatial barcode UMIs per nucleus, the spread of spatial barcode coordinates per nucleus (e.g., average pairwise distance or the like), and fluorescence measurements of fluorescently labeled spatial barcodes retained within the nuclei.

[0146] kit The present disclosure also provides kits containing agents of the present disclosure for use in the methods of the present disclosure. The kits of the present disclosure may include one or more containers containing agents (e.g., capture materials such as liquid electrical tape) and / or compositions (e.g., slide capture bead arrays) of the present disclosure. In exemplary embodiments, the kits include beads containing spatially barcoded nucleic acids described herein. In exemplary embodiments, the beads are configured for specific single-cell genomics assays described herein. In some embodiments, the kits even include instructions for use in accordance with the methods of the present disclosure. In some embodiments, the instructions include instructions for preparing tissue cryosections, forming spatially defined (or simply spatially definable, pending the performance of steps that define the spatial resolution of the bead array) bead arrays, contacting tissue cryosections with the spatially defined bead arrays, and using tagged nuclei in subsequent single-cell assays.

[0147] The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions stored on a storage drive or provided over the internet) are also acceptable. The label or package information indicates that the composition is used for staging frozen sections and / or diagnosing analyte patterns in frozen sections. Instructions can be provided for practicing any of the methods described herein.

[0148] The kits of the present disclosure are contained in suitable packaging, including but not limited to vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The container may further contain a pharmaceutically active agent.

[0149] Kits may optionally provide additional components, such as buffers and instructional information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0150] Further embodiments are illustrated in the following examples, which are for illustrative purposes only and do not limit the scope of the invention. [Example]

[0151] Example 1 - Slide Tag Protocol Preparation: Stock 500 mL of dissociation buffer (DB) using ultrapure nuclease-free water and the following reagents: Na2SO4 - 5.83g K2so4 - 2.615g Glucose - 0.905g Hepes - 1.2g Mgcl2 - 2.5mL Extraction buffer: ExB DB-15mL 1% Kollidon - 0.150g 1% TX-100-150ul 10% BSA - 15 μL RNase inhibitor (Lucigen) 1 bottle Washing buffer: WB (20 ml per sample) DB-20mL 10% BSA - 20ul RNase inhibitor 50ul Steps: Prepare tissue sections of 1.3 mm x 20 μm. NOTE: Use a paintbrush to gently press the tissue slices flat. 2. Transfer the tissue to a 3mm pack and dissolve the tissue with your fingers. NOTE: Place the puck on a glass slide. Place this glass slide on ice until the tissue is transferred. Immediately before transferring the tissue to the puck, place the glass slide in the cryostat. Transfer the cut tissue to the puck, position the tissue in the center of the puck with a brush, and melt the tissue with your fingers by gently tapping the tissue. This step must be performed as quickly as possible, or the tissue will begin to bend. 3. Add 5 ul of DB to the pack, photocleave for 3 minutes, then incubate on ice for 5 minutes. 4. Transfer the pack to a well plate. Note: Transfer pack by grasping the edge with pointed tweezers. 5. Dissociate the tissue on the pack using extraction buffer with a 200 / 1000ul pipette. NOTE: Use a 200 / 1000µl pipette to place the extraction buffer onto the pack. The total volume of extraction buffer used is 2ml. Pipetting pressure should be moderate. After every 15-20 pipetting cycles, observe under a microscope to see if the tissue is dissociated. Repeat this process until the tissue is dissociated. 6. Once the tissue is dissociated, remove the pack from the well. 7. Transfer the dissociated sample to 20 ml of wash buffer and split this into two 50 ml tubes. 8. Centrifuge at 600g for 10 minutes at 4°C. 9. Remove the supernatant, leaving 500ul of pellet in each tube, and pool the pellets to make 1ml. 10. Filter the solution using a 40 micron filter. 11. Add 1:1000 DAPI to the filtered solution and incubate for 8-10 minutes. Centrifuge at 200g for 10 minutes at 12.4°C. 13. Remove the supernatant, leaving 50ul of pellet. 14. Count nuclei using a hemocytometer and load 43.3ul into a 10X.

