Tissue spatial omics

Abstract

Provided herein are compositions, kits and methods for the collection, integration and analysis of spatially-based information from tissues at the cellular or subcellular level. Layers of information include, without limitation, gene expression, morphology, nucleic acid and protein. In some workflows, light-based technology is incorporated for selective spatial barcoding of regions. In a further step, such barcoded tags are analyzed by high-throughput next-generation sequencing, if necessary.

Description

[Technical field]

[0001] cross reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 323,290, filed March 24, 2022, U.S. Provisional Patent Application No. 63 / 326,384, filed April 1, 2022, and U.S. Provisional Patent Application No. 63 / 346,454, filed May 27, 2022, the entire contents of each of which are hereby incorporated by reference herein.

[0003] Sequence Listing

[0004] This application has been submitted electronically in ST.26xml format and contains a Sequence Listing, which is hereby incorporated by reference in its entirety. The xml copy, created on Mar. 14, 2023, is named 205590-701601_SL.xml and is 3,445 bytes in size. [Background technology]

[0005] background

[0006] The integration of cell imaging-based analysis and high-throughput screening allows spatial selection of information from tissue samples.In many published methodologies, the limitations on the number of recorded visual features or the manner of pooling information from such features result in the loss of certain cell or subcellular location-specific information, including the spatial information of one feature compared to another.Therefore, there is a need for improved compositions, devices, methods and systems for collecting multiple channels of spatial information at cellular and subcellular levels, including but not limited to morphological data, genomic data, transcriptomic data and proteomic data. Summary of the Invention [Means for solving the problem]

[0007] overview

[0008] Provided herein is a method of generating biological cellular information, the method comprising the steps of: contacting a population of cells with a nucleic acid polymerase and a primer to generate an amplification product; placing DNA barcodes on the population of cells, where the DNA barcode comprises a region complementary to a region of the amplification product, and where the DNA barcode comprises a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating the population of cells to form covalently linked DNA barcode-amplification product complexes; washing the population of cells to remove DNA barcodes that are not complexed with an amplification product; and repeating the placing, selectively irradiating, and washing steps, where the DNA barcode in a first placing step of the placing steps comprises a different sequence than the DNA barcode in a second placing step of the placing steps, and the selectively irradiating step is performed at different locations for the first selectively irradiating step and the second selectively irradiating step.

[0009] Provided herein is a method for generating biological cellular spatial information, the method comprising the steps of contacting a population of cells with a capture probe, where the capture probe comprises a DNA binding region and a target binding region; placing a DNA barcode on the population of cells, where the DNA barcode comprises a region that binds to the DNA binding region, and where the DNA barcode comprises a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating the population of cells to form a DNA barcode-amplification product complex; washing the population of cells to remove DNA barcodes that are not complexed with an amplification product; and repeating the placing, selectively irradiating, and washing steps, where the DNA barcode in a first placing step of the placing steps comprises a different sequence than the DNA barcode in a second placing step of the placing steps, and the selectively irradiating step is performed at different locations for the first selectively irradiating step and the second selectively irradiating step.

[0010] Provided herein is a method for generating biological cellular spatial information, the method comprising the steps of capturing an image of a three-dimensional tissue sample; contacting the tissue sample with a nucleic acid polymerase and a primer to generate an amplification product; placing a DNA barcode on the tissue sample, the DNA barcode comprising a region complementary to a region of the amplification product, the DNA barcode comprising a photoreactive nucleic acid base capable of crosslinking to another nucleic acid base; selectively irradiating the three-dimensional tissue sample to form a DNA barcode-amplification product complex; washing the tissue sample to remove DNA barcodes that are not complexed with an amplification product; and repeating the placing, selectively irradiating, and washing steps, wherein the DNA barcode in a first one of the placing steps comprises a different sequence than the DNA barcode in a second one of the placing steps, and the selectively irradiating step is performed at different locations of the tissue sample for the first selectively irradiating step and the second selectively irradiating step.

[0011] Provided herein is a method for generating biological cellular spatial information, the method comprising the steps of capturing an image of a population of cells, the population of cells being immobilized on a substrate; reverse transcribing RNA from the population of cells to generate cDNA; placing a DNA barcode on the population of cells; hybridizing the DNA barcode to the cDNA, the DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating the population of cells to form a crosslinked barcode; washing to remove uncrosslinked DNA barcodes; and repeating the placing, hybridizing, selectively irradiating, and washing steps, wherein the DNA barcodes in the placing step are varied.

[0012] Provided herein is a method of generating biological cellular spatial information, the method comprising the steps of: placing DNA barcodes on a population of cells, where the DNA barcodes hybridize to a target region; the DNA barcodes comprise photoreactive nucleobases capable of crosslinking to another nucleobase; selectively irradiating the population of cells in the region of interest to form covalently linked DNA barcode-target complexes, where the irradiating step is selectively varied by intensity or duration; washing the population of cells to remove DNA barcodes that are not complexed to the target region; and repeating the placing, selectively irradiating, and washing steps, where the DNA barcodes in a first one of the placing steps comprise a different sequence than the DNA barcodes in a second one of the placing steps.

[0013] Provided herein is a method of generating biological information, the method comprising the steps of: disposing, on a population of nucleic acids, a primer that hybridizes to a target sequence of the nucleic acid to form a nucleic acid primer-complex; disposing a DNA barcode on the nucleic acid-primer complex, where the DNA barcode hybridizes to the primer to form a nucleic acid concatemer; the DNA barcode comprises a photoreactive nucleic acid base capable of crosslinking to another nucleic acid base; irradiating the nucleic acid concatemer to form covalently linked concatemers; and removing the DNA barcodes that are not in the covalently linked concatemers.

[0014] Provided herein is a method for generating biological cell information, comprising: disposing DNA barcodes on a population of cells, where the DNA barcodes comprise photoreactive nucleobases capable of crosslinking to another nucleobase; forming concatemers via a light-based process comprising repeating the following steps: a) selectively irradiating the population of cells with a first wavelength to form covalently linked complexes of DNA barcodes hybridized to a substrate nucleic acid sequence; and b) selectively irradiating the population of cells with a second wavelength to cleave the DNA barcodes hybridized to the substrate nucleic acid sequence; and collecting the concatemers, where the collecting step is before or after the step of selectively irradiating the population of cells with the second wavelength, and where the concatemers contain information about the population of cells.

[0015] Provided herein is a method of generating spatial organization information, the method comprising the steps of at least partially removing paraffin from a sample comprising paraffin-embedded tissue; heating the sample to a temperature of at least about 50° C.; contacting the sample with a nucleic acid polymerase and a primer to generate amplification products; disposing a DNA barcode on the sample, the DNA barcode comprising a region complementary to a region of the amplification product, the DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating the sample to form covalently linked DNA barcode-amplification product complexes, the selectively irradiating steps being performed at different locations for the first selectively irradiating step and the second selectively irradiating step; washing the sample to remove DNA barcodes not complexed with amplification products; and determining the spatial organization information based at least in part on the generated amplification products and DNA barcodes.

[0016] Provided herein is a method of generating biological cellular spatial information, the method comprising the steps of: placing a DNA barcode on a population of cells, where the DNA barcode comprises a photoreactive nucleobase capable of crosslinking to another nucleobase, and where the DNA barcode hybridizes to a target region; selectively irradiating the population of cells in at least one region of interest (ROI) to form covalently linked DNA barcode-target complexes, where the irradiating step is selectively varied by intensity or duration; washing the population of cells to remove DNA barcodes that are not complexed to the target region; and repeating the placing, selectively irradiating, and washing steps, where the DNA barcode in a first one of the placing steps comprises a different sequence than the DNA barcode in a second one of the placing steps, and where the second selectively irradiating step is applied at the same or different duration or intensity as the first selectively irradiating step.

[0017] Provided herein are nucleic acid libraries produced according to the methods described herein.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief description of the drawings]

[0020] [Figure 1A] 1A and 1B show a process workflow incorporating sample preparation, light-based in situ barcoding of populations of cells, and record extraction and sequencing.

[0021] [Figure 1B]1A and 1B show a process workflow incorporating sample preparation, light-based in situ barcoding of populations of cells, and record extraction and sequencing.

[0022] [Figure 1C] FIG. 1C shows a workflow that provides fractional spatial barcoding.

[0023] [Diagram 2] 2A and 2B show a fractional barcoding scheme that follows the workflow described in FIG. 1C.

[0024] [Diagram 3] FIG. 3 illustrates the photoinduced crosslinking reaction described herein.

[0025] [Figure 4] FIG. 4 shows an iterative barcode addition method in which a barcode strand is added to the 5′ end of the cDNA.

[0026] [Figure 5A] FIG. 5A shows light-induced cross-linking to generate barcode concatemers.

[0027] [Figure 5B] FIG. 5B shows data reconstruction from barcode concatemers.

[0028] [Figure 6] FIG. 6 shows the process of cross-junction synthesis.

[0029] [Figure 7] Figure 7 shows the workflow for cross-junction synthesis across concatemers of barcodes to generate contiguous nucleic acids.

[0030] [Figure 8]FIG. 8 shows a coding combination with four barcode strands using a binary encoding method.

[0031] [Figure 9] FIG. 9 shows a coding combination with two barcode strands using the 4-ary encoding method.

[0032] [Figure 10] FIG. 10 shows a coding combination with two barcode strands using the fractional encoding method.

[0033] [Figure 11] FIG. 11 shows representative images of paraformaldehyde-fixed (PFA), paraformaldehyde with Triton® X-100-fixed (PFA-Tx), and formalin-fixed paraffin-embedded (formalin) brain, spleen, and liver sections after light-induced transcriptome barcoding with fluorescently labeled barcodes, demonstrating that transcriptome barcoding is comparable across tissue types and fixation conditions; the bar in the bottom right corner of the images corresponds to 100 μm.

[0034] [Figure 12A] FIG. 12A is an image of an FFPE liver section showing fluorescently labeled barcoding in the region of interest marked by the dashed line.

[0035] [Figure 12B] FIG. 12B is a heat map showing z-scores of detected expression of 50 genes in PFA-Tx fixed brain sections and PFA-Tx and FFPE fixed liver samples, showing increased expression of canonical organ marker genes and consistent detection levels across preparation methods.

[0036] [Figure 12C]FIG. 12C is a volcano plot showing the relative expression levels of a selected set of genes with canonical organ-specific expression described in FIG. 12B in samples.

[0037] [Figure 12D] FIG. 12D is a heat map showing the z-scores of the relative expression levels of 2,653 differentially expressed genes that show increased detected expression in canonical organ marker genes.

[0038] [Figure 12E] FIG. 12E is a heatmap showing Pearson correlations calculated to compare mRNA expression values ​​in FFPE-fixed versus PFA-Tx-fixed tissues, showing high correlation between liver or brain samples, and low correlation when comparing liver samples to brain samples.

[0039] [Figure 13A-B] Figures 13A and 13B are images showing liver sections containing rectangular areas of light-induced barcoding after heat-induced epitope retrieval (HIER) treatment at 70°C (Figure 13A) and 95°C (Figure 13B).

[0040] [Figure 13C] FIG. 13C is a bar plot of the average barcode fluorescence intensity in the samples shown in FIGS. 13A and 13B, showing 3-fold higher fluorescence intensity in the samples treated at 95° C. compared to the samples treated at 70° C.

[0041] [Figure 13D] FIG. 13D is a bar plot of the average Cq values ​​of generated cDNA libraries collected from sections treated with HIER at 70° C. and 95° C. with and without UV treatment, showing that samples treated at 95° C. were amplified in fewer cycles than samples treated at 70° C., and control samples (not UV treated) required at least 50% more amplification cycles than treated samples.

[0042] [Figure 14] FIG. 14A is an image of a mouse liver section with areas of light-induced barcoding after illumination with a DMD using a 380 nm LED (lower rectangle) or a 405 nm laser (upper rectangle). FIG. 14B is an image showing autofluorescence in the same liver section shown in FIG. 14A. FIG. 14C is an overlay of images from FIG. 14A and FIG. 14B. FIG. 14D is a close-up of the dashed rectangle in FIG. 14A showing the intensity of barcoding in four areas exposed to different power levels of illumination from a 405 nm laser. FIG. 14E is an image showing autofluorescence in the same liver section shown in FIG. 14D. FIG. 14F is an overlay of images from FIG. 14D and FIG. 14E.

[0043] [Figure 15A] FIG. 15A is an image of a mouse retina showing FITC-labeled barcoding in five regions of interest (ROIs) treated with increasing power levels of illumination in each ROI, from left to right.

[0044] [Figure 15B] FIG. 15B is an image of the same retinal section as FIG. 15A, showing Cy3-labeled barcoding in the same five ROIs after processing with illumination at a consistent power level across all ROIs.

[0045] [Figure 15C] FIG. 15C is an overlay of the images from FIGS. 15A and 15B.

[0046] [Figure 15D] FIG. 15D is a bar plot of the image intensity ratios of the ROIs shown in FIGS. 15A and 15B.

