Methods and compositions for solid capture-based spatial cut&tag

Abstract

Compositions, methods, and kits for performing multiplexed, spatially-resolved chromatin analysis. In certain embodiments, a transposome complex comprising a first transposase and a second transposase are provided, the first transposase having a first adapter comprising a T7 promoter and the second transposase having a second adapter comprising a capture sequence, the first transposase and the second transposase each being a fusion protein comprising an antibody-binding ligand.

Description

[0001] Attorney Docket: NYG-LIPP-249PCT

[0002] METHODS AND COMPOSITIONS FOR SOLID CAPTURE-BASED

[0003] SPATIAL CUT&TAG

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under HG01 1014 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The electronic sequence listing filed herewith named “NYG- LIPP249PCT_SeqList” with size of 15,615 bytes, created on date of November 21, 2025, and the contents of the electronic sequence listing (e.g., the sequences and text therein) are incorporated herein by reference in entirety'.

[0008] BACKGROUND OF THE INVENTION

[0009] Tissue organization and function require an intricate and dynamic interplay of multiple cell types and cell states. Such local and tissue scale cellular relationships are the product of interacting gene regulatory networks within cells and signal transduction between cells. This complexity' must be finely tuned to maintain homeostasis, but must also be reconfigurable in response to changing environmental or physiological needs. Epigenetic marks, transcriptional activity', RNA processing, translation, post-translational modification, and regulated degradation are all required to achieve this. Currently, no method exists for mapping all of these modalities simultaneously in tissues with spatial resolution. Such multimodal profiling can be conducted in single cells, but tissue dissociation into a cell suspension is prone to introduction of technical artifacts and inherently destroys spatial information needed for understanding the cellular microenvironment and tissue architecture. As such, our understanding of how chromatin states relate to gene expression is limited, and we have yet to measure how tissue - specific and -independent factors influence these processes. Technologies for spatially resolved -omics scale profiling are prohibitively expensive, requiring costly consumables or specialized capital equipment. Overcoming these technical and financial barriers holds the potential to democratize research involving spatial profiling, and thereby catalyze advances across a broad swath of biological investigation. Attorney Docket: NYG-LIPP-249PCT

[0010] What is needed is improved compositions and methods for performing spatially resolved, genome-wide chromatin profding.

[0011] SUMMARY OF THE INVENTION

[0012] In one aspect, provided herein is a transposome complex comprising a first transposase and a second transposase, the first transposase having a first adapter comprising a T7 promoter and the second transposase having a second adapter comprising a capture sequence, the first transposase and the second transposase each being a fusion protein comprising an antibody-binding ligand. In another aspect, provided herein is a transposome complex comprising a first transposase and a second transposase, the first transposase having a first adapter comprising a T7 promoter and the second transposase having a second adapter, the first transposase and the second transposase each being a fusion protein comprising an antibody -binding ligand. In certain embodiments, the second adapter comprises a capture sequence. In certain embodiments, the capture sequence is 1 to lOObp, optionally 30 bp. In certain embodiments, the capture sequence comprises a poly(d)T sequence or poly(d)T VN sequence. In certain embodiments, the capture sequence comprises a mosaic end sequence. In certain embodiments, the first adapter and / or the second adapter further comprise a mosaic end sequence. In certain embodiments, the first transposase and the second transposase are Tn5 or TnY. In certain embodiments, the antibody-binding ligand is protein A, protein G, or a nanobody. IN certain embodiments, the transposome complex is bound to an antibody or fragment thereof bound to a chromatin epitope in situ, optionally wherein the chromatin epitope is present in a biological sample mounted on a capture surface comprising an array of capture regions. In certain embodiments, the chromatin epitope comprises a histone modification. In certain embodiments, the histone modification comprises a histone acylation, a histone phosphorylation, or a histone methylation, optionally wherein the histone acylation is an H3K27ac and / or the histone methylation is an H3K4me3 mark or an H3K27me3 mark.

[0013] In another aspect, provided herein a method for in situ analysis of protein-DNA interactions in a biological specimen, the method comprising: a) mounting a biological specimen to a solid substrate having a capture surface comprising an array of capture regions, each capture region comprising capture oligonucleotides, the individual capture regions being identifiable by one or more unique spatial barcodes of the capture Attorney Docket: NYG-LIPP-249PCT oligonucleotides; b) optionally, labeling or staining the biological specimen for visualization; c) permeabilizing the biological specimen; d) contacting the biological specimen with an antibody or fragment thereof capable of binding a chromatin epitope, optionally a histone modification, and a tagmentation complex, as described herein, and incubating the biological specimen under conditions suitable for tagmentation to occur, wherein tagmented genome fragments comprising the T7 promoter and the capture sequence are generated; e) performing a gap-filling reaction; f) contacting the biological specimen with a reaction mixture comprising a T7 polymerase to amplify the tagmented genome fragments: g) generating a library of spatially barcoded tagmented genome products, wherein the tagmented genome fragments hybridized to the capture oligonucleotides are barcoded with the one or more unique spatial barcodes of the capture oligonucleotides; and h) sequencing and analysis of the library' of step g), wherein sequencing and analysis includes mapping protein-DNA interactions to spatial locations of the biological specimen using the one or more distinct spatial barcodes of the capture oligonucleotides.

[0014] In yet another aspect, provided herein a method for in situ analysis of protein- DNA interactions and protein-protein interactions in a biological specimen, the method comprising: a) mounting a biological specimen to a solid substrate having a capture surface comprising an array of capture regions, each capture region comprising capture oligonucleotides, the individual capture regions being identifiable by one or more unique spatial barcodes of the capture oligonucleotides: b) optionally, fixing the biological specimen; c) optionally, labeling or staining the biological specimen for visualization and for protein-protein labeling; d) contacting the biological specimen with one or antibodies or fragments thereof having linked oligonucleotides comprising a barcode and anchor sequence; e) permeabilizing the biological specimen; f) contacting the biological specimen with an antibody or fragment thereof capable of binding a chromatin epitope, optionally a histone modification, and a tagmentation complex, as described herein, and incubating the biological specimen under conditions suitable for tagmentation to occur, wherein tagmented genome fragments comprising the T7 promoter and the capture sequence are generated; g) performing a gap-filling reaction; h) contacting the biological specimen with a reaction mixture comprising a T7 polymerase to amplify the tagmented genome fragments; i) generating 1) a library of spatially barcoded tagmented genome products, wherein tagmented genome fragments hybridized to the capture Attorney Docket: NYG-LIPP-249PCT oligonucleotides are barcoded with the one or more unique spatial barcodes of the capture oligonucleotides, and 2) a library of spatially barcoded oligonucleotides comprising i) the barcodes of the oligonucleotides linked to the one more antibodies or fragments thereof of step d). and ii) the one or more unique spatial barcodes of the capture oligonucleotides; and j) sequencing and analysis of the libraries of step i), wherein sequencing and analysis includes mapping protein-DNA interactions and protein-protein interactions to spatial locations of the biological specimen using the one or more distinct spatial barcodes of the capture oligonucleotides.

[0015] In another aspect, provided herein a method for spatially resolving DNA-protein interactions in a biological sample, the method comprising: a) mounting a biological specimen to a solid substrate having a capture surface comprising an array of capture regions, each capture region comprising capture oligonucleotides, the individual capture regions being identifiable by one or more unique spatial barcodes of the capture oligonucleotides; b) optionally, labeling or staining the biological specimen for visualization; c) permeabilizing the biological specimen; d) contacting the biological specimen with an antibody or fragment thereof capable of binding a chromatin epitope, optionally a histone modification, and a tagmentation complex, as described herein, and incubating the biological specimen under conditions suitable for tagmentation to occur, wherein tagmented genome fragments comprising the T7 promoter and the capture sequence are generated; e) performing a gap-filling reaction; and f) contacting the biological specimen with a reaction mixture comprising a T7 polymerase to amplify the tagmented genome fragments. In certain embodiments, the method further comprises fixing the biological specimen. In certain embodiments, staining the biological sample comprises hematoxylin and eosin (H&E) staining. In certain embodiments, labeling the biological specimen comprises contacting the biological specimen with fluorescently- labeled antibodies or fragments thereof capable of binding epitopes in the biological sample. In certain embodiments, the method further comprises imaging the biological specimen using brightfield microscopy or immunofluorescence microscopy. In certain embodiments, permeabilizing the biological specimen comprises contacting the biological specimen comprises with a solution comprising perm / wash buffer, digitonin buffers, and / or HC1. In certain embodiments, the capture oligonucleotides of the capture surface comprise a polyT or a polyT VN sequence. In certain embodiments, the capture oligonucleotides of the capture surface comprise a Mosaic End (ME) sequence. In certain Attorney Docket: NYG-LIPP-249PCT embodiments, incubating the biological specimen under conditions suitable for tagmentation to occur comprises incubating the biological specimen with about 0.1 mg of the tagmentation complex and / or incubating the biological specimen for a period of time at about 37°C and a period of time at about 55°C. In certain embodiments, amplification of the genome fragments comprises i) incubating the biological specimen with about 0. 1 to about 0.4 p / pl T7 RNA polymerase, and / or ii) incubating the biological specimen at about 37°C for about 14 hours or at least about 8 hours, about 10 hours, or about 12 hours. In certain embodiments, the gap-filling reaction comprises gap-filling via ligation or gap filling via polymerization. In certain embodiments, gap-filling via ligation is performed using about 0.015 units of T4 DNA polymerase and about 2 units T4 DNA ligase and / or at about 24°C for about 30 minutes. In certain embodiments, gap filling via polymerization is performed using about 0.143 units Klenow (exo-) polymerase and / or at about 37°C for about 30 minutes.

[0016] Other aspects and advantages of these compositions and methods are described further in the following detailed description of the preferred embodiments thereof.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows spatial multi-modal CUT&Tag workflow. FIG. 1-(I) shows workflow starting with native histone modifications in the tissue genome, FIG. 1 -(II) tissues are to be permeabilized and stained with primary antibodies against target proteins including 1 cell typing proteins (1) and (2) large protein-protein interactions panels such as CITE-seq. Labeling of cell-type specific protein can occur using both fluorescently labeled (1) or (2) DNA barcoded (CITE-seq) antibodies or both simultaneously. Then, tethered to a fusion protein, FIG. 1 -(III) protein A-Tn5 or nanobody-Tn5, will bind to the primary histone antibodies and the Tn5 will mediate a “CUT” and “Tag” of the genome, thus both fragmenting and barcoding specific locations in the genome. FIG. l-(IV) shows that these fragments can then be amplified in situ via in vitro transcription to generate sufficient molecules for targeted capture. FIG. 1-(V) shows that sequencing libraries can then be constructed using an established protocol similar to ST and CITE-seq, followed by sequencing and FIG. 1-(VI) mapping of protein interactions and epigenome data with spatial resolution.

[0019] FIG. 2 shows detailed overview of the Tn5 constructs. FIG. 2A shows a diagram of adaptor engineering method. All the red adaptors are engineered sequences and blue Attorney Docket: NYG-LIPP-249PCT parts are original nextera sequences. FIG. 2B shows sequence composition of adaptor sequences, the composition of Capture Sequences denoted in red can be customized according to the need of applications. FIG. 2C shows a diagram and sequence compositions of engineered PA-Tn5 constructs used in spatial multiome CUT&Tag. FIG. 2D shows that after tagmention of the genome with engineered PA-Tn5 construct, in vitro transcription can be performed.

[0020] FIG. 3A shows that a two-step reaction, 37°C+55°C, significantly increases tagmentation efficiency. Condition 1-4 corresponding to different histone marks and primary antibody staining time. FIG. 3B shows that on another optimization. Megashort T7 reactions result in most complexities in the final library.

[0021] FIG. 4 shows spatial CUT&Tag signals achieved using the in situ amplification approach. FIG. 4A shows IF signals using the optimized tissue processing workflows for: MAP2. NeuN and GFAP. Scale bar: 250pm. FIG. 4B shows fluorescent tissue activity signatures were synthesized on a spatial capture surface using the same tissue staining and permeabilization conditions in all three experiments. In the first experiment (left panel), we successfully present mRNA-only signals confirming feasibility of mRNA capture using tissue processing for SM-Omics. In the second experiment (middle panel), we stained the tissue with H3K27 antibody, performed our CUT&Tag protocol and capture both mRNA and H3K27-specific signals on the spatial surface which additionally confirmed the feasibility7of performing CUT&Tag using tissue processing for SM-Omics. In the third experiment (right panel), we successfully show successful localized in situ amplification of CUT&Tag signals using the same tissue processing conditions as in the previous experiment. FIG. 4C shows IGV view of peaks mapped to genomics regions encompassing Ptma and Micalll. FIG. 4D shows ‘Tornado plot’ of signal at peak regions called in ENCODE H3K27ac ChlP-seq. FIG. 4E shows a spatially resolved H3K27ac data (color scale) using the Visium 10X Genomics platform.

[0022] FIG. 5 shows spatial CUT&Tag signals for specific histone marks for the Srgapl gene in the adult mouse brain. Annotation color code represents distinct morphological regions in the mouse brain while the Srgapl color scale represents the calculate gene activity scores.

[0023] FIG. 6 shows representative CUT&Tag signal traces (H3K27ac) for a signature mouse cortex gene: Satb2 shown in the top panel. The spatial CUT&Tag signals (two red traces at the bottom) demonstrate strong concordance with reference ENCODE data (grey Attorney Docket: NYG-LIPP-249PCT traces). Spatial CUT&Tag signals for specific histone marks for the Satb2 gene in the adult mouse brain. Annotation color code represents distinct morphological regions in the mouse brain while the Satb2 color scale represents the calculated gene activity scores. Allen Brain atlas represents the IHC image of the Satb2 staining in the adult mouse brain in the reference mouse atlas.

[0024] FIG. 7 shows bulk-level validation of the spatial multi-modal CUT&Tag. FIG. 7A shows representative CUT&Tag signal traces (H3K4me3) across two signature genes. The spatial CUT&Tag signals (two red traces at the bottom) demonstrate strong concordance with reference ENCODE data (grey traces). FIG. 7B shows genome-wide comparison of CUT&Tag signals (H3K4me3) across the top 40,000 genes from ENCODE data. The spatial CUT&Tag data shows consistent signal patterns when compared to ENCODE data at a global scale, further validating the robustness of the approach.

[0025] FIG. 8 shows spatial-level validation of the spatial CUT&Tag. FIG. 8A shows UMAP showing the unsupervised clustering of Spatial CUT&Tag data. FIG. 8B shows HE image (left) of the mouse embryo profded and the spatial projection (right) of cell clusters identified in spatial multi-modal CUT&Tag. Colored arrows show different organs, and the color code matches FIG. 8A.

[0026] FIG. 9A shows methodology of surface modification. FIG. 9B shows spatial CUT&Tag performed on the ligated surface, and compares the resulting data with ENCODE.

[0027] FIG. 10 shows SPACE-Tag method overview and performance. FIG. 10A shows schematic overview of the SPACE-Tag workflow. The process begins by placing a fresh frozen tissue section onto a solid-phase capture spatial transcriptomics slide. Following steps are then performed: I) Engineered transposome adaptors (containing a T7 promoter and a poly(d)T sequencing) are loaded onto pA-Tn5. II) Primary antibody, secondaryantibody, and pA-Tn5 are sequentially applied. Ill) Tagmentation and gap filling are followed by in vitro T7 transcription (IVT) to generate amplified RNA fragments. IV) The IVT-generated RNA is captured by poly-T probes, reverse transcribed, and processed into sequencing libraries. FIG. 10B shows Per-spot unique fragment counts (left; y-axis) and FRiP scores (right: y-axis) obtained by SPACE-Tag and other published spatial chromatin profiling methods (x-axis). FIG. 10C shows expert-annotated brain regions (left) compared with spatial maps derived from unsupervised topic clustering of histone Attorney Docket: NYG-LIPP-249PCT modification profiles (right). Each row shows a different histone mark: H3K27ac (top), H3K4me3 (middle), and H3K27me3 (bottom). FIG. 10D shows spatial projections of inferred gene activity (color scale) (for H3K27ac and H3K4me3) or repression (for H3K27me3). Columns show representative genes (left to right: Satb2, Tcf712, Zeb2). while rows show different histone modifications: H3K27ac (top), H3K4me3 (middle), and H3K27me3 (bottom). FIG. 10E shows MERFISH gene expression (color scale) for representative genes (left to right: Satb2, Tcf712, Zeb2). For each gene, the image pair shows MERFISH expression data from the Allen Brain Atlas (left) and the illustrated Allen Brain Atlas spatial regions where the gene is expected to be expressed (right). Spatial region color code is shared with expert annotations in (10C). FIG. 10F shows spatial cell ty pe projections (color scale) in MERFISH (left) and SPACE-Tag (right) data. Four cell types (columns) are highlighted (from left to right: inhibitory neurons, oligodendrocytes, non-telencephalon astrocytes, and telencephalon astrocytes).

[0028] FIG. 11 shows SPACE-Tag analysis of regulatory elements and their spatial organization. FIGs. 11A and 1 IB show normalized signal intensity of peaks identified with SPACE-Tag (top) and fraction of peaks overlapping with SEdb SEs (bottom) for (FIG. 11 A) H3K27ac and (FIG. 1 IB) H3K4me3. Peaks are ordered by normalized epigenomic signals. Marker color indicates peak overlaps with known super-enhancers (SEs) from the SEdb database (blue: overlapping; red: not overlapping). FIG. 11C shows spatial distribution (color scale) of identified SE modules across brain sections. Module 1 occupies the cortex regions. Module 2 occupies the hypothalamus region. Module 3 occupies white matter tracts. FIG. 1 ID shows GO enrichment scores (color scale) of genes regulated by different SE modules. GO terms (rows) overrepresented in the gene sets for each module (columns) are shown. FIG. HE shows enrichment scores (color scale) of differentially enriched TF motifs (rows) within each SE module (columns). FIG. 1 IF shows expert annotation of a mouse brain section and the cortical subregion. Cortical subplate regions: color code. Shaded: non-cortical area. FIG. 11G shows spatial maps of topic scores (color scale) for CRE-derived topics overlaid on cortical functional regions (left), and violin plot of topic scores for each cortical subregion (right). Cortical region color code shared with (FIG. 11G). FIG. 11H shows topics identified by SPACE- Tag exhibit higher chromatin accessibility scores (color scale) in corresponding cortex regions as previously in Zu et al. Attorney Docket: NYG-LIPP-249PCT

[0029] FIG. 12 shows optimized antibody staining for adult mouse brain sections. Fluorescence images for nuclear (DAPI; blue) and antibody stains (red) for H3K27ac (top), H3K4me3 (middle) and H3K27me3 (bottom). Channels overlay shown on the right. Scale bar: 400pm.

[0030] FIGs. 13A and 13B show comparison of gap-fdling and tagmentation conditions on SPACE-Tag library complexity. Computationally estimated library sizes (Methods) defined as the estimated number of unique fragments in the full cDNA 1 ibrary (y axis) across three tested experimental conditions (T4 ligation, Klenow extension and tagmentation temperature) and shown for two histone marks (H3K4me3 (FIG. 13 A) and H3K27ac (FIG. 13B)). Gap-filling using Klenow-based extensions significantly increases library complexity compared to using T4-based DNA ligation. Additionally, performing the tagmentation reaction at 55°C (right) further enhanced complexity relative to the conventional 37°C reaction temperature (left, mid). Significance testing: one-sided t-test; * denotes p<0.05.

[0031] FIG. 14 shows the effect of T7 amplification on signal capture in SPACE-Tag. FIG. 14A shows schematic of the workflow for signal quantification. (I) Brain sections are deposited on a pseudospatial array with a poly(d)T capture area and stained for H&E histology (II) The sections are then stained for primary and secondary antibodies (III) In situ tissue reactions are performed: pA-Tn5 tethering, tagmentation, gap-filling and HC1 permeabilization. The SPACE-Tag procedure next utilizes T7-based amplification to generate amplified RNA molecules that are subsequently captured on the pseudospatial slide. cDNA molecules are then generated with a simultaneous staining using fluorescently labeled nucleotides. (IV) Signals are captured by fluorescent imaging. FIG. 14B shows H&E images (top) and cDNA signal intensity (white) of sections with or without the T7 amplification step. Scale bars are 400pm. FIG. 14C shows quantification of the fluorescence signals from FIG. 14B. Significance testing: one-sided t-test; * denotes p<0.05.

[0032] FIG. 15 shows SPACE-Tag reproducibility. FIGs. 15A to 15C show reproducibility7of SPACE-Tag experiments across biological replicates for H3K27ac (FIG. 15A), H3K4me3 (FIG. 15B) and H3K27me3 (FIG. 15C). The genome was split into lOkb tiles, and a scatterplot of normalized signal between replicate experiments was plotted for all tiles. The Spearman correlation coefficient was calculated for each modification; significance was determined by two-sided t-test (p values shown). Attorney Docket: NYG-LIPP-249PCT

[0033] FIG. 16 shows SPACE-Tag signal in cortical regions correlates with ENCODE ChlP-seq mouse cortex data. FIGs. 16A and 16B show cumulative normalized epigenomic signal for ENCODE ChlP-seq and SPACE-Tag across all ENCODE-defined peaks at + / - 2kb from peak centers (top), and normalized signal intensities (color scale) over ENCODE-defined peaks are shown for both ENCODE ChlP-seq data and corresponding SPACE-Tag data (columns), demonstrating concordant enrichment patterns for (a) H3K4me3 and (b) H3K27ac histone modifications (bottom). Each row7represents a single peak in ENCODE ChlP-seq; columns represent normalized signal intensities at + / - 2kb from each peak center. FIGs. 16C and 16D show heatmap of correlations between ENCODE and SPACE-Tag. The genome was split into lOkb tiles, and a Spearman correlation coefficient was calculated on the normalized signals across all tiles (Methods). The Spearman correlation coefficient was calculated for each modification: significance was determined by two-sided t-test (p values shown).

[0034] FIG. 17 shows SPACE-Tag signal in cortical regions correlates with bulk CUT&Tag. FIGs. 17A to 17C show cumulative normalized epigenomic signal for bulk CUT&Tag and SPACE-Tag across all SPACE-Tag-defined peaks at + / - 2kb from peak centers (top), and normalized epigenomic signal (color scale) over SPACE-Tag-defined peaks are shown for both bulk CUT&Tag data and corresponding SPACE-Tag data (columns), demonstrating concordant enrichment patterns for H3K27ac (FIG. 17A), H3K4me3 (FIG. 17B) and H3K27me3 (FIG. 17C) histone modifications (bottom). Each row represents a single peak in SPACE-Tag; columns represent normalized signal intensities at + / - 2kb from each peak center. FIGs. 17D to 17F show heatmap of correlations between bulk CUT&Tag and SPACE-Tag for H3K27ac (FIG. 17D), H3K4me3 (FIG. 17E) and H3K27me3 (FIG. 17F) histone modifications. The genome was split into lOkb tiles, and Spearman’s correlation coefficient was calculated on the normalized signals across all tiles (Methods); significance was determined by two-sided t- test (p values shown).

[0035] FIG. 18 shows genome coverage tracks of region-specific key neuronal marker genes profiled by SPACE-Tag. Genome coverage tracks showing normalized epigenomic signal (y-axis; left: H3K27ac; middle: H3K4me3; right: H3K27me3) across the gene body and flanking regions (x-axis) of marker genes (top: Satb2, middle: Tcf712; bottom: Zeb2). The corresponding gene body is depicted beneath each track, along with genomic coordinates. Attorney Docket: NYG-LIPP-249PCT

[0036] FIG. 19 shows super-enhancer modules are active in specific cell types. FIG. 19A shows UMAP of cell t pes identified in a previously published H3K27ac single-cell CUT&Tag dataset of the postnatal mouse brain. FIG. 19B shows H3K27ac signal (color scale) of SE modules (columns) across cell types (color scale); Module 1 (left) is primarily enriched in excitatory and inhibitor}' neurons. Module 2 (middle) in astrocytes, and Module 3 (right) in oligodendrocytes.