[0152] Example 2 - Slide Tag Description and Results The slide tag method is described in Figures 1, 2, and 3. In the first step of slide tag, fresh frozen tissue slices are placed on a barcoded bead array. Each bead contains tens of thousands of oligos with the same spatial barcode, and the sequence and location of each spatial bead barcode are known. These oligos are photocleaved and released into the tissue, where they "tag" nuclei. These tagged nuclei are then isolated. Finally, these nuclei can be used as input for established single-cell sequencing techniques. This allows users to obtain true single-cell resolution, with data quality indistinguishable from snRNA-seq-only experiments, while also providing the spatial coordinates of each nucleus. This protocol is easy to implement, and the tagging process adds only approximately 10 minutes to the workflow.

[0153] Slide-tag offers many advantages over other truly spatial methods (Figure 4). Slide-tag is optimized to increase the proportion of cells that can be spatially located (Figure 6). Slide-tag can accurately profile the mouse hippocampus (Figures 5 and 7). Slide-tag recovers embryonic cell types at assigned locations with high probability and recapitulates known anatomy with de novo clusters that show clear regional patterns (Figures 8 and 11). Slide-tag can recapitulate dynamic events during development (Figures 9 and 10). Slide-tag can provide 3D spatial data (Figure 12). The thinner the tissue section, the higher the spatial assignment (Figure 13). Slide-tag can be used for spatially resolved single-cell multi-omics experiments (Figure 14).

[0154] Slide Tags can be used with the 10X single-cell sequencing technology (Figure 15). To benchmark Slide Tags, we applied it to profiling the mouse hippocampus, which has a highly stylized structure, allowing us to clearly evaluate the technology's performance (Figure 16). The standard output from an snRNA-seq experiment is a cell x gene matrix, from which we can create a dimensionality reduction plot highlighting cell type populations, with cell type populations indicated by shading. Using Slide Tags, each of these cells is associated with a cluster of spatial barcodes. We can then take the centroid of these points to obtain the cell's precise location. This allows for a spatial coordinate matrix to accompany each Slide Tags experiment. Slide Tags enables single-nucleus spatial transcriptomics in the mouse hippocampus (Figure 17). After spatially mapping all profiled cells, Slide Tags reproduces the expected organizational structure of the mouse hippocampus.

[0155] To quantify spatial positioning accuracy, we first compared the width of the hippocampal subfield, area cornu ammonis (CA1), in slide tags with Nissl-stained serial sections and found that the width of the slide tag features matched the Nissl images (Figure 18). CA1 is divided into deep and superficial sublayers. Different gene expression patterns are observed in the deep and superficial layers (Dong HW, Swanson LW, Chen L, Fanselow MS, Toga AW. Genomic-anatomic evidence for distinct functional domains in hippocampal field CA1. Proc Natl Acad Sci U S A. 2009;106(28):11794-11799; Cid E, Marquez-Galera A, Valero M, et al. Sublayer- and cell-type-specific neurodegenerative transcriptional trajectories in hippocampal sclerosis. Cell Rep. 2021;35(10):109229). Furthermore, applicants were able to precisely identify cell subtypes in the deep and superficial layers of CA1 (FIG. 19).

[0156] To verify whether slide tags affect the quality of transcriptome data, we compared standard snRNA-seq experiments with experiments in which slide tags were used followed by snRNA-seq. We found little difference in data quality in terms of cell type proportions, average UMI per cell, and average overall gene expression (Figure 21). Furthermore, because each nucleus receives many spatial barcode oligos from many different beads, spatial resolution is not limited to the 10-micron size of the beads (Figure 20). For example, cell X received four spatial barcodes from two beads, three spatial barcodes from another bead, and one barcode each from two other beads. Using this information, the location of cell X can be determined to within 10 microns. Instead, we estimate that using slide tags results in spatial resolution below 10 microns, between 3 and 4 microns (Figure 20). Slide tags generate high-quality spatial single-cell data that outperform traditional spatial methods (Figure 22). Slide tags are compatible with existing single-nucleus sequencing technologies and amino acid analysis tools (Figure 23). Applicants have demonstrated this in the applications shown.