[0047] [Figure 16]Figure 16A is an image of a mouse brain section stained with DRAQ5 used to generate a photomask, with the dashed line indicating the area that was drawn to generate the photomask. Figure 16B is an image of the same mouse brain section as Figure 16A showing the bound fluorescently barcoded areas after light-induced barcoding using a photomask generated from the image of Figure 16A. Figure 16C is an overlay of the images from Figures 16A and 16B showing the barcoded areas relative to the nuclear stained features on the sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Detailed Description

[0049] Provided herein are compositions, devices, methods and systems for use in accessible and scalable visual selection methods that can directly correlate multi-dimensional high-resolution cellular phenotypes (including morphology, protein markers and spatial organization) with transcriptome, genomic or proteomic profiles for a variety of sample types.The processes described herein are applicable to the analysis of cellular states in tissue, synthetic tissue or solution.Described herein are methods of sample preparation that provide imaging and region selection, methods for in situ barcoding and imaging, and methods of record extraction and sequencing to provide spatially defined data from samples.

[0050] Provided herein is a method for spatial indexing of transcribed nucleic acid in selected regions of interest (ROI) in cells and tissues. In some embodiments, the transcribed nucleic acid is reverse transcribed DNA or transcribed RNA. In some embodiments, barcode sequences from sequencing reads provide spatial indexing data. In some embodiments, spatial indexing data combined with imaging methods previously described herein provides a link between cell phenotype information and transcriptome information.

[0051] Workflow An exemplary workflow for the analysis of biological cells is provided in FIG. 1A. Briefly, a step of sample preparation (e.g., tissue sample or cell population) is performed. Sample preparation step may include cell culture or tissue or organoid acquisition and subsequent imaging. Dyes and / or probes to enhance the visualization of cells and / or structures therein may be applied to the sample. Subsequent imaging provides morphological and architectural information of the sample to aid in region selection, cell segmentation and mask generation. The sample is then permeabilized as necessary. Permeabilization may be performed before imaging the sample or simultaneously with the fixation of the sample.

[0052] According to the workflow, reverse transcription of sample RNA is performed to analyze biological cells in situ, followed by tailing of the resulting target cDNA, if necessary. Tail addition adds non-templated nucleotides to the 3' end of the nucleic acid strand. In some embodiments, tail addition includes ligation of nucleic acid fragments. In some embodiments, tail addition includes extension of the nucleic acid strand. Then, in some examples, spatially directed barcoding in selected regions of interest is performed utilizing a photomask, and region selection for unmasking is based on prior sample imaging. The biological cells are optionally further imaged to show incorporation of the barcode. According to the process described in FIG. 1A, record extraction and sequencing includes displacement of the barcoded nucleic acid. Release is optionally achieved by application of RNaseH. Cross-junction synthesis to generate consecutive nucleic acids can also result in the release of the barcoded nucleic acid. The enzymatic release and synthesis reactions can be combined or performed sequentially in any order. After sequences are extracted from the sample, further imaging or other assays (e.g., mass spectrometry, H&E staining) can be performed on the sample. Subsequent polymerase chain reaction (PCR) amplification and optional purification are followed by optional library preparation and next generation sequencing. The resulting sequence analysis is merged with the captured imaging data to provide an integrated spatial and sequence-based information output.

[0053] Further exemplary workflow is shown in FIG. 1B. Briefly, a step of sample preparation (e.g., tissue sample or cell population) is performed. Sample preparation step may include cell culture or tissue or organoid acquisition followed by imaging. Dyes and / or probes to enhance the visualization of cells and / or structures therein may be applied to the sample. Subsequent imaging provides morphological and architectural information of the sample to aid in region selection, cell segmentation and mask generation. The sample is then permeabilized as required. In this exemplary embodiment, no reverse transcription step is performed. Alternatively, targets in the sample are bound to a recognition binding agent that may be fused or otherwise connected to the nucleic acid that is then barcoded as described in FIG. 1A.

[0054] The fractional spatial barcoding workflow is shown in FIG. 1C. Sample preparation and optional imaging steps include similar methodology to the previous workflow. In this case, a unique ratio of barcodes or barcode permutations is assigned to a specific cell or region of interest (ROI). The duration or power of the applied irradiation energy is controlled to define the fraction of available targets that crosslink with the contacted barcode. FIG. 2A shows an exemplary depiction of controllably increasing the illumination power or time to induce more crosslinking events in a specific region, in this case a cell. Multiple rounds of fractional barcode crosslinking can be applied across a sample, allowing differentiation and identification of cells or regions of interest. FIG. 2B shows an example of a combination of four different barcode sequences applied across a cell or region. After subsequent barcoding and sequencing of a single cell or region, each cell or region of interest can be assigned an individualized split of barcode sequences across its reads, such that the original spatial location of the cell can be recovered based on the fraction of reads from each barcode measured using next-generation sequencing. In some cases, cells or regions are barcoded separately after dissociation of samples.In some embodiments, before sequencing, a sorting method is applied to separate separate regions, such as split pool barcoding, microfluidics, barcoded beads or other single cell barcoding techniques.Each cell or ROI can be identified based on the fraction of photocrosslinked barcode or combinations thereof.

[0055] Sample preparation Cell culture / sample acquisition The spatial barcoding method described herein can be applied to a wide range of materials. Methods for preparing samples for downstream biological analysis steps are provided herein. In some embodiments, the samples described herein comprise a population of cells. In some embodiments, the samples described herein comprise a tissue sample. In some embodiments, the samples described herein comprise a layered cell culture. Non-limiting examples of samples that can be used include: intact tissue, dissected tissue, dissociated cells, organoids, engineered tissue, cultured cells, subcellular compositions, cell suspensions, organs, tissue biopsies, or any combination thereof. In some embodiments, the samples described herein comprise a population of cultured cells. Organoids comprise small self-organized three-dimensional tissue cultures. In some embodiments, the organoids are derived from stem cells. Cultured cells can include, but are not limited to, primary cell cultures, dissociated cells, dissected cells, passaged cell lines, and / or transformed cells. In some embodiments, the population of cells provided herein comprises engineered tissue. In some embodiments, the engineered tissue comprises a population of in vitro differentiated human cells. In some embodiments, the in vitro differentiated human cells are derived from human induced pluripotent stem cells (hiPSCs), embryonic stem cells, or adult stem cells. In some embodiments, the in vitro differentiated human cells are in vitro differentiated cardiomyocytes, in vitro differentiated neurons, in vitro differentiated hepatocytes, in vitro differentiated endothelial cells, in vitro differentiated epithelial cells, in vitro differentiated smooth muscle cells, in vitro differentiated skeletal muscle cells, or in vitro differentiated kidney cells. In some embodiments, the population of cells provided herein comprises retinal cells. In some embodiments, the population of cells provided herein is obtained from a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a primate, a non-human primate, a human, a mouse, a rat, a goat, a rabbit, a dog, a camelid, or a cat.

[0056] In some embodiments, the population of cells comprises a homogenous population of cells (e.g., cells of the same type or species). In some embodiments, the population of cells comprises a heterogenous population of cells (e.g., cells of different types, different species, or any combination thereof). In some embodiments, the population of cells comprises cells from one species. In some embodiments, the population of cells comprises cells from two or more species. In some embodiments, the population of cells comprises one cell type. In some embodiments, the population of cells comprises cells from two or more different cell types. In some embodiments, the population of cells comprises cells from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 250, at least 500, at least 1000 or more species. In some embodiments, the population of cells comprises cells from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 250, at least 500, at least 1000 or more different cell types.

[0057] In some embodiments, the sample used in the methods described herein is a tissue sample. In some embodiments, the tissue sample provided herein comprises connective tissue, epithelial tissue, muscle tissue, or nerve tissue. In some embodiments, the tissue sample provided herein comprises a combination of tissues. An organ comprises different types of tissues and has a specific function. In some embodiments, the combination of tissues is an organ. In some embodiments, the combination of tissues is an organoid. In some embodiments, the tissue sample is obtained from a subject. In some embodiments, the tissue sample provided herein is obtained from the brain, eye, lung, liver, bladder, kidney, heart, stomach, intestine, lymph node, skeletal muscle, smooth muscle, pharynx, larynx, artery, liver, gallbladder, bone, spleen, vein, pancreas, reproductive organ, tumor, infected tissue, or any combination thereof. In some embodiments, the tissue sample provided herein is a whole organ or biopsy tissue. In some embodiments, the biopsy tissue comprises tissue that has or is suspected of having abnormal proliferation or growth. In some embodiments, the biopsy tissue comprises cancer cells.

[0058] In some embodiments, the population of cells described herein comprises one or more microorganisms. In some embodiments, the population of cells comprises prokaryotic or eukaryotic cells. In some embodiments, the population of cells provided herein comprises bacterial cells, fungal cells, archaic cells, eukaryotic cells, or any combination thereof. In some embodiments, the population of cells comprises mammalian cells. In some embodiments, the mammalian cells are human, non-human primate, primate, porcine, equine, ovine, feline, rat, mouse, canine, llama, rabbit, or caprine cells. In some embodiments, the population of cells further comprises a virus. In some embodiments, the virus is a pathogenic virus. In some embodiments, the population of cells comprises a viral vector. In some embodiments, the population of cells is genetically modified. Non-limiting examples of genetic modifications include base editing, gene silencing (e.g., by CRISPR / Cas9 system), gene insertion, and gene deletion.

[0059] In some embodiments, the sample comprises cells. In some embodiments, the sample comprises cells from a suspension. In some embodiments, the sample comprises a whole organism. In some embodiments, the whole organism is a multicellular organism. In some embodiments, the whole organism is a unicellular organism. Examples of multicellular organisms include, without limitation, the roundworm (Caenorhabditis elegans), zebrafish (Danio rerio), and Drosophila. Examples of unicellular organisms include, without limitation, bacteria and yeast.

[0060] Imaging Imaging of the sample is used to guide photomasking for light-induced barcoding. Imaging is also used to visualize barcoding with fluorophore-labeled barcodes. Methods for image capture and enhancement are provided herein that incorporate the application of imaging reagents. The imaging reagents can be used to identify cells, cell types, and / or subcellular structures. In some embodiments, the tissue sample or cell population provided herein is contacted with an imaging reagent. In some embodiments, the imaging reagent is a dye or stain. In some embodiments, the dye or stain comprises 7-AAD, acridine orange, Bismarck brown, calcein, CFSE, carmine, Coomassie blue, cresyl violet, crystal violet, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hoechst stain, iodine, malachite green, methyl green, methylene blue, neutral red, Nile blue, Nile red, Osmium tetroxide (formal name: osmium tetraoxide), propidium iodide, resazurin, rhodamine, safranine, trypan blue, tetrazolium salts, or any combination thereof.