[0037] FIG. 20 shows definition of a dorsal-ventral trajectory' along the cortical plate axis. Spatial plot of cortical plate trajectory ordering (color scale) for spots belonging to the cortex. N on-cortical spots are grayed out. Black line: principal curve definition of the cortical plate axis (Methods).

[0038] FIG. 21 shows super-enhancer topics do not distinguish fine cortical functional sub-regions. Spatial plots (color scale) show topics (columns) identified along the cortical plate axis.

[0039] FIG. 22 shows reproducibility of cortex sub-region-specific topics identified by SPACE-Tag profiling of mouse brain. Spatial plots show topic scores (color scale) corresponding to the retrosplenial area (Topic 1), visual cortex (Topic 2), auditory / somatosensory cortex (Topic 3), perirhinal area (Topic 4), and piriform cortex (Topic 5) across replicate sections (columns).

[0040] FIG. 23 shows epigenetic differences between functional regions of the cortical plate are primarily driven by glutamatergic neuronal subty pes. Violin plots show' accessibility scores (y-axis) from a published single-cell ATAC-seq dataset across different cell types (x-axis. blue: Glutamatergic Neurons; red: GABAergic neurons; Grey: non-neuronal cells). Five plots correspond to topics 1 to 5, as in FIG. 1 1. Significance testing: two-sided t-test.

[0041] DETAILED DESCRIPTION

[0042] The compositions and methods described herein provide improved reagents and methods for performing spatially resolved chromatin analysis.

[0043] Current technologies for chromatin profiling, such as Chromatin immunoprecipitation followed by sequencing (ChlP-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag), lack spatial resolution. This limitation prevents these methods from retaining tissue architecture and cellular context while studying the Attorney Docket: NYG-LIPP-249PCT chromatin landscape, making it unable to understand the regulatory mechanisms in spatial context in complex tissues.

[0044] While Spatial Transcriptomics (ST), exemplified by the Visium product (lOx Genomics, offers spatial transcriptomic data, spatial chromatin profiling remains a critical unmet need in the field. This is particularly true for the study of tissues of genetic diseases with high intrinsic heterogeneity, as seen in diseases like cancer and neurodegenerative disorders. In these contexts, diseased and healthy cells are often interspersed, and the ability to spatially resolve chromatin states is crucial for identify ing driving mutations (in coding and non-coding genomic regions) and epigenetic (DNA- protein interactions) alterations that are either disease-causative or driving disease progression.

[0045] Existing spatial chromatin profiling techniques, such as microchannel-based spatial CUT&Tag developed by Deng, et al., (Deng et al.. 2022. "Spatial-CUT&Tag: Spatially Resolved Chromatin Modification Profiling at the Cellular Level.’’ Science 375 (6581): 681-86) require specialized and custom-fabricated devices. These equipment demands pose a significant obstacle to the widespread adoption of such technologies within the scientific community. While there have been attempts to present and optimize this technology as a multimodal (simultaneous) RNA and CUT&Tag technology by the same group, this has not performed using an imaging-based platform on the same tissue section as when measuring the CUT&Tag signal. This is crucial for understanding biology as protein-protein interactions are these present potential targets for therapyinterventions.

[0046] The methods disclosed herein preserve the spatial architecture of tissues, allowing for chromatin modification profiling without the need for tissue dissociation, as is common in bulk chromatin or single-cell assays. By maintaining the structural integrity of the tissue, the method allows for the identification of cell type-specific regulatory- mechanisms within their native spatial contexts, a capability' not feasible with traditional or single-cell chromatin profiling techniques. This spatial context is crucial for understanding complex tissues, such as tumors and brain tissues, where cellular interactions and the microenvironment significantly influence gene regulation. In plain terms, we collect data that is our much higher resolution and of benefit to functional genomics as compared to standard assays. Attorney Docket: NYG-LIPP-249PCT

[0047] Unlike other spatial CUT&Tag technologies that require specialized device, such as DBIT-seq, which can be difficult for many researchers to access, the methods described herein leverage commercially available reagents and equipment. This makes the technology more accessible to a broader audience in the scientific community, facilitating widespread adoption without the need for specialized hardware. This technology is fully compatible with three leading spatial platforms on the market: Curio, 10X Genomics, and Stereo-seq, which immediately enables its wide use as the commercial vendors currently do not provide a kit for any spatial epigenome profiling. The only current device on the market for spatial CUT&Tag assays is based on the DBIT- seq technology (AtlasXOmics); however, this product suffers from intrinsic problems associated with PDMS device failure when applied to tissue sections. These platforms also do not utilize multi-modal capabilities or a T7 amplification step, as described herein.

[0048] Unless defined otherwise in this specification, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0049] As used throughout this specification and the claims, the terms “comprising”, “containing”, “including”, and its variants are inclusive of other components, elements, integers, steps and the like. Conversely, the term “consisting” and its variants are exclusive of other components, elements, integers, steps and the like.

[0050] It is to be noted that the term “a” or “an”, refers to one or more, for example, “an oligonucleotide”, is understood to represent one or more oligonucleotides. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.

[0051] The term “and / or” means one or more of the listed elements or a combination of any two or more of the listed elements.

[0052] As used herein, the term “about” means a vari ability of plus or minus 10% from the reference given, unless otherwise specified.

[0053] As used herein, the phrase “consisting essentially of’ limits the scope of a described composition or method to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the described or claimed method or composition. Attorney Docket: NYG-LIPP-249PCT

[0054] Wherever in this specification, a method or composition is described as “comprising” certain steps or features, it is also meant to encompass the same method or composition consisting essentially of those steps or features and consisting of those steps or features.

[0055] A “nucleic acid”, “nucleic acid sequence”, or “nucleotide sequence” as described herein, can be RNA, DNA, or a modification thereof, and can be single or double stranded.

[0056] The terms “nucleotide”, “nucleic acid”, “nucleotide residue”, and “nucleic acid residue” are used interchangeably, referring to a nucleotide in a nucleic acid polymer.

[0057] Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. As used herein, RNA may be any RNA molecule which may occur in a cell. Thus, it may be mRNA, tRNA, rRNA, viral RNA. small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), ribozymal RNA, antisense RNA or non-coding RNA. Preferably, it is mRNA.

[0058] As used herein, deoxyribonucleic acid (DNA) is a polymeric molecule formed by deoxyribonucleic acid, including, but not limited to. genomic DNA, double-strand DNA. single-strand DNA, DNA packaged with a histone protein, complementary DNA (cDNA which is reverse-transcribed from an RNA), mitochondrial DNA, and chromosomal DNA.

[0059] Nucleic acid sequences described herein can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and / or molecular cloning (e.g. GeneArt, GenScript, Life Technologies, Eurofins). dNTP stands for deoxy ribonucleotide triphosphate. Each dNTP is made up of a phosphate group, a deoxyribose sugar and a nitrogenous base. There are four different dNTPs and can be split into two groups: the purines (including dATP, deoxyadenosine 5'- triphosphate, and dGTP, deoxy guanine 5 '-triphosphate) and the pyrimidines (including dTTP, deoxythymidine 5'-triphosphate, and dCTP, deoxycytidine 5'-triphosphate). As used herein, dNTP Mix (also referred to as dNTPs herein) is a mixture (normally in a solution containing sodium salts) of dATP, dCTP, dGTP and dTTP, suitable for use in Attorney Docket: NYG-LIPP-249PCT polymerase chain reaction (PCR), sequencing, fill-in reactions, nick translation, cDNA synthesis, and TdT-tailing reactions.

[0060] As used herein, “complementary DNA” or “cDNA” can refer to a synthetic DNA reverse transcribed from RNA through the action of a reverse transcriptase. The cDNA may be single-stranded or double-stranded and can include strands that have either or both of a sequence that is substantially identical to a part of the RNA sequence or a complement to a part of the RNA sequence.

[0061] As used herein, the term “oligonucleotide” or “oligo” refers to short DNA or RNA molecules. In one embodiment, an oligo can be at least about 1 to 500 monomeric components, e.g., nucleotides, in length. In a further embodiment, an oligo can be about 20 to about 80 nucleotides in length. Thus, in various embodiments, an oligo is formed of at least 1. 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

[0062] 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. 38. 39. 40. 41, 42, 43, 44, 45, 46, 47, 48, 49,

[0063] 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,

[0064] 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97,

[0065] 98, 99, 100 nucleotides. However, in some instances, where the oligo is comprised of several smaller oligos, the resultant oligo may be longer than 100 nucleotides.

[0066] Some embodiments include the use of primers. As used herein, a “primer” can refer to a short polynucleotide, generally with a free 3'-OH group, that binds to a target or template polynucleotide present in a sample by hybridizing with the target or template, and thereafter promoting extension of the primer to form a polynucleotide complementary to the target or template. Primers can include polynucleotides ranging from 5 to 1000 or more nucleotides. In some embodiments, the primer has a length of at least 4 nucleotides, 5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, or a length within a range of any two of the foregoing lengths.

[0067] As used herein, a “barcode” describes a defined polymer, e.g., a polynucleotide, which when it is a functional element of the polymer construct, is specific for a compartment, a single cell, or cell nucleus or cellular components (for example, DNA, RNA and / or mitochondria and ribosomes) thereof. In one embodiment, the barcode is about 2 to 4 monomeric components, e g., nucleotide bases, in length. In other embodiments, the barcode is at least about 1 to 100 monomeric components, e.g., Attorney Docket: NYG-LIPP-249PCT nucleotides, in length. Thus, in various embodiments, the barcode is formed of a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

[0068] 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,

[0069] 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60. 61. 62. 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,

[0070] 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96,

[0071] 97, 98, 99, or up to 100 monomeric components, e.g., nucleotides. In one embodiment, the barcode is 1 Ont long. A barcode can be an artificial sequence or a naturally occurring sequence. In certain embodiments, each barcode within a population of barcodes is different. In other embodiments, a portion of barcodes in a population of barcodes is different, e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%, or any values therebetween, of the barcodes in a population of barcodes is different. A population of barcodes may be randomly generated or non-randomly generated. In certain embodiments, a population of barcodes are error correcting barcodes. Barcodes can be used to computationally deconvolute the multiplexed sequencing data and identify sequence reads derived from an individual cell, compartment, etc. A barcode can also be used for deconvolution of a collection of cells or cell nuclei or cellular components thereof that have been distributed into small compartments for enhanced mapping.

[0072] In certain embodiments, the term “barcode” and “barcoded” also refers to a process of introducing a barcode to a DNA or RNA. An example of introducing a barcode to an RNA is illustrated in the reverse transcriptase step, wherein the substrate oligonucleotide is a template for extension of the captured RNA.

[0073] The terms “another,” “first,” “second,” “third,” “fourth,” “fifth,” and “sixth,” are used throughout this specification as reference terms to distinguish between various forms and components of the compositions and methods, for example, barcodes.

[0074] As used herein, the term “array” or “capture array” refers to a population of oligonucleotides or sites on a solid substrate that can be differentiated from each other according to relative location. Different oligonucleotides that are at different sites or features of an array can be differentiated from each other according to the locations of the sites or features in the array. An individual site or feature of an array can include one or more molecules of a particular type (e.g. species of capture probe). For example, a site or feature can include a single nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence. Attorney Docket: NYG-LIPP-249PCT

[0075] Oligonucleotides may be attached to the solid substrate, e.g. array, of the invention by any suitable means. As used herein, the terms “attached” or “bound” refers to the state of two things being j oined, fastened, adhered, connected or bound to each other. For example, 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 pairs of electrons between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of pairs of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions and hydrophobic interactions. In certain embodiments, nucleic acids are bound by hybridization or annealing to one another.

[0076] The terms “solid substrate,” “solid surface” and other grammatical equivalents herein refer to any material that is appropriate for or can be modified to be appropriate for the attachment of materials for the processing of nucleic acids, including, for example, materials for nucleic acid library preparation. As will be appreciated by those in the art, the number of possible solid substrate materials is very large. Possible materials include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers. In certain embodiments, the solid substrate is a commercially available product having bound capture oligonucleotides (for example, Visium from lOx Genomics, Curio Seeker or Curio Trekker from Curio Bioscience, etc.)

[0077] In certain embodiments, a solid substrate includes silica-based substrates, such as glass, fused silica, or other silica-containing materials. In certain embodiments, silica- based substrates can also be silicon, silicon dioxide, silicon nitride, or silicone hydrides. In some examples, a solid substrate includes plastic materials such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylons, polyesters, polycarbonates, cyclic olefin polymers, or poly(methyl methacrylate). In certain embodiments, the solid substrate is a silica-based material or plastic material. In certain embodiments, the solid substrate has at least one surface comprising glass.

[0078] In certain embodiments, the solid substrate comprises a patterned surface. A “patterned surface” refers to an arrangement of different capture regions in or on an Attorney Docket: NYG-LIPP-249PCT exposed layer of a solid support. In certain embodiments, the pattern can be an x-y format of features that are in rows and columns. In certain embodiments, the pattern can be a repeating arrangement of features and / or interstitial regions. In some examples, the pattern can be a random arrangement of features and / or interstitial regions.

[0079] In certain embodiments, a solid substrate is ‘'functionalized’’, i.e., coated with a surface polymer comprising functional groups capable of forming covalent bonds with oligonucleotides or modified oligonucleotides, such as those described in PCT Publ. Nos. WO 2013 / 184796 or WO2016 / 066586.

[0080] In certain embodiments, the solid substrate is a glass slide, functionalized with a surface polymer comprising amino groups, e.g., capable of forming bonds with oligonucleotides that have been functionalized, e.g., on the 5’ end. The solid substrate can be of any size. In certain embodiments, the substrate is a glass slide having the standard dimensions of approximately 75mm x 25mm x 1 mm.

[0081] As used herein, an “antibody” is a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a multi-specific binding construct that can bind two or more targets, a dual specific antibody, a bi-specific antibody or a multi-specific antibody, or an affinity matured antibody, a single antibody chain or an scFv fragment, a diabody, a single chain comprising complementary scFvs (tandem scFvs) or bispecific tandem scFvs, an Fv construct, a disulfide-linked Fv, a Fab construct, a Fab1construct, a F(ab')2 construct, an Fc construct, a monovalent or bivalent construct from which domains non- essential to monoclonal antibody function have been removed, a single-chain molecule containing one VL, one VH antigen-binding domain, and one or two constant “effector” domains optionally connected by linker domains, a univalent antibody lacking a hinge region, a single domain antibody, a dual variable domain immunoglobulin (DVD-Ig) binding protein or a nanobody. Also included in this definition are antibody mimetics such as affibodies, i.e., a class of engineered affinity proteins, generally small (—6.5 kDa) single domain proteins that can be isolated for high affinity and specificity to any given protein target. In certain embodiments, the ligand is a single domain antibody.

[0082] The term “biological specimen” is intended to mean one or more cell, tissue, organism or portion thereof. It will be evident that a biological specimen from any organism may be used, e.g. plant, animal or fungal. The compositions and methods described herein allow the capture of any nucleic acid, e.g. mRNA molecules present in Attorney Docket: NYG-LIPP-249PCT cells or tissues. The compositions and methods are particularly suitable for isolating and analyzing the transcriptome or genome of cells within a biological specimen, e.g. a tissue sample, wherein spatial resolution of the transcriptomes is desirable, e.g. where the cells are interconnected or in contact directly with adjacent cells. However, it will be apparent to a person of skill in the art that the compositions and methods may also be useful for the analysis of the transcriptome of different cells or cell types within a sample even if said cells do not interact directly, e.g. a blood sample. In other words, the cells do not need to present in the context of a tissue and can be applied to the array as single cells (e.g. cells isolated from a non-fixed tissue, e.g. a blood sample). Such single cells, while not necessarily fixed to a certain position in a tissue, are nonetheless applied to a certain position on the array and can be individually identified. Thus, in the context of analyzing cells that do not interact directly, or are not present in a tissue context, the spatial properties of the described methods may be applied to obtaining or retrieving unique or independent transcriptome information from individual cells.

[0083] The biological specimen may be a harvested or biopsied tissue sample, or a cultured sample. Representative samples include clinical samples e.g. whole blood or blood-derived products, blood cells, tissues, biopsies, or cultured tissues or cells etc. including cell suspensions. Artificial tissues may for example be prepared from cell suspension (including for example blood cells). Cells may be captured in a matrix (for example a gel matrix e.g. agar, agarose, etc.) and may then be sectioned in a conventional way. Such procedures are known in the art in the context of immunohistochemistry (see e.g. Andersson et al 2006, J. Histochem. Cytochem. 54(12): 1413-23. Epub 2006 Sep 6).

[0084] Compositions

[0085] In certain embodiments, the compositions and methods utilize tagmentation reagents and reactions, some of which are known in the art. Some of these reagents and / or methodologies have been modified or adapted as described herein.

[0086] The compositions and methods utilize a transposome complex that includes a first transposase and a second transposase. In certain embodiments, the first transposase is loaded with a first adapter comprising a T7 promoter and the second transposase is loaded with a second adapter comprising a capture sequence. The first transposase and the second transposase are each fusion proteins comprising an antibody-binding ligand.

[0087] Transposome Complexes Attorney Docket: NYG-LIPP-249PCT

[0088] Transposon based technology can be utilized for fragmenting DNA, for example, as exemplified in the workflow for NEXTERA™ XT and FLEX DNA sample preparation kits (Illumina, Inc.), wherein target nucleic acids, such as genomic DNA, are treated with transposome complexes that simultaneously fragment and tag (“tagmentation’’) the target, thereby creating a population of fragmented nucleic acid molecules tagged with unique adaptor sequences at the ends of the fragments.

[0089] A “transposome complex” is comprised of at least one transposase enzyme loaded with a transposon that comprises an adapter. The transposase facilitates the random insertion of a transposon into target DNA. Efficient transposition requires the presence of a specific 19-bp transposase recognition sequence (Mosaic End or ME sequence) at each end of the transposon.

[0090] The transposition process catalyzed by the transposase involves a multi-step “cut and paste” mechanism. Initially, the enzyme binds to the 19-bp ME of the transposon, forming a complex known as the transposome. Subsequently, the transposome initiates random attacks on the phosphodiester backbone of the target DNA, leading to cleavage. Finally, the transposase catalyzes the covalent linkage of the 3'-OH ends of the transposon to the exposed 5 '-phosphorylated ends of the target DNA. This transposition event results in the creation of a 9-bp sequence duplication immediately flanking the site of transposon insertion that contains the remainder of the adapter sequences.

[0091] Exemplary transposition procedures and systems that can be readily adapted for use with the transposases of the present disclosure are described, for example, in PCT Publ. No. W010 / 048605, U.S. Pat. Publ. No. 2012 / 0301925, U.S. Pat. Publ. No. 2012 / 13470087, or U.S. Pat. Publ. No. 2013 / 0143774, each of which is incorporated herein by reference in its entirety.

[0092] Transposases

[0093] Exemplary transposases that can be used with certain embodiments provided herein include (or are encoded by): Tn5 transposase (see Reznikoff et al., Biochem. Biophys. Res. Commun. 2000, 266, 729-734), Vibrio harveyi (transposase characterized by Agilent and used in SureSelect QXT product), MuA transposase and a Mu transposase recognition site comprising R1and R2end sequences (Mizuuchi, K., Cell, 35: 785, 1983; Savilahti, H, et al., EMBO J., 14:4893, 1995), Staphylococcus aureus Tn552 (Colegio, O. et al., J. Bactenol., 183:2384-8, 2001; Kirby, C. et al., Mol. Microbiol., 43: 173-86, 2002), Tyl (Devine & Boeke, Nucleic Acids Res., 22:3765-72, 1994 and PCT Publ. No. Attorney Docket: NYG-LIPP-249PCT

[0094] WO95 / 23875), Transposon Tn7 (Craig, N. L , Science, 271 : 1512, 1996; Craig, N. L„ Curr. Top. Microbiol. Immunol., 204:27-48, 1996), Tn / O and IS10 (Kleckner N. et al., Curr. Top. Microbiol. Immunol., 204:49-82, 1996), Mariner transposase (Lampe, D. J. et al., EMBO J., 15:5470-9, 1996), Tel (Plasterk, R. H., Curr. Top. Microbiol. Immunol.. 204: 125-43, 1996), P Element (Gloor, G. B„ Methods Mol. Biol., 260:97-1 14, 2004), Tn3 (Ichikawa & Ohtsubo, J. Biol. Chem., 265: 18829-32, 1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol. 204:1-26, 1996), retroviruses (Brown et al., Proc. Natl. Acad. Sci. USA. 86:2525-9, 1989), and retrotransposon of yeast (Boeke & Corces, Ann. Rev. Microbiol. 43:403-34, 1989). More examples include IS5, TnlO, Tn903, IS911, and engineered versions of transposase family enzymes (Zhang et al., (2009) PLoS Genet. 5:el000689. Epub October 16; Wilson C. et al. (2007) J. Microbiol. Methods 71:332-5). The compositions described herein also include combinations of transposases, and not just a single transposase.

[0095] In certain embodiments, the transposase is a Tn5, MuA, or Vibrio harveyi transposase, or an active mutant thereof. In certain embodiments, the transposase is a Tn5 transposase or an active mutant thereof. In certain embodiments, the Tn5 transposase is a hyperactive Tn5 transposase (see, e g., Reznikoff et al.. PCT Publ. No. WO2001 / 009363, U.S. Pat. Nos. 5,925,545, 5,965,443, 7,083,980, and 7,608,434, and Goryshin and Reznikoff, J. Biol. Chem. 273:7367, 1998), or an active mutant thereof. In certain embodiments, the Tn5 transposase is a Tn5 transposase as described in PCT Publ. No. WO2015 / 160895, which is incorporated herein by reference. In certain embodiments, the Tn5 transposase is a fusion protein.

[0096] An exemplary coding sequence for Tn5 transposase is shown in SEQ ID NO: 1 : atgattaccagtgcactgcatcgtgcggcggattgggcgaaaagcgtgttttctagtgctgcgctgggtgatccgcgtcgtaccg cgcgtctggtgaatgttgcggcgcaactggccaaatatagcggcaaaagcattaccattagcagcgaaggcagcaaagccatg caggaaggcgcgtatcgttttattcgtaatccgaacgtgagcgcggaagcgattcgtaaagcgggtgccatgcagaccgtgaaa ctggcccaggaatttccggaactgctggcaattgaagataccacctctctgagctatcgtcatcaggtggcggaagaactgggca aactgggtagcattcaggataaaagccgtggttggtgggtgcatagcgtgctgctgctggaagcgaccacctttcgtaccgtggg cctgctgcatcaagaatggtggatgcgtccggatgatccggcggatgcggatgaaaaagaaagcggcaaatggctggccgct gctgcaacttcgcgtctgagaatgggcagcatgatgagcaacgtgattgcggtgtgcgatcgtgaagcggatattcatgcgtatct gcaagataaactggcccataacgaacgttttgtggtgcgtagcaaacatccgcgtaaagatgtggaaagcggcctgtatctgtat gatcacctgaaaaaccagccggaactgggcggctatcagattagcattccgcagaaaggcgtggtggataaacgtggcaaacg taaaaaccgtccggcgcgtaaagcgagcctgagcctgcgtagcggccgtattaccctgaaacagggcaacattaccctgaacg Attorney Docket: NYG-LIPP-249PCT cggtgctggccgaagaaattaatccgccgaaaggcgaaaccccgctgaaatggclgctgctgaccagcgagccgglggaaag tctggcccaagcgctgcgtgtgattgatatttatacccatcgttggcgcattgaagaatttcacaaagcgtggaaaacgggtgcgg gtgcggaacgtcagcgtatggaagaaccggataacctggaacgtatggtgagcattctgagctttgtggcggtgcgtctgctgca actgcgtgaatcttttactccgccgcaagcactgcgtgcgcagggcctgctgaaagaagcggaacacgttgaaagccagagcg cggaaaccgtgctgaccccggatgaatgccaactgctgggctatctggataaaggcaaacgcaaacgcaaagaaaaagcggg cagcctgcaatgggcgtatatggcgattgcgcgtctgggcggctttatggatagcaaacgtaccggcattgcgagctggggtgc gctgtgggaaggttgggaagcgctgcaaagcaaactggatggctttctggccgcgaaagacctgatggcgcagggcattaaaa tc

[0097] The amino acid sequence for Tn5 transposase is shown in SEQ ID NO: 2: MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKA MQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEE LGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESG KWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRK DVESGLYLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRIT LKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEF HKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLL KEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGF MDSKRTGIASWGALWEGWEALQSKLDGFLAAKDLMAQGIKI

[0098] In certain embodiments, the transposase is TnY. TnY is a hyperactive mutant of the transposase from Vibrio parahemolyticus (ViPar) with P50K and M53Q mutations. The inside and outside ends (IE and OE, respectively) of the ViPar transposon utilize the same sequence as the IE and OE of the Tn5 transposon (see, WO 2021 / 011433, which is incorporated herein by reference).