[0157] Applicant applied Slide Tag to a human metastatic melanoma sample (Figure 24). The H&E image shows two histopathological lobes separated by a line. As before, Applicant was able to identify transcriptome clusters, here confirming three clusters of tumor cells, but using Slide Tag pinpointed the location of these clusters. Applicant observed two transcriptionally distinct subpopulations of profiled cancer cells. Applicant spatially mapped these subpopulations and found them to be spatially separated (Figure 25A). Applicant asked whether there were cytogenetic differences between the subpopulations and used inferCNV, a method for inferring copy number variation from transcriptome data. Applicant found many common copy number variations between the subpopulations, but also some inferred alterations unique to each subpopulation (Figure 25B). Using single-cell data, Applicant could ask how this heterogeneity relates to the cell-cell interactions that form these tumor clusters. Applicants calculated the change in the proportion of cells near these tumor cells and determined that CD8 T cells were enriched in the vicinity of tumor 2, depleted in tumor 1b, and neither enriched nor depleted in tumor 1a (Figure 26A). Applicants demonstrate this spatially on a single cell basis when the enrichment of the proportion of CD8 T cells is plotted within the vicinity of each tumor (Figure 26B).

[0158] Applicant asked: What are the T cell states and receptors in different neighborhoods? Applicant observed that T cells in Tumor 2 had a more cytotoxic phenotype (i.e., cell state) (Figure 27A). Applicant sequenced TCR receptors from slide tags and observed that TCR clones had distinct spatial localization (Figure 27B). Slide tags are compatible with sequencing TCR receptors from 3' single-cell libraries (Liu S, Iorgulescu JB, Li S, et al. Spatial maps of T cell receptors and transcriptomes reveal distinct immune niches and interactions in the adaptive immune response. Immunity. 2022;55(10):1940-1952.e5).

[0159] Applicant observed differences in T cell infiltration and status between tumor 1 and tumor 2. Applicant hypothesized that HLA loci were downregulated with copy number loss on Chr6 in tumor 1 but not in tumor 2. Applicant observed a predicted copy number loss on Chr6 (Figure 28). Furthermore, gene set enrichment analysis of differentially expressed genes between tumor populations revealed that the endogenous antigen presentation machinery was downregulated in tumor 1. Slide-tag multiome recovered expression and epigenomic profiles (Figures 29A-C).

[0160] Another hypothesis is that melanoma cells in Tumor 1 are undergoing a cell state transition, i.e., dedifferentiation accompanied by the loss of endogenous antigen expression. Applicant observed differentially expressed and accessed genes between the two tumor populations in RNA and ATAC data and discovered TNC, a gene associated with a cell state transition toward a more aggressive melanoma phenotype (Figures 30A-30C). Applicant hypothesized that Tumor 1 lost endogenous antigens associated with melanocyte identity, resulting in a more suppressive T cell microenvironment. Applicant assessed tumor cells based on the expression of mesenchymal-like cell state genes (Figure 31A). Consistent with their hypothesis, Applicant found that Tumor 1 exhibited a more mesenchymal-like phenotype compared to Tumor 2, beginning to lose melanocyte markers such as PMEL and MLANA and increasing expression of TNC and AXL. Applicant then referenced the same nuclear ATAC data to identify transcription factor motifs associated with this mesenchymal-like state. We found that transcription factors such as FOS, JUN, and IRF9 were positively correlated with the mesenchymal-like score, whereas transcription factors such as MITF were negatively correlated (or more correlated with the melanocyte-like score) (Figure 31B). Finally, spatial mapping of these TF motif scores revealed that some TF motifs spatially clustered in correlation with mesenchymal-like cell states. This suggests pressures in the tumor microenvironment or epigenetic inheritance through cell division.

[0161] Human tissue often poses unique challenges for technology development. For example, nuclei are more sparsely packed in the human brain, necessitating a different scale for the sequences used for barcoding. Applicant performed slide tagging on a 5.5x5.5 area of ​​human prefrontal cortex, where laminar structure is well characterized (Figure 32). Applicant sequenced a total of 14,000 nuclei, of which 6,500 were spatially located. Applicant recovered all known cortical layer types (see, e.g., Bakken TE, Jorstad NL, Hu Q, et al. Comparative cellular analysis of motor cortex in human, marmoset, and mouse [published correction appears in Nature. 2022 Apr;604(7904):E8]. Nature. 2021;598(7879):111-119).

[0162] Human tonsils are highly dynamic, dense organs in which many cell differentiation events occur simultaneously. Human tonsils contain germinal centers where B cell maturation occurs. Memory B cells stimulate germinal centers, while naive cells are excluded from them. Tonsils possess a rich immune receptor repertoire. Tonsils have traditionally been difficult to profile with spatial tools due to their small size and tightly packed cell types. We demonstrate the utility of using slide tags (Figure 33). Applicant used spatially mutated genes to identify dark and light regions in germinal centers (Figure 34).