[0061] In some embodiments, the sample is visualized using immunofluorescence (IF) staining. In some embodiments, the IF staining reagent comprises fluorophore-conjugated streptavidin. In some embodiments, the imaging reagent is a fluorophore. Exemplary fluorophores for inclusion in the workflow described herein include 1,5 IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein (pH10); 5-carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-carboxyfluorescein); 5-hydroxytryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-CR 6G;6-JOE;7-amino-4-methylcoumarin;7-aminoactinomycin D (7-AAD);7-hydroxy-4-methylcoumarin;9-amino-6-chloro-2-methoxyacridine;ABQ;Acid Fuchsin;ACMA (9-amino-6-chloro-2-methoxyacridine);Acridine Orange;Acridine Red;Acridine Yellow;Acriflavine;Acriflavine Feulgen SITSA;Aequorin (photoprotein);Alexa Fluor 350(TM);Alexa Fluor 430(TM);Alexa Fluor 488(TM);Alexa Fluor 532(TM);Alexa Fluor 546(TM);Alexa Fluor 568(TM);Alexa Fluor 594(TM);Alexa Fluor 633(TM);Alexa Fluor 647(TM);Alexa Fluor 660(TM);Alexa Fluor 680 (trademark); Alizarin Complexone; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R;Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG(TM) CBQCA; ATTO-TAG(TM) FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine sulfate; Beta-lactamase; BFP Blueshift GFP (Y66H); BG-647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV; BOBO(TM)-1; BOBO(TM)-3; BodiPi 492 / 515; BodiPi 493 / 503; BodiPi 500 / 510; BodiPi 505 / 515; BodiPi 530 / 550; BodiPi 542 / 563; BodiPi 558 / 568; BodiPi 564 / 570; BodiPi 576 / 589; BodiPi 581 / 591; BodiPi 630 / 650-X; BodiPi 650 / 665-X; BodiPi 665 / 676; BodiPi FI; BodiPi FL ATP; BodiPi Fl-Ceramide; BodiPi R6G SE; BodiPi TMR; BodiPi TMR-X Conjugate; BodiPi TMR-X, SE; BodiPi TR; BodiPi TR ATP; BodiPi TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green-1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+;Calcium Orange;Calcium Orange;Calcofluor White;Carboxy-X-Rhodamine (5-ROX);Cascade Blue™;Cascade Yellow;Catecholamines;CFDA;CFP-Cyan Fluorescent Protein;Chlorophyll;Chromomycin A;CMFDA;Coelenterazine;Coelenterazine cp;Coelenterazine f;Coelenterazine fcp;Coelenterazine h;Coelenterazine hep;Coelenterazine ip;Coelenterazine O;Coumarin phalloidin;CPM methylcoumarin;CTC;Cy2™;Cy3.18;Cy3.5™; Cy3™; Cy5.18; Cy5.5™; Cy5™; Cy7™; Cyan GFP; Cyclic AMP Fluorosensor (FiCRhR); d2; Dabsyl; Dansyl; Dansylamine; Dansylcadaverine; Dansyl chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein diacetate) Diacetate);DDAO;DHR(dihydrorhodamine 123);Di-4-ANEPPS;Di-8-ANEPPS(non-ratio);DiA(4-Di-16-ASP);DIDS;dihydrorhodamine 123(DHR);DiO(DiOC18(3));DiR;DiR(DiIC18(7));dopamine;DsRed;DTAF;DY-630-NHS;DY-635-NHS;EBFP;ECFP;EGFP;ELF 97;Eosin;Erythrosine;Erythrosine ITC;Ethidium homodimer-1 (EthD-1);Euchrysin;Europium(III) chloride;Europium;EYFP;Fast Blue;FDA;Feulgen (Pararoseaniline);FITC;FL-645;Flazo Orange;Fluo-3;Fluo-4;Fluorescein diacetate;Fluoro-Emerald;Fluoro-Gold (Hydroxystilbamidine);Fluor-Ruby;FluorX;FM 1-43 (trademark);FM 4-46;Fura Red™ (high pH);Fura-2, high calcium;Fura-2, low calcium;Genacryl Brilliant Red B;Genacryl Brilliant Yellow 10GF;Genacryl Pink 3G;Genacryl Yellow 5GF;GFP (S65T);GFP Redshift (rsGFP);GFP wild type, non-UV excited (wtGFP);GFP wild type, UV excited (wtGFP);GFPuv;Gloxalic Acid;Granular Blue;Hematoporphyrin;Hoechst 33258;Hoechst 33342;Hoechst 34580;HPTS;Hydroxycoumarin;Hydroxystilbamidine (Fluorogold);Hydroxytryptamine;Indodicarbocyanine (DiD);Indotricarbocyanine (DiR);Intrawhite Cf;JC-1;JO-JO-1;JO-PRO-1;LaserPro;Laurodan;LDS 751;Leucophor PAF;Leucophor SF;Leucophor WS;Lissamine Rhodamine;Lissamine Rhodamine B;LOLO-1;LO-PRO-1;Lucifer Yellow;Mag Green;Magdala Red (Phloxine B);Magnesium Green;Magnesium Orange;Malachite Green;Marina Blue;Maxilon Brilliant Flavin 10 GFF;Maxilon Brilliant Flavin 8 GFF; Merocyanine; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD-amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Irvine Iavin) E8G; Oregon Green™; Oregon Green 488-X; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararoseaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-Texas Red (Red 613); Phloxine B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26; PKH67; PMIA;Pontochrome Blue Black;POPO-1;POPO-3;PO-PRO-1;PO-PRO-3;Primulin;Procion Yellow;Propidium Iodide (PI);PyMPO;Pyrene;Pyronine;Pyronine B;Pyrozal Brilliant Flavin 7GF;QSY 7;Quinacrine Mustard;Resorufin;RH 414;Rhod-2;Rhodamine;Rhodamine 110;Rhodamine 123;Rhodamine 5 GLD;Rhodamine 6G;Rhodamine B 540;Rhodamine B 200;Rhodamine B Extra;Rhodamine BB;Rhodamine BG;Rhodamine Green;Rhodamine Phallicidine;Rhodamine Phalloidin;Rhodamine Red;Rhodamine WT;Rose Bengal;R-Phycoerythrin (PE);Redshift GFP (rsGFP, S65T);S65A;S65C;S65L;S65T;Sapphire GFP;Serotonin;Sevron Brilliant Red 2B;Sevron Brilliant Red 4G;Sevron Brilliant Red B;Sevron Orange;Sevron Yellow L;sgBFP(TM);sgBFP(TM) (super glow glow (BFP); sgGFP™; sgGFP™ (Superglow GFP); SITS; SITS (primulin); SITS (stilbene isothiosulfonic acid); SPQ (6-Methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulforhodamine B can C; Sulforhodamine G Extra; Tetracycline; Tetramethylrhodamine; Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5);TRITC (TetramethylRodamine isothiocyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOY; O-1; YOYO-3; or any combination thereof. Many suitable forms of these fluorescent compounds are available and can be used.

[0062] In some embodiments, the sample described herein is imaged using a microscope. In some embodiments, the microscope used in the method described herein includes a compound microscope, a fluorescent microscope, a confocal microscope, a light sheet microscope, a Raman microscope, a digital microscope, or any combination thereof.

[0063] Sample fixation The methods described herein are not limited by sample fixation conditions. In some embodiments, the population of cells is fixed and then frozen. In some embodiments, the population of cells is frozen and then fixed after thawing and / or sectioning. In some embodiments, the fixed population of cells is sectioned and then fixed a second time. In some embodiments, the population of cells is stored at room temperature after fixation. In some embodiments, the population of cells is fixed using organic solvents, such as alcohol and acetone. In some embodiments, the population of cells (e.g., tissue) is embedded in paraffin before fixation. In some embodiments, the population of cells is fixed using a cross-linking reagent. In some embodiments, the cross-linking reagent comprises formalin, formaldehyde, paraformaldehyde, dithio-bis(succinimidyl propionate) (DSP), or any combination thereof.

[0064] In some embodiments, the samples provided herein are frozen at -5°C to -200°C. In some embodiments, the samples provided herein are frozen at about -5°C, about -10°C, about -15°C, about -20°C, about -25°C, about -30°C, about -35°C, about -40°C, about -45°C, about -50°C, about -55°C, about -60°C, about -65°C, about -70°C, about -75°C, about -80°C, about -85°C, about -90°C, about -95°C, about -100°C, or any combination thereof. In some embodiments, the tissue samples or cell populations provided herein are flash frozen. In some embodiments, the tissue samples or cell populations provided herein are frozen by slow freezing or equilibrium freezing.

[0065] In some embodiments, the sample comprises a tissue. In some embodiments, the sectioned tissue is sliced. In some embodiments, the slices are up to about 5 um, about 10 um, about 15 um, about 20 um, about 25 um, about 30 um, about 35 um, about 40 um, about 45 um, about 50 um or thicker.

[0066] In some embodiments, the samples described herein comprise cells. In some embodiments, the cells are attached to a surface. In some embodiments, the cells are in solution. In some embodiments, the cells are attached to a hydrogel. In some embodiments, the cells are attached to a synthetic scaffold. In some embodiments, the cells are attached directly or indirectly to the synthetic scaffold. In some embodiments, the synthetic scaffold comprises beads or a semi-solid gel. In some embodiments, the synthetic scaffold comprises an extracellular matrix protein. In some embodiments, the extracellular matrix protein comprises collagen or fibronectin.

[0067] In some embodiments, the samples provided herein are fixed on a microscope slide or a cover slip. In some embodiments, the microscope slide is a chamber slide. In some embodiments, the chamber slide comprises one or more chambers. In some embodiments, the slide comprises polystyrene. In some embodiments, the slide comprises glass.

[0068] In some embodiments, the slide comprises a coating to promote attachment of the tissue sample or cell population provided herein to the surface of the slide. In some embodiments, the coating comprises poly-L-lysine or poly-D-lysine. In some embodiments, the coating comprises gelatin. In some embodiments, the coating comprises 3-aminopropyltriethoxysilane (APES). In some embodiments, the sample is attached to the slide using adhesive tape. In some embodiments, the adhesive tape is polyvinylidene chloride, polyester / silicone, polypropylene film, cellophane, synthetic rubber-resin hot melt, low density polyethylene film, polyester film, or any combination thereof.

[0069] In some embodiments, the sample is incorporated into a hydrogel. In some embodiments, the hydrogel comprises a synthetic gel or a polyepoxide. In some embodiments, the embedding of the sample occurs in a tissue-hydrogel matrix. In some embodiments, the embedding of the sample provides for the expansion of a tissue sample or a population of cells provided herein. In some embodiments, the hydrogel is a natural hydrogel, a synthetic hydrogel, or a synthetic / natural hybrid hydrogel. In some embodiments, the natural hydrogel is a protein, a polysaccharide, a protein / polysaccharide, DNA, or any combination thereof. In some embodiments, the protein hydrogel comprises collagen, elastin, fibrin, silk, lysozyme, Matrigel® (Corning Inc., Corning, NY), a genetically engineered protein, or any combination thereof. In some embodiments, the polysaccharide comprises hyaluronic acid (HA), alginate, chitosan, dextran, or any combination thereof. In some embodiments, the protein / polysaccharide comprises collagen / HA, laminin / cellulose, fibrin / alginate, gelatin / agarose, chitosan, alginate, dextran, or any combination thereof. In some embodiments, the DNA hydrogel comprises X-DNA, Y-DNA, T-DNA, linear plasmid DNA, or any combination thereof. In some embodiments, the synthetic hydrogel is a non-biodegradable hydrogel, a biodegradable hydrogel, a bioactive hydrogel, or any combination thereof.In some embodiments, the non-biodegradable hydrogel is selected from the group consisting of poly(2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate (HEMA), poly(2-hydroxypropyl methacrylate) (PHPMA), poly(N-isopropylacrylamide (PNIPAm), poloxamer (Pluronic®, ThermoFisher In some embodiments, the biodegradable hydrogel comprises degradable polyethylene glycol (PEG), polypropylene fumarate-PEG (PPF-PEG), poly(2-hydroxyethyl methacrylate)-poly(ε-caprolactone)-poly(p ... In some embodiments, the bioactive hydrogel comprises a cell-adhesive hydrogel, an enzyme-sensitive hydrogel, a growth factor-bearing hydrogel, another bioactive hydrogel, or any combination thereof. In some embodiments, the synthetic / natural hybrid hydrogel comprises PEG / dextran, heparin, HA, chondroitin sulfate (CS), one or more proteins, PNIPAm / protein, chitosan, alginate, synthetic peptide / protein, polysaccharide, PVA / DNA, Pluronic / dextran, PHPMA / protein, or any combination thereof.

[0070] In some embodiments, the samples described herein comprise nucleic acids. In some embodiments, the samples comprise nucleic acids directly or indirectly bound or tethered to a surface. In some embodiments, the samples comprise nucleic acids in solution. In some embodiments, the samples comprise nucleic acids connected to a hydrogel. In some embodiments, the samples comprise nucleic acids in a scaffold. In some embodiments, the nucleic acids are directly or indirectly bound to a synthetic matrix. In some embodiments, the synthetic matrix comprises beads or semi-solid gels.

[0071] Analysis using the methods described herein can be applied to fixed nucleic acids. In some embodiments, the fixed nucleic acids are fixed to a substrate. In some embodiments, the fixation is via formalin / formaldehyde treatment. In some embodiments, the fixation is via ethanedialdehyde or oxalaldehyde treatment. In some embodiments, the fixed nucleic acids are in tissues that are formalin-fixed and paraffin-embedded (FFPE), paraformaldehyde (PFA)-fixed, or fixed using PFA with Triton X-100 (PFA-Tx). Analysis using the methods described herein can be applied to any fixed nucleic acid. The methods described can be used to characterize nucleic acids, proteins, or any combination thereof.

[0072] The immobilized nucleic acid can be conjugated to an affinity molecule or immobilized on a scaffold, substrate, surface, bead or column.The methods described herein can be used to profile in situ hybridization (ISH) probes, DNA conjugated to antibodies, DNA-encoded libraries, CRISPR libraries, viral libraries and / or genomes, nucleic acids covalently linked to cleaned hydrogels, nucleic acid-conjugated proteins, nucleic acids localized on surfaces (e.g., glass slides or microarrays), or sequences generated in situ (e.g., by in vitro transcription (IVT)).

[0073] In some embodiments, the immobilized nucleic acid is conjugated to an affinity molecule. In some embodiments, the affinity molecule comprises a component of a ligand / receptor complex. In some embodiments, the affinity molecule comprises an antibody or a component thereof. In some embodiments, the antibody component is an antibody fragment, a nanobody or other affinity reagent. In some embodiments, the antibody fragment described herein is a F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, an IgG fragment, an Fc fragment, or any combination thereof. In some embodiments, the antibody is an IgA, IgG or IgM antibody or a functional fragment thereof. In some embodiments, the affinity molecule is an aptamer, a probe, an siRNA or a peptide small molecule.

[0074] In some embodiments, the immobilized nucleic acid is conjugated to a scaffold. In some embodiments, the scaffold is biodegradable. In some embodiments, the scaffold is non-biodegradable. In some embodiments, the scaffold is biocompatible. In some embodiments, the scaffold comprises a synthetic material. In some embodiments, the synthetic material comprises silicone, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly-lactic-co-glycolic acid (PLGA), or any combination thereof. In some embodiments, the scaffold comprises a natural material. In some embodiments, the natural material comprises a derivative of an extracellular matrix, collagen, fibrin, polysaccharides including chitosan or glycosaminoglycans (GAG), or any combination thereof. In some embodiments, the glycosaminoglycans (GAG) comprise hyaluronic acid.

[0075] In some embodiments, the immobilized nucleic acid is conjugated to a surface comprising a flexible material. In some embodiments, the flexible material includes, without limitation, modified nylon, unmodified nylon, nitrocellulose, polypropylene, or any combination thereof. In some embodiments, the surface comprises a rigid material. In some embodiments, the rigid material includes, without limitation, glass, fused silica, silicon, silicon dioxide, silicon nitride, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof), metals (e.g., gold, platinum), or any combination thereof. The surfaces described herein can be made of materials including silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), glass, or any combination thereof.

[0076] In some embodiments, the immobilized nucleic acid is conjugated to a bead. In some embodiments, the bead is a magnetic bead. In some embodiments, the bead is an immunoaffinity bead.

[0077] optimization Additional steps may be applied to optimize hybridization or barcoding in the analyzed sample. In some embodiments, a permeabilization step is performed on the sample. In some embodiments, permeabilization is not performed. In some embodiments, permeabilization is performed simultaneously with fixation. In some embodiments, permeabilization is performed after fixation. In some embodiments, the permeabilization reagent comprises acetone, alcohol, detergent, or any combination thereof.