[0099] An exemplar)' coding sequence for TnY transposase is shown in SEQ ID NO: 3: atgacccact ccgatgcgaa actgtgggct caggagcaat tcggtcaggc ccaactgaaagatccgcgcc cacccagcg cctgatttct ctggcgacca gcattgctaa ccagccgggtgttagcgttg cgaaactgcc gttttctaaa gccgatcagg agggcgcgta ccgtttcattcgtaacgata acatcgacgc gaaagacatc gctgaagcag gctttcagtc caccgtatcccgcgctaacg aacacaaaga gctgctggcg ctggaagaca ctacgaccct gtctttcccgcatcgttcca tcaaagaaga actgggccat acgaaccagg gtgatcgcac ccgcgccctgcacgttcact ctaccctgct gttcgcgccg cagaaccaga ctatcgtggg tctgatcgag cagcagcgtt ggtctcgtga tattactaaa cgcggtcaga aacatcagca cgctacccgt ccttataaag aaaaagaatc ctataaatgg gagcaggctt cccgtcgtgt tgtggagcgc ctgggtgata aaatgctgga tgtcatttct gtttgcgacc gcgaggcaga tctgtttgaa tacctgacct acaaacgtca acaccagcag cgtttcgttg ttcgtagcat gcagtctcgc tgtctggaag aacacgctca gaaactgtat gactacgcac aggcgctgcc Attorney Docket: NYG-LIPP-249PCT atctgtaaaa acgaaggcac tgaccatccc tcaaaaaggt ggccgtaaag cacgtgacgt taaactggac gttaaatacg gccaggttac tctgaaagcg ccggccaaca aaaaggagca cgcaggcatt ccggtttact acgtgggctg cctggaacag ggtacttcca aagataaact ggcgtggcac ctgctgacct ctgaacctat taacaacgtc gaggatgcca tgcgtatcat cggctactac gaacgtcgtt ggctgatcga ggattttcac aaagtatgga aatccgaagg tactgacgta gaatccctgc gtctgcagag caaagacaac ctggaacgtc tgtccgttat ctacgcgttt gttgctaccc gcctgctggc actgcgtttt atcaaggaag ttgatgaact gaccaaagaa agctgtgaaa aagttctggg ccagaaagcg tggaaactgc tgtggctgaa gctggaatct aaaaccctgc cgaaagaggt accggacatg ggttgggctt ataaaaacct ggctaaactg ggtggctgga aggacactaa gcgtaccggt cgcgcttcta tcaaagttct gtgggagggt tggttcaaac tgcagaccat cctggagggc tatgaactgg cgatgtccct ggaccac

[0100] The amino acid sequence for TnY transposase is shown in SEQ ID NO: 4: MTHSDAKLWAQEQFGQAQLKDPRRTQRLISLATSIANQPGVSVAKLPFSKADQE GAYRFIRNDNIDAKDIAEAGFQSTVSRANEHKELLALEDTTTLSFPHRSIKEELGH TNQGDRTRALHVHSTLLFAPQNQTIVGLIEQQRWSRDITKRGQKHQHATRPYKE KESYKWEQASRRVVERLGDKMLDVISVCDREADLFEYLTYKRQHQQRFVVRSM QSRCLEEHAQKLYDYAQALPSVKTKALTIPQKGGRKARDVKLDVKYGQVTLKA PANKKEHAGIPVYYVGCLEQGTSKDKLAWHLLTSEPINNVEDAMRIIGYYERRW LIEDFHKVWKSEGTDVESLRLQSKDNLERLSVIYAFVATRLLALRFIKEVDELTKE SCEKVLGQKAWKLLWLKLESKTLPKEVPDMGWAYKNLAKLGGWKDTKRTGR ASIKVLWEGWFKLQTILEGYELAMSLDH

[0101] Other useful transposases include those having sequences set forth in the table below. Attorney Docket: NYG-LIPP-249PCT

[0102] In certain embodiments, the transposome complex is a dimer of two molecules of a transposase. In certain embodiments, the compositions and methods described herein employ two populations of transposome complexes. In some embodiments, the transposome complexes are heterodimers, wherein the first transposase has a first adaptor sequence and the second population has a different adaptor sequence.

[0103] Adaptor

[0104] The transposons further include adapter sequences loaded thereon. See, e.g., FIG.

[0105] 2. As used herein, the term "transposon" is used interchangeably with mosaic-end DNA sequence (MEDS) adapter, referring to a nucleic acid molecule that is capable of being incorporated into a nucleic acid by a transposase enzyme. The MEDS adapter includes two transposon ends (also termed “arms7’ and “mosaic end” or “ME”, for example, a double-stranded mosaic end). In one embodiment, the two transposon ends are linked by a sequence that is sufficiently long to form a loop in the presence of a transposase. The formation of a complex between the Tn5 transposase and the 19-bp MEs is necessary for the transposition to occur, and the intervening DNA must be long enough to bring 2 of these sequences close together to form an active transposase homodimer. Transposons can be double-, single-stranded, or mixed, containing single- and double-stranded region(s), depending on the transposase used to insert the transposon. As show n in FIG. 2, Attorney Docket: NYG-LIPP-249PCT a portion of each adapter is double stranded with a single strand overhang, that comprises a T7 promoter or a customizable capture sequence.

[0106] In one embodiment, each adapter comprises a mosaic end sequence (or other transposase recognition sequence). Such mosaic end sequences are known in the art, for example, for use with the Tn5 transposase. The top strand of an exemplary ME sequence for use with Tn5 transposase is: 5’-AGATGTGTATAAGAGACAG- 3’ (SEQ ID NO: X). In one embodiment, the ME sequence is contained on the 5' end of the adapter, the 3’ end, or both. In one embodiment the ME sequence is contained on the 3’ end of the adapter. See, e.g., Picelli et al.. Genome Research, July 30, 2014, 24:2033-40, which is incorporated herein by reference. Other sequences which may be used in place of a ME include inverted 19-bp end sequences (ESs), including outside end (OE) and inside end (IE) sequences of the transposon. An example of an OE sequence is: 5’- CTGACTCTTATACACAAGT - 3’ (SEQ ID NO: X). An example of an IE sequence is: 5’ CTGTCTCTTGATCAGATCT - 3’ (SEQ ID NO: X). See, e g., Rezmkoff, Molecular Microbiology', 47(5): 1199-1206 (February72003), which is incorporated herein by reference.

[0107] The first adapter also includes a T7 promoter sequence. The T7 promoter is a DNA sequence that is recognized by the T7 RNA polymerase and is used to regulate the expression of recombinant proteins. An exemplary T7 promoter sequence is 5 ’ — TAATACGACTCACTATAG 3’ (SEQ ID NO: X) Other T7 promoter sequences are known and are useful herein, such as 5’ - AATTCTAATACGACTCACTATAGGGA - 3' (SEQ ID NO: X), known as T7Max (see, Deich, C., Cash, B., Sato, W. et al. T7Max transcription system. J Biol Eng 17, 4 (2023). doi.org / 10.1186 / sl 3036-023-00323-1), which is incorporated herein by reference.

[0108] In certain embodiments, the second adapter contains a capture sequence. The capture sequences are fully modular (up to 30 bp) and can (for example) include a poly(d)T or a nextera Mosaic End (ME) sequence. This modification allows for the compatibility7with commercially available poly(d)T based spatial transcriptomics (ST) platforms (such as Nextera) or with academically run ST platforms with different spatial surface capture sequences. The capture sequence is a sequence complementary to a sequence found in a target nucleic acid. In certain embodiments, the capture sequence is a polyT (polythymine) sequence of about 30 nucleotides. Attorney Docket: NYG-LIPP-249PCT

[0109] The adapters may comprise one or more additional sequences selected from the group consisting of universal sequences, primer sequences, index sequences, capture sequences, barcode sequences (used, e.g., for counting or error correction), cleavage sequences, sequencing-related sequences, protection sequences and combinations thereof. A skilled artisan will recognize additional sequences which may be of use for library preparation and next generation sequencing.

[0110] In other embodiments, the capture sequence is bound to the capture surface, as further described below.

[0111] Ligand

[0112] The transposase is a fusion protein that includes an antibody-binding ligand. As used herein, the term ligand refers to any molecule that specifically binds to another molecule, which is sometimes referred to herein as the partner molecule or target. In one embodiment, the ligand binds to an antibody that is bound to a desirable target molecule on the genomic DNA or chromatin.

[0113] In certain embodiments, the ligand is protein A, such as that used with CUT&Tag described by Kaya-Okur et al. Nat Protoc. 2020 Oct;15(10):3264-3283, which is incorporated herein by reference. Protein A is a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. Each domain is able to bind proteins from many mammalian species, most notably IgGs. It binds the heavy chain within the Fc region of most immunoglobulins and also within the Fab region in the case of the human VH3 family.

[0114] In certain embodiments, the ligand is to protein G. such as that used with CUT&Tag described by Steven Henikoff, Jorja G Henikoff, Hatice S Kaya-Okur, Kami Ahmad (2020) Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation eLife 9:e63274, which is incorporated herein by reference. Protein G is an immunoglobulin-binding protein expressed in group C and G streptococcal bacteria much like protein A but with differing binding specificities.

[0115] In another embodiment, the ligand is a nanobody. Nanobodies are single domain antibodies derived from llama, alpaca, shark heavy-chain only antibodies, or from other animal models engineered to produce camelidae-like VHHs, that have unique properties such as nanoscale size, robust structure, stable and soluble behaviors in aqueous solution, high affinity and specificity for only one cognate target. Nanobodies achieve comparable binding affinities and specificities to classical antibodies, despite comprising only a single Attorney Docket: NYG-LIPP-249PCT

[0116] 15 kDa variable domain. The camelid VHH domain that forms the Nb is homologous to the Ab VH domain and contains three highly variable loops Hl, H2, and H3. See, e.g., Muyldermans S., Nanobodies: natural single-domain antibodies. Annu Rev Biochem. 2013:82:775-97 and Mitchell, Laura S, and Lucy J Colwell. Proteins vol. 86,7 (2018): 697-706, which are incorporated herein by reference. In certain embodiments, the nanobody binds an immunoglobulin.

[0117] Target Molecule

[0118] In certain embodiments, the transposome complex is bound to a ligand that is bound a target molecule on the genomic DNA. In certain embodiments, the ligand that is bound to a target molecule is an antibody and may be referred to as the primary antibody. Such target molecules include, without limitation, peptides, proteins, antibodies or antibody fragments, affibodies, a ribonucleic acid sequence or deoxyribonucleic acid sequence, aptamers, lipids, polysaccharides, lectins, a chimeric molecule formed of multiples of the same or different moieties.

[0119] In certain embodiments, the target molecule is a protein found on, or associated with, chromatin found in the biological specimen, sometimes referred to as a “chromatin epitope7’. Chromatin is composed of a cell’s DNA and associated proteins. Histone proteins and DNA are found in approximately equal mass in eukaryotic chromatin, and nonhistone proteins are also in great abundance. The basic unit of organization of chromatin is the nucleosome, a structure of DNA and histone proteins that repeats itself throughout an organism’s genetic material. Histones are highly conserved basic proteins, whose positively charged character helps them to bind the negatively charged phosphate backbone of DNA.

[0120] Exemplary target molecules include histones, including Hl, H2A, H2B, H3, H4, and H5. See, Annunziato, A. (2008) DNA Packaging: Nucleosomes and Chromatin. Nature Education 1 (1 ):26, which is incorporated herein by reference. Post-translationally modified histones may also be targeted, such as phosphorylation on serine or threonine residues, methylation on lysine or arginine, acetylation and deacetylation of lysines, ubiquitylation of lysines and sumoylation of lysines. Histone modifications that may be targeted include, without limitation, H3K27ac, H3K27me3, H3K4mel, H2BK5ac, H3K4me3, H3K27ac, H3K4me3, and H3K9me2. Attorney Docket: NYG-LIPP-249PCT

[0121] In other embodiments, the target molecule is a transcription factor (TF), or a suspected transcription factor. A list of 1639 known and likely human transcription factors have been described in the art, and cataloged by Lambert SA, et al. (2018) The Human Transcription Factors. Cell. 172(4):650-665. doi: 10. 1016 / j. cell.2018.01.029.

[0122] Methods

[0123] Provided herein in another aspect, are methods of using the compositions described herein. In one aspect is provided a method for in situ analysis of protein-DNA interactions in a biological specimen. The method includes one or more of the following steps.

[0124] A biological specimen is mounted to a solid substrate having a capture surface comprising an array of capture regions. The solid substrate, for example, is modified to include a plurality of barcoded capture oligonucleotides in distinct regions, called capture regions, wherein the oligos of each capture region contain the same barcode. Such solid substrates are known in the art and include beads, glass or other slides, plates, chips, chambers, etc. In one embodiment, the substrate is a glass slide. For example, the Visium Spatial Gene Expression Slide is an example of a substrate useful with the methods described herein.

[0125] Each capture region comprises capture oligonucleotides, the individual capture regions being identifiable by one or more unique spatial barcodes of the capture oligonucleotides. Capture oligos attached to the substrate allow capture of the tagmented DNA (or product thereof) via binding to the capture sequence included in the adapter. Thus, the capture oligo includes a capture sequence, which may by a polyT. When present on the adapter, the capture sequence on the substrate oligonucleotide must be complementary to the capture compatible sequence in the adapter. The sequence may be any unique sequence, as long as the capture sequence and the capture compatible sequence are complementary7. In certain embodiments, the capture sequence is a polyT. In other embodiments, the capture sequence comprises a polyT and a second sequence appended thereto, such as a ME sequence. See, FIG. 9A. In another embodiment, the capture sequence is between 10 nt to 30 nt in length. In one embodiment, the capture sequence 10. 11. 12. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23. 24. 25. 26. 27, 28. 29, or 30 nt in length. Attorney Docket: NYG-LIPP-249PCT

[0126] The capture sequence may include a universal capture sequence and, optionally, a unique UMI, that identifies a specific capture event, i.e., the binding of a single oligo to its target molecule.

[0127] The substrate oligonucleotide contains a barcode sequence that is used to identify the spatial location of the sample, such that all oligos on a specific capture region on a slide share the same barcode. Such barcode may be termed a “spatial barcode”. Similarly, the spatial barcode sequence is, in one embodiment, between 5 nt to 100 nt in length. In another embodiment, the spatial barcode sequence is between 10 nt to 20 nt in length. In one embodiment, the spatial barcode is 10 nt in length. In another embodiment, the spatial barcode is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nt in length.

[0128] In certain embodiments, the substrate oligonucleotide includes a PCR handle or priming region to enable PCR amplification subsequent to tagmentation. Optionally, the PCR handle is compatible with another capture sequence that is attached to a bead, glass slide, or other solid support. In some embodiments, the primer can comprise an R1 primer sequence for Illumina sequencing. In some cases, the primer can comprise an R2 primer sequence for Illumina sequencing. Other priming regions for use with other systems are known and may be used. Any suitable nucleic acid sequencing method can be used to sequence the nucleic acids described herein, and / or to detect the presence, absence or amount of the various nucleic acids, constructs, targets, oligonucleotides, amplification products and barcodes described herein.

[0129] In certain embodiments, the substrate oligonucleotide includes a sequencing primer (e.g., read 1 sequencing primer), a spatial barcode, optionally a UMI, and a polyT sequence. In other embodiments, a desired capture sequence is appended to the end of the polyT sequence. See, FIG. 9A. An example of a desirable capture sequence is an ME sequence, for capturing the product of a cut and tag reaction.

[0130] The methods described herein may also, in some embodiments include cell fixing, histology and imaging, cell permeabilizing, staining, template switching, transcript extension, single strand synthesis, gap filling, denaturing double strand nucleic acids, hybridization, PCR, and sequencing steps. These procedures are know n in the art, and relevant protocols can be found, e.g., Corces et al., Nat Methods. 2017 Oct;14(10):959- 962; Kaya-Okur et al., Nat Commun. 2019 Apr 29;10(l): 1930; Mimitou EP, et al. Nat Biotechnol. 2021 Oct;39(10): 1246-1258.; Meers MP et al., Multifactorial chromatin regulatory landscapes at single cell resolution. BioRxiv 2021 :2021.07.08.451691.; Deng Attorney Docket: NYG-LIPP-249PCT

[0131] Y et al. Spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissues at genome scale and cellular level. BioRxiv 2021 :2021.06.06.447244.; Fan R et al., Nature. 2022 Sep;609(7926):375-383; Stahl PL et al. Science 2016;353:78-82. Cho C-S et al. Cell 2021;184:3559-3572.e22.; Chen A et al. Large field of view-spatially resolved transcriptomics at nanoscale resolution. Cold Spring Harbor Laboratory 2021:2021.01.17.427004. Fu X, et al. Continuous Polony Gels for Tissue Mapping with High Resolution and RNA Capture Efficiency. Cold Spring Harbor Laboratory' 2021:2021.03.17.435795, each of which is incorporated herein by reference.

[0132] In certain embodiments, the methods include contacting the biological sample containing the genomic DNA with the transposase enzyme (e.g., transposome or transposome antibody complex, e.g., a reaction mixture (e.g., solution)) including a transposase, transposome, or transposome antibody complex), under any suitable conditions. In certain embodiments instances, such suitable conditions result in the tagmentation of the genomic DNA of cells present in the biological sample. Typical conditions will depend on the transposase enzyme and / or antibody complexed to the transposome used and can be determined using routine methods known in the art. Therefore, suitable conditions can be conditions (e.g., buffer, salt, concentration, pH. temperature, time conditions) under which the transposase enzyme is functional, e.g., in which the transposase enzyme displays transposase activity, particularly tagmentation activity7, in the biological sample.

[0133] Various modes of preparation of the biological specimen, e.g. tissue sample, may be utilized. For example, tissue samples will have different physical characteristics and it is well within the skill of a person in the art to perform the necessary manipulations to yield a biological specimen, e.g. tissue sample, for use herein. However, it is evident from the disclosure herein that any method of sample preparation may be used to obtain a biological specimen, e.g. tissue sample, that is suitable for use herein. For instance, any layer of cells with a thickness of approximately 1 cell or less may be used. In one embodiment, the thickness of the biological specimen, e.g. tissue sample, may be less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1, or any values therebetween, of the crosssection of a cell. In certain embodiments, the methods allow for single cell resolution and the biological specimen, e.g. a tissue sample having a thickness of one cell diameter or less. In certain embodiments, thicker biological specimens, e.g. tissue samples, are used. For example, cry ostat sections may be used, which may be e.g. 10-20 pm thick. Attorney Docket: NYG-LIPP-249PCT

[0134] The thickness of the biological specimen, e.g. tissue sample section, for use herein may be dependent on the method used to prepare the sample and the physical characteristics of the tissue. Thus, any suitable section thickness may be used. In certain embodiments, the thickness of the tissue sample section will be at least 0. 1 pm, further preferably at least 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10pm. In certain embodiments, the thickness of the tissue sample section will be at least 0.1 pm, further preferably at least 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10pm, or any values therebetween. In other embodiments the thickness of the tissue sample section is at least 10, 12, 13, 14, 15, 20, 30, 40 or 50pm. In other embodiments the thickness of the tissue sample section is at least 10, 12, 13, 14, 15, 20, 30, 40, 50pm, or any values therebetween. However, the thickness is not critical and these are representative values only. Thicker samples may be used if desired or convenient e.g. 70 or 100 pm or more. Typically, the thickness of the tissue sample section is between 1-100 pm, 1-50 pm, 1-30 pm, 1-25 pm, 1-20 pm, 1-15 pm, 1-10 pm, 2-8pm, 3-7pm or 4-6pm, but as mentioned above thicker samples may be used.

[0135] The biological specimen, e.g. tissue sample, may be prepared in any convenient or desired way. Fresh, frozen, fixed or unfixed tissues may be used. Any desired convenient procedure may be used for fixing or embedding the biological specimen, e.g. tissue sample, as described and known in the art. Thus, any known fixatives or embedding materials may be used.

[0136] In certain embodiments, a tissue is prepared by deep freezing at a temperature suitable to maintain or preserve the integrity (i.e. the physical characteristics) of the tissue structure, e.g. less than -20°C and preferably less than -25, -30, -40, -50, -60, -70 or -80 °C. The frozen tissue sample may be sectioned, i.e. thinly sliced, onto the solid substrate, e.g. array surface by any suitable means. For example, a tissue sample may be prepared using a chilled microtome, a cryostat, set at a temperature suitable to maintain both the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample, e.g. to less than -15°C and preferably less than -20 or -25°C. Thus, the sample should be treated so as to minimize the degeneration or degradation of the nucleic acid, e.g. RNA, in the tissue. Such conditions are well-established in the art and the extent of any degradation may be monitored through nucleic acid extraction, e.g. total RNA extraction, and subsequent quality analysis at various stages of the preparation of the tissue sample. Attorney Docket: NYG-LIPP-249PCT

[0137] In certain embodiments, a tissue is prepared using standard methods of formalinfixation and paraffin-embedding (FFPE), which are well-established in the art. Following fixation of a tissue sample and embedding in a paraffin or resin block, the tissue sample may be sectioned, i.e. thinly sliced, onto the solid substrate, e g. array. As noted above, other fixatives and / or embedding materials can be used.

[0138] In certain embodiments, the biological specimen, e.g. tissue sample section, will need to be treated to remove the embedding material e.g. to deparaffinize, i.e. to remove the paraffin or resin, from the sample prior to carrying out the methods. This may be achieved by any suitable method and the removal of paraffin or resin or other material from tissue samples is well established in the art, e g. by incubating the sample (on the surface of the solid substrate, e.g. array) in an appropriate solvent e.g. xylene, e.g. twice for 10 minutes, followed by an ethanol rinse, e.g. 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes.

[0139] As used herein, the term ’‘hybridize” is used in its broadest sense to mean the formation of a stable nucleic acid duplex. A duplex can be ‘'perfectly matched”, such that the polynucleotide and / or oligonucleotide strands making up the duplex form a double stranded structure with one another such that every nucleotide in each strand undergoes Watson-Crick base pairing with a nucleotide in the other strand. A duplex can comprise at least one mismatch, wherein the term “mismatch” means that a pair of nucleotides in the duplex fail to undergo Watson-Crick bonding. Thus, two sequences need not have perfect homology to be “complementary” under the invention. Usually two sequences are sufficiently complementary when at least about 90% (preferably at least about 95%) of the nucleotides share base pair organization over a defined length of the molecule.

[0140] In certain embodiments, the method includes labeling or staining the biological specimen for visualization. Any stain useful for dying tissue may be used, such as Hematoxylin and eosin (H&E) staining.

[0141] In order to correlate the sequence analysis or transcriptome information obtained from each capture region of the array with the region (i.e. an area or cell) of the biological specimen, e.g. tissue sample, the biological specimen is oriented in relation to capture regions on the array. In certain embodiments, this step includes visualization of the fiducial pixels. In other words, the tissue sample is placed on the array such that the position of a substance oligonucleotides on the array may be correlated with a position in the biological specimen, e.g. tissue sample. Thus it may be identified where in the Attorney Docket: NYG-LIPP-249PCT biological specimen, e.g. tissue sample, the position of each species of capture probe (or each feature of the array) corresponds. In other words, it may be identified to which location in the biological specimen, e.g. tissue sample, the position of each capture region.