[0163] Slide tags can be used to specify spatially significant receptor-ligand interactions (Figure 35). Traditional methods for inferring receptor-ligand interactions using single-cell data cannot determine whether single cells are spatially close together (Figure 35A). Using spatial information to curate receptor-ligand interactions in germinal centers allows us to identify nearby single cells that express the ligand-receptor pair (Figure 35B). For example, a single cell expresses CD40LG and CD40 in a bright area.

[0164] Next, we investigated whether we could perform spatial multi-omics sequencing by testing both ATAC and RNA-seq on the same single cells using slide tags (Figure 36). We profiled a region of mouse p1 brain (Figure 36A). We collected high-quality ATAC data: enrichment of TSSs, fragment lengths corresponding to nucleosome peaks, and a high number of unique fragments per cell (Figure 36B).

[0165] While Applicants can embed cells with both RNA expression and ATAC peak profiles, Applicants can now also use combined mutations (WNN) to better inform cell clustering (Figure 37A). Applicants can plot these clusters spatially, which reveals many spatially defined clusters (Figure 37B).

[0166] Applicant used slide tags to identify spatially restricted transcriptome and epigenetic signatures in developing L2 / 3 (Figure 38). Applicant zoomed in on one of the clusters belonging to the outer layers of the developing cortex, where layers are defined but regions along these layers are not yet fully defined. Applicant performed an autocorrelation analysis using the RNA data, identifying broad regions: the isocortex, the posterior callosal cortex, and the hippocampal column, with several representative top hits (Figures 38A and 38B). Applicant then asked the same question in the ATAC data and found peaks identifying subregions of the isocortex (Figure 38C). Applicant sought to further understand this by identifying transcription factor motifs that vary along this axis and discovered four transcription factors, three of which have known roles in cortical development (Figure 38D).

[0167] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the present invention has been described in connection with specific embodiments, it will be understood that the invention is capable of further modification, and that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the present invention. This application is intended to cover any variations, uses, or adaptations of the present invention, including departures from the present disclosure, that generally follow the principles of the invention, are within known practice in the art to which the invention pertains, and can be applied to the essential features described hereinabove.

Claims

1. 1. A method for generating spatially tagged nuclei for use in single cell genomics, comprising: a) placing a tissue sample on a sequence-verified spatial array, the spatial array comprising nucleic acid sequences comprising spatial barcodes attached to the array via cleavable linkers, the spatial barcodes being the same for each location on the array but different for any other location on the spatial array; b) cleaving the linker and delivering the spatially barcoded nucleic acid to nuclei in the tissue sample; c) isolating the tagged nuclei from the tissue sample.

2. 10. The method of claim 1, further comprising preparing a single-cell genomics sequencing library using the isolated tagged nuclei, wherein nucleic acid sequences each comprising a cellular barcode sequence identifying a cell of origin and optionally a unique molecular identifier (UMI) are captured from each spatially tagged nucleus to create a nucleic acid sequence comprising a spatial barcode and a cellular barcode, such that genomics data of each single cell can be identified by the cellular barcode, and the spatial location of the same single cell in the tissue can be identified by the same cellular barcode.

3. 3. The method of claim 1 or 2, wherein the spatial barcode is delivered to the nucleus by diffusion.

4. 4. The method of claim 1, wherein prior to step (a), the spatial array is sequence verified by in situ sequencing of nucleic acid sequences comprising spatial barcodes, thereby generating an index of the spatial barcodes on the array.

5. 5. The method of claim 4, wherein the in situ sequencing is performed by sequencing by ligation or sequencing by synthesis.

6. 6. The method of any one of claims 1 to 5, wherein the spatial array comprises a solid support fixed at each location on the array and linked via the cleavable linker to the nucleic acid sequence comprising a spatial barcode, the spatial barcode being the same for each individual solid support but different for any other solid support in the spatial array.

7. The method of claim 6 , wherein the solid support is covalently immobilized in the spatial array.

8. The method of claim 6 , wherein the solid support is immobilized in the spatial array by a vinyl polymer.

9. The method of any one of claims 6 to 8, wherein the solid support is a bead.

10. The method of claim 9 , wherein the beads are polystyrene beads.

11. 11. The method of claim 9 or 10, wherein the beads have a diameter of 50 μm or less.