[0078] Fixation may affect protein biochemistry, in some cases masking epitopes or binding sites. The methods described herein may provide for unmasking of binding sites, making the binding sites more available for binding. In some embodiments, the samples described herein are further treated with an antigen retrieval method. In some embodiments, the antigen retrieval method is protease-induced epitope retrieval (PIER). PIER treatment may utilize enzymes, such as proteinase K, trypsin, or pepsin, to unmask antigens in the sample. In some embodiments, the antigen retrieval method is heat-induced epitope retrieval (HIER). The sample may be heated using a microwave, pressure cooker, steamer, autoclave, heat block, hot plate, water bath, or another heat source. The HIER-treated sample may be heated to 50°C to 120°C. In some embodiments, the sample is heated to 75°C to 100°C. In some embodiments, the sample is heated to about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, about 100°C, about 105°C, about 110°C, about 115°C or about 120°C. In some embodiments, the antigen retrieval method is any other method that can reverse the masking of epitopes. In some embodiments, the antigen retrieval step is combined with a permeabilization step.

[0079] In some embodiments, antigen retrieval step is carried out independently.Additional processing can be carried out in conjunction with epitope retrieval.For example, paraffin-embedded samples can be deparaffinized before PIER or HIER.Deparaffinization can be achieved by applying organic solvent, such as xylene, naphthenic solvent, d-limonene or any suitable solvent.

[0080] Barcoding Barcoding can be applied directly to a target in a sample, to a transcription product of the sample, to a reverse transcribed product of the sample, or to another element (e.g., an antibody) that specifically binds to the target, or to a transcription product of the sample. The targets described herein can be anything to which a specific binding product can be applied. The specific binding can be affinity binding, hybridization, ligand / receptor binding, aptamer binding, immunobinding, or any other type of specific binding or combinations thereof. In some embodiments, the nucleic acid in the sample is transcribed to generate a target nucleic acid. In some embodiments, the sample nucleic acid is mRNA. In some embodiments, the target nucleic acid is DNA. In some embodiments, the DNA is nuclear DNA. In some embodiments, the DNA is mitochondrial DNA. In some embodiments, the target nucleic acid is RNA. In some embodiments, the target nucleic acid is an intron. In some embodiments, the target nucleic acid is an exon. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the target nucleic acid is a regulatory RNA. In some embodiments, the RNA is circular RNA (circRNA), ribosomal RNA (rRNA), 16S ribosomal RNA (16S rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), non-coding RNA, long non-coding RNA (lncRNA) or microRNA (miRNA).

[0081] In some embodiments, the target nucleic acid is an amplification product. Nucleic acid amplification may be performed at a constant temperature ("isothermal") to reduce the use of complex external equipment. In some embodiments, the target nucleic acid is generated by isothermal amplification. In some embodiments, the isothermal amplification is performed at ambient and / or room temperature. In some embodiments, the ambient and / or room temperature is about 20°C to 25°C. In some embodiments, the isothermal amplification is rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA). In some embodiments, the target nucleic acid is generated by signal amplification by exchange reaction (SABER). In some embodiments, the target nucleic acid is generated from a synthesis reaction. In some embodiments, the target nucleic acid is generated by primer exchange reaction (PER) or RCA. In some embodiments, the PER is a terminal deoxynucleotidyl transferase (Tdt)-based PER assay. In some embodiments, the target nucleic acid is a long repetitive single-stranded RNA or DNA sequence. In some embodiments, the amplification is performed at multiple temperatures, e.g., in cycles. In some embodiments, the amplification is performed using a non-isothermal, linear or exponential amplification method. In some embodiments, the amplification is PCR. In some embodiments, the PCR is asymmetric PCR. In some embodiments, the amplification is isothermal. Examples of isothermal amplification methods include strand displacement amplification (SDA), rolling circle amplification (RCA), whole genome amplification (WGA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA) and multiple displacement amplification (MDA).

[0082] In some embodiments, the target nucleic acid is conjugated to a molecule. In some embodiments, the target nucleic acid and / or the molecule conjugated to the target nucleic acid is detected by proximity ligation assay (PLA). PLA allows for detection of protein-protein interactions in situ at distances less than about 40 nanometers. In some embodiments, the target nucleic acid is a PLA oligonucleotide. In some embodiments, the barcode nucleic acid provided herein further comprises a PLA oligonucleotide. In some embodiments, the target nucleic acid comprises a first PLA oligonucleotide, and the first PLA oligonucleotide is conjugated to a first protein of interest. In some embodiments, the first PLA oligonucleotide hybridizes to a second PLA oligonucleotide, and the second PLA oligonucleotide is conjugated to a second protein of interest. In some embodiments, the first protein of interest or the second protein of interest is selected from the group consisting of an antibody, an antibody fragment, a cell surface receptor, a cell surface protein and an intracellular protein, a mitochondrial protein, a nuclear protein, a transcription factor, or a scaffolding protein. In some embodiments, the first PLA oligonucleotide and the second PLA oligonucleotide are ligated to form a circular DNA. In some embodiments, the circular DNA is amplified. In some embodiments, the circular DNA comprises a barcode strand provided herein. In some embodiments, the circular DNA is contacted with a barcode strand provided herein. In the PLA reaction, the barcode can be used to identify specific protein-protein interactions or nucleic acid-protein interactions in a sample.

[0083] In some embodiments, the target nucleic acid is exposed to a DNA or RNA polymerase. In some embodiments, polymerase refers to an enzyme that performs template-guided synthesis of polynucleotides. In some embodiments, the DNA polymerase is isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. In some embodiments, the polymerase enzyme includes, but is not limited to, Klenow fragment (New England Biolabs Inc.), Taq DNA polymerase (QIAGEN), 9°N™ DNA polymerase (New England Biolabs Inc.), Deep Vent™ DNA polymerase (New England Biolabs Inc.), Manta DNA polymerase (Enzymatics), Bst DNA polymerase (New England Biolabs Inc.), or phi29 DNA polymerase (New England Biolabs Inc.). In some embodiments, the polymerase is DNA-dependent. In some embodiments, the polymerase is RNA-dependent. In some embodiments, the polymerase is a reverse transcriptase.

[0084] In some embodiments, the primer is bound to an affinity molecule, a scaffold, a substrate, a surface, a bead, or a column. In some embodiments, the primer is in suspension, in solution, or in a hydrogel.

[0085] Reverse transcription The generation of cDNA complementary to the target mRNA may include in situ reverse transcription. In some embodiments, the in situ reverse transcription includes the incorporation of a primer. In some embodiments, the primer describes a short nucleic acid sequence. In some embodiments, the primer is DNA. In some embodiments, the primer is RNA. In some embodiments, the primer has a region complementary to a strand of DNA or RNA. In some embodiments, the primer provides a free 3'-OH at which DNA polymerase initiates synthesis of a deoxyribonucleotide strand. In some embodiments, the primer contains about 5 to about 50 or more nucleotides. In some embodiments, the primer contains about 10 to about 30 nucleotides. In some embodiments, the primers described herein include the 3' sequence NNNNNNN (7N), NNNNNGGG (5N and 3G), NNNNNCCC (5N and 3C), NNNNNAAA (5N and 3A) or NNNNNTTT (5N and 3T).

[0086] In some embodiments, overhangs are added to cDNA by adding poly-A tails. In some embodiments, 3' overhangs are added to cDNA transcripts. In some embodiments, 5' overhangs are added to cDNA transcripts. In some embodiments, tail addition is accomplished through the use of terminal transferase enzymes and dXTP (dATP, dCTP, dGTP, dTTP, or any combination thereof). Addition of ddXTP (ddATP, ddCTP, ddGTP, ddTTP, or any combination thereof) or another terminating nucleotide to the 3' or 5' end prevents subsequent extension during the subsequent cross-junction synthesis step. In some embodiments, ddXTP or another terminating nucleotide is included at a low concentration to randomly terminate the 3' or 5' end. In some embodiments, other strategies are used to add 3' or 5' overhangs. In some embodiments, overhangs are added by ligation. In some embodiments, overhangs are added by extension.

[0087] target binding As described in FIG. 1B, the barcoding of the sample includes target binding. In some embodiments, the target binding is affinity binding. The affinity binding may include binding to a ligand, epitope, aptamer, protein, receptor, small molecule, or any other specific recognition binding. In some embodiments, the target binding is hybridization. The hybridization may include hybridization of nucleic acid strands, including but not limited to deoxyribonucleic acid (DNA) strands, ribonucleic acid (RNA) strands, peptide nucleic acid (PNA) strands, locked nucleic acid (LNA) strands, and combinations thereof. For example, the hybridization may include hybridization of RNA to RNA, DNA to DNA, LNA to LNA, RNA to DNA, RNA to PNA, RNA to LNA, DNA to LNA, DNA to PNA, PNA to PNA, etc.

[0088] In some embodiments, the target is recognized by an antibody, an antibody fragment, a protein, a peptide, a nanobody, a small molecule, a nucleic acid, a probe, an aptamer, siRNA, a lipid, a nanoparticle, a lipid nanoparticle or other affinity reagent.

[0089] Barcode A method of information labeling by nucleic acid is provided herein. Information labeling as described herein provides non-genetic information. In some embodiments, the non-genetic information includes spatial information in the sample. In some embodiments, the sample is a two-dimensional sample. In some embodiments, the sample is a three-dimensional sample. In some embodiments, the non-genetic information includes sample information, such as tube number, well number, sample source, charge, mass or other sample characteristics, or any combination thereof. In some embodiments, the non-genetic information includes digital information. The method provided herein allows for the placement of barcodes on a surface. Such barcodes can carry information about target identity, substrate, timing, location, strength, charge, affinity, etc. In this way, the barcode provides a nucleic acid medium for digital information storage. In some embodiments, the barcode further includes a unique molecular identifier (UMI).

[0090] In some embodiments, the population of cells for analysis provided herein are live cells. In some embodiments, the live cells are contacted with the barcode sequence directly or indirectly through a linking molecule. In some embodiments, the linking molecule binds to a cell surface receptor. In some embodiments, the linking molecule binds to a cell surface molecule. In some embodiments, the cell surface molecule is a protein or a nucleic acid. The nucleic acid can be RNA or DNA. The protein can be a cell receptor. In some embodiments, the cell surface molecule is located at a cell attachment point. In some embodiments, the barcode sequence is introduced intracellularly into the live cells. In some embodiments, the barcode sequence is attached to a lipid nanoparticle (LNP) as a carrier for delivery into the cells. In some embodiments, the cells are transfected or transformed to receive the barcode sequence. In some embodiments, the cells are mechanically perturbed to allow cellular uptake of the barcode sequence.

[0091] In some embodiments described herein, the method includes barcoding a target in a sample. In some embodiments, the target is a nucleic acid, a protein, an antigen, a lipid, a sugar, a cellular structure, a scaffold, or any combination thereof. In some embodiments, the target is a nucleic acid and is generated in situ. In some embodiments, cDNA is generated from a nucleic acid in a sample via in situ transcription. In some embodiments, the in situ transcription is reverse transcription. In some embodiments, RNA is generated in situ from a partially or fully double-stranded DNA via transcription.

[0092] Barcodes provided herein generally encode non-genetic data. In some embodiments, the barcode strand is a single stranded nucleic acid. The introduction of hybridization domains into the barcode strand allows for any arrangement of barcodes via repeated hybridization, cross-linking and washing steps. In some embodiments, the nucleic acid barcode strand comprises a barcode domain. In some embodiments, the barcode domain is about 5 to about 10 bases in length. In some embodiments, the barcode domain is flanked by a set of two alternating hybridization domains. In some embodiments, the barcode domain is flanked by separate hybridization domains. In some embodiments, the barcode domain is flanked by an alternating set of three, four, five, six or more hybridization domains. In some embodiments, the hybridization domain is a cross-linking domain. In some embodiments, the hybridization domain is a complementary strand. In some embodiments, the set of two alternating hybridization domains comprises one cross-linking strand and one complementary strand. In some embodiments, the nucleic acid barcode comprises a barcode domain linked to (i) a bridge strand comprising photoactivated nucleotides and (ii) a complementary strand. In some embodiments, the alternating hybridization domains can be at least 5, at least 10, at least 15, or at least 20 bases in length. In some embodiments, the alternating hybridization domains comprise bases for hybridizing to a hybridization domain of a second nucleic acid barcode. In some embodiments, the bridge strand of one nucleic acid barcode hybridizes to the complementary strand of another nucleic acid barcode.

[0093] Methods for barcode placement provided herein include, without limitation, the application of a force to move the barcode from a reservoir to a sample. Examples of forces for application include laminar pressure, capillary pressure, slip flow pressure, magnetic force, electrostatic force, peristaltic force, acoustic force, vibration force, centripetal force, centrifugal force, or any combination thereof.

[0094] In some embodiments, a buffer exchange process is employed for barcode placement. In some embodiments, an electrophoretic process is employed for barcode placement. In such a process, the movement of charged particles (e.g., nucleic acid barcode molecules) in an electric field is due to electrophoresis, and an electrode is in the vicinity of a substrate containing cells. In some embodiments, electrophoresis is applied to remove nucleic acids from a surface. In some embodiments, electrophoresis is applied to remove concatemers described herein from a surface. The electrodes may create a uniform field across the surface, or specific electrodes may be used for each region of interest. In some embodiments, a temperature change is employed for barcode placement. In further embodiments, the barcode sequence includes a temperature-sensitive feature, and an increase in temperature can make available hybridization regions that are unavailable at lower temperatures. In some embodiments, the ROI includes a heating unit or electrode adjacent to the ROI sufficient to increase the temperature in the ROI. In some embodiments, the barcode comprises a melting temperature and the ROI is heated to reach the barcode melting temperature, thereby allowing selective hybridization of the barcode(s).