[0142] In certain embodiments, the biological specimen, e.g. tissue sample, may be imaged following its contact with the array. In certain embodiments, the biological specimen, e.g. tissue sample, is imaged prior to substrate oligonucleotide cleavage and / or reverse transcription steps. Generally speaking, imaging may take place at any time after contacting the biological specimen, e.g. tissue sample, with the solid substrate, but before any step which degrades or removes the biological specimen, e.g. tissue sample. As noted above, this may depend on the biological specimen, e.g. tissue sample.

[0143] The methods described herein include a reverse transcription step, particularly reverse transcription using a reverse transcriptase. A reverse transcriptase is an enzyme that uses RNA as a template for DNA synthesis.

[0144] The following is an overview of exemplary conditions suitable for performing reverse transcription reaction on an array surface - RT reaction is loaded with IX RT buffer, 0.2-0.4 mg BSA, 0.5-1 pmol dNTPs. 5-15 nmol template switch oligo, 0. IX PEG 8000 (50%w / v), 8pL RT Maxima H- enzyme and 4ul RNAOUT (Invitrogen) inhibitor. The reaction volumes are 80 pl. Reaction conditions may be run overnight at 42°C.

[0145] In certain embodiments, the method includes permeabilizing the biological specimen. Any suitable buffer can be used for permeabilization, including any of the commercially available reagents, such as from Beckman Coulter, etc. In one embodiment, permeabilizing the biological specimen comprises contacting the biological specimen comprises with a solution comprising perm / wash buffer, digitonin buffers, and / or HC1. In one embodiment, the permeabilization buffer comprises 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 3 mM MgC12, 0. 1 % NP40. 0.1 % Tween-20. 1 % BSA, 1 x protease inhibitors.

[0146] In certain embodiments, the method includes contacting the biological specimen with an antibody or fragment thereof capable of binding a target molecule. In certain embodiments, the target molecule is a chromatin epitope, optionally a histone modification.

[0147] The method includes contacting the biological specimen with a tagmentation complex as described herein, and incubating the biological specimen under conditions suitable for tagmentation to occur, wherein tagmented genome fragments comprising the Attorney Docket: NYG-LIPP-249PCT

[0148] T7 promoter, optionally, and the capture sequence, are generated. Tagmentation is initiated using magnesium or other divalent cation, such as cobalt. In certain embodiments, MgCh. is added, optionally at a concentration of lOmM.

[0149] In certain embodiments, a gap-filling reaction is performed. In certain embodiments, the gap-filling is done via ligation. In some embodiments, the gap-filling is done via polymerization. Gap-filling via ligation may be performed using about 0.015 units of T4 DNA polymerase and about 2 units T4 DNA ligase and / or at about 24°C for about 30 minutes. Other useful ligases include T3 ligase, T7 ligase, PBCV-1 DNA ligase, or E. coli ligase.

[0150] Gap-filling via polymerization may be performed using about 0.143 units Klenow (exo-) polymerase and / or at about 37°C for about 30 minutes. This may be accomplished using a Klenow reaction, which has been described previously. See, e.g., Lotstedt, B., Strazar, M., Xavier, R. et al. Spatial host-microbiome sequencing reveals niches in the mouse gut. Nat Biotechnol 42, 1394-1403 (2024).

[0151] In certain embodiments, the methods include a step that requires a T7 RNA polymerase (also “T7 polymerase”), a bacterial enzyme that synthesizes RNA and replicates DNA. Such reagents are known in the art and available commercially, for example MEGAshortscript™ T7 Transcription Kit (Invitrogen). In one embodiment, the method include contacting the biological specimen with a reaction mixture comprising a T7 polymerase to amplify the tagmented genome fragments.

[0152] In certain embodiments, the method includes generating a library of spatially barcoded tagmented genome products, wherein the tagmented genome fragments hybridized to the capture oligonucleotides are barcoded with the one or more unique spatial barcodes of the capture oligonucleotides; and sequencing and analyzing the library. In certain embodiments, sequencing and analysis includes mapping protein-DNA interactions to spatial locations of the biological specimen using the one or more distinct spatial barcodes of the capture oligonucleotides. Sequencing libraries can be constructed using an established protocol similar to ST and CITE-seq, followed by sequencing and mapping of protein interactions and epigenome data with spatial resolution. See, Patrik L. Stahl et al., Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science353,78-82(2016).D01: 10.1126 / science.aaf2403 and Mimitou, E.P., Cheng, A., Montalbano, A. et al. Multiplexed detection of proteins, transcriptomes, Attorney Docket: NYG-LIPP-249PCT clonotypes and CRISPR perturbations in single cells. Nat Methods 16, 409-412 (2019). doi.org / 10. 1038 / s41592-019-0392-0, both incorporated herein by reference.

[0153] As used herein, the term “library” refers to a collection of members. In certain embodiments, the library includes a collection of nucleic acid members, for example, a collection of whole genomic, subgenomic fragments, cDNA, cDNA fragments, RNA, RNA fragments, or a combination thereof. In certain embodiments, a portion or all library members include an adaptor and / or barcode sequence. The adaptor sequence and / or barcode sequence can be located at one or both ends. The adaptor sequence can be used in, for example, a sequencing method (for example, an NGS method), for amplification, for reverse transcription, or for cloning into a vector.

[0154] As used herein, “sequencing” generally refers to determining the order of nucleotides (base sequences) in a nucleic acid sample, e.g., DNA or RNA. Many techniques are available, such as Sanger sequencing or High Throughput Sequencing technologies (HTS). Sanger sequencing may involve sequencing via detection through (capillary) electrophoresis, in which up to 384 capillaries may be sequence analyzed in one run. High throughput sequencing involves the parallel sequencing of thousands or millions or more sequences at once. HTS can be defined as Next Generation sequencing (NGS), i.e. techniques based on solid phase pyrosequencing or as Next-Next Generation sequencing based on single nucleotide real time sequencing (SMRT). HTS technologies are available such as offered by Roche, Illumina and Applied Biosystems (Life Technologies). Further high throughput sequencing technologies are described by and / or available from Helicos, Pacific Biosciences. Complete Genomics, Ion Torrent Systems, Oxford Nanopore Technologies, Nabsys, ZS Genetics, GnuBio. Sequencing platforms such as Nanopore direct RNA sequencing (DRS) allow direct sequencing of full-length native RNA molecules without the need for RT or amplification.

[0155] As used herein, “next generation sequencing” refers to high-throughput sequencing methods that allow the sequencing of millions to billions of molecules in parallel. Examples of next generation sequencing methods include sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, nanopore sequencing, and pyrosequencing. By attaching primers to a solid substrate and a complementary sequence to a nucleic acid molecule, a nucleic acid molecule can be hybridized to the solid substrate via the primer and then multiple copies can be generated in a discrete area on the solid substrate by using Attorney Docket: NYG-LIPP-249PCT polymerase to amplify (these groupings are sometimes referred to as polymerase colonies or polonies). Consequently, during the sequencing process, a nucleotide at a particular position can be sequenced multiple times (e.g., hundreds or thousands of times) — this depth of coverage is referred to as “deep sequencing.” Examples of high throughput nucleic acid sequencing technology include platforms provided by Illumina, BGI, Qiagen, ThermoFisher, and Roche, including formats such as parallel bead arrays, sequencing by synthesis, sequencing by ligation, capillary electrophoresis, electronic microchips, “biochips,” microarrays, parallel microchips, and single-molecule arrays, as reviewed by Service (Science 311: 1544-1546, 2006).

[0156] In certain embodiment, sequencing is performed using nanopore sequencing (e.g. as described in Soni GV and Meller A. Clin Chem 53: 1996-2001, 2007). Nanopore sequencing is a single-molecule sequencing technology whereby a single molecule of RNA is sequenced directly as it passes through a nanopore. The technology allows for characterization and quantification of full-length RNA transcripts, splice variants, and fusions using short to ultra-long fragment sequencing A nanopore is a small hole, of the order of 1 nanometer in diameter. Immersion of a nanopore in a conducting fluid and application of a potential (voltage) across it results in a slight electrical current due to conduction of ions through the nanopore. The amount of current which flows is sensitive to the size and shape of the nanopore. As an RNA molecule passes through a nanopore, each nucleotide on the RNA molecule obstructs the nanopore to a different degree, changing the magnitude of the current through the nanopore in different degrees. Thus, this change in the current as the RNA molecule passes through the nanopore represents a reading of the RNA sequence. See, e.g., WO 2016059436 Al , which is incorporated herein by reference.

[0157] Any method of nucleic acid analysis may be used to analyze sequence information obtained according to the disclosed methods. Typically, the sequence analysis information obtained is used to obtain spatial information as to the protein / DNA in the biological specimen, e.g. tissue sample. In other words, the sequence analysis information may provide information as to the location of the protein / DNA in the biological specimen, e.g. tissue sample. Spatial information may conveniently be obtained by correlating the sequence analysis data to an image of the biological specimen, e.g. tissue sample. Accordingly, in a preferred embodiment the method also includes a step of correlating sequence analysis information with an image of the biological specimen. Attorney Docket: NYG-LIPP-249PCT

[0158] In another embodiment, the target epitope is bound by a primary antibody, and the ligand of the fusion protein recognizes a primary- antibody that recognizes the target epitope, thus indirectly binding the target epitope. Thus, in certain embodiments, the ligand of the fusion protein is specific to the primary antibody's species and isotype. For example, the ligand may be anti- IgA, IgD, IgE, IgG, or IgM. In addition, the ligand may be raised against a primary antibody of any species including human, mouse, rat, rabbit, etc. The ligand and the primary7antibody are independently selected from any type of antibody / ligand, as described herein and known in the art. For example, in one embodiment, the primary’ antibody is a monoclonal antibody, and the ligand is a nanobody. In another embodiment, the primary antibody is a scFv, and the ligand is a nanobody. As a non-limiting example, the primary antibody may be an anti-IgGl, IgG2A, IgG2B, IgG2C or IgG3 mouse antibody, or universal mouse antibody.

[0159] Provided herein, in one aspect, are compositions which contain one or more of the components described above, optionally in addition to other features, molecules or components. In one embodiment, a composition is provided yvhich alloyvs for interaction mapping of molecules found in a biological sample. The selection of the components of the composition will depend upon the identity of the partner molecule sought, the methodology being employed and interactions being elucidated. The method used may dictate the selection and compositions of the various components described above which make up the composition. Thus the following description of compositions is not exhaustive, and one of skill in the art can design many different compositions based on the teachings provided herein. The composition may also contain the constructs in a suitable buffer, diluent, carrier or excipient. The elements of each composition wall depend upon the assay format in yvhich it will be employed. Several embodiments of compositions are described below, but are not to limit the compositions encompassed herein, which are intended to extend to compositions comprising any component(s) herein described.

[0160] In one embodiment, a composition is provided which comprises a reagent. The reagent includes fusion protein as described herein yvhich includes a transposome complex comprising an adapter comprising a T7 promoter. In another embodiment, a composition is provided which includes the adapter-loaded transposome complex bound to chromatin by a protein-specific primary- antibody, to which the transposome complex binds. Attorney Docket: NYG-LIPP-249PCT

[0161] In yet another embodiment, a composition is provided which includes the adapter- loaded transposome complex bound to chromatin by a protein-specific primary' antibody, to which the transposome complex binds, wherein the chromatin-bound composition is bound to a substrate, e.g., a glass slide.

[0162] Kits containing the compositions are also provided. Such kits will contain one or more of the following: fusion proteins as described herein, adapters, substrates, substrate oligonucleotides, one or more preservatives, stabilizers, or buffers, and such suitable assay and amplification reagents depending upon the amplification and analysis methods and protocols with which the composition will be used. Still other components in a kit include optional reagents for cleavage of the linker, fixative, ligase, wash buffer, detectable labels, immobilization substrates, optional substrates for enzymatic labels, as well as other laboratory' items.

[0163] Specific Embodiments

[0164] 1. A transposome complex comprising a first transposase and a second transposase, the first transposase having a first adapter comprising a T7 promoter and the second transposase having a second adapter comprising a capture sequence, the first transposase and the second transposase each being a fusion protein comprising an antibody-binding ligand.

[0165] 2. A transposome complex comprising a first transposase and a second transposase, the first transposase having a first adapter comprising a T7 promoter and the second transposase having a second adapter, the first transposase and the second transposase each being a fusion protein comprising an antibody-binding ligand.

[0166] 3. The transposome complex of embodiment 1, wherein the second adapter comprises a capture sequence.

[0167] 4. The transposome complex of embodiment 2 or embodiment 3, wherein the capture sequence is 1 to lOObp, optionally 30 bp.

[0168] 5. The transposome complex of embodiment 2 or 3, wherein the capture sequence comprises a poly(d)T sequence or poly(d)T VN sequence. Attorney Docket: NYG-LIPP-249PCT

[0169] 6. The transposome complex of embodiment 2 or 3, wherein the capture sequence comprises a mosaic end sequence.

[0170] 7. The transposome complex of any one of embodiments 1 to 6, wherein the first adapter and / or the second adapter further comprise a mosaic end sequence.

[0171] 8. The transposome complex of any one of embodiments 1 to 7, where the first transposase and the second transposase are Tn5 or TnY.

[0172] 9. The transposome complex of any one of embodiments 1 to 8, wherein the antibody -binding ligand is protein A, protein G, or a nanobody.

[0173] 10. The transposome complex of any one of embodiments 1 to 9, wherein the transposome complex is bound to an antibody or fragment thereof bound to a chromatin epitope in situ, optionally wherein the chromatin epitope is present in a biological sample mounted on a capture surface comprising an array of capture regions.

[0174] 1 1. The transposome complex of any one of embodiments 1 to 10, wherein the chromatin epitope comprises a histone modification.

[0175] 12. The transposome complex of embodiment 11, where the histone modification comprises a histone acylation, a histone phosphorylation, or a histone methylation, optionally wherein the histone acylation is an H3K27ac and / or the histone methylation is an H3K4me3 mark or an H3K27me3 mark.

[0176] 13. A method for in situ analysis of protein-DNA interactions in a biological specimen, the method comprising: a) mounting a biological specimen to a solid substrate having a capture surface comprising an array of capture regions, each capture region comprising capture oligonucleotides, the individual capture regions being identifiable by one or more unique spatial barcodes of the capture oligonucleotides; b) optionally, labeling or staining the biological specimen for visualization; Attorney Docket: NYG-LIPP-249PCT c) permeabilizing the biological specimen; d) contacting the biological specimen with an antibody or fragment thereof capable of binding a chromatin epitope, optionally a histone modification, and a tagmentation complex of any one of embodiments 1 to 12, and incubating the biological specimen under conditions suitable for tagmentation to occur, wherein tagmented genome fragments comprising the T7 promoter and the capture sequence are generated; e) performing a gap-filling reaction; f) contacting the biological specimen with a reaction mixture comprising a T7 polymerase to amplify the tagmented genome fragments; g) generating a library of spatially barcoded tagmented genome products, wherein the tagmented genome fragments hybridized to the capture oligonucleotides are barcoded with the one or more unique spatial barcodes of the capture oligonucleotides; and h) sequencing and analysis of the library of step g), wherein sequencing and analysis includes mapping protein-DNA interactions to spatial locations of the biological specimen using the one or more distinct spatial barcodes of the capture oligonucleotides.

[0177] 14. A method for in situ analysis of protein-DNA interactions and protein-protein interactions in a biological specimen, the method comprising: a) mounting a biological specimen to a solid substrate having a capture surface comprising an array of capture regions, each capture region comprising capture oligonucleotides, the individual capture regions being identifiable by one or more unique spatial barcodes of the capture oligonucleotides; b) optionally, fixing the biological specimen; c) optionally, labeling or staining the biological specimen for visualization and for protein-protein labeling; d) contacting the biological specimen with one or antibodies or fragments thereof having linked oligonucleotides comprising a barcode and anchor sequence; e) permeabilizing the biological specimen; f) contacting the biological specimen with an antibody or fragment thereof capable of binding a chromatin epitope, optionally a histone modification, and a tagmentation complex of any one of embodiments 1 to 12, and incubating the biological Attorney Docket: NYG-LIPP-249PCT specimen under conditions suitable for tagmentation to occur, wherein tagmented genome fragments comprising the T7 promoter and the capture sequence are generated; g) performing a gap-filling reaction; h) contacting the biological specimen with a reaction mixture comprising a T7 polymerase to amplify the tagmented genome fragments; i) generating 1) a library of spatially barcoded tagmented genome products, wherein tagmented genome fragments hybridized to the capture oligonucleotides are barcoded with the one or more unique spatial barcodes of the capture oligonucleotides, and 2) a library of spatially barcoded oligonucleotides comprising i) the barcodes of the oligonucleotides linked to the one more antibodies or fragments thereof of step d), and ii) the one or more unique spatial barcodes of the capture oligonucleotides; and j) sequencing and analysis of the libraries of step i), wherein sequencing and analysis includes mapping protein-DNA interactions and protein-protein interactions to spatial locations of the biological specimen using the one or more distinct spatial barcodes of the capture oligonucleotides.

[0178] 15. A method for spatially resolving DNA-protein interactions in a biological sample, the method comprising: a) mounting a biological specimen to a solid substrate having a capture surface comprising an array of capture regions, each capture region comprising capture oligonucleotides, the individual capture regions being identifiable by one or more unique spatial barcodes of the capture oligonucleotides; b) optionally, labeling or staining the biological specimen for visualization; c) permeabilizing the biological specimen; d) contacting the biological specimen with an antibody or fragment thereof capable of binding a chromatin epitope, optionally a histone modification, and a tagmentation complex of any one of embodiments 1 to 12, and incubating the biological specimen under conditions suitable for tagmentation to occur, wherein tagmented genome fragments comprising the T7 promoter and the capture sequence are generated; e) performing a gap-filling reaction; and f) contacting the biological specimen with a reaction mixture comprising a T7 polymerase to amplify the tagmented genome fragments. Attorney Docket: NYG-LIPP-249PCT

[0179] 16. The method of embodiment 13 or embodiment 15, further comprising fixing the biological specimen.

[0180] 17. The method of any one of embodiments 13 to 16, wherein staining the biological sample comprises hematoxylin and eosin (H&E) staining.

[0181] 18. The method of any one of embodiments 13 to 17, wherein labeling the biological specimen comprises contacting the biological specimen with fluorescently-labeled antibodies or fragments thereof capable of binding epitopes in the biological sample.

[0182] 19. The method of any one of embodiments 13 to 18, further comprising imaging the biological specimen using brightfield microscopy or immunofluorescence microscopy.

[0183] 20. The method of any one of embodiments 13 to 19, wherein permeabilizing the biological specimen comprises contacting the biological specimen comprises with a solution comprising perm / wash buffer, digitonin buffers, and / or HC1.

[0184] 21. The method of any one of embodiments 13 to 20, wherein the capture oligonucleotides of the capture surface comprise a polyT or a polyT VN sequence.

[0185] 22. The method of any one of embodiments 13 to 20, wherein the capture oligonucleotides of the capture surface comprise a Mosaic End (ME) sequence.

[0186] 23. The method of any one of embodiments 13 to 22, wherein incubating the biological specimen under conditions suitable for tagmentation to occur comprises incubating the biological specimen with about 0. 1 mg of the tagmentation complex and / or incubating the biological specimen for a period of time at about 37°C and a period of time at about 55°C.

[0187] 24. The method of any one of embodiments 13 to 23, wherein amplification of the genome fragments comprises i) incubating the biological specimen with about 0. 1 to about 0.4 p / pl T7 RNA polymerase, and / or ii) incubating the biological specimen at Attorney Docket: NYG-LIPP-249PCT about 37°C for about 14 hours or at least about 8 hours, about 10 hours, or about 12 hours.

[0188] 25. The method of any one of embodiments 13 to 24, wherein the gap-filling reaction comprises gap-filling via ligation or gap filling via polymerization.

[0189] 26. The method of embodiment 25, wherein gap-filling via ligation is performed using about 0.015 units of T4 DNA polymerase and about 2 units T4 DNA ligase and / or at about 24°C for about 30 minutes.

[0190] 27. The method of embodiment 25, wherein gap filling via polymerization is performed using about 0.143 units Klenow (exo-) polymerase and / or at about 37°C for about 30 minutes.

[0191] EXAMPLES

[0192] Example 1 : Solid-capture-based spatial CUT&Tag Platform Description

[0193] In this example we describe a devised new platform of technologies for mapping cellular connections and signal transmission within complex tissues. Our strategy for technology development is carefully designed such that each new capability we build can be employed independently, often enhancing capabilities of spatial technologies already in widespread use. However, they are to function synergistically as an integrated platform.

[0194] In this example, we describe a solid-capture based spatial CUT&Tag to profile genome-wide Protein-DNA interactions and protein-protein interactions simultaneously in tissues (hereinafter referred as spatial multi-modal CUT&Tag), which provides a widely accessible, scalable solution for spatially resolved chromatin profiling, with the potential to significantly advance our understanding of epigenetic regulation in diseased tissues, thus offering a broader applicability in cancer, neurological disorders, and beyond. We have also used this technology to simultaneously measure protein-protein and DNA-protein interactions simultaneously in tissue, which has not been attempted previously. Attorney Docket: NYG-LIPP-249PCT

[0195] The Solid-capture based spatial CUT&Tag (spatial CUT&Tag) is a novel method that combines Cleavage Under Targets and Tagmentation (CUT&Tag) with Spatial transcriptomics (ST) to enable genome-wide protein-DNA and protein-protein interaction profiling with spatial resolution (Example 2, FIG. 1).

[0196] The spatial CUT&Tag method includes engineering of specific Tn5 adaptor sequences. The Tn5 adaptors have been modified to include a T7 promoter and a capture sequence. The capture sequences are fully modular (up to 30 bp) and can (for example) include a poly(d)T or a nextera Mosaic End (ME) sequence. This modification allows for the compatibility with commercially available poly(d)T based ST platforms or with academically run ST platforms with different spatial surface capture sequences (Example 2, FIG. 2).

[0197] The spatial CUT&Tag method includes optimization of in situ ProteinA-Tn5 or nanobody-Tn5 tagmentation conditions with engineered custom adaptors. These complexes can be bound before any tethered to the antibodies in antibody-stained tissue, where it will initiate the tagmentation process upon the addition of magnesium. To optimize the tagmentation reaction, in our platform method, we perform the tagmentation in situ with a 0.01 mg / ml concentration of the loaded protein at two different temperatures: 37°C and 55°C. Our results indicate that this two-step reaction significantly increases tagmentation efficiency (Example 2, FIG. 3).

[0198] The spatial CUT&Tag method also includes optimization of tissue handling conditions for efficient in situ DNA-protein and protein-protein interactions.

[0199] Optimizing tissue staining conditions includes, but are not limited to, staining with primary and secondary-specific antibodies, for specific antibody-antigen specific binding either performed simultaneously for multiplexed protein-protein or protein-DNA interactions. In our platform method, we use a cocktail of 3 antibodies during the primary and secondary antibody staining (Example 2, FIG. 4A).

[0200] Optimizing tissue permeabilization conditions for tissue staining includes DNA- protein material release to the spatial capture surface and protein-protein interaction detection, or both simultaneously. In our platform method, we use a permeabilization / wash buffer, digitonin buffers and HC1 for efficient reaction conditions which enables us to either image or sequence protein-protein and protein-DNA interactions simultaneously (Example 2, FIG. 4B, briefly, a frozen mouse brain section was placed on the spatial capture surface, stained for hematoxylin and eosin (H&E) and Attorney Docket: NYG-LIPP-249PCT imaged). Using our tissue permeabilization and processing protocols that we successfully optimized, we labeled tagmented material in situ with fluorescent nucleotides. These fluorescent patterns are considered to be “tissue activity footprints’" which can quantitatively be assessed (e.g. signal intensities from different cell types in respect to different reaction conditions; signal spread and diffusion as compared to cell boundaries).