12. 12. The method of claim 11, wherein the beads are 1, 3, 10, 15, or 20 μm.

13. The method of any one of claims 1 to 12, wherein the linker is a photocleavable, chemically cleavable, or enzymatically cleavable linker.

14. 14. The method of any one of claims 2 to 13, wherein the spatial barcode nucleic acid comprises a polyA sequence for capture by a cellular barcode nucleic acid comprising a polyT sequence.

15. The method of any one of claims 1 to 14, wherein the tissue sample is treated to permeabilize the nuclei.

16. 16. The method of claim 15, wherein the tissue sample is permeabilized in a buffer that increases diffusion of the spatially barcoded nucleic acids.

17. The method of any one of claims 1 to 16, wherein the spatial barcode nucleic acids are of different lengths.

18. 18. The method of any one of claims 1 to 17, wherein the spatial barcode nucleic acid further comprises one or more modifications that facilitate diffusion into the nucleus.

19. 20. The method of claim 18, wherein the spatial barcode nucleic acid is modified by the addition of one or more lipid or cholesterol groups.

20. 20. The method of any one of claims 1 to 19, wherein the spatial barcode nucleic acid further comprises one or more fluorescent labels that can be used to identify tagged nuclei.

21. The method of any one of claims 1 to 20, wherein the tissue sample is a fresh frozen tissue section.

22. The method of any one of claims 1 to 20, wherein the tissue sample is a fresh, unfixed tissue section.

23. The method of any one of claims 1 to 20, wherein the tissue sample is a fixed tissue section.

24. The method of any one of claims 2 to 23, wherein the location of each cell in the tissue sample is determined computationally based on sequencing of the library.

25. 25. The method of claim 24, wherein the cellular barcode nucleic acid comprises a UMI sequence, and the location of each cell in the tissue is determined based on the number of UMIs sequenced for each spatial barcode that has the same cellular barcode sequence.

26. 26. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-nucleus RNA sequencing library (snRNA-seq).

27. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell DNA accessibility library.

28. 26. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell ATAC sequencing library (ATAC-seq).

29. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell chromatin immunoprecipitation (ChIP) sequencing library.

30. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell genome sequencing library.

31. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell DNA methylation sequencing library.

32. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell Hi-C sequencing library.

33. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell CUT&Tag sequencing library.

34. 26. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell genome and transcriptome sequencing library (G&T-seq).

35. The method of any one of claims 2 to 25, wherein the single-cell genomics sequencing library is a single-cell proteome library.

36. 1. A kit comprising a plurality of solid supports attached via cleavable linkers to nucleic acid sequences comprising spatial barcodes and capture sequences, wherein the spatial barcodes are the same for each solid support but different for any other solid support in the spatial array, and the capture sequences are the same across all of the solid supports.

37. 37. The kit of claim 36, wherein the solid support further comprises a chemical linking moiety for covalently immobilizing the solid support to an array.

38. 38. The kit of claim 36 or 37, wherein the solid support is a bead.

39. 39. The kit of claim 38, wherein the beads are polystyrene beads.

40. 40. The kit of claim 38 or 39, wherein the beads have a diameter of 50 μm or less.

41. 41. The kit of claim 40, wherein the beads are 1, 3, 10, 15, or 20 μm, preferably 10 μm.

42. The kit of any one of claims 36 to 41, wherein the linker is a photocleavable, chemically cleavable, or enzymatically cleavable linker.

43. The kit of any one of claims 36 to 42, wherein the capture sequence comprises a polyA sequence.

44. 44. The kit of any one of claims 36 to 43, wherein the spatial barcode nucleic acids attached to the solid support are of different lengths.

45. 45. The kit of any one of claims 36 to 44, wherein the spatial barcode nucleic acid further comprises one or more modifications that facilitate diffusion into the nucleus.

46. 46. ​​The kit of claim 45, wherein the spatial barcode nucleic acid is modified by the addition of one or more lipid or cholesterol groups.

47. 47. The method of any one of claims 36 to 46, wherein the spatial barcode nucleic acid further comprises one or more fluorescent labels that can be used to identify tagged nuclei.

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