[0095] The exemplary methods described herein can be further combined to incorporate any combination of the methods described. For example, samples barcoded with an iterative construction of barcodes can be further tagged using the fractional barcoding methods described herein.

[0096] The nucleic acids described herein may be non-specifically or specifically conjugated to another molecule (e.g., a protein, an epitope, or another nucleic acid) in the sample. In some embodiments, the barcode is conjugated to a molecule. In some embodiments, the barcode comprises a nucleic acid conjugated to an antibody, an antibody fragment, a protein, a peptide, a nanobody, a small molecule, a nucleic acid therapeutic, a probe, an aptamer, an siRNA, a lipid, a nanoparticle, a lipid nanoparticle, or another affinity reagent. In some embodiments, the antibody fragment described herein is a F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, an IgG fragment, an Fc fragment, or any combination thereof. In some embodiments, the lipid is an emulsion, a liposome, a particle, or any combination thereof. In some embodiments, the antibody is an IgA, IgG, or IgM antibody or a functional fragment thereof. In some embodiments, the tissue sample or population of cells provided herein is imaged after barcoding.

[0097] In some embodiments, the barcodes provided herein are disposed on the sample at a concentration of about 20 nM to about 500 nM. In some embodiments, the barcodes described herein are disposed on the sample at a concentration of up to about 1 nM, 10 nM, 20 nM, 100 nM, 250 nM, 500 nM, 1000 nm or more. Barcode binding to the target region can be stable or transient.

[0098] In some embodiments, the nucleic acid barcode domain is about 5-10 bases in length. In some embodiments, the nucleic acid barcode domain is 4, 5, 6, 7, 8, 9, 10 or more bases in length. In some embodiments, the nucleic acid barcode domain is about 10 or about 10-15 bases in length. Individual barcode domains can be assigned a bit value of "0" or "1". In some embodiments, a concatenated string of nucleic acid barcodes is equivalent to a string of 0s and 1s.

[0099] Photoinduced crosslinking The nucleic acid barcode strands may be concatenated and covalently fixed by a photochemical cross-linking reaction. In some embodiments, the cross-linking reaction is light-driven. In some embodiments, the cross-linking reaction is chemically driven. In some embodiments, the cross-linking reaction is pH-driven. In some embodiments, the cross-linking reaction may be carried out in an aqueous solution. In some embodiments, a photoreactive cross-linker is used in the reaction. In some embodiments, a photoreactive nucleobase is used in the reaction. In such embodiments, the cross-linked strands of the nucleic acid barcode include at least one photoreactive nucleobase. In some embodiments, the photoreactive nucleobase may be any modified nucleobase capable of forming a cross-link with another nucleobase in the presence of light. In some embodiments, the photoreactive nucleobase is a modified pyrimidine or purine nucleobase. In some embodiments, the photoreactive nucleobase includes 2-hydroxycarbazole, 3-cyanovinylcarbazole phosphoramidite (CNVK) or 3-cyanovinylcarbazole modified d-threoninol (CNVD). In some embodiments, the photoreactive nucleobase comprises vinyl, acrylate, N-hydroxysuccinimide, amine, carboxylate or thiol chemical groups. In some embodiments, the photoreactive nucleobase comprises bromo-deoxyuridine. In some embodiments, the CNVK crosslinking base exhibits the highest crosslinking efficiency with the thymine (T) base located adjacent to it on the complementary strand, and can be directly incorporated into the DNA hybridization domain itself as a base replacement.

[0100] The nucleic acid barcode strands may be linked and covalently fixed by a photochemical cross-linking reaction. In some embodiments, the cross-linking reaction is light-driven. In some embodiments, the cross-linking reaction is chemically driven. In some embodiments, the cross-linking reaction is pH-driven. In some embodiments, the cross-linking reaction may be carried out in an aqueous solution. In some embodiments, a photoreactive cross-linker is used in the reaction. In some embodiments, a photoreactive nucleobase is used in the reaction. In such embodiments, the cross-linked strand of the nucleic acid barcode comprises at least one photoreactive nucleobase. In some embodiments, the photoreactive nucleobase may be any modified nucleobase capable of forming a cross-link with another nucleobase in the presence of light. In some embodiments, the photoreactive nucleobase is a modified pyrimidine or purine nucleobase. In some embodiments, the photoreactive nucleobase comprises 2-hydroxycarbazole, 3-cyanovinylcarbazole phosphoramidite (CNVK) or 3-cyanovinylcarbazole modified d-threoninol (CNVD). In some embodiments, the photoreactive nucleobase comprises a vinyl, acrylate, N-hydroxysuccinimide, amine, carboxylate or thiol chemical group. In some embodiments, the photoreactive nucleobase comprises a bromo-deoxyuridine. In some embodiments, the CNVK crosslinking base exhibits the highest crosslinking efficiency with a thymine (T) base on the complementary strand located adjacent to it, and can be directly incorporated into the DNA hybridization domain itself as a base replacement. In some embodiments, the crosslinking reaction is carried out using 365 nm wavelength light, as shown in FIG. 3, and is completed within one second. In some embodiments, multiple photocrosslinker molecules are incorporated such that different barcodes contain different photocrosslinker molecules capable of crosslinking at different wavelengths. Such differences in the barcodes allow for a method of varying the crosslinking events in one or more regions of interest. In some embodiments, a reverse crosslinking reaction is carried out. For example, application of a 312 nm wavelength for several minutes can provide a reversal of the crosslinking. In some embodiments, a series of crosslinking, followed by decrosslinking reactions is carried out to form concatemers.In some embodiments, molecules are disposed on substrates provided herein to locally generate wavelengths of light to support a light-sensitive crosslinking reaction.

[0101] In some embodiments, the irradiation is provided at a specific wavelength or range of wavelengths. The crosslinking reaction can be designed to use any wavelength of visible or ultraviolet light. In some embodiments, the irradiation is ultraviolet (UV) irradiation. In some embodiments, the irradiation is provided at a wavelength ranging from about 300 nanometers (nm) to about 450 nm. In some embodiments, the irradiation is provided at a wavelength of about 300 nm, about 305 nm, about 310 nm, about 315 nm, about 320 nm, about 325 nm, about 330 nm, about 335 nm, about 340 nm, about 345 nm, about 350 nm, about 355 nm, about 360 nm, about 365 nm, about 370 nm, about 375 nm, about 380 nm, about 385 nm, about 390 nm, about 395 nm, about 400 nm, about 405 nm, about 410 nm, about 415 nm, about 420 nm, about 425 nm, about 430 nm, about 435 nm, about 440 nm, about 445 nm, about 450 nm, or any combination thereof. In some embodiments, the irradiation is provided at a wavelength of about 365 nm. In some embodiments, the irradiation is provided at a wavelength of about 405 nm.

[0102] In some embodiments, the illumination is provided for a particular amount of time. In some embodiments, the illumination is provided for between about 1 second and 10 seconds. In some embodiments, the illumination is provided for between about 100 milliseconds (ms) and 10 seconds. In some embodiments, the illumination is provided for at least about 10 ms or more, at least about 100 ms or more, at least about 500 ms or more, at least about 600 ms or more, at least about 700 ms or more, at least about 800 ms or more, at least about 900 ms or more, at least about 1 second or more, at least about 2 seconds or more, at least about 3 seconds or more, at least about 4 seconds or more, at least about 5 seconds or more, at least about 6 seconds or more, at least about 7 seconds or more. , at least about 8 seconds or more, at least about 9 seconds or more, at least about 10 seconds or more, at least about 11 seconds or more, at least about 12 seconds or more, at least about 13 seconds or more, at least about 14 seconds or more, at least about 15 seconds or more, at least about 16 seconds or more, at least about 17 seconds or more, at least about 18 seconds or more, at least about 19 seconds or more, at least about 20 seconds or more, or at least about 30 seconds or more. In some embodiments, the crosslinking reaction can be completed within 0.01, 0.1, 0.25, 0.5, 1, 5, or 10 seconds. In some embodiments, the crosslinking reaction can be completed within 0.5, 1, 5, 10, 20, 30, 40, 50, or 60 minutes.

[0103] In some embodiments, the crosslinking reaction is carried out using 365 nm wavelength light, as shown in FIG. 3, and is completed within 1 second. In some embodiments, multiple photocrosslinker molecules are incorporated such that different barcodes contain different photocrosslinker molecules capable of crosslinking at different wavelengths. Such differences in the barcodes allow for a method of varying the crosslinking events in one or more regions of interest. In some embodiments, a reverse crosslinking reaction is carried out. For example, application of a 312 nm wavelength for several minutes can provide a reversal of the crosslinking. In some embodiments, a series of crosslinking, followed by decrosslinking, is carried out to form concatemers. In some embodiments, molecules are placed on the substrates provided herein to locally generate wavelengths of light to support the photosensitive crosslinking reaction.

[0104] In some embodiments, the crosslinking reaction has negligible effect on bases adjacent to the photoreactive nucleobase. In some embodiments, other photochemical nucleic acid crosslinkers, including psoralens, are used in combination with nucleic acid barcodes.

[0105] In some embodiments, a cross-linking reaction is applied to the nucleic acid barcode strand hybridized to the 5' end of the cDNA. In some embodiments, a cross-linking reaction is applied to the nucleic acid barcode strand hybridized to the 3' end of the cDNA. In some embodiments, additional barcode strands are iteratively added to either the 5' or 3' end of the cDNA-nucleic acid barcode structure using binding and cross-linking as previously described. Figure 4 shows a process involving iterative addition of barcode strands to the 5' end of the cDNA according to the methods described herein. In some embodiments, a cross-linking reaction is performed simultaneously with hybridization.

[0106] Cycles of hybridization and cross-linking can be repeated without a washing step to remove uncross-linked barcode strands. In such embodiments, the barcode strands are designed with different or alternating hybridization domains such that only subsequently added barcode strands hybridize and cross-link to existing target / barcode concatemers.

[0107] In some embodiments, the crosslinked strand is a single stranded nucleic acid 200 nucleotides or less in length. In some embodiments, the crosslinked strand can be 5-100, 5-75, 5-50, 5-25, 5-15, or 5-10 nucleotides in length. In some embodiments, the crosslinked strand can be 10-100, 10-50, 10-25, 10-20, 15-25, or 15-25 nucleotides in length. In some embodiments, the crosslinked strand can be 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the crosslinked strand comprises at least one photoreactive nucleotide. In some embodiments, the crosslinked strand comprises at least one 3-cyanovinylcarbazole nucleoside (CNVK) or 3-cyanovinylcarbazole modified d-threoninol (CNVD) nucleotide. In some embodiments, the crosslinking strand is complementary to the complementary strand, the crosslinking strand being a component of a first nucleic acid barcode, and the complementary strand being a component of a second nucleic acid barcode. In some embodiments, the crosslinking strand is DNA or RNA. In some embodiments, the crosslinking strand is further functionalized with a moiety such as a fluorophore, a quantum dot, biotin, streptavidin, a functional chemical group, an acrydite, a maleimide, a lipid, a metal isotope, another tag (e.g., an antibody) or a nanoparticle.

[0108] In some embodiments, the nucleic acid barcode strand is at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In some embodiments, the nucleic acid barcode strand is 20 nucleotides in length. In some embodiments, the nucleic acid barcode strand comprises a barcode domain 2-10 nucleotides in length, a bridging strand 2-12 nucleotides in length, and a complementary strand 2-12 nucleotides in length. In some embodiments, the nucleic acid barcode strand comprises the following structure in the 5' to 3' direction: 5'-bridging strand-barcode domain-complementary strand-3'. In some embodiments, the nucleic acid barcode strand comprises the following structure in the 5' to 3' direction: 5'-complementary strand-barcode domain-bridging strand-3'.

[0109] The crosslinking strand may be designed to stably or transiently bind to the complementary strand. In some embodiments, the crosslinking strand may be covalently or non-covalently bound to the complementary strand. In some embodiments, the crosslinking strand may be complementary to the complementary strand. The crosslinking strand may be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% complementary to the complementary strand. Furthermore, the crosslinking strand may bind to complementary domains of different nucleic acid barcodes. In some embodiments, the crosslinking strand may bind to complementary domains of a nucleic acid that includes its own crosslinking strand but does not include a barcode strand.

[0110] Area Selection Methods are provided herein in which cells (or subcellular compartments, e.g., nuclei) of a sample are sorted by defined features before or after barcoding or tagging. In some embodiments, cells are sorted before every iteration of barcoding. In some embodiments, cells are sorted before any barcoding step. In some embodiments, cells are sorted after every barcoding step. In some embodiments, cells are sorted in one step based on a feature, e.g., a surface receptor. In further embodiments, cells are sorted in a second step based on a second feature, e.g., gene expression. In some embodiments, cells are sorted based on a first feature and barcoded using the methods described herein. In further embodiments, barcoded cells are further sorted based on a second feature and, if necessary, further barcoded.