[0201] The spatial CUT&Tag method also includes in vitro amplifying strategy to enhance the DNA-protein interaction signal detection.

[0202] To initiate an efficient gap filling reaction, we have optimized two different strategies: gap filling via ligation and gap filling via polymerization. This step is needed for efficient coupling of a fragmented DNA fragment denoting the location of a DNA- protein interaction to the specifically engineered adapters that we inserted via tagmentation. Ligation-based gap filling was performed in lx T4 DNA ligase buffer, 0.05mM dNTP mix, and 0.015 units of T4 DNA polymerase and 2 units T4 DNA ligase at 24°C for 30 minutes. The polymerase-based gap filling was performed using 0.143 units Klenow (exo-) polymerase in a lx NEB2 buffer at 37°C for 30 minutes (Example 2, FIG. 3A).

[0203] After the DNA has been tagmented and the fragments made full-length in the previous step at the specific DNA-protein location, we can use the T7 promoters inserts to amply these specific DNA fragments. We can do this by addition of a specific T7-based polymerase that binds only to this bacterial promoter sequence inserted in the human genome to make linear copies of the fragmented DNA template (Example 2, FIG. 2).

[0204] While we tested for an in vitro amplification strategy, this principle can be applied through an in situ PCR reaction as well. However, due to intrinsic temperatures used for an efficient PCR reaction (high temperatures make it difficult to work with tissues in situ), we chose to only focus on a T7-based amplification. We have optimized this assay to work with a specific type of T7 polymerase that preferentially works with amplifying short-inserted fragments (Example 2, FIG. 3B). In vitro amplification was performed lx T7 RNA Polymerase buffer, 2mM each rNTP, 0.4 p / pl T7 RNA polymerase. Alternatively, to facilitate the short-inserted fragments, in vitro transcription (IVT) can also be earned out through Megashort scripts from Thermo Fisher (e.g., thermofisher.com / order / catalog / product / AM1354). Both reactions can be incubated at 37°C for 14 hours. Attorney Docket: NYG-LIPP-249PCT

[0205] We show the successful impact of the T7 amplification in FIG. 4B (Example 2). To note is that we also show in the same figure that we can also capture spatial CUT&Tag signals (1) post detecting protein-protein interactions and (2) with and without a T7 amplification step.

[0206] The surface capture can then occur either by DNA-DNA or DNA-RNA hybridization processes, dependent on insert fragment lengths, which we have optimized. Among 2 types of capturing sequences we used (ME and poly(d)T(30)), we found a buffer condition: 50 mM Tris-HCl (pH 8.3 at 25°C), 75 mM KC1. 3 mM MgCh and 10 mM DTT, that achieved the best hybridization of to the array surface. This part of the method is modular and adaptable to any inserted spatial capture sequence. We show results using an inserted (via tagmentation) poly(d)T sequence in FIGs. 4B-E (Example 2), but this is adaptable to any DNA or RNA sequence structure.

[0207] Reverse transcription or DNA polymerization or a hybrid RNA-dependent DNA polymerization and DNA-helicase process is then applied to spatially barcode the material (that can be amplified in the previous step, but does not have to be amplified and be directly bound to the surface as well hybridized material), followed by standard Illumina sequencing library preparation using commercially available reagents. See, Example 2, FIGs. 4E, 5 and 6.

[0208] In a complementary spatial multi-modal alternative approach we modified the capture surface via ligation to attach a different capture oligo (non poly(d)T) to the commercially available platform (Example 2, FIG. 9A). To fine-tune tissue processing and array production for efficient co-capture of these molecules on the spatial surface, we also tested an alternative approach to the pA-Tn5 constructs described above. In this version of the technology, we only add the T7 promoter to the standard Tn5 mosaic end (ME) sequences. We perform the optimized reactions as described above. However, to bind these DNA fragments to a commercial spatial surface, we need to modify the spatial surface itself. Drawing from our prior experiments when developing the spatial hostmicrobiome sequencing approach (with co-capture of poly adenylated transcripts and 1 S sequences), we recognize the critical importance of this step in establishing a spatial sequence co-capture saturation curve, enabling us to quantitatively assess the surface's capacity to capture each of our targeted molecules effectively. Here, we chose to perform a ligation reaction to attach a ME capture sequence on the spatial oligos. We then Attorney Docket: NYG-LIPP-249PCT performed Spatial CUT&Tag with the modified surface, and we also reached genomewide correlation with ENCODE data (Example 2, FIG. 9B).

[0209] Example 2. Solid-capture based spatial CUT&Tag Platform Workflow

[0210] In this example, we tested that the tissue processing workflows for simultaneous spatial CUT&Tag and protein-protein co-staining are compatible using frozen mouse brain samples and confirmed the quality' and spatial distribution of the spatial CUT&Tag signals by sequencing. Provided below is preliminary data and the method.

[0211] FIG. 1 shows spatial multi-modal CUT&Tag workflow. FIG. 1-(1) shows workflow starting with native histone modifications in the tissue genome, FIG. 1 -(II) tissues are to be permeabilized and stained with primary antibodies against target proteins including 1 cell ty ping proteins (1) and (2) large protein-protein interactions panels such as CITE-seq. Labeling of cell-type specific protein can occur using both fluorescently labeled (1) or (2) DNA barcoded (CITE-seq) antibodies or both simultaneously. Then, tethered to a fusion protein, FIG. 1 -(III) protein A-Tn5 or nanobody-Tn5, will bind to the primary' histone antibodies and the Tn5 will mediate a “CUT” and “Tag” of the genome, thus both fragmenting and barcoding specific locations in the genome. FIG. l-(IV) shows that these fragments can then be amplified in situ via in vitro transcription to generate sufficient molecules for targeted capture. FIG. 1-(V) shows that sequencing libraries can then be constructed using an established protocol similar to ST and CITE-seq, followed by sequencing and FIG. 1 -(VI) mapping of protein interactions and epigenome data with spatial resolution.

[0212] FIG. 2 shows detailed overview of the Tn5 constructs. FIG. 2A shows a diagram of adaptor engineering method. All the red adaptors are engineered sequences and blue parts are original nextera sequences. FIG. 2B shows sequence composition of adaptor sequences, the composition of Capture Sequences denoted in red can be customized according to the need of applications. FIG. 2C shows a diagram and sequence compositions of engineered PA-Tn5 constructs used in spatial multiome CUT&Tag. FIG. 2D shows that after tagment, the genome with engineered PA-Tn5 construct, in vitro transcription can be performed.

[0213] FIG. 3A shows that a two-step reaction, 37°C+55°C, significantly increases tagmentation efficiency. Condition 1-4 corresponding to different histone marks and Attorney Docket: NYG-LIPP-249PCT primary antibody staining time. FIG. 3B shows that on another optimization, Megashort T7 reactions result in most complexities in the final library.

[0214] FIG. 4 shows spatial CUT&Tag signals achieved using the in situ amplification approach. FIG. 4A shows IF signals using the optimized tissue processing workflows for: MAP2, NeuN and GFAP. Scale bar: 250pm. FIG. 4B shows fluorescent tissue activity signatures were synthesized on a spatial capture surface using the same tissue staining and permeabilization conditions in all three experiments. In the first experiment (left panel), we successfully present mRNA-only signals confirming feasibility of mRNA capture using tissue processing for SM-Omics. In the second experiment (middle panel), we stained the tissue with H3K27 antibody, performed our CUT&Tag protocol and capture both mRNA and H3K27-specific signals on the spatial surface which additionally confirmed the feasibility of performing CUT&Tag using tissue processing for SM-Omics. In the third experiment (right panel), we successfully show successful localized in situ amplification of CUT&Tag signals using the same tissue processing conditions as in the previous experiment. FIG. 4C shows IGV view of peaks mapped to genomics regions encompassing Ptma and Micalll. FIG. 4D shows "Tornado plot’ of signal at peak regions called in ENCODE H3K27ac ChlP-seq. FIG. 4E shows a spatially resolved H3K27ac data (color scale) using the Visium 10X Genomics platform.

[0215] More specifically, in FIG. 4A, we show that the tissue staining and permeabilization workflows we developed successfully stained several marker targets in the mouse brains (MAP2, NeuN and GFAP). Then, in FIG. 4B, using the same protocol, we successfully generated tissue activity footprints, where molecules were labeled during a cDNA synthesis, but now instead of only showing successful capture of mRNA molecules as shown in our previous work (Stah el al., [Stahl, P., et al., Visualization and analysis of gene expression in tissue sections by spatial transcriptomics, Science, 2016 Jul 1; 353(6294):78-82]. Vickovic et al), we show successful localized capture of amplified CUT&Tag genomic regions. With such success with developing our in situ amplification approach, we first applied this approach to fresh frozen mouse brains, targeting active promoters, enhancers (H3K27ac). We sequenced and compared the resulting CUT&Tag signals with published bulk and single cell CUT&Tag data (FIGs. 4C and 4D). FIG. 4C illustrates two representative traces of marker genes for mature oligodendrocytes (mOL) marker genes, Ptma and Micalll . Across both regions, the H3K27ac CUT&Tag signals exhibit consistency between published and our CUT&Tag data. In both datasets, the Attorney Docket: NYG-LIPP-249PCT signal peak localized to the TSS region, in accordance with the fact that H3K27ac are active chromatin markers and localized to the promoter and enhancer regions in the genome. From a genome-wide standpoint, both previously published (Deng et al., [Deng, Y., et al., Spatial-CUT&Tag: Spatially resolved chromatin modification profiling at the cellular level, Science, 2022 Feb 11;375(6581):681-686]) and our H3K27ac CUT&Tag exhibit a robust spearman correlation (p) value of 0.84 when compared to the gold standard (ENCODE) H3K27ac ChiP-seq dataset (FIG. 4D), while the single cell CUT&Tag data exhibited much lower efficiency and correlated poorly with the ENCODE data (p = 0.70). Additionally, we successfully generated spatial CUT&Tag H3K27ac libraries (FIG. 4E) using a low resolution spatial capture platform (Visium 10X Genomics) for a proof-of-concept. In summary, we show that we can capture spatially resolved genome-wide histone modification signals that are highly consistent with published single cell and bulk data in mouse brain tissue. These preliminary data collectively indicate that we can already (i) effectively run an in situ amplification reaction and capture chromatin material similar to other published and gold standard data and (ii) we have an efficient tissue processing that has yielded satisfactory' results for simultaneous capture mRNA and chromatin tags in parallel from the same tissue.

[0216] FIG. 5 shows spatial CUT&Tag signals for specific histone marks for the Srgapl gene in the adult mouse brain. Annotation color code represents distinct morphological regions in the mouse brain while the Srgapl color scale represents the calculate gene activity scores.

[0217] FIG. 6 shows representative CUT&Tag signal traces (H3K27ac) for a signature mouse cortex gene: Satb2 shown in the top panel. The spatial CUT&Tag signals (two red traces at the bottom) demonstrate strong concordance with reference ENCODE data (grey traces). Spatial CUT&Tag signals for specific histone marks for the Satb2 gene in the adult mouse brain. Annotation color code represents distinct morphological regions in the mouse brain while the Satb2 color scale represents the calculated gene activity scores. Allen Brain atlas represents the IHC image of the Satb2 staining in the adult mouse brain in the reference mouse atlas

[0218] FIG. 7 shows bulk-level validation of the spatial multi-modal CUT&Tag. FIG. 7A shows representative CUT&Tag signal traces (H3K4me3) across two signature genes. The spatial CUT&Tag signals (two red traces at the bottom) demonstrate strong concordance with reference ENCODE data (grey traces). FIG. 7B shows genome-wide Attorney Docket: NYG-LIPP-249PCT comparison of CUT&Tag signals (H3K4me3) across the top 40,000 genes from ENCODE data. The spatial CUT&Tag data shows consistent signal patterns when compared to ENCODE data at a global scale, further validating the robustness of the approach.

[0219] To apply the spatial multi-modal CUT&Tag method outside of the mouse brain, we applied it to mouse embryonic tissue. This is to demonstrate capability, robustness and quick adaptability' of the method to the maj ority of organs present (and not only the mouse brain). We used the low resolution Visium 10X Genomics platform to use as a spatial platform for profiling a mouse embry o at El 8 (embryonic day). After initial data processing, we performed dimensionality reduction on the data, enabling the identification of distinct cell clusters. When these cell clusters yvere projected onto the mouse embryo, they exhibited clear and distinct spatial distributions, confirming that spatial epigenomic profiling with this method can effectively differentiate various tissue types in the mouse embryo (FIG. 8).

[0220] FIG. 8 shoyvs spatial-level validation of the spatial CUT&Tag. FIG. 8A shoyvs UMAP showing the unsupervised clustering of Spatial CUT&Tag data. FIG. 8B shoyvs HE image (left) of the mouse embryo profiled and the spatial projection (right) of cell clusters identified in spatial multi-modal CUT&Tag. Colored arrows show different organs, and the color code matches FIG. 8A.

[0221] FIG. 9A shows methodology' of surface modification. FIG. 9B shows spatial CUT&Tag performed on the ligated surface, and comparing the resulting data with ENCODE.

[0222] Alternative Starting Materials: A variety of target proteins could be profiled, depending on the specific chromatin state or factor of interest. For instance, antibodies targeting active histone marks such as H3K4me3 (active promoters) or repressive marks like H3K27me3 (silenced regions) could be used. In addition, transcription machinery antibodies, such as those targeting CTCF, G-quadruplex or RNA Polymerase II (Pol II), could be employed to investigate transcriptional regulation in different tissue contexts. And a lot more beyond mentioned above can be adopted in this assay.

[0223] Alternative ST platforms: This platform method can be integrated with solidcapture spatial transcriptomics (ST) to profile genome-yvide epigenomics within tissue environments. Furthermore, any sequencing-based spatial technology', such as Slide-seq, Attorney Docket: NYG-LIPP-249PCT

[0224] Stereo-seq, or DBIT-seq, can be employed in conjunction with this method, expanding its utility for spatially resolved epigenomics analysis.

[0225] Tissue Variations: The platform method is highly adaptable to a broad spectrum of tissues, including, but not limited to. tumor biopsies, brain tissue sections, and samples from vast kinds of solid tissues. This versatility enables its application across multiple domains of research, including oncology, neuroscience, and developmental biology.

[0226] Example 3: Further Studies

[0227] Further studies focus on applying this technology to various disease tissues, aiming to tackle important scientific challenges related to epigenetic regulation in the context of aging and cancer.

[0228] Senescent neurons in brain aging: Characterizing neuronal senescence remains a challenge despite its pivotal role in brain aging. The conventional hallmarks of cellular senescence often prove ineffective when applied to neurons. Due to the postmitotic and highly specialized nature of neurons, senescence manifestations in this cell type may diverge from classical hallmarks found in other cell types. Furthermore, senescent neurons are scarce in space and notoriously difficult to identify, posing significant barriers for their characterization.

[0229] However, changes in chromatin structure landscape manifest prominently during the aging process across various organisms (Feser, J., and Tyler, J., Chromatin structure as a mediator of aging, Epigenetics, 2011, 585(13):2041-2048). Notably, several of these alterations have been associated with the onset of age-related phenotypes (Dang, J., et al., Cancer-associated IDH1 mutations produce 2-hydroxyglutarate, Nature, 2009 Dec 10;462(7274):739-44). Significantly, shifts in chromatin profiles is one of the most striking changes observed in aging brains, leading to shifts in gene expression and cellular functions (Harman M. F., Martin M. G., Epigenetic mechanisms related to cognitive decline during aging. J. Neurosci. Res., 2020, 98, 234-246; Booth, L., and Brunet, A., The Aging Epigenome, Mol Cell, 2016 Jun 2;62(5):728-44). Given this context, it becomes essential to profile aging brain tissue for the chromatin landscape change of senescent neurons. We plan to perform spatial CUT&Tag on human brain samples and mouse brain samples across different ages. We anticipate that spatially profiling of aging brain tissue will offer crucial insights into understanding senescent neurons, thus providing valuable information for studying brain aging. Attorney Docket: NYG-LIPP-249PCT

[0230] Identify driving factors for tumorigenesis in colorectal cancer samples: Tumor heterogeneity presents a long-standing challenge for diagnosis and treatment. Colorectal cancer, in particular, develops with a non-random accumulation of genomic mutations over time, creating distinct spatial and functional tumor structures. While bulk sequencing has identified a collective of mutations, it lacks spatial context, which is crucial for understanding how these mutations drive tumor development and interact with the surrounding microenvironment.

[0231] Our spatially resolved chromatin profiling approach provides insight into the chromatin dynamics of cancer cells in relation to their local environment. By combining the profiling of active and silent histone marks, along with spatial transcriptomics data, we aim to refine cell-type clustering and establish a mutational genomic library' that highlights mutation hotspots specific to cancer cells. The spatial CUT&Tag approach enables deeper insights into gene regulatory mechanisms in cancer by allowing the identification of cell-type-specific chromatin landscapes and lineage relationships within cancerous cells. This comprehensive spatial profiling yields deeper insights into the molecular drivers of tumorigenesis, aiding in the development of more targeted therapeutic interventions.

[0232] Example 4: Methods

[0233] Described below is an exemplary' protocol for solid-capture based spatial CUT&Tag (spatial CUT&Tag).

[0234] On Day 1 :

[0235] A. Load Tn5 freshly each time before experiment:

[0236] 1) Start with 0.5mg / ml PA-Tn5.

[0237] 2) Mix. a) 5 pl Tn5 or 6 pl pA-Tn5 (0.5mg / mL) b) 1 pl Transposon (50 pM Stock)

[0238] 3) Incubate at room temperature for 30 minutes to aboutlhour.

[0239] B. Formaldehyde Tissue Fixation Before experiment: a. Prepare 0.2% formaldehyde (methanal-free, Sigma F8775-25ML (sigmaaldrich.com / US / en / product / sigma / f8775?srsltid=AfmBOooo6o8uICWORg 91SdKM7fabOgUV9-PJV2uFva7Y5h9CzwTnOovj)) in PBS. Attorney Docket: NYG-LIPP-249PCT b. Set thermos-mixer at 37°C and equilibrate for 5 min. A thermo-top is recommended.

[0240] Tissue fixation:

[0241] 1) Place a slide with freshly sectioned tissue on the thermomixer, the active surface facing up, and incubate for 5 minutes at 37°C.

[0242] 2) Remove the slide from Thermocycler adapter and, if necessary, wipe excess liquid from the back of the slide, without touching the tissue sections.

[0243] 3) Apply 1 ml 0.2% FA on the slide, and incubate at room temperature (RT) for 10 minutes.

[0244] 4) Wash with 1 ml IX PBS with 1.25 glycine and remove it.

[0245] 5) Quick Wash with 1 ml IX PBS for about 2 minutes at room temperature (RT).

[0246] C. Permeabilization:

[0247] 1) Prepare lx BD permeabilization buffer.

[0248] 2) Permeabilizing for 30 minutes at 4°C (fridge).

[0249] D. 1st antibody staining:

[0250] 1) After removing the BD Perm buffer, apply 1st antibody in IX BD Perm buffer.

[0251] 2) Incubate at room temperature (RT) for 1 hour.

[0252] E. 2nd antibody staining

[0253] 1) Add 2ndantibody in digi-CT wash buffer (1 :50), 70 pl / well, incubate at room temperature (RT) for 30 min.

[0254] 2) Wash with digi-CT wash buffer for 5 minutes at room temperature (RT).

[0255] F. Tagmentation:

[0256] 1) Loaded PA-Tn5 in 70 pl digi-300-wash buffer was added and incubated for 1 hour at room temperature (RT). Attorney Docket: NYG-LIPP-249PCT

[0257] 2) Wash with digi-300-wash buffer for 5 minutes at room temperature (RT).

[0258] 3) Add tagmentation buffer: lOmM MgCh + digi-300-Wash buffer. i Tagmentation buffer digi-300-wash buffer 1000

[0259] I M Mg(’l2

[0260] 4) incubate at 37°C for 1 hour OR at 55°C for 1 hour OR at 37°C for 30 minutes and at 50°C for 30 minutes.

[0261] 5) Stop tagmentation with 40mM EDTA, incubate at room temperature RT for 5 minutes (70 pl). 6) Wash section with IX T4 buffer and remove it at room temperature (RT).

[0262] G. Gap filling and In vitro transcription (from ChiL / Tipseq):

[0263] A sealing reaction was performed in 70 pl reaction mixture

[0264] T4 gap filling:

[0265] A sealing reaction was performed in 70 pl reaction mixture: 1) Wash with IX T4 ligation buffer for 5 minutes at room temperature

[0266] (RT).

[0267] 2) Prepare gapfill solution (according to the number). Attorney Docket: NYG-LIPP-249PCT

[0268] 3) After removing the buffer, add 70 pl of fdl-in solution. Incubate at RT (24°C on thermomixer) for 30 minutes with gentle shaking.

[0269] Klenow Gap filling: Pre-wash with lxNEB2 for 5 minutes.

[0270] Prepare fill-in solution (according to the number of samples)

[0271] After removing the buffer, add 70 pl of fill-in solution. Incubate at 37°C for 30 minutes with gentle shaking.

[0272] H. Tissue clearing: 1) Remove the gap fill mixture.

[0273] 2) Add 70 pl 0. 1 M HC1 (diluted from stock; ensure accurate dilution) to each well.

[0274] 3) Incubate 2 minutes at room temperature.

[0275] 4) Using a pipette, remove HC1 from the wells. 5) Add 100 pl IX T7 buffer to each well, wash for 5 minutes room temperature (RT).

[0276] I. In vitro transcription:

[0277] 1) Prepare in vitro transcription solution (according to the number of wells).

[0278] Thermo T7 : Attorney Docket: NYG-LIPP-249PCT

[0279] Megashort Solution:

[0280] 2) After removing the IX buffer, add 70 pL of in vitro transcription solution. Seal the wells with film. Incubate at 37° C overnight (aboutl4 hours) with gentle shaking. On Day 2:

[0281] J. Methanol fixation

[0282] Night before: Ensure that the methanol (40 ml / slide) dispensed in a 50-ml centrifuge tube is chilled to -20°C.

[0283] 1) Completely immerse the slide in the pre-chilled methanol. Secure the tube cap to prevent methanol loss. Incubate upright for 30 minutes at -20°C.

[0284] 2) Remove slide from methanol and wipe excess liquid from the back of the slide, without touching the tissue sections. Place on a flat, clean, non-absorbent work surface. Some residual droplets may remain.

[0285] 3) Add 500 pL isopropanol to uniformly cover all tissue sections on the slide. Incubate 1 minute at room temperature (RT). Discard reagent by draining and / or holding the slide at an angle with the bottom edge in contact with a laboratory wipe. Wipe excess liquid.

[0286] 4) Air dry the slide. To prevent tissue section from over drying, inspect slide after 5 minutes. DO NOT exceed 10 minutes.

[0287] K. HE Staining (optional)

[0288] Before experiment:

[0289] 1) Dispense the following volumes of Milli-Q water. Attorney Docket: NYG-LIPP-249PCT

[0290] Dispensed volume in each beaker can be used for two slides.

[0291] 2) Prepare Eosin Mix. DO NOT add pure eosin to tissue sections. a) Add 1 mL Hematoxylin to uniformly cover all tissue sections on the slide. Incubate 7 minutes at room temperature (RT). Discard reagent by draining and / or holding the slide at an angle with the bottom edge in contact with a laboratory wipe. b) Immerse the slide 5x in the water in centrifuge tube.

[0292] Immerse the slide 15x in the water in Beaker 1.

[0293] Immerse the slide 15x in the water in Beaker 2.

[0294] Quickly spin dry the slide in slide spinner. c) Add 1 mL Bluing Buffer to uniformly cover all tissue sections. Incubate 2 minutes at room temperature. Discard reagent. d) Immerse the slide 5x in the water in Beaker 2. Quickly spin dry the slide in slide spinner. e) Add 1 mL Eosin Mix to uniformly cover all tissue sections. Incubate 1 minute at room temperature. Discard reagent. f) Immerse the slide 15x in the water in Beaker 3. Quickly spin dry the slide in slide spinner. g) Incubate slide on the Thermocycler Adaptor with the thermal cycler lid open for 1 min at 37°C.

[0295] Proceed to tissue imaging on metafer scope.

[0296] L. Tissue Wash

[0297] 1) After imaging, wash with IX RT (Reverse Transcriptase) buffer (mmulv (neb . com / en-us / products / m0253 -m-mulv-reverse- transcriptase?srsltid=AfmBOoogTXQwI4Vp7NMXz5YuDkHDVC4FQn4_iXu48odq_JD _BIdPorKx) or maxima (thermofisher.com / order / catalog / product / EP0741) or ssIII (hermofisher.com / order / catalog / product / 18080093)) at 37°C for 30 minutes.

[0298] 4) Remove lx RT buffer Attorney Docket: NYG-LIPP-249PCT

[0299] M. cDNA synthesis

[0300] 1) Prepare and pre-heat (42°C) a RT mixture (70 pL / well):

[0301] MMulv (NEB)

[0302] 2) Add 70 pL of the RT mix to each well, make sure you do not induce any bubbles in the wells.

[0303] 3) Cover wells with a plate sealer.

[0304] 4) Incubate at 42°C overnight or for 6 hours in an Eppendorf thermomixer. Note: Alternative formulation for the cDNA synthesis can be applied such as using Maxina H- or Superscript III at the same w / v as Mmulv.

[0305] On Day 3:

[0306] N. Denaturation:

[0307] 1) Remove the RT mixture.

[0308] 2) Add 70 pL 0.08 M KOH (freshly diluted from stock; ensure accurate dilution) to each well.

[0309] 3) Incubate 5 min at room temperature.

[0310] 4) Using a pipette, remove KOH from the wells.

[0311] 5) Add 100 pL EB (10 mM Tris-Cl, pH 8.5) to each well.

[0312] O. 2nd strand synthesis:

[0313] 1) Primer annealing: make lx primer annealing mix

[0314] Primer annealing mix: Attorney Docket: NYG-LIPP-249PCT

[0315] 2) Add 70ul primer annealing mix to each well; incubate @RT for 30min

[0316] 3) Wash with EB by pipetting 100 ul in each well and immediately pipette it off. 4) KI enow 2nd strand synthesis: a) Add 70 pl KI enow mix to each well, make sure you do not induce any bubbles in the wells. Cover wells with a plate sealer and incubate for 1 hour at 37°C. b) Remove the Klenow mix. Alternative to "O":

[0317] 0.1 Q5 second strand synthesis:

[0318] 1) Prepare second strand mix on ice: Attorney Docket: NYG-LIPP-249PCT

[0319] 2) Using a pipette, remove Buffer EB from the wells.

[0320] 3) Add 70 pL second strand mix to each well.

[0321] 4) Apply slide seal on clamp and place on the thermocycler:

[0322] Primer extension 65°C 60 minutes, Ramp down to 37°C.

[0323] P. Denaturation:

[0324] 1) Remove reagents from the wells.

[0325] 2) Add 100 pL Buffer EB to each well.

[0326] 3) Using a pipette, remove Buffer EB from the wells.

[0327] 4) Add 35 pL 0.08 M KOH (freshly diluted from stock) to each well.

[0328] 5) Incubate 10 min at room temperature with 300 rpm shaking.

[0329] 6) Add 5 pE Tris (1 M, pH 7.0) to an 8-tube strip.

[0330] 7) Transfer 35 pL sample from each well to a corresponding tube containing Tris in the 8-tube strip. DO NOT discard sample. Aboutl-2 pL volume variation is expected.

[0331] 8) Vortex, centrifuge briefly, and place on ice.

[0332] Q. PCR Amplify the library

[0333] 1) Amplify the library with (PCR1 for HD slides, mouse brain (-70% of capture area), run around 18 cycles we can see the CT product bands):

[0334] Program: Attorney Docket: NYG-LIPP-249PCT

[0335] Example gel:

[0336] 2) 0.65x SPRI to purify, elute with 40 pL, take 20 pL for the following PCR

[0337] 3) Index PCR with SIPCR and N7 indexing primers (DME)

[0338] 4) PCR for 5 cycles.

[0339] Code link slides capture sequence:

[0340] TSO in visium kit: 80 nmol in 80 pl TE, resulting in 1 pM of oligos Second strand primer 509 ng / ul approximately 58uM. cDNA primer: mix of tw o, 15 pM.

[0341] Example 5. SPACE-Tag Enables Spatial Chromatin Profiling With CUT&Tag

[0342] A. Abstract Spatial epigenomics maps chromatin states within tissue context, yet current methods remain limited by accessibility and scale. We developed SPACE-Tag, a spatial CUT&Tag framework that converts epigenetic marks into poly-adenylated RNA via T7- based amplification, seamlessly integrating our methods with standard spatial Attorney Docket: NYG-LIPP-249PCT transcriptomic workflows. Applied to the mouse brain, SPACE-Tag resolved activating (H3K27ac, H3K4me3) and repressive (H3K27me3) chromatin landscapes recapitulating gene-regulatory and anatomical features, and showed strong correlation with existing bulk and single-cell reference data. Spatial module analysis partitioned the brain into distinct regulatory domains, each defined by co-active super-enhancers and enriched transcription-factor motifs governing neuronal, astrocytic, or oligodendrocytic identity. Within the cortex, finer-scale cis-regulatory element topics resolved functional subregions such as visual, auditor}’, and somatosensory areas, illuminating the epigenomic underpinnings of regional specialization. SPACE-Tag thus links chromatin state to tissue topology, providing an accessible, scalable framework to chart the spatial grammar of gene regulation.

[0343] B. Introduction

[0344] Cellular activity in tissues arises from intrinsic gene-regulatory programs modulated by extrinsic microenvironmental signals, resulting in highly ordered spatial organization (Bissell, M.J. & Hines, W.C. Nat. Med. 17, 320-329 (2011)). Spatial-omics methods increasingly enable the capture and analysis of these complex patterns directly within tissues across multiple molecular layers, including the genome, trans criptome, and proteome (To, K. et al. Nature 635, 657-667 (2024); Steyn, C. et al. Nat. Genet. 56, 2718-2730 (2024); Ounadjela, J.R. et al. Nat. Med. 30, 3495-3508 (2024); Oliver, A. J. et al. Nature 635, 699-707 (2024); Rozenblatt-Rosen, O. et al. Nat. Biotechnol. 39, 149-153 (2021); Kang, S.H. et al. Nat. Neurosci. 16, 571-579 (2013).

[0345] RNA, being more abundant and more readily accessible than chromatin, has been more extensively profiled with high-throughput single-cell and spatial approaches. Many of these efforts have empowered widespread exploration of transcriptomic snapshots of cell states involved in tissue organization, intercellular communication, development, and disease (To, K. et al. Nature 635, 657-667 (2024); Steyn, C. et al. Nat. Genet. 56, 2718— 2730 (2024); Ounadjela, J.R. et al. Nat. Med. 30, 3495-3508 (2024); Oliver, A. J. et al. Nature 635, 699-707 (2024); Rozenblatt-Rosen, O. et al. Nat. Biotechnol. 39, 149-153 (2021); Kang, S.H. et al. Nat. Neurosci. 16, 571-579 (2013)). Although more challenging to profile, the epigenome varies substantially across cell types, shaping transcriptional identity' and cellular function (Stadhouders, R., Filion, G J. & Graf, T. Nature 569, 345- 354 (2019)). As such, it offers insights into cellular programming and memory that Attorney Docket: NYG-LIPP-249PCT extend beyond the transient snapshots captured by RNA profiling. Among the key epigenetic regulators of gene expression are chromatin features, particularly histone modifications, which mark active, repressed, or poised regulatory elements. To address this challenge, a suite of single-cell epigenomic techniques has been developed to profile higher-order chromatin architecture (Nagano, T. et al. Nature 502, 59-64 (2013)), chromatin accessibility (Jin, W. et al. Nature 528, 142-146 (2015); Cusanovich, D.A. et al. Science 348, 910-914 (2015); Buenrostro, J.D. et al. Nature 523, 486-490 (2015)), histone modifications, and transcription factor binding (Rotem, A. et al. Nat. Biotechnol. 33. 1165-1 172 (2015); Kaya-Okur, H.S. et al. Nat. Commun. 10. 1930 (2019); Carter, B. et al. Nat. Commun. 10, 3747 (2019); Wang, Q. et al. bioRxiv (2019).doi:10.1101 / 590661; Ai, S. et al. Nat. Cell Biol. 21, 1164-1172 (2019); Skene, P.J. & Henikoff, S. Elife 6, (2017)). CUT&Tag (Cleavage Under Targets and Tagmentation) (Kaya-Okur, H.S. et al. Nat. Commun. 10. 1930 (2019)) is one of the most widely used methods for profiling histone modifications. Its ease of use is based on histone mark-specific antibodies fused to a protein A-Tn5 transposase recognizing histone-specific modification and then inserting sequencing adapters into DNA near epigenetically modulated target sites. These adapters serve as signposts in the subsequent sequencing step, which maps the genomic neighborhood of the targeted histone modifications. Since its introduction, CUT&Tag has been applied across a range of biological contexts due to its scalability, base-pair resolution, and high sensitivity7.

[0346] While single-cell multi-omics and spatial transcriptomics methods are now well established, techniques for spatially resolved chromatin profiling remain limited. Existing methods for in situ mapping of histone modifications or chromatin accessibility often depend on custom microfluidics (Deng, Y. et al. Science 375, 681-686 (2022); Zhang, D. et al. Nature 1-10 (2023); Guo, P. et al. Nat. Methods 1-10 (2025); Thornton, C.A. et al. Nat. Commun. 12, 1274 (2021).), which constrain scalability and broader adoption. Other approaches, such as spatial ATAC-seq (Llorens-Bobadilla, E. et al. Nat. Biotechnol. 1-4 (2023); Huang, Y.-H. et al. Proc. Natl. Acad. Sci. U. S. A. 122, e2424070122 (2025)), adapted for use with commercial platforms, profile chromatin accessibility but do not directly resolve histone modifications. Finally, both approaches are limited by sensitivity, specificity, and scalability, which restricts their ability to generate comprehensive maps across large tissues or cohorts. Attorney Docket: NYG-LIPP-249PCT

[0347] To address these limitations, and building on our previous work in spatial transcriptomics (Stahl, P.L. et al. Science 353, 78-82 (2016); Vickovic, S. et al. Nat. Methods 16, 987-990 (2019)), we developed spatially resolved CUT&Tag sequencing (SPACE-Tag) — a method that combines the specificity of CUT&Tag-based histone profiling with the scalability and accessibility of the Visium (lOx Genomics) platform (FIG. 10A). To realize SPACE-Tag, we engineered a protein A-Tn5 construct bearing a polyT tail (for compatibility with widely available spatial array technologies) and a T7 promoter (for in situ genomic DNA signal amplification). And to further enhance SPACE-Tag efficiency, we optimized SPACE-Tag reaction conditions (for efficient and localized spatial fragment capture). Briefly, frozen tissue sections were placed onto barcoded slides, permeabilized, and sequentially stained with primary antibodies, secondary' antibodies, and ProteinA-Tn5. Finally, gap filling, histone removal, and T7- mediated transcription convert tagged DNA fragments into polyadenylated RNA molecules, which are captured and processed using workflows analogous to those established for spatial transcriptomics (Stahl, P.L. et al. Science 353, 78-82 (2016)). To demonstrate and validate our technology , we applied SPACE-Tag to profile the adult mouse brain.

[0348] C. Results

[0349] Motivated by' the issue of low sensitivity7, we first standardized tissue processing steps, including tissue fixation, nuclei permeabilization, and antibody staining (FIG. 12). We also optimized reaction conditions specific to the SPACE-Tag workflow, including Tn5 tagmentation and gap filling, resulting in more than a two-fold increase in library yield (FIGs. 13A and 13B). Most importantly, we introduced a T7-based in situ linear amplification step, which locally increased the copy number of tagmented fragments. This resulted in a 70 fold increase in signal intensity (Methods, FIG. 14), while avoiding off-target amplification and background noise associated with exponential amplification (Chen, C. et al. Science (New York, N.Y.) 356, 189 (2017)).

[0350] FIG. 12 shows optimized antibody' staining for adult mouse brain sections. Fluorescence images for nuclear (DAPI; blue) and antibody stains (red) for H3K27ac (top), H3K4me3 (middle) and H3K27me3 (bottom). Channels overlay shown on the right. Scale bar: 400pm.

[0351] FIGs. 13A and 13B show comparison of gap-filling and tagmentation conditions on SPACE-Tag library7complexity7. Computationally estimated library' sizes (Methods) Attorney Docket: NYG-LIPP-249PCT defined as the estimated number of unique fragments in the full cDNA library (y axis) across three tested experimental conditions (T4 ligation, Klenow extension and tagmentation temperature) and shown for two histone marks (H3K4me3 (FIG. 13 A) and H3K27ac (FIG. 13B)). Gap-filling using Klenow-based extensions significantly increases library complexity compared to using T4-based DNA ligation. Additionally, performing the tagmentation reaction at 55°C (right) further enhanced complexity relative to the conventional 37°C reaction temperature (left, mid). Significance testing: one-sided t-test; * denotes p<0.05.

[0352] FIG. 14 shows the effect of T7 amplification on signal capture in SPACE-Tag. FIG. 14A shows schematic of the workflow for signal quantification. (I) Brain sections are deposited on a pseudospatial array with a poly(d)T capture area and stained for H&E histology (II) The sections are then stained for primary' and secondary' antibodies (III) In situ tissue reactions are performed: pA-Tn5 tethering, tagmentation, gap-filling and HC1 permeabilization. The SPACE-Tag procedure next utilizes T7-based amplification to generate amplified RNA molecules that are subsequently captured on the pseudospatial slide. cDNA molecules are then generated with a simultaneous staining using fluorescently labeled nucleotides. (IV) Signals are captured by fluorescent imaging. FIG. 14B shows H&E images (top) and cDNA signal intensity (white) of sections with or without the T7 amplification step. Scale bars are 400pm. FIG. 14C shows quantification of the fluorescence signals from FIG. 14B. Significance testing: one-sided t-test; * denotes p<0.05.

[0353] Sensitivity and specificity of the workflow were evaluated by profiling two activating (H3K27ac and H3K4me3) and one repressive (H3K27me3) histone modification in mouse brain tissues. We assessed the performance of SPACE-Tag relative to other spatial chromatin profiling methods (Deng, Y. et al. Science 375, 681-686 (2022); Guo, P. et al. Nat. Methods 1-10 (2025); Llorens-Bobadilla, E. et al. Nat. Biotechnol. 1-4 (2023); Huang, Y.-H. et al. Proc. Natl. Acad. Sci. U. S. A. 122, e2424070122 (2025)) using two metrics: the per-spot number of unique fragments within annotated tissue boundaries as a proxy for sensitivity', and the fraction of reads in peaks (FRiP) as a proxy' for specificity’. Comparable to other solid-capture methods (Llorens- Bobadilla, E. et al. Nat. Biotechnol. 1-4 (2023); Huang, Y.-H. et al. Proc. Natl. Acad. Sci. U. S. A. 122, e2424070122 (2025)), SPACE-Tag captured approximately -15,000 fragments per spot for H3K27ac, -6,000 for H3K4me3, and -6000 for H3K27me3. The Attorney Docket: NYG-LIPP-249PCT

[0354] FRiP for these modifications was 0.78. 0.71. and 0.66, respectively, exceeding the specificity reported by most methods (FIG. 10B). In addition, the detected fragments exhibited strong reproducibility' across biological replicates (Spearman’s R > 0.94, FIG. 15), and demonstrated robust enrichment over ENCODE ChlP-seq peaks (H3K27ac Spearman’s R > 0.79; H3K4me3 Spearman’s R > 0.87) (FIG. 16). A high degree of concordance was also observed between SPACE-Tag and bulk CUT&Tag histone profiles (H3K27ac Spearman’s R > 0.85; H3K4me3 Spearman’s R > 0.93; H3K27me3 Spearman’s R > 0.79) (FIG. 17). further highlighting the robustness of our method.

[0355] FIG. 15 shows SPACE-Tag reproducibility. FIGs. 15A to 15C show reproducibility of SPACE-Tag experiments across biological replicates for H3K27ac (FIG. 15A), H3K4me3 (FIG. 15B) and H3K27me3 (FIG. 15C). The genome was split into lOkb tiles, and a scatterplot of normalized signal between replicate experiments was plotted for all tiles. The Spearman correlation coefficient was calculated for each modification; significance was determined by two-sided t-test (p values shown).

[0356] FIG. 16 shows SPACE-Tag signal in cortical regions correlates with ENCODE ChlP-seq mouse cortex data. FIGs. 16A and 16B show7cumulative normalized epigenomic signal for ENCODE ChlP-seq and SPACE-Tag across all ENCODE-defined peaks at + / - 2kb from peak centers (top), and normalized signal intensities (color scale) over ENCODE-defined peaks are shown for both ENCODE ChlP-seq data and corresponding SPACE-Tag data (columns), demonstrating concordant enrichment patterns for (a) H3K4me3 and (b) H3K27ac histone modifications (bottom). Each row represents a single peak in ENCODE ChlP-seq; columns represent normalized signal intensities at + / - 2kb from each peak center. FIGs. 16C and 16D show heatmap of correlations between ENCODE and SPACE-Tag. The genome was split into lOkb tiles, and a Spearman correlation coefficient w as calculated on the normalized signals across all tiles (Methods). The Spearman correlation coefficient was calculated for each modification; significance was determined by two-sided t-test (p values shown).

[0357] FIG. 17 shows SPACE-Tag signal in cortical regions correlates with bulk CUT&Tag. FIGs. 17A to 17C show7cumulative normalized epigenomic signal for bulk CUT&Tag and SPACE-Tag across all SPACE-Tag-defined peaks at + / - 2kb from peak centers (top), and normalized epigenomic signal (color scale) over SPACE-Tag-defined peaks are shown for both bulk CUT&Tag data and corresponding SPACE-Tag data (columns), demonstrating concordant enrichment patterns for H3K27ac (FIG. 17A), Attorney Docket: NYG-LIPP-249PCT

[0358] H3K4me3 (FIG. 17B) and H3K27me3 (FIG. 17C) histone modifications (bottom). Each row represents a single peak in SPACE-Tag; columns represent normalized signal intensities at + / - 2kb from each peak center. FIGs. 17D to 17F show heatmap of correlations between bulk CUT&Tag and SPACE-Tag for H3K27ac (FIG. 17D), H3K4me3 (FIG. 17E) and H3K27me3 (FIG. 17F) histone modifications. The genome was split into lOkb tiles, and Spearman’s correlation coefficient was calculated on the normalized signals across all tiles (Methods); significance was determined by two-sided t- test (p values shown).

[0359] Having established SPACE-Tag’s technical performance, we evaluated whether SPACE-Tag can capture the distinct epigenetic landscapes that shape cellular identity and function across different anatomical structures. To this end, we identified spatial domains defined by distinct histone modification profiles using an unsupervised, spatially aware topic model (Methods) (Zhong, C., Ang, K.S. & Chen, J. Nat. Methods 21. 2072-2083 (2024)). Based on the modification profile of H3K27ac, the model successfully identified distinct regions corresponding to isocortex, piriform cortex, thalamus, hypothalamus, dorsal thalamus, fiber tracts, and the hippocampus (FIG. 10C). Comparable clustering was also observed for H3K4me3 and H3K27me3 (FIG. 10C).

[0360] Because histone modifications are closely linked to gene expression, the captured modification landscapes can be used to infer gene activity. Indeed, the activation and repression scores inferred from our epigenome dataset (Methods) recapitulated established spatial gene expression (Zhang, M. et al. Nature 624, 343-354 (2023)) (FIG. 10D, FIG. 18). For example, the neuronal transcription factor Saib 2. which drives formation of cortical neuronal projections (Alcamo, E. A. et al. Neuron 57, 364-377 (2008)), showed increased H3K27ac and H3K4me3 activation scores in the isocortex, while its corresponding H3K27me3 repression scores were, as expected, reduced in this region (FIG. 10D). Consistent patterns were likewise observed for Tcf7l2 and Zeb2, established markers of the thalamus (Lipiec, M.A. et al. Development 147, devl90181 (2020)) and a joint region representing the isocortex, fiber tracts and the hippocampus respectively (FIG. 10D). These expression patterns were consistent with MERFISH data (Zhang, M. et al. Nature 624, 343-354 (2023)) from the Allen Brain Atlas (FIG. 10E).

[0361] FIG. 18 shows genome coverage tracks of region-specific key neuronal marker genes profiled by SPACE-Tag. Genome coverage tracks showing normalized epigenomic signal (y-axis; left: H3K27ac; middle: H3K4me3; right: H3K27me3) across the gene Attorney Docket: NYG-LIPP-249PCT body and flanking regions (x-axis) of marker genes (top: Satb2, middle: Tcf712; bottom: Zeb2). The corresponding gene body is depicted beneath each track, along with genomic coordinates.

[0362] Finally, given the close association between histone modifications and cell identity, we evaluated whether SPACE-Tag can resolve spatial patterns of distinct cell types. To this end, we relied on a previously published single cell CUT&Tag (scCUT&Tag) dataset (Barcenas -Walls, J.R. et al. Nat. Protoc. 19, 791-830 (2024)). Integrating scCUT&Tag-defined cell-types with the SPACE-Tag data, we observed that distinct cell types were consistently predicted in the expected anatomical regions (FIG. 10D). Inhibitory neurons were predominantly detected in the isocortex, oligodendrocytes in the fiber tract white matter, and astrocyte subtypes segregated between the hippocampus and thalamus. This correspondence between inferred cell types, gene activity and their known anatomical distributions (Zhang. M. et al. Nature 624. 343-354 (2023)) validated the spatial resolution of SPACE-Tag, and highlighted its power in resolving complex tissue heterogeneity based solely on epigenomic features.

[0363] FIG. 10 shows SPACE-Tag method over iew and performance. FIG. 10A shows schematic overview of the SPACE-Tag workflow. The process begins by placing a fresh frozen tissue section onto a solid-phase capture spatial transcriptomics slide. Following steps are then performed: I) Engineered transposome adaptors (containing a T7 promoter and a poly(d)T sequencing) are loaded onto pA-Tn5. II) Primary antibody, secondary antibody, and pA-Tn5 are sequentially applied. Ill) Tagmentation and gap filling are followed by in vitro T7 transcription (IVT) to generate amplified RNA fragments. IV) The IVT-generated RNA is captured by poly-T probes, reverse transcribed, and processed into sequencing libraries. FIG. 10B shows Per-spot unique fragment counts (left; y-axis) and FRiP scores (right: y-axis) obtained by SPACE-Tag and other published spatial chromatin profiling methods (x-axis). FIG. 10C shows expert-annotated brain regions (left) compared with spatial maps derived from unsupervised topic clustering of histone modification profiles (right). Each row shows a different histone mark: H3K27ac (top), H3K4me3 (middle), and H3K27me3 (bottom). FIG. 10D shows spatial projections of inferred gene activity (color scale) (for H3K27ac and H3K4me3) or repression (for H3K27me3). Columns show representative genes (left to right: Satb2, Tcf712, Zeb2). while rows show different histone modifications: H3K27ac (top), H3K4me3 (middle), and H3K27me3 (bottom). FIG. 10E shows MERFISH gene expression (color scale) for Attorney Docket: NYG-LIPP-249PCT representative genes (left to right: Satb2, Tcf712, Zeb2). For each gene, the image pair shows MERFISH expression data from the Allen Brain Atlas (left) and the illustrated Allen Brain Atlas spatial regions where the gene is expected to be expressed (right). Spatial region color code is shared with expert annotations in (10C). FIG. 10F shows spatial cell type projections (color scale) in MERFISH (left) and SPACE-Tag (right) data. Four cell types (columns) are highlighted (from left to right: inhibitory neurons, oligodendrocytes, non-telencephalon astrocytes, and telencephalon astrocytes).