[0111] Provided herein is a method for nucleic acid barcoding, including cell sorting. In some embodiments, cell sorting is performed using fluorescence-activated cell sorting (FACS) analysis. In some embodiments, cell sorting is performed using a microfluidic device. In some embodiments, cell sorting is performed using flow cytometry. In some embodiments, cell sorting is performed using magnetic cell sorting. In some embodiments, nuclei or assortments of multiple cells are sorted, rather than individual cells.

[0112] In some embodiments, sample imaging provides a basis for identification of a cellular region of interest (ROI). In some embodiments, the ROI comprises an area of ​​the sample targeted for spatially defined barcoding of cDNA. In some embodiments, the ROI is based on the morphology of the sample, for example, cells, cellular structures, tissue layers, or subcellular structures. In some embodiments, the ROI has regular or irregular boundaries. In some embodiments, the ROI describes boundaries for spatially defined barcoding in the sample. In some embodiments, the ROI is based on a grid. In some embodiments, the ROI is two-dimensional (2D). In some embodiments, the ROI is three-dimensional (3D).

[0113] In some embodiments, the barcodes are placed in the ROI in a preselected pattern across the surface provided herein. In some embodiments, the pattern forms a grid of repeating shapes across an area, or alternating shapes are preselected for barcode placement. In some embodiments, the pattern forms a grid of repeating shapes across an area, and less than all shapes are preselected for barcode placement. In some embodiments, the shapes are squares, rectangles, triangles, ellipses, diamonds, or spheres. In other embodiments, the shapes can be complex geometric or non-geometric patterns (e.g., tissue or cell feature outlines, alphanumeric characters).

[0114] In some embodiments, light-induced crosslinking is selectively applied to the cell sample. Crosslinking is selectively performed by shielding areas from irradiation, allowing crosslinking only in the unshielded areas. The photomask provides a template for shielding areas of the sample that receive the irradiated light. In some embodiments, the photomask is generated to allow irradiation of one or more regions of interest (ROIs). In some embodiments, the photomask is manually generated or machine generated. In some embodiments, one or more photomasks are generated. In some embodiments, a photomask is generated in association with each ROI. In some embodiments, the photomask is generated according to the sample imaging previously described herein. In some embodiments, the photomask is applied to the sample prior to irradiation. In some embodiments, each crosslinking step includes a different photomask. In some embodiments, multiple crosslinking steps use the same photomask.

[0115] The ROI defines an area for crosslinking of one or more targets within the sample. In some embodiments, the one or more ROIs define separate areas. In some embodiments, the one or more ROIs are congruent. In some embodiments, the one or more ROIs overlap. In some embodiments, the one or more ROIs are isolated. In some embodiments, the one or more ROIs are not isolated.

[0116] In some embodiments, in addition to or instead of a photomask as described herein, light is focused on one or more ROIs. In some embodiments, light is focused using a digital micromirror device (DMD). In some embodiments, light is focused using a confocal microscope. In some embodiments, the confocal microscope is a laser scanning confocal microscope. In some embodiments, light is focused using a DVD writer. In some embodiments, light is focused using a Blu-ray disc writer. In some embodiments, the light source is an entity separate from another device, e.g., the microscope. In some embodiments, the light source is a laser. In some embodiments, the laser has a dominant wavelength of about 180 nm to about 1 mm. In some embodiments, the laser has a dominant wavelength of about 180 nm to about 400 nm. In some embodiments, the laser has a dominant wavelength of about 400 nm to about 700 nm. In some embodiments, the laser has a dominant wavelength of about 700 nm to about 1 mm.

[0117] The optical system used with the methods described herein can provide selective z-plane illumination. In some embodiments, the optical system can provide light in the z-plane having a resolution of about 2 nm to about 10 μm, about 5 nm to about 9 μm, about 10 nm to about 8 μm, about 15 nm to about 7 μm, about 20 nm to about 6 μm, about 25 nm to about 5 μm, about 30 nm to about 4 μm, about 35 nm to about 3 μm, about 40 nm to about 2 μm, about 45 nm to about 1 μm, about 50 nm to about 800 nm, about 100 nm to about 600 nm, about 200 nm to about 400 nm, or any range of resolution therebetween. In some embodiments, the light in the z-plane has a resolution of 10 μm or greater. In some embodiments, the light is focused on the surface of the tissue sample or population of cells provided herein. In some embodiments, the light is focused on a plane that includes at least a portion of the tissue sample or cell population provided herein. In some embodiments, the light is focused on one or more planar surfaces on or within the tissue sample or cell population provided herein. In some embodiments, the light-induced crosslinking described herein is applied to the surface of the tissue sample or cell population provided herein. In some embodiments, the light-induced crosslinking described herein is applied across multiple planes within the tissue sample or cell population. In some embodiments, the light-induced crosslinking described herein is applied on multiple planes of the three-dimensional tissue sample or cell population provided herein. Exemplary devices for illumination restricted to the z-plane include two-photon microscopes and light sheet microscopes.

[0118] In some embodiments, the optical system used with the methods described herein can provide selective xy-plane illumination. In some embodiments, the optical system can provide light in the xy-plane with a resolution of about 2 nm to about 10 μm, about 5 nm to about 9 μm, about 10 nm to about 8 μm, about 15 nm to about 7 μm, about 20 nm to about 6 μm, about 25 nm to about 5 μm, about 30 nm to about 4 μm, about 35 nm to about 3 μm, about 40 nm to about 2 μm, about 45 nm to about 1 μm, about 50 nm to about 800 nm, about 100 nm to about 600 nm, about 200 nm to about 400 nm, or any range therebetween. In some embodiments, the light in the xy-plane has a resolution of 10 μm or greater.

[0119] In some embodiments, the nucleic acids described herein are covalently linked to at least one other nucleic acid via a single photoreactive element of the hybridization domain.

[0120] Fraction Barcoding By irradiating for longer time durations or with higher intensities, the number of crosslinks formed in 1D, 2D or 3D regions can be controllably increased in specific regions, cells, or on samples or strands in test tubes. Intensity is measured in watts per square centimeter (W / cm 2 ) is a radiometric quantity measured in W / cm. Because irradiation exposure time and power levels can be precisely controlled, the fraction of crosslinks formed within a particular area or volume can be precisely controlled (e.g., on a microscope, with an LED or other light source onto a sample or test tube). Such adjustment allows for region-specific light-sensitive crosslinking of barcode events, thereby allowing for preselected gradations ("fractionation") for any given barcode (or tag) for attachment to targets within a particular region of interest (tissue region, cell, subcellular region, microwell, tube, etc.). In some embodiments, the light intensity is from about 1 to about 5000 W / cm. 2In some embodiments, the light intensity is up to about 1 W / cm 2 In some embodiments, the light intensity is up to about 5000 W / cm 2 In some embodiments, the light intensity is at least 2 In some embodiments, the light intensity is 5000 W / cm 2 The above intensities are for focusing the intensity into a planar light. Similar intensities can be applied in 3D, in which case the intensities are expressed in W / cm 3 In the case of irradiating a solution, the volume of the sample is irradiated. In such instances, the intensity is applied per volume, for example with a UV gun.

[0121] The duration or power of the irradiation exposure allows for controllable adjustment of the fraction of crosslinks formed in a particular region of interest (ROI). In other words, the fraction of sequences crosslinked to barcodes in a particular ROI can be controlled by the irradiation exposure time or intensity level. In some embodiments, up to about 100% of the barcodes are crosslinked after irradiation. In some embodiments, 1, 5, 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99% or any percentage therebetween of the barcode sequences in the ROI are photocrosslinked to the target sequence(s) after irradiation. In some embodiments, about 10% of the barcodes in the ROI are photocrosslinked to one or more target sequences after irradiation. In some embodiments, about 20% of the barcodes in the ROI are photocrosslinked to one or more target sequences after irradiation. In some embodiments, about 30% of the barcodes in the ROI are photocrosslinked to one or more target sequences after irradiation. In some embodiments, about 40% of the barcodes in the ROI are photocrosslinked to one or more target sequences after irradiation. In some embodiments, about 50% of the barcodes in the ROI are photocrosslinked to one or more target sequences after irradiation. In some embodiments, about 60% of the barcodes in the ROI are photocrosslinked to one or more target sequences after irradiation. In some embodiments, about 70% of the barcodes in the ROI are photocrosslinked to one or more target sequences after irradiation. In some embodiments, about 80% of the barcodes in the ROI are photocrosslinked to one or more target sequences after irradiation. In some embodiments, about 90% of the barcodes in the ROI are photocrosslinked to one or more target sequences after irradiation.

[0122] The duration or power of illumination, or both, may be modulated across a gradient to provide a graded series of increasing or decreasing fractions of barcoding response across one or more ROIs. The gradient of duration or power may range from 0% to 100%, 0% to 50%, 50% to 100%, 0% to 20%, 20% to 40%, 40% to 60%, 60% to 80%, 80% to 100%, 0% to 35%, 35% to 70%, 70% to 100%, or any combination or interval therein.

[0123] Barcoding can also be modulated through the introduction of an amount of interfering nucleic acid strand in solution during the hybridization step, the irradiation step, or both steps. In some embodiments, the interfering nucleic acid binds to the target nucleic acid so as to prevent the barcoding reaction. In some embodiments, the interfering nucleic acid binds to the barcode strand so as to prevent the barcoding reaction. Modulation of the barcoding reaction can be effected by varying the amount of interfering nucleic acid strand added. In some embodiments, the amount of interfering nucleic acid strand is a percentage of the calculated target nucleic acid in the sample. In some embodiments, the amount of interfering nucleic acid strand is a percentage of the barcode strand added. In some embodiments, the interfering nucleic acid strand is added in an amount of about 1% to about 1000% of either the target nucleic acid or the barcode strand. In some embodiments, the interfering nucleic acid strand is added in an amount of about 1% to about 100%, about 100% to about 500%, about 500% to about 1000%, about 1% to about 10%, about 10% to about 20%, about 20% to about 30%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% of either the target nucleic acid or the barcode strand.

[0124] A population of targets described herein within a sample may be coded for identification using one or more fractional applications of barcodes. Binary, ternary, quaternary or higher-ary encoding may be used. In some embodiments, any combination of fractional applications of distinct barcodes that allows for the differentiation of barcode fractions may be used. The fractional barcodes may be read using sequencing techniques, fluorescent imaging, spectrophotometry or any other detection method. In an exemplary embodiment, one ROI in a sample contains 40% barcode1 + 20% barcode2 + 20% barcode3, while another ROI contains 20% barcode1 + 40% barcode2 + 20% barcode3. In some embodiments, the fractions do not sum to 100%. In some embodiments, the fractions do sum to 100%. In some embodiments, the fractions are distinguishable and deconvoluted.

[0125] Barcode Processing The methods described herein include application of reagents, including barcode strands, washing sequences, and the like, to the sample. In some embodiments, the application method includes conventional microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handlers (e.g., Echo Liquid Handler®, Beckman Coulter, Indianapolis, IN) for depositing new solutions, inkjet printers with modified content, automated liquid handlers incorporating pipetters as needed, robotic arms for handling slides between solution baths, or any combination thereof. In some embodiments, the application method is performed on the stage of a device (e.g., a microscope). In some embodiments, the application method is performed off the stage of a device (e.g., a microscope).

[0126] In some embodiments, the diffusion of nucleic acids is enhanced by the addition of a molecular crowding agent. In some embodiments, the crowding agent comprises a polymer, a protein, or any combination thereof. In some embodiments, the polymer comprises polyethylene glycol. In some embodiments, the protein comprises serum albumin, e.g., bovine serum albumin. In some embodiments, the diffusion of nucleic acids is enhanced by heating the tissue sample or population of cells provided herein. In some embodiments, the diffusion is enhanced by heating to a temperature greater than 30°C, greater than 35°C, greater than 40°C, greater than 45°C, or greater than 50°C. In some embodiments, the diffusion is enhanced by heating to about 30°C, about 35°C, about 37°C, about 40°C, about 45°C, or about 50°C.

[0127] The sample may be treated to prevent or reduce non-specific binding of the barcode strand. In some embodiments, a blocking agent is added to the tissue sample or cell population provided herein prior to application of the barcode strand. In some embodiments, a blocking agent is added to the tissue sample or cell population provided herein concurrently with application of the barcode strand. In some embodiments, the blocking agent comprises a non-specific nucleic acid sequence, sheared salmon sperm DNA, tRNA, yeast tRNA, single-stranded oligonucleotide, double-stranded oligonucleotide, polysaccharide, charged polysaccharide, negatively charged polysaccharide, negatively charged molecule, charged molecule bovine serum albumin (BSA), dry milk, detergent, non-ionic polymeric surfactant (e.g., poly(ethylene oxide) / poly(propylene oxide) triblock copolymer-PLURONIC™ F-127, Gibco® Pluronic® F-68), crowding agent, or any combination thereof. In some embodiments, ribosomal depletion or targeted depletion of highly abundant sequences is performed before any of the following: reverse transcription, barcoding, sequence extraction, cross-junction synthesis or PCR amplification. In some embodiments, ribosomal depletion or targeted depletion of highly abundant sequences is performed after PCR amplification. In some embodiments, in situ hybridization (ISH) probes targeting highly abundant sequences are included before or during reverse transcription to block certain highly abundant sequences from acting as binding sites for reverse transcription primers. In some embodiments, ISH probe binding is combined with nuclease activity (e.g., RNaseH) to specifically digest highly abundant sequences in situ before reverse transcription. In some embodiments, double-stranded specific nuclease (e.g., dsDNAse) is applied after PCR to specifically de-enrich highly abundant sequences.