[0364] After establishing SPACE-Tag’ s spatial fidelity, we turned to assess whether the method could resolve epigenomic features associated with gene regulation. In particular, we focused on super-enhancers (SE), key regulatory elements which are marked by broad and high-intensity H3K27ac signals and Medl binding (Whyte, W.A. et al. Cell 153, 307-319 (2013)). To this end, we compared the H3K27ac signals detected with SPACE- Tag to SE annotations curated in SEdb, a comprehensive database of mouse and human Ses (Wang, Y. et al. Nucleic Acids Res. 51, D280-D290 (2023)). We observed a strong concordance between high-intensity H3K27ac peaks and SEdb-defined SEs, demonstrating SPACE-Tag’s ability to capture these key regulatory regions (FIG. 11 A). Notably, this strong concordance was not observed for H3K4me3 (FIG. 1 IB), in accordance with recent studies showing that H3K27ac-marked SEs and broad H3K4me3 domains occupy distinct genomic intervals (Benayoun, B.A. et al. Cell 158, 673-688 (2014)).

[0365] To investigate the spatial arrangement of active SEs, we performed a spatial module analysis (Methods), by identifying groups of SEs that co-varied across functional tissue domains. This analysis revealed three distinct spatial SE modules: the isocortex (Module 1), the hypothalamus (Module 2), and the white matter tracts (Module 3) (FIG. 11C, (SEs associated with distinct brain regions)). Cataloguing the SE-proximal genes within each module by Gene Ontology (GO) enrichment analysis revealed that Modules 1 and 2 were enriched for processes including axonogenesis, neurotransmitter secretion, and forebrain development, whereas Module 3 was enriched for glial processes, particularly gliogenesis (FIG. 1 ID). To validate the cell-type specificity of these biological programs, we leveraged the previously published scCUT&Tag H3K27ac dataset from the postnatal mouse brain (Bartosovic, M. & Castelo-Branco, G. Nature Biotechnology 41, 794-805 (2022)) and found that each SE module displayed distinct activity' patterns across cell types: Module 1 was enriched in both excitatory and Attorney Docket: NYG-LIPP-249PCT inhibitory neurons, while Modules 2 and 3 showed enrichment specific to astrocytes and oligodendrocytes, respectively (FIG. 19). Given that SEs are preferentially bound by master transcription factors (TFs) that establish and maintain cell identity (Whyte, W.A. et al. Cell 153, 307-319 (2013)). we asked which TFs are specifically associated with these modules. The analysis revealed that: (i) Module 1 was enriched for Gata4, a transcription factor known to regulate axon regeneration; (ii) Module 2 was enriched for K119 and Smadl, both of which regulate axon regeneration and neuronal activity37; and (iii) Module 3 was enriched for Mbdl and Srebf2 (Apara, A. et al. J. Neurosci. 37, 9632- 9644 (2017)), which are transcription factors implicated in the maintenance of neural stem cell identity7(Jobe, E.M. et al. J. Neurosci. 37, 523-536 (2017)), and the regulation of cholesterol metabolism in astrocytes, respectively (FIG. 1 IE). Notably, Srebf2 is a master regulator of astrocytic cholesterol metabolism essential for myelination (Ho, W.Y. et al. J Cell Biol 220. (2021)). Overall, the spatial SE modules identified by SPACE-Tag closely recapitulated known brain-specific regulatory programs, and provided independent validation of the method’s ability7to resolve spatially organized enhancer landscapes across the mouse brain.

[0366] FIG. 19 shows super-enhancer modules are active in specific cell types. FIG. 19A shows UMAP of cell types identified in a previously published H3K27ac single-cell CUT&Tag dataset of the postnatal mouse brain. FIG. 19B show s H3K27ac signal (color scale) of SE modules (columns) across cell types (color scale); Module 1 (left) is primarily enriched in excitatory and inhibitory neurons, Module 2 (middle) in astrocytes, and Module 3 (right) in oligodendrocytes.

[0367] Finally, to determine whether SE-associated chromatin landscapes can resolve finer-scale functional organization beyond broad anatomical regions, we performed a focused analysis of the mouse cortex. The mouse cortex, composed of the isocortex and piriform cortex, consists of multiple spatially organized functional subregions, including motor, somatosensory, auditory, and olfactory-associated domains (FIG. 1 IF), which share broadly similar cellular compositions but exhibit distinct gene expression profiles and connectivity7patterns (Lein, E.S. et al. Nature 445, 168-176 (2007); Tasic, B. et al. Nat. Neurosci. 19, 335-346 (2016); Yao, Z. et al. Cell 184, 3222-3241. e26 (2021); Zhang, Z. et al. Nature 598, 167-173 (2021)). These differences suggest that regionspecific regulatory programs underlie their distinct identities, although the epigenomic mechanisms driving this specialization remain unexplored. Using trajectory-based Attorney Docket: NYG-LIPP-249PCT analysis (DeTomaso, D. & Yosef, N. Cell Syst. 12, 446-456.e9 (2021 )) (Methods, FIG. 20), we found that SEs alone were insufficient to delineate the functional subregions of the cortex (FIG. 21). Extending the analysis to all civ-regulatory elements (CREs) defined by H3K27ac SPACE-Tag profiling, we identified multiple CRE topics, whose spatial boundaries corresponded to known functional subregions of the cortex, such as the retrosplenial area (topic 1), visual cortex (topic 2), auditory / somatosensory cortex (topic 3), perirhinal area (topic 4), and piriform cortex (topic 5) (FIG. 11G). This separation into distinct topics was reproducible across replicate tissue sections. (FIG. 22. (CREs associated with cortical substructures)). To validate these candidate CRE topics, we examined their chromatin accessibility in a cortical-region-resolved single-cell ATAC-seq atlas of the postnatal mouse brain (Zu, S. et al. Nature 624, 378-389 (2023); Li, Y.E. et al. Nature 598, 129-136 (2021)). Notably, peaks within each topic exhibited preferential accessibility in the anatomically matched cortical regions, supporting their functional significance (FIG. 11H). Further, cell type-specific analysis revealed that region-specific topics were more accessible in glutamatergic neurons than in GABAergic neurons and non-neuronal cell ty pes (FIG. 23), consistent with the well-defined spatial distribution of glutamatergic neurons subtypes across cortical subregions (Lein, E.S. et al. Nature 445, 168-176 (2007); Tasic, B. et al. Nat. Neurosci. 19, 335-346 (2016); Yao, Z. et al. Cell 184, 3222-3241. e26 (2021); Zhang, Z. et al. Nature 598, 167-173 (2021).).

[0368] FIG. 11 shows SPACE-Tag analysis of regulatory7elements and their spatial organization. FIGs. 11A and 11B show normalized signal intensity of peaks identified with SPACE-Tag (top) and fraction of peaks overlapping with SEdb SEs (bottom) for (FIG. 1 1 A) H3K27ac and (FIG. 1 IB) H3K4me3. Peaks are ordered by normalized epigenomic signals. Marker color indicates peak overlaps with known super-enhancers (SEs) from the SEdb database (blue: overlapping; red: not overlapping). FIG. 11C shows spatial distribution (color scale) of identified SE modules across brain sections. Module 1 occupies the cortex regions. Module 2 occupies the hypothalamus region. Module 3 occupies white matter tracts. FIG. 1 ID shows GO enrichment scores (color scale) of genes regulated by different SE modules. GO terms (rows) overrepresented in the gene sets for each module (columns) are shown. FIG. 1 IE shows enrichment scores (color scale) of differentially enriched TF motifs (rows) within each SE module (columns). FIG. 1 IF shows expert annotation of a mouse brain section and the cortical subregion. Cortical subplate regions: color code. Shaded: non-cortical area. FIG. 11G shows spatial Attorney Docket: NYG-LIPP-249PCT maps of topic scores (color scale) for CRE-derived topics overlaid on cortical functional regions (left), and violin plot of topic scores for each cortical subregion (right). Cortical region color code shared with (FIG. 11G). FIG. 11H shows topics identified by SPACE- Tag exhibit higher chromatin accessibility scores (color scale) in corresponding cortex regions as previously in Zu et al.

[0369] FIG. 20 shows definition of a dorsal-ventral trajectory along the cortical plate axis. Spatial plot of cortical plate trajectory ordering (color scale) for spots belonging to the cortex. Non-cortical spots are grayed out. Black line: principal curve definition of the cortical plate axis (Methods).

[0370] FIG. 21 shows super-enhancer topics do not distinguish fine cortical functional sub-regions. Spatial plots (color scale) show topics (columns) identified along the cortical plate axis.

[0371] FIG. 22 shows reproducibility of cortex sub-region-specific topics identified by SPACE-Tag profiling of mouse brain. Spatial plots show topic scores (color scale) corresponding to the retrosplenial area (Topic 1), visual cortex (Topic 2), auditory / somatosensory cortex (Topic 3), perirhinal area (Topic 4), and piriform cortex (Topic 5) across replicate sections (columns).

[0372] FIG. 23 shows epigenetic differences between functional regions of the cortical plate are primarily driven by glutamatergic neuronal subtypes. Violin plots show accessibility scores (y-axis) from a published single-cell ATAC-seq dataset across different cell types (x-axis. blue: Glutamatergic Neurons; red: GABAergic neurons; Grey: non-neuronal cells). Five plots correspond to topics 1 to 5, as in FIG. 11. Significance testing: two-sided t-test.

[0373] D. Discussion

[0374] In conclusion, SPACE-Tag provides a scalable and accessible platform for mapping of histone modifications in intact tissues. By integrating CUT&Tag chemistry’ with a commercial spatial transcriptomics platform, SPACE-Tag bridges the gap between epigenomic profiling and spatial context. Using the postnatal mouse brain, we demonstrated that SPACE-Tag can resolve fine-scale tissue architecture, infer gene regulatory activity, and uncover spatially organized enhancer landscapes and transcriptional programs. These findings both validate the method’s technical performance and establish its power to illuminate the spatial logic of gene regulation. As spatial genomics moves toward integrative, multimodal maps of tissue function, SPACE- Attorney Docket: NYG-LIPP-249PCT

[0375] Tag offers a versatile framework for dissecting the regulatory architecture underlying development, disease, and cellular identity' in situ.

[0376] E. Materials and Methods

[0377] Murine tissue collection

[0378] C57BL / 6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained following ethical guidelines monitored by the Institutional Animal Care and Use Committees (IACUC) established by the Division of Comparative Medicine at Columbia University, under protocol AACE0752. All mice were maintained under SPF conditions on a 12-h light-dark cycle at an ambient temperature of 21 .5 ± 1 °C with relative humidity between 30% and 70% and were provided food and water ad libitum. Brains were collected subsequently after cervical dislocation and washed with ice-cold IX PBS. Tissues were dried and embedded in Optimal Cutting Temperature (23-730-571, Fisher Scientific) in 22x22x20 mm disposable molds (15160-215, VWR). Samples were then plunged onto a pre-chilled metal plate and left on top of dry ice for 2 min or until completely frozen. Samples w ere transferred and stored at -80°C until use. Immunostaining and epifluorescent microscopy

[0379] To optimize histone antibody concentrations, we performed the following experiments. Frozen 10 pm tissue sections were mounted on microscopic slides and incubated at 37°C for 5 minutes on an Eppendorf ThermoMixer C with athermal block. After fixation in 0.2% formaldehyde (F8775, Sigma Aldrich) at room temperature (r.t.) for 10 minutes, tissue sections were quenched with 1.25M Glycine in IX PBS, followed by a IX PBS wash. Tissue sections were permeabilized in IX Perm / Wash buffer (554723, BD) for 30 min at 4°C. Antibodies were added at desired dilutions in IX Perm / Wash buffer and incubated for 1 hr r.t.. Subsequently, tissues were washed with IX PBS and incubated for 30 min with fluorescent-labeled secondary antibody (711-165-152, Jackson ImmunoResearch) in CT wash buffer (20 mM HEPES. 150 mM NaCl. 0.5 mM spermidine, proteinase inhibitors, and 0.01% digitonin). Stained tissues were then washed for 5 min in the 100 pl CT wash buffer and then proceeded to microscopy. Samples were imaged using a Zeiss Axioscope inverted microscope with a 20X objective (MetaSystems). Images were stitched using Vslide (v 1.1. 132) and extracted with VS Viewer in a JPEG format. The following antibodies were optimized and used in spatial CUT&Tag experiments: anti-H3K4me3 (ab213224, Abeam) anti-H3K27me3 (9733S, Cell Signaling) and anti-H3K27ac (ab4729, Abeam). Attorney Docket: NYG-LIPP-249PCT

[0380] Pseudo-spatial slide production

[0381] Amino-binding CodeLink high-density (HD) glass slides (DHD1-0023, Surmodics) were printed with an equimolar mix of four amine-modified oligonucleotides containing a poly(d)T segment (Amine-visium2-l / 2 / 3 / 4, (Oligonucleotide primer sequences)), following an established protocol (Vickovic, S. et al. Nat Commun 7, 13182 (2016).). Briefly, slides were placed in 16-well hybridization cassettes (AHC4X16, Arraylt), and a printing solution (150 mM Sodium Phosphate, 0.06% sarkosyl, pH 8.5) containing 20 pM of amine-modified oligonucleotides was added to each well and incubated for 1 min. After removing the solution, slides were air-dried at room temperature for 5h, followed by a 14h incubation in a high (>80%) humidity chamber at r.t.. The slides were then blocked for 30 min in a blocking solution (50 mM Tris, 50 mM Tris-HCl (pH 9), and 50 mM ethanolamine) at 50 °C, followed by a 30 min wash in 4X SSC with 0.1% SDS at 50 °C. Finally, the slides were washed in Milli-Q water, spun dried, and stored in a desiccator (RH<10%) until use. These slides were used in all optimization experiments (FIG. 14). Transposome assembly

[0382] Firstly, each modified Tn5 adaptor (T7L_MedsB or dual_ME19_polyT_v2, 25 pl of 100 pM) was mixed separately with 25pl mosaic ends oligonucleotides (ME_19_Phos, lOOpM) and Ipl 50X annealing buffer (0.5 M Tris pH 8, 2.5 M NaCl). The mixtures were annealed by heating to 95°C for 5 min, followed by gradual cooling to 14 °C at -0. 1 °C / s. ProteinA-Tn5 was diluted to 0.5 mg / ml in dilution buffer (50% glycerol, lOmM Tris-HCl, pH 7.5. 100 mM NaCl. 0. 1 mM EDTA, and 1 mM DTT) and then combined with the annealed adaptor at a 5: 1 ratio. The reaction was incubated at r.t. for Ih to form the transposome. The transposome was stored at -20 °C until use. SPACE-Tag procedure

[0383] Tissue fixation, permeabilization, and antibody staining

[0384] Frozen tissue sections were prepared using a Leica CM1950 Cryostat at -17°C and sectioned at a thickness of 10 pm to be mounted onto capture areas on Visium slides (1000184, lOx Genomics). The slides were then incubated at 37°C on an Eppendorf ThermoMixer C with a thermal block for 5 min to enhance tissue adhesion. For fixation, tissue sections were treated with 0.2% formaldehyde (F8775, Sigma Aldrich) in IX PBS for 10 minutes, followed by quenching with 1.25M Glycine in PBS for 5 min. Sections were then washed with IX PBS for 2 min. Visium slides were then mounted on Arraylt Attorney Docket: NYG-LIPP-249PCT reaction cassettes for the following experiment (AHC 1X16, Arraylt). Permebilization was performed with 70 pl IX BD Perm / Wash buffer (554723, BD) for 30 minutes at 4°C. Primary' antibody staining was conducted in 70 pl IX BD Perm / Wash buffer for Ih at r.t.. Tissue sections were then washed with IX PBS for 5 min r.t. and incubated with secondary antibody in 70 pl CT wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, proteinase inhibitors, and 0.01% digitonin) for 30 min at r.t.. pA-Tn5 Tethering and tagmentation

[0385] Antibody-stained sections were incubated with preloaded ProteinA-Tn5 at a concentration of O.Olmg / ml in 70 pl 300-wash buffer (20 mM HEPES, 300 mM NaCl, 0.5 mM spermidine, proteinase inhibitors, and 0.01% digitonin ) at r.t. for one hour. Unbound Protein A-Tn5 was then removed by washing with 100 pl of the 300-wash buffer for 5 minutes. Following this, tagmentation was initiated by adding 70 pl of tagmentation buffer (10 mM MgCh in 300 wash buffer) and incubated at 55°C for 1 hour. Alternatively, we tested tagmentation with 37°C for 1 hour, and these results are presented in FIGs. 13A and 13B. Tagmentation was stopped by adding 40mM EDTA for 5 minutes, followed by a wash with IX NEBuffer 2 (B7002S, NEB). Gap filling and in vitro transcription

[0386] The gap-filling reaction was performed on the tissue using a 70 pl reaction mixture containing IX NEBuffer 2 (B7002S, NEB), 0.5 mM dNTPs (N0447S, NEB), 28 U RNaseOUT (10777019, Invitrogen), and 10 U exo- Klenow Fragment (M0212L, NEB). The reaction was incubated at 37°C for 30 minutes and removed. Tissue sections were treated with 0. 1 M HC1 (H9892, Sigma- Aldrich) for 2 minutes at r.t. to remove histones from genomic DNA. The sections were washed with 1 X transcription buffer (EP0111, Thermo Fisher Scientific) for 5 minutes at r.t. In vitro transcription (IVT) was initiated by adding 70 pl IVT reaction mixture containing IX transcription buffer, 2 mM rNTPs (N0466S, NEB), 140 U RNaseOUT (10777019, Thermo Fisher Scientific), and 28 U T7 RNA Polymerase (EP0111, Thermo Fisher Scientific) to the tissue sections. The reaction was incubated overnight (~14 hours) at 37°C. We alternatively tested T4-based gap filling using IX T4 ligase buffer, 0.05 mM dNTPs, 2 U / pL T4 DNA ligase (M0202S, NEB), and 0.015 U / pL T4 DNA polymerase (M0203S, NEB) in 70 pl reaction volume at r.t for 30 minutes. These comparison results were presented in FIGs. 13A and 13B. H&E staining Attorney Docket: NYG-LIPP-249PCT

[0387] After removing the TVT mixture, tissue sections were removed from the reaction cassette and fixed in methanol at -20°C for 30 minutes, and an abbreviated hematoxylin and eosin protocol was used for histological staining. Briefly, tissue sections were incubated in isopropanol for 1 minute and in Mayer's hematoxylin (S330930, Agilent) for 7 mins. Sections were then rinsed by immersing the slides in deionized water. Subsequently, the slides were incubated for 2 minutes in Bluing Buffer (CS70230, Agilent) and rinsed in deionized water. Finally, the slides were incubated for 1 minute in Eosin Y solution (100 pl Eosin Y (HT110216. Sigma Aldrich) in 900 pl 0.45M Trisacetate, pH 6) and rinsed in deionized water. Finally, the slides are dried and ready for imaging. Tissues were imaged using a Zeiss Axioscope inverted microscope with a 20X objective. Images were stitched with Vslide (vl.1.132) and extracted with VSViewer in a JPEG format.

[0388] Reverse transcription (library preparation)

[0389] After imaging, the slides were mounted again on the reaction cassettes and washed with IX Maxima H Minus Reverse Transcriptase buffer at 37°C for 30 minutes. The buffer was then removed, and a reverse transcription (RT) mixture was prepared according to the Visium protocol with slight modifications. Briefly. 43.06 pL H2O. 18.75 pL RT reagent, 1.5 pL reducing reagent B, and 11.69 pL RT enzyme D are mixed for each well of reaction. After adding the RT mixture, tissue sections were incubated at 53°C for 1.5 hrs for cDNA synthesis.

[0390] Reverse transcription (fluorescent cDNA gene activity print)

[0391] After imaging, the slides were remounted on the reaction cassettes and washed with IX Maxima H Minus Reverse Transcriptase buffer at 37°C for 30 minutes. The buffer was then removed, and a 70 pL reverse transcription (RT) mixture was prepared as followed: 39.6 pL Water, 7 pL lOxRT buffer (B0253S, NEB), 0.65 pL BSA (20mg / ml), 3.5 pL QC dNTPs (lOmM dATP, lOmM dGTP. lOmM dTTp, 0.25mM dCTP), 1.75 pL cy3-dCTP (0.2 mM, PA53021, Cytiva), 3.5 pL RNaseOUT (10777019, Thermo Fisher Scientific), 7 pL M-MuLV Reverse Transcriptase (M0253S, NEB). The RT mixture was added to the tissue sections, which were incubated at 37°C overnight for cDNA synthesis. Next day, the RT mix was removed and 70 pL proteinase K mixture was added per well (9 pL proteinase K. [EO0491, Thermo Fisher Scientific] and 61 pL PKD buffer [1034963, Qiagen]). Tissue removal was performed by incubating 55 °C for Ihr. Slides were then washed sequentially in 2xSSC with 0.1% SDS at 50 °C for 10 min, 0.2X SSC at r.t. for 1 Attorney Docket: NYG-LIPP-249PCT min and 0.1X SSC at r.t. for 1 min. Finally, the slides were imaged on a fluorescent microscope to capture the cDNA print. Finally, the slides were imaged using a Dragonfly 600 confocal microscope system (Andor, Belfast, UK) to capture the cDNA print, with image acquisition performed in Fusion software (v2.4.0.14). Imaging was carried out using a 594 nm laser at 20% intensity with an exposure time of 200 ms.

[0392] Second-strand synthesis

[0393] The RT mixture was removed, and 70 pl 0.08 M KOH (freshly diluted from the 8 M KOH stock, 11-102-0056. Thermo Fisher Scientific) was added to the tissue sections and incubated for 5 minutes at r.t. to remove the RNA strand. KOH was then removed, and Elution Buffer (EB; 10 mM Tris-Cl, pH 8.5) was added to wash the tissue sections. A primer annealing mix of 70 pl containing 2* SSC, 20% formamide (221198, Sigma- Aldrich), 0.1% Tween-20, and 0.02 pM PF-nextera-R2 was applied to the tissue sections and incubated at r.t. for 30 minutes. The unbound primer was washed away with EB. Second-strand cDNA synthesis was performed using 70 pl second-strand mix containing 5 U / pl Klenow Fragment (M0210L, NEB), 1 pM PF-nextera-R2 primer, ImM dNTP mix, and IX Maxima H Minus Reverse Transcriptase buffer. The reactions were incubated at 37°C for 1 hour. Following the reaction, tissue sections were first washed with EB. Then, 35 pL 0.08 M KOH is added to the tissue, incubated for 10 minutes at r.t., and the solution volume collected. 5 pL of Tris buffer (1 M, pH 7.0, BBT-70, Boston Biosciences) was added to each sample to neutralize the KOH.

[0394] Library preparation and indexing

[0395] PCR amplification was performed by adding PCR mix and primers to the neutralized sample to reach a concentration of IX NEBNext High-Fidelity PCR Master Mix, 0.5 pM cDNA Oligo 1 primer, and 0.5 pM PF-NexteraR2. After amplification, libraries were cleaned using a 0.65: 1 ratio with AMPure XP beads (A63880, Beckman) and eluted in 40 pL of water. Indexing PCR was performed using 10 pL of the PCR product in IX NEBNext High-Fidelity PCR Master Mix and 0.5 pM indexing primers (SIPCR-T501-T504, N701-N722). Indexed cDNA libraries were then purified using AMPure XP beads at a 0.6: 1 ratio, following the manufacturer’s protocol, and eluted in 40 pL of EB. All samples were sequenced on the Illumina Novaseq X sequencing platform using a 28-cycle forward read (Rl), 8-cycle index read, and 100-cycle reverse read (R2) configuration (final loading concentration of 190 pM). Bulk CUT&Tag data generation Attorney Docket: NYG-LIPP-249PCT

[0396] To generate bulk CUT&Tag profiles, tissue fixation, permeabilization, and antibody staining were performed as in the SPACE-Tag procedure denoted above, except that frozen tissue sections were mounted on a Superfrost plus slide (22-037-246, Fisher) instead of a Visium array. ProteinA-Tn5 tethering and tagmentation was also performed similarly to SPACE-Tag, except that the transposome was loaded with MedsA / B annealed with ME19Phos. This was followed by procedures denoted below.