[0128] In some embodiments, after irradiation and crosslinking reaction, the non-crosslinked barcodes are removed. In some embodiments, the non-crosslinked barcodes are removed by washing. In some embodiments, the non-crosslinked barcodes are not removed before additional cycles of crosslinking. In some embodiments, the sample is washed with a chemical denaturant solution. In some embodiments, the chemical denaturant solution includes one or more salts. In some embodiments, the salt is NaCl. In some embodiments, the salt concentration is 0.1M to 2M. In some embodiments, the salt concentration is about 0.1M, about 0.2M, about 0.3M, about 0.4M, about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1.0M, about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.8M, about 1.9M, about 2.0M. In some embodiments, the chemical denaturant is formamide. In some embodiments, the chemical denaturant is ethylene carbonate. In some embodiments, the formamide solution is 50-60% formamide. In some embodiments, the denaturant solution is in PBS buffer. In some embodiments, the denaturant solution includes a detergent. In some embodiments, the concentration of the detergent in the denaturant solution is about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1.0%, about 2%, about 3%, about 4%, or about 5% by volume. In some embodiments, the detergent is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 (TWEEN® 20, Merck KGaA, Darmstadt, Germany) or polysorbate 80 (TWEEN® 80, Merck KGaA, Darmstadt, Germany). In some embodiments, the detergent is a non-ionic surfactant. In some embodiments, the non-ionic surfactant is a non-ionic polyoxyethylene detergent. In some embodiments, the detergent is polyethylene glycol (PEG), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (CAS number: 9002-93-1), N,N dimethyltetradecylamine N-oxide (TDAO, CAS number: 3332-27-2).In some embodiments, the cleaning agent is Triton X-100, Nereid, TDAO, or a combination thereof. In some embodiments, the cleaning is performed at room temperature. In some embodiments, the cleaning is performed at a temperature warmer or colder than room temperature. In some embodiments, the cleaning is performed at 4°C, 16°C, 25°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or higher.

[0129] In situ hybridization In some embodiments, in situ hybridization (ISH) is applied. The ISH step generally involves placing small nucleic acids (e.g., oligomers of 15-40 nucleotides). In some embodiments, the nucleic acid probe recognizes a region or regions of the genome. In some embodiments, the nucleic acid probe of ISH recognizes RNA. In the case of single molecule in situ hybridization (smISH), many small nucleic acids together recognize RNA, and the binding of a single probe results in a weak signal, but the ability to obtain a signal from the ensemble of all probes (directly or via the linkage of a label to the probe) results in a stronger signal compared to the binding of a single probe. In some embodiments, an ISH reaction or a modified ISH reaction is applied to the sample described herein before or after antigen retrieval (e.g., an amplification reaction). In further embodiments, the probe used for ISH is further used for concatemer formation, either as a barcode or for barcode binding, followed by the methods described herein.

[0130] The iterative addition of nucleic acid barcodes to cDNA can produce nucleic acid concatemers. In some embodiments, the nucleic acid concatemers include at least three nucleic acid barcode strands. The nucleic acid concatemers can include nucleic acid barcode strands covalently linked to each other via photoreactive nucleotides. In some embodiments, the nucleic acid concatemers can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more nucleic acid barcode strands. In some embodiments, the nucleic acid concatemers can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more barcode domains, each of which incorporates data (e.g., each barcode domain can be uniquely / independently assigned to a bit value). One embodiment of digital data storage using light-induced concatemerization of barcodes is shown in Figures 5A-5B. A concatemer of nucleic acid barcodes can include at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 50 nucleic acid barcodes, or any number in between.

[0131] The number of additional barcode strands that can be added is not limited. For example, 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 or more additional barcode strands can be added. In some embodiments, 75, 100, 150, 200, 250 or more additional barcode strands can be added.

[0132] In some embodiments, the CNVK-labeled strand may hybridize to the docking strand by 1-10 bases or 10-20 bases. In some embodiments, salt concentration, temperature, denaturant concentration, or strand concentration affect the stability of strand binding. In some embodiments, the stability of strand binding is adjusted such that the CNVK-labeled strand binds for less than 1 second, between 1 second and 10 seconds, or less than 1 minute on average. In some embodiments, the CNVK-labeled strand may have domains of increased homology to the docking strand. In some embodiments, the CNVK-labeled strand has homology over about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65 or more bases. In some embodiments, the salt is increased or the temperature is decreased, thereby adjusting strand binding for times greater than 1 minute on average. In some embodiments, conditions can be adjusted to determine the average binding time of the CNVK-labeled strand to the docking strand.

[0133] The concatemers of nucleic acid barcodes can be at various positions on the substrate described herein.For example, the concatemers can be in a linear relationship with each other, can have two different orientations with respect to the target, or can have multiple orientations in different directions.The barcodes can be positioned along x, y and z coordinates in space.

[0134] In some embodiments, the concatemers described herein are linked to a protein. In further embodiments, a proteinase is applied to release the concatemers from the protein. In some embodiments, the barcodes described herein are linked to a protein. In further embodiments, a proteinase is applied to release the barcodes from the protein. Examples of proteinases for inclusion include, without limitation, proteinase K. In some embodiments, the proteinase is applied before the antigen retrieval reaction (e.g., reverse transcription), before the barcoding reaction, before the concatemerization reaction, and / or before the concatemer retrieval reaction.

[0135] Imaging Nucleic acid barcodes optionally include additional detectable labels, such as fluorophores, luminescent and bioluminescent markers (e.g., biotin, luciferases (e.g., bacterial, firefly, click beetle, etc.), luciferin and aequorin), radioactive labels (e.g., 3H, 125I, 35S, 14C, 33P or 32P), enzymes (e.g., galactosidase, glucorinidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase) and cholinesterase), and calorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene and latex) beads. In some embodiments, the barcodes described herein are modified to include the addition of detectable labels described herein.

[0136] The resolution of detection using the barcoding method described herein is not limited by optical diffraction.Such barcoding method allows detection of samples with very high density as well as very low density targets.Moreover, the barcoding method described herein can be applied in two or three dimensions without loss of resolution or significant adaptation of the method.

[0137] In some embodiments, fluorescent in situ hybridization (FISH) is applied. The FISH step generally involves placing small nucleic acids (e.g., oligomers of 15-40 nucleotides) with a label (e.g., a fluorescent label). In some embodiments, the nucleic acid fluorescent probe of the FISH recognizes a region or regions of the genome. In some embodiments, the nucleic acid fluorescent probe of the FISH recognizes RNA. In some embodiments, a FISH reaction or a modified FISH reaction is applied to the sample described herein before or after antigen retrieval (e.g., an amplification reaction). In further embodiments, the probes used for FISH are further used for concatemer formation, either as barcodes or for barcode binding, followed by the methods described herein.

[0138] After washing to remove uncrosslinked DNA barcodes, the bound barcodes can be visualized or detected, in some embodiments, by colorimetric, fluorescent, ultraviolet, radiometric or other means.

[0139] The barcoding methods described herein may include single cell or single nucleus labeling methods. In some embodiments, split pool barcoding is used. In such embodiments, samples are distributed and pooled in replicates into wells for barcoding. The final library contains separate combinations of barcodes.

[0140] Record extraction and sequencing In some embodiments, barcoded DNA strands are pooled (e.g., combined for batch processing and / or analysis) across multiple ROIs. In some embodiments, DNA strands from individual ROIs are collected in a region-specific manner. In some embodiments, DNA strands from individual cells are pooled separately. In some embodiments, DNA from individual cells or other defined ROIs is barcoded and sequenced separately.

[0141] In some embodiments, the release of the barcoded nucleic acid includes application of a reagent to release the barcode-containing nucleic acid from the surface. The reagent to release the barcode-containing nucleic acid may include an RNAse, a DNAse, and / or a proteinase. In some embodiments, the record extraction and sequencing includes the release of the barcoded nucleic acid. The release is optionally achieved by application of an RNA hydrolase. In some embodiments, the RNA hydrolase is RNaseH. Cross-junction synthesis to generate consecutive nucleic acids may also result in the release of the barcoded nucleic acid. The enzymatic release and synthesis reactions may be combined in any order or performed sequentially. After the sequence is extracted from the sample, further imaging or other assays (e.g., mass spectrometry, H&E staining) may be performed on the sample. Subsequent polymerase chain reaction (PCR) amplification and optional purification are followed by library preparation and next-generation sequencing as required. The resulting sequence analysis may be merged with the captured imaging data to provide an integrated spatial and sequence-based information output.

[0142] The generated barcode concatemers are released from the tissue sample or cell population provided herein and synthesized into a continuous DNA strand.These steps can be performed sequentially, in any order, or simultaneously.The continuous DNA strands are optionally pooled.The nucleic acid is then optionally amplified and sequenced.The sequencing data is optionally incorporated with the previous imaging data to provide spatially defined sequencing information.

[0143] Methods for concatemer release are provided herein. In some embodiments, the concatemers bound to RNA are released from the tissue sample or population of cells provided herein. In some embodiments, the concatemers are chemically released. In some embodiments, the concatemers are enzymatically released. In some embodiments, the concatemers are released using an enzyme that specifically cleaves RNA (e.g., RNase H). In some embodiments, the concatemers are released using an enzyme that specifically cleaves DNA, DNAse (e.g., DNAse I). In some embodiments, the concatemers are physically released. In some embodiments, the concatemers are released by application of energy. In some embodiments, the energy is chemical energy, electrical energy, radiant energy, mechanical energy, thermal energy, nuclear energy, or any combination thereof. In some embodiments, the concatemers are released by application of heat. In some embodiments, the concatemers are released by electrophoresis. In some embodiments, the concatemers are released through the process of cross-junction synthesis. In some embodiments, the cross-junction synthesis is performed before the release of the barcoded DNA. In some embodiments, the cross-junction synthesis is performed after the release of the barcoded DNA. In some embodiments, the release and the cross-junction synthesis are performed simultaneously.

[0144] Cross-junction synthesis Provided herein is a method for cross-junction synthesis, in which a nucleic acid combined with one or more barcode sequences is copied onto a new single strand of DNA. Cross-junction synthesis is performed directly in situ or during / after release. The basic strategy for cross-junction synthesis is shown in FIG. 6. Two nucleic acids hybridized together to form a junction are shown. A primer is attached on the first nucleic acid (left) before the junction. A strand-displacing polymerase is used to copy the x domain (also referred to herein as the junction domain) until it reaches a stopper (shown in black, also referred to herein as the blocking domain). The new x domain and the old x domain compete in a random walk branch migration process. Eventually, the new x domain will migrate to the exposed x domain on the second template strand (right, also referred to herein as the synthesis region). * Polymerization can then continue on the second template strand, copying along the new backbone.

[0145] In some embodiments, the cross-junction synthesis produces DNA, RNA, PNA or LNA. In some embodiments, the method of cross-junction synthesis comprises contacting a target nucleic acid or barcode provided herein with a strand-displacing polymerase. In some embodiments, the cross-junction synthesis is performed at room temperature (e.g., at least about 20°C to about 25°C). In some embodiments, the cross-junction synthesis is performed at a temperature below room temperature (e.g., about 15°C, about 10°C, about 4°C). In some embodiments, the cross-junction synthesis is performed at a temperature above room temperature (e.g., about 30°C, about 37°C). In some embodiments, the nucleic acid amplification or cross-junction synthesis is performed at a constant temperature. In some embodiments, the nucleic acid amplification or cross-junction synthesis is performed at different or varying temperatures.

[0146] Provided herein are methods in which a polymerase is prevented or stopped from continuing along the backbone of a template strand and / or copying the template strand by a blocking domain. In some embodiments, the blocking domain is a T m In some embodiments, the modified nucleotides include one or more modified nucleotides known to increase the binding affinity of the nucleic acid to the nucleic acid. In some embodiments, the modified nucleotides include, but are not limited to, locked nucleic acids (LNAs), 2'-O-methoxy-ethyl (2'-MOE) nucleotides, 2,6-diaminopurines, G-clamps (analogues of C with four hydrogen bonds), and guanidinium G-clamp nucleotides.

[0147] The compositions and methods described herein can be used for the synthesis of designated sequences. An exemplary embodiment is shown in FIG. 7. As shown in FIG. 7, by hybridizing multiple barcode strands together, cross-junction synthesis reactions can be chained together to form longer sequences. Each junction between barcode strands exhibits the same domain motif, such that the copied strand domains (b, c, d, e) on the 3' end of the growing strand before reaching the stopper can extend across the junction and reach the exposed complementary sequence (b, c, d, e) on the neighboring barcode strand. * , c * , d * , e * ) Any nucleic acid sequence can be added into the template regions (shown in grey) between the motif domains to allow longer sequences to be assembled.

[0148] In some embodiments, at least one of the blocking domains comprises a polymonomer stretch. For example, the blocking domain may comprise a stretch of polyA, polyT, polyC or polyG nucleotides.

[0149] In some embodiments, the blocking domain or stopper may comprise a covalent cross-linking of two barcode strands. In some embodiments, the blocking domain or stopper comprises one or more proteins. In some embodiments, the one or more proteins are bound to one or more of the barcode strands. In some embodiments, the one or more proteins are cross-linked to one or more of the barcode strands. In some embodiments, the covalent cross-linking is photocross-linking or chemical cross-linking. In some embodiments, the covalent cross-linking comprises cross-linking the 5'-terminal nucleotide of the first barcode strand to the 3'-terminal nucleotide of the second template strand. In some embodiments, the cross-linking is at an oligonucleotide or single nucleic acid backbone linkage. In some embodiments, the cross-linking is by a phosphodiester bond. In some embodiments, the cross-linking reaction is between a CNVK nucleotide and a complementary nucleotide. In some embodiments, the cross-linked nucleotide acts as a stopper for the polymerase.