[0397] Tissue digestion and genomic DNA purification

[0398] Seventy microliters of Proteinase K mixture (9 pL Proteinase K [EO0491, Thermo Fisher Scientific] and 61 pL PKD buffer [1034963, Qiagen]) was added to each well, and the cassettes were incubated at 55 °C for 1 h. The digested solution was then purified for genomic DNA using Ampure XP beads at a 2:1 ratio and eluted in 20 pL EB buffer. Library preparation and sequencing

[0399] PCR amplification was carried out by adding PCR mix and primers to the eluate to achieve final concentrations of l x NEBNext High-Fidelity PCR Master Mix, 0.5 pM MedsA primer, and 0.5 pM MedsB primer. Amplification was performed for 15 cycles, and the product was purified with Ampure XP beads at a 0.65: 1 ratio. Indexing PCR was then performed using 10 pL of the amplified product in 1 x NEBNext High-Fidelity PCR Master Mix with 0.5 pM indexing primers (S502-S508, N701-N722), for a total of 5 cycles. Libraries were sequenced on an Illumina NovaSeq X platform using a 28-cycle forward read (Rl), an 8-cycle index read, and a 100-cycle reverse read (R2), with a final loading concentration of 190 pM.

[0400] Data preprocessing and statistical quantification Primary data processing

[0401] A custom computational workflow was implemented using the Snakemake workflow manager v7.32.4 to perform data preprocessing. Reads were first trimmed with cutadapt v4.5 (Martin, M. EMBnet J. 17, 10 (2011)). Spatial barcode sequences and UMIs are next extracted from the forward read (Rl), and barcode filtering was performed using the 10X Visium VI barcode list (github.com / heruiyang / spatial_multiome_processing / blob / main / refs / visium-vl.txt) as a reference with umi-tools vl. 1.4 (Smith, T., Heger. A. & Sudbery, I. Genome Res. 27, 491-499 (2017)). The reverse read (R2) was then aligned using local alignment with bowtie2 v2.5.2 (Langmead, B. & Salzberg, S.L. Nat. Methods 9, 357-359 (2012)) to the mmlO mouse genome reference. Aligned sam files are deduplicated using the spatial Attorney Docket: NYG-LIPP-249PCT barcode and alignment coordinates before peak calling using macs2 v2.2.9. 1 (Zhang, Y. et al. Genome Biol. 9, R137 (2008)) with custom parameters slocal 5000 —local 50000 —broad -max-gap 1000' to enable identification of broad histone modification domains. The resulting peaks list and alignment files were used to create a fragments file and raw peak-by-spot data matrix using sinto vO. 10.0 (github.com / timoast / sinto). The raw peak matrix was next filtered to remove peaks associated with polyadenylated genome sequences. A custom script was implemented in R v4.3.1 to identify peaks associated with poly-A and poly-T sequences of length 20 within 70 bp of the peak, and these peaks are filtered out. In addition, the tissue region was annotated using manual fiducial alignment using the Loupe browser v6.3.0 (lOx Genomics), and spots outside the tissue were filtered out. We further manually annotated H&E images of the mouse brain using a custom in-house interactive tool26, using annotation labels corresponding to the retrosplenial area, visual cortex, somatosensory cortex, auditory cortex, perirhinal area, piriform cortex, fiber tracts, hippocampus, thalamus, hypothalamus, striatum, and amygdala. The resulting filtered peak-by -tissue spot matrix was used for downstream analysis.

[0402] Comparison of estimated library complexity across conditions

[0403] To compare differences in library complexity between conditions for protocol optimization (FIGs. 13A and 13B), we used Preseq (Daley, T. & Smith, A.D. Nature Methods 10, 325-327 (2013)), an empirical Bayesian method for estimating the total number of unique fragments in a cDNA library conditioned on the observed frequency of duplicated fragments. Specifically, we used the ‘bound_pop’ method to compute the total library complexity estimate. Tissue registration

[0404] To register mouse tissue sections, we leveraged manual spot annotations together with the highly stereotyped architecture of the brain. This annotation-guided strategy provides greater robustness than direct alignment of H&E images, which are often confused by incomplete or disrupted morphologies. Our registration workflow proceeds as follows. First, for each replicate, we identify7centroids corresponding to annotated spots in the retrosplenial area and thalamus. We then rotate the spatial coordinates of spots within each replicate to minimize the distance between corresponding centroids across replicates. After rotation, we translate the coordinates so that the point equidistant Attorney Docket: NYG-LIPP-249PCT between the centroids lies at the origin of the coordinate system. Finally, all registered sections are manually inspected to confirm alignment accuracy.

[0405] Gene activity quantification

[0406] The ‘Gene Activity’ function in Signac vl.13.0 (Stuart, T.. Srivastava. A.. Madad, S., Lareau, C.A. & Satija, R. Nat. Methods 18, 1333-1341 (2021)) was used to estimate gene activities from histone modification levels. To that end, reads aligning in the gene body and within 2kb upstream of the gene body were aggregated to give a gene activity' score for each gene. Joint quantification of gene activity across histone modifications and experimental replicates was then performed using Splotch vO. 1.0, a hierarchical Bayesian model for spatial counts data (Maniatis, S. et al. Science 364, 89-93 (2019)). Splotch models counts data across multiple spatial experiments using a hierarchical model, wherein the transcript count for each gene (or, in our case, gene activity of each gene) i in spot k for sample .7 is assumed to be sampled from a Poisson distribution. The model specification is given in Eq. 1 below:

[0407] Here, ^-3^ is an error term to account for spot-level variability', and is a size factor, corresponding to the total UMIs in spot z (i.e. i ), to normalize for per-spot read depth. A two-level hierarchical prior is placed on the latent rate parameter A to account for differences between animals and distinct histone modifications (Eq. 2). Specifically, this is done through A, which quantifies the contribution of each annotation level in the one-hot encoded annotation vector to the expression of gene i in spot k, for sample 7:

[0408] AJ172 ~ ,h ’ ^i' ) Attorney Docket: NYG-LIPP-249PCT

[0409] (Eq. 2)

[0410] Here, we set the upper-level li annotation to be the histone modification identity, and the L annotation to the identity of the experimental animal. Model inference was performed using Markov Chain Monte Carlo sampling with Stan v2.36.0 (mc~stan.org), with 4 chains of 500 bum-in and 500 sampling iterations each. To plot inferred gene activity across replicates, registered coordinates (see above) were used. The mean gene activity of each spot was computed using the inferred posterior means of A and plotted. Comparisons to published data

[0411] To establish the sensitivity and specificity of SPACE-Tag, we performed comparisons against a variety of bulk, single-cell, and spatial datasets. Comparison with bulk ChlP-seq and bulk CUT&Tag

[0412] To compare with bulk ENCODE Chip-seq data of H3K27ac in the adult mouse cortex, we downloaded aligned bam files and replicated peak bed files from the ENCODE data portal (Luo, Y. et al. Nucleic Acids Res. 48, D882-D889 (2020)). For comparison to bulk ChlP-seq, we filtered spots annotated as the isocortex in the SPACE-Tag H3K27ac data. The aligned bam files corresponding to the annotated spots were then extracted, and both our SPACE-Tag and the ENCODE CHIP-seq cortex data were normalized by genome coverage to account for differences in sequencing depth using the 'bamCoverage’ function in deepTools v3.5.5 (Ramirez, F. et al. Nucleic Acids Res. 44, W160-5 (2016)). We then plotted the normalized signal at replicated ENCODE peaks using the ‘plotHeatmap’ function in deepTools v3.5.5. To quantity7this correlation, we calculated pairwise spearman correlation coefficients between the normalized signal across SPACE-Tag replicates and ENCODE ChlP-seq. For comparison to bulk CUT&Tag, we used the same procedure, replacing the ENCODE replicated peaks with peaks called using mac2 v2.2.9.1 (Zhang, Y. et al. Genome Biol. 9, R137 (2008)) with custom parameters ‘-slocal 5000 —local 50000 —broad -max-gap 1000’ from our bulk CUT&Tag data.

[0413] Comparisons with spatial ATAC-seq and CUT&Tag

[0414] To compare with existing spatial CUT&Tag and ATAC-seq methods, we downloaded processed fragments files from the GEO repositories for the Deng et al., Attorney Docket: NYG-LIPP-249PCT

[0415] Zhang et al., and Guo et al. studies (Deng, Y. et al. Science 375, 681-686 (2022); Zhang, D. et al. Nature 1-10 (2023); Guo, P. et al. Nat. Methods 1-10 (2025)) (GSE165217, GSE205055, and GSE263333, respectively); we then computed the number of fragments per barcode using the fragments file. In the case of Huang et al. (GSE279771), we identified a number of issues in the provided fragments file, including fragments of implausible length (>100kb). As this is likely due to a processing artifact arising from the single-end nature of the read, we reprocessed the data from the raw sequencing files using our data processing pipeline, using default parameters. We compared the total fragment count and FRiP score between these methods and our SPACE-Tag data.

[0416] Comparison with single-cell CUT&Tag

[0417] To compare with existing single-cell CUT&Tag methods, we used the publicly available preprocessed peak-by-cell matrix (accessed from the GEO repository GSE198467). We first merged this data with SPACE-Tag data by re-quantifying the counts matrix of SPACE-Tag data using peaks identified in the scCUT&Tag dataset. Next, we performed anchor-based integration with Seurat v5.2.1 (Stuart, T., Srivastava, A., Madad, S., Lareau, C.A. & Satija, R. Nat. Methods 18, 1333-1341 (2021)) for our SPACE-Tag data with ’FindTransfer Anchors' and 'TransferData' functions using the joint Latent Semantic Indexing (LSI) embedding of the merged data to compute neighbourhood distances, and plotted the resulting projection scores for the high- resolution cell subtypes identified in the original manuscript. Comparison with single-cell ATAC-seq

[0418] To compare with the previously published single-cell ATAC-seq atlas of postnatal mouse brain (Zu, S. et al. Nature 624, 378-389 (2023)), we utilized two different methods for the pseudobulk comparison and single-cell comparison. To investigate the pseudobulked region-specific AT AC accessibility of CRE topics identified in our analysis, we identified scATAC-seq datasets from functional regions corresponding to those annotated in our coronal brain section, downloaded raw sequencing data for these datasets from GEO (GSE246791), aligned to the mmlO mouse reference genome, and computed the sequencing depth-normalized coverage at each peak in a given topic. We then took the mean of these coverage scores for each topic and computed aZ-score across pseudobulked samples to derive the overall AT AC signal score for each CRE topic in each pseudobulked sample. To compare the single-cell accessibility of our CRE topics between cell types in each region, we downloaded processed genomic bin-by-cell Attorney Docket: NYG-LIPP-249PCT matrices from GEO (GSE246791), subset the cells corresponding to that region, identified the set of genomic bins overlapping the peaks in each topic, and computed percell topic scores using the Signac ‘AddChromatinModule’ function.

[0419] Analysis of gene regulation in the postnatal mouse brain Topic modelling and clustering

[0420] To identify spatial domains in SPACE-Tag profiling, we performed topic modelling with STAMP vO. l.O, a spatially-aware probabilistic topic model (Zhong, C., Ang, K.S. & Chen. J. Nat. Methods 21, 2072-2083 (2024)). Briefly, ajoint topic model with n=10 topics was constructed with the registered data (see above) of all replicates of the adult brain, using the peak-by-spot count matrix with Poisson likelihoods for peak counts, and Simplified Graph Convolutions (option ‘sgc’) to account for the spatial locations of spots for inference of spatial domains. To ensure a consistent feature set between replicates, the union of peak sets across all replicates was used. To account for batch effects, the sample ID was included in the model as a categorical covariate. Training was performed for 2000 epochs with a learning rate of 0.01. To assign the resulting probabilistic topic proportions to discrete clusters, we labelled each spot using the topic with the highest proportion in that spot. Analysis of cortical plate axis

[0421] To investigate functional region-specific H3K27ac peaks in the cortex of the P28 postnatal mouse brain, we first manually annotated functional regions across the brain and extracted functional regions corresponding to the cortex. We next defined a spatial cortical plate axis along the dors al -ventral axis of the cortex. To do this, we first identified a set of anchor points in the tissue using K-means clustering (K=10), and then defined the axis by fitting a principal curve for the anchor point set. We then performed spatial module analysis on all peaks with greater than 100 counts across all spots, using Hotspot vO.9.1 (DeTomaso, D. & Yosef, N. Cell Syst. 12. 446-456. e9 (2021)), which estimates local correlations between spots given a pre-defined measure of similarity between spots. For cortical subregion analysis, the projection of each spot along the cortical plate principal curve was used as the similarity measure, from which a KNN neighbourhood graph (K=300) was constructed. The resulting peak-peak correlation matrix was then clustered using hierarchical clustering to obtain spatial modules. Identification and analysis of super-enhancer elements Attorney Docket: NYG-LIPP-249PCT

[0422] To identify putative super-enhancer elements, we adapted the strategy used in the original Whyte et al. publication (Whyte, W.A. et al. Cell 153, 307-319 (2013)), by ranking H3K27ac and H3K4me3 peaks based on their aggregated normalized signal. The standard normalization method used in analysis of single-cell and spatial epigenomic data is the term frequency -inverse document frequency (TF-IDF) transformation, which is unsuitable for our purposes as it weights peaks by their inverse frequency; we therefore used an alternate normalization scheme whereby the raw fragment counts for each peak in a spot is normalized by a scaling factor equal to the ratio of the total number of unique fragments in that spot to the median unique fragments across all spots. This accounts for biases caused by sequencing depth and the number of nuclei per spot. Super-enhancer elements were then defined as peaks in the top decile of normalized signal that overlapped with an element in the SEdb database (Wang, Y. et al. Nucleic Acids Res. 51, D280-D290 (2023)). To assess spatial coherence in H3K27ac peaks, we divided the peak set into 10 deciles, with super-enhancer elements as the top decile as described above. We then used the ‘calculateUnivariate’ function in Voyager vl. 10.0 (Moses, L. et al. bioRxivorg 2023.07.20.549945 (2023)), a package for geospatial analysis of spatial omics data, to compute Moran’s I. We performed spatial module analysis for super-enhancer elements using Hotspot as described above; all parameters were unchanged except for the similarity metric used to construct the spot-spot neighbourhood graph, which was changed to the spatial coordinates of each spot. Motif enrichment in spatial modules was computed by comparing the number of motifs for TFs in the Cis-BP database (Weirauch, M.T. et al. Cell 158, 1431-1443 (2014)) in peaks associated with a spatial module versus a background set of 40000 GC-matched regions across the genome using the ‘FindMotifs’ function in Signac. Finally, GO enrichment of candidate genes regulated by each spatial module was performed by identifying a candidate gene set using the closest gene to each SE in a given module; this gene set was then assessed for enrichment of biological process terms in the gene ontology (’GO: BP’) using an FDR-corrected hypergeometric test, as implemented in clusterProfiler v4.10.1 (Yu, G., Wang, L.-G., Han, Y. & He, Q.-Y. OMICS 16, 284-287 (2012)). GO enrichment and motif enrichment scores were calculated as -loglO(p.adj), where p.adj is the FDR-corrected p-value.

[0423] Multiple hypothesis testing

[0424] In all cases, false discovery rate correction for multiple hypothesis testing was performed using the Benj amini-Hochberg procedure for FDR adjustment. Attorney Docket: NYG-LIPP-249PCT

[0425] References

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[0486] Each and every patent, patent application, and publication, including websites cited throughout the specification, and sequence identified in the specification, is incorporated herein by reference. US Provisional Patent Application No. 63 / 723,294, filed November 21, 2024 is incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

Attorney Docket: NYG-LIPP-249PCTClaims:

1. A transposome complex comprising a first transposase and a second transposase, the first transposase having a first adapter comprising a T7 promoter and the second transposase having a second adapter comprising a capture sequence, the first transposase and the second transposase each being a fusion protein comprising an antibody -binding ligand.

2. A transposome complex comprising a first transposase and a second transposase, the first transposase having a first adapter comprising a T7 promoter and the second transposase having a second adapter, the first transposase and the second transposase each being a fusion protein comprising an antibody -binding ligand.

3. The transposome complex of claim 1, wherein the second adapter comprises a capture sequence.

4. The transposome complex of claim 2 or claim 3. wherein the capture sequence is 1 to lOObp, optionally 30 bp.

5. The transposome complex of claim 2 or 3, wherein the capture sequence comprises a poly(d)T sequence or poly(d)T VN sequence.

6. The transposome complex of claim 2 or 3, wherein the capture sequence comprises a mosaic end sequence.

7. The transposome complex of any one of claims 1 or 2, wherein the first adapter and / or the second adapter further comprise a mosaic end sequence.

8. The transposome complex of any one of claims 1 or 2, where the first transposase and the second transposase are Tn5 or TnY.

9. The transposome complex of any one of claims 1 or 2, wherein the antibodybinding ligand is protein A, protein G, or a nanobody.Attorney Docket: NYG-LIPP-249PCT10. The transposome complex of any one of claims 1 or 2, wherein the transposome complex is bound to an antibody or fragment thereof bound to a chromatin epitope in situ, optionally wherein the chromatin epitope is present in a biological sample mounted on a capture surface comprising an array of capture regions.

11. The transposome complex of any one of claims 1 or 2, wherein the chromatin epitope comprises a histone modification.

12. The transposome complex of claim 11 , where the histone modification comprises a histone acylation, a histone phosphorylation, or a histone methylation, optionally wherein the histone acylation is an H3K27ac and / or the histone methylation is an H3K4me3 mark or an H3K27me3 mark.

13. A method for in situ analysis of protein-DNA interactions in a biological specimen, the method comprising: a) mounting a biological specimen to a solid substrate having a capture surface comprising an array of capture regions, each capture region comprising capture oligonucleotides, the individual capture regions being identifiable by one or more unique spatial barcodes of the capture oligonucleotides; b) optionally, labeling or staining the biological specimen for visualization; c) permeabilizing the biological specimen; d) contacting the biological specimen with an antibody or fragment thereof capable of binding a chromatin epitope, optionally a histone modification, and a tagmentation complex of any one of claims 1 or 2, and incubating the biological specimen under conditions suitable for tagmentation to occur, wherein tagmented genome fragments comprising the T7 promoter and the capture sequence are generated; e) performing a gap-filling reaction; f) contacting the biological specimen with a reaction mixture comprising a T7 polymerase to amplify the tagmented genome fragments; g) generating a library of spatially barcoded tagmented genome products, wherein the tagmented genome fragments hybridized to the capture oligonucleotides areAttorney Docket: NYG-LIPP-249PCT barcoded with the one or more unique spatial barcodes of the capture oligonucleotides; and h) sequencing and analysis of the library of step g), wherein sequencing and analysis includes mapping protein-DNA interactions to spatial locations of the biological specimen using the one or more distinct spatial barcodes of the capture oligonucleotides.

14. A method for in situ analysis of protein-DNA interactions and protein-protein interactions in a biological specimen, the method comprising: a) mounting a biological specimen to a solid substrate having a capture surface comprising an array of capture regions, each capture region comprising capture oligonucleotides, the individual capture regions being identifiable by one or more unique spatial barcodes of the capture oligonucleotides; b) optionally, fixing the biological specimen; c) optionally, labeling or staining the biological specimen for visualization and for protein-protein labeling; d) contacting the biological specimen with one or antibodies or fragments thereof having linked oligonucleotides comprising a barcode and anchor sequence; e) permeabilizing the biological specimen; f) contacting the biological specimen with an antibody or fragment thereof capable of binding a chromatin epitope, optionally a histone modification, and a tagmentation complex of any one of claims 1 or 2, and incubating the biological specimen under conditions suitable for tagmentation to occur, wherein tagmented genome fragments comprising the T7 promoter and the capture sequence are generated; g) performing a gap-filling reaction; h) contacting the biological specimen with a reaction mixture comprising a T7 polymerase to amplify the tagmented genome fragments; i) generating 1) a library of spatially barcoded tagmented genome products, wherein tagmented genome fragments hybridized to the capture oligonucleotides are barcoded with the one or more unique spatial barcodes of the capture oligonucleotides, and 2) a library of spatially barcoded oligonucleotides comprising i) the barcodes of the oligonucleotides linked to the one more antibodies or fragments thereof of step d), and ii) the one or more unique spatial barcodes of the capture oligonucleotides; andAttorney Docket: NYG-LIPP-249PCT j) sequencing and analysis of the libraries of step i), wherein sequencing and analysis includes mapping protein-DNA interactions and protein-protein interactions to spatial locations of the biological specimen using the one or more distinct spatial barcodes of the capture oligonucleotides.

15. A method for spatially resolving DNA-protein interactions in a biological sample, the method comprising: a) mounting a biological specimen to a solid substrate having a capture surface comprising an array of capture regions, each capture region comprising capture oligonucleotides, the individual capture regions being identifiable by one or more unique spatial barcodes of the capture oligonucleotides; b) optionally, labeling or staining the biological specimen for visualization; c) permeabilizing the biological specimen; d) contacting the biological specimen with an antibody or fragment thereof capable of binding a chromatin epitope, optionally a histone modification, and a tagmentation complex of any one of claims 1 or 2, and incubating the biological specimen under conditions suitable for tagmentation to occur, wherein tagmented genome fragments comprising the T7 promoter and the capture sequence are generated; e) performing a gap-filling reaction; and f) contacting the biological specimen with a reaction mixture comprising a T7 polymerase to amplify the tagmented genome fragments.

16. The method of claim 13, further comprising fixing the biological specimen.

17. The method of claim 16, wherein staining the biological sample comprises hematoxylin and eosin (H&E) staining.

18. The method of claim 13, wherein labeling the biological specimen comprises contacting the biological specimen with fluorescently-labeled antibodies or fragments thereof capable of binding epitopes in the biological sample.

19. The method of claim 13, further comprising imaging the biological specimen using brightfield microscopy or immunofluorescence microscopy.Attorney Docket: NYG-LIPP-249PCT20. The method of claim 13, wherein permeabilizing the biological specimen comprises contacting the biological specimen comprises with a solution comprising perm / wash buffer, digitonin buffers, and / or HC1.

21. The method of claim 13, wherein the capture oligonucleotides of the capture surface comprise a polyT or a polyT VN sequence.

22. The method of claim 13, wherein the capture oligonucleotides of the capture surface comprise a Mosaic End (ME) sequence.

23. The method of claim 13, wherein incubating the biological specimen under conditions suitable for tagmentation to occur comprises incubating the biological specimen with about 0. 1 mg of the tagmentation complex and / or incubating the biological specimen for a period of time at about 37°C and a period of time at about 55°C.

24. The method of claim 13, wherein amplification of the genome fragments comprises i) incubating the biological specimen with about 0. 1 to about 0.4 p / pl T7 RNA polymerase, and / or ii) incubating the biological specimen at about 37°C for about 14 hours or at least about 8 hours, about 10 hours, or about 12 hours.

25. The method of claim 13, wherein the gap-filling reaction comprises gap-filling via ligation or gap filling via polymerization.

26. The method of claim 25, wherein gap-filling via ligation is performed using about 0.015 units of T4 DNA polymerase and about 2 units T4 DNA ligase and / or at about 24°C for about 30 minutes.

27. The method of claim 25, wherein gap filling via polymerization is performed using about 0.143 units Klenow (exo-) polymerase and / or at about 37°C for about 30 minutes.

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