[0150] In some embodiments, the blocking region comprises any desired nucleotide sequence or any desired number of nucleotides. In some embodiments, each blocking domain can be any length. In some embodiments, each blocking domain can be 1 to 100 nucleotides long. In some embodiments, each blocking domain is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides long. It is noted that a blocking domain can be a single nucleotide.

[0151] In embodiments where the nucleic acid template comprises two or more cross-junctions, the first nucleic acid of the cross-junction and the second nucleic acid of the cross-junction can have complementary sequences (e.g., the 3' end of the first nucleic acid is complementary to the 5' end of the second nucleic acid). Similarly, the 3' end of the second nucleic acid of the cross-junction and the 5' end of the first nucleic acid of the cross-junction can have complementary sequences. In this manner, nucleic acids can be cross-linked in alternating arrangements to generate concatemers as described herein.

[0152] Please note that the nucleic acids described herein may optionally include a barcode domain. In an exemplary embodiment, the second nucleic acid of the junction includes a barcode domain at the 5' end. In some embodiments, one of the first or second synthetic regions of the nucleic acid includes a barcode domain.

[0153] purification The methods and systems described herein may include purification of the sample. In some embodiments, the barcoded sample is purified by chemical means, e.g., by precipitating nucleic acids. In some embodiments, the barcoded sample is purified using column chromatography, e.g., ion exchange, reverse phase, or size exclusion chromatography. In some embodiments, on-bead binding is used to purify the barcoded sample. In some embodiments, the barcoded sample is purified using electrophoretic properties.

[0154] PCR amplification A method of amplification is provided herein. A method of PCR amplification is provided herein. PCR amplification uses DNA polymerase to synthesize DNA from deoxynucleotide substrates on single-stranded templates. In some embodiments described herein, PCR methods include real-time PCR, quantitative real-time PCR (Q-RT PCR), reverse transcriptase PCR (RT-PCR), multiplex PCR, nested PCR, long-range PCR, single-cell PCR, fast cycling PCR, methylation specific PCR (MSP), hot-start PCR, high-fidelity PCR, in situ PCR, variable number of tandem repeats (VNTR) PCR, asymmetric PCR, repeat sequence-based PCR, overlap extension PCR, assemble PCR, intersequence-specific PCR (ISSR), ligation-mediated PCR, methylation specific PCR, mini-primer PCR, solid-phase PCR, touchdown PCR, or any combination thereof. In some embodiments, reverse transcriptase is applied for the amplification described herein. In some embodiments, the reverse transcriptase is MuLV reverse transcriptase or avian myeloblastosis virus (AMV) reverse transcriptase. In some embodiments, a DNA polymerase is applied for amplification as described herein. In some embodiments, the DNA polymerase is T7 DNA polymerase, thermophilic eubacterial microorganism Thermus aquaticus DNA polymerase, Pfu DNA polymerase or Bst DNA polymerase. In some embodiments, a DNA ligase is applied to join the strands. In some embodiments, the DNA ligase is EC 6.5.1.1. In some embodiments, the DNA ligase is T4 DNA ligase.

[0155] In some embodiments, the barcode sequence, or a primer complementary to such a sequence, comprises a polymerase promoter sequence. In some embodiments, the promoter is an RNA polymerase promoter. In some embodiments, the polymerase promoter sequence includes, without limitation, a T7 RNA polymerase promoter region (SEQ ID NO:1: 5' TAATACGACTCACTATAG 3'), a T3 RNA polymerase promoter region (SEQ ID NO:2: 5' AATTAACCCTCACTAAAG 3') or a SP6 RNA polymerase promoter region (SEQ ID NO:3: 5' ATTTAGGTGACACTATAG 3').

[0156] Methods for amplicon preparation are provided herein. In some embodiments, the amplicons are purified. In some embodiments, the amplicons are analyzed via gel electrophoresis. In some embodiments, the amplicons are isolated. In some embodiments, the amplicons are fragmented. In some embodiments, the amplicons are end-repaired. In some embodiments, the amplicons are covalently linked to adapters.

[0157] Samples may be further prepared for sequencing using any combination of one or more of tagmentation, fragmentation, adapter ligation, rRNA reduction or other library preparation steps.

[0158] Sequencing Methods for sequencing are provided herein. In some embodiments, the sequencing comprises next-generation sequencing (NGS) technology. In some embodiments, the sequencing comprises parallel sequencing. The sequencing may comprise paired-end, non-paired-end or long-read sequencing. In some embodiments, the sequencing comprises high-throughput sequencing, such as single molecule real-time sequencing, ion semiconductor sequencing (Ion Torrent), pyrosequencing, sequencing by synthesis, combinatorial probe anchor synthesis, sequencing by ligation (SOLiD), nanopore sequencing, genapsys sequencing or chain termination (Sanger) sequencing. In some embodiments, the sequencing comprises massively parallel sequencing.

[0159] Image and sequence analysis The density of barcoded cDNA targets can be measured by the number of unique sequences detected. In some embodiments, raw sequencing reads are filtered for those that map to the transcriptome only once. The reads are "deduplicated" to identify identical molecules that have the same UMI sequence and are sequenced more than once as reads according to those that map to the same transcript in the transcriptome. All identical sequencing reads are collapsed into a single "deduplicated" read. A gene expression matrix is ​​generated to summarize the unique counts sequenced for each gene in the transcriptome. The difference in the number of counts across all genes can then be compared across samples.

[0160] In some embodiments, the density of the barcoded cDNA targets is 1 micrometer squared (μm 2 ) of a tissue sample, population of cells or population of cells. In some embodiments, the density of barcoded cDNA targets is 1 cubic micrometer (μm 3) of a tissue sample, population of cells, organoid or organ. In some embodiments, the methods provided herein provide for the number of barcodes generated within one square micrometer (μm 2 In some embodiments, the methods provided herein generate approximately 55 tags or barcodes per 1 μm 2 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 5000, 5000, 60000, 70000, 80000, 90000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 50000, 60000, 70000, 80000, 90000, 10000, 15000, 15000, 16000, 17000, 18000, 19 ... Generate 550, approximately 600, approximately 650, approximately 700, approximately 800, approximately 900, approximately 1000, approximately 1200, approximately 1400, approximately 1600, approximately 1800, approximately 2000, approximately 2250, approximately 2500, approximately 2750, approximately 3000, approximately 3500, approximately 4000, approximately 4500, approximately 5000 tags.

[0161] In some embodiments, the methods provided herein involve measuring a volume of 1 cubic micrometer (μm 3 ) with a maximum of approximately 5 tags per cubic micrometer (μm 3) per 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, Generates 650 tags, up to about 700 tags, up to about 800 tags, up to about 900 tags, up to about 1000 tags, up to about 1200 tags, up to about 1400 tags, up to about 1600 tags, up to about 1800 tags, up to about 2000 tags, up to about 2250 tags, up to about 2500 tags, up to about 2750 tags, up to about 3000 tags, up to about 3500 tags, up to about 4000 tags, up to about 4500 tags, up to about 5000 tags, up to about 6000 tags, up to about 70,000 tags, up to about 8000 tags, up to about 9000 tags, and up to about 10,000 tags.

[0162] Further analysis Methods for protein analysis are provided herein.In some embodiments, the proteins recovered from the samples described herein are analyzed by mass spectrometry, which is an analytical tool that measures the mass-to-charge ratio (m / z) of one or more molecules present in a sample.Such measurements can be used to calculate the molecular weight, abundance and / or identity of components in a sample.

[0163] Further provided herein is a method for screening an area of ​​interest that is affected by an intervention, compared to an area of ​​interest that is not affected. In some embodiments, the method comprises screening one or more areas of interest in a sample. In some embodiments, the method comprises screening one or more areas of interest across one or more samples. In some embodiments, one or more areas of interest are affected by an intervention before screening, after screening, or between multiple screenings. In some embodiments, the affecting comprises application of a small molecule, a peptide, an antibody, a cell, a protein, energy, or any combination thereof. In some embodiments, the affecting comprises application of thermal energy, radiant energy, chemical energy, nuclear energy, electrical energy, kinetic energy, acoustic energy, or any combination thereof.

[0164] Applications of the methods described herein include, but are not limited to, drug target identification, biomarker identification, profiling, characterization of cells and disease models, characterization of differentiation status and cell states, tissue mapping, and multidimensional analysis.

[0165] Exemplary embodiments Provided herein is a method for generating biological cell information, comprising the steps of contacting a population of cells with a nucleic acid polymerase and a primer to generate an amplification product; disposing a DNA barcode on the population of cells, the DNA barcode comprising a region complementary to a region of the amplification product, the DNA barcode comprising a photoreactive nucleic acid base capable of crosslinking to another nucleic acid base; selectively irradiating the population of cells to form a covalently linked DNA barcode-amplification product complex; washing the population of cells to remove DNA barcodes that are not complexed with the amplification product; and repeating the disposing, selectively irradiating and washing steps, wherein the DNA barcode in a first disposing step of the disposing steps comprises a different sequence than the DNA barcode in a second disposing step of the disposing steps, and the selectively irradiating step is performed at different locations for the first selectively irradiating step and the second selectively irradiating step. Further provided herein is a method further comprising determining biological cell spatial information based at least in part on the amplification product and the DNA barcode. Further provided herein is a method further comprising synthesizing the continuous DNA strand, the synthesizing comprising contacting the DNA barcode-amplification product complex with a polymerase. Further provided herein is a method further comprising synthesizing comprising cross-junction synthesis. Further provided herein is a method further comprising releasing the continuous DNA strand from the population of cells. Further provided herein is a method wherein the synthesizing and releasing steps are performed simultaneously. Further provided herein is a method wherein the released continuous DNA strands are pooled (e.g., combined for batch processing and / or analysis). Further provided herein is a method wherein the pooled continuous DNA strands are amplified. Further provided herein is a method wherein the pooled continuous DNA strands are sequenced. Further provided herein is a method further comprising identifying targets associated with the continuous DNA strands.Further provided herein is a method further comprising a step of mapping the target to different locations. Further provided herein is a method wherein the primer is a random primer. Further provided herein is a method wherein the primer is a non-random primer. Further provided herein is a method wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK). Further provided herein is a method wherein the selectively irradiating step is performed using a photomask. Further provided herein is a method wherein the photomask is varied in different selectively irradiating steps. Further provided herein is a method wherein the photomask is manually generated. Further provided herein is a method wherein the photomask is machine generated. Further provided herein is a method wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 300 nm to 450 nm. Further provided herein is a method wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 350 nm to 420 nm. Further provided herein is a method, wherein the selectively irradiating step includes applying irradiation at a wavelength of about 365 nm to 405 nm. Further provided herein is a method, wherein the selectively irradiating step includes applying irradiation to a preselected region of interest (ROI). Further provided herein is a method, wherein the ROI is cell-specific or sub-cellular-specific. Further provided herein is a method, wherein the ROI is based on morphology. Further provided herein is a method, wherein the positioning step is performed manually, automatically, robotically, or any combination thereof. Further provided herein is a method, wherein the positioning step is performed using a modified inkjet printer. Further provided herein is a method, wherein the positioning step or the washing step is performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handler, inkjet printer, automated liquid handler, or any combination thereof. Further provided herein is a method, wherein the positioning step or the washing step is performed using a pipettor.Further provided herein is a method in which the repeating step is completed at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more times. Further provided herein is a method in which at least 2, 3, 4, 5, 6, 7, 8 or 9 different DNA barcode strands are arranged. Further provided herein is a method in which the method further comprises contacting the population of cells with a cell permeabilizing agent. Further provided herein is a method in which the method further comprises contacting the population of cells with an agent that generates a population of fixed cells. Further provided herein is a method in which the agent that generates a population of fixed cells comprises formalin, formaldehyde, paraformaldehyde, methanol, acetic acid, or any combination thereof. Further provided herein is a method, wherein the fixed cell population is fixed to a substrate. Further provided herein is a method, wherein the fixed cell is a formalin-fixed paraffin-embedded (FFPE) cell, fixed with paraformaldehyde (PFA), or fixed with paraformaldehyde with Triton X-100 (PFA-Tx). Further provided herein is a method, wherein the cell population comprises one or more of: cultured cells; tissue obtained from a subject; engineered tissue; or organoid. Further provided herein is a method, wherein the tissue is an intact tissue or a dissected tissue. Further provided herein is a method, wherein the cultured cells are dissociated cells. Further provided herein is a method, wherein the engineered tissue comprises a population of human cells differentiated in vitro. Further provided herein is a method, wherein the human cells differentiated in vitro are derived from human induced pluripotent stem cells (hiPSC), embryonic stem cells, or adult stem cells. Further provided herein is a method, wherein the cell population is retinal cells. Further provided herein is a method, further comprising imaging the population of cells after the washing step.Further provided herein is a method, wherein the washing step comprises disposing a solution comprising a chemical denaturant and a detergent. Further provided herein is a method, wherein the chemical denaturant comprises formamide. Further provided herein is a method, wherein the formamide is present in an amount of about 40% (v / v) to about 60% (v / v). Further provided herein is a method, wherein the formamide is present in an amount of about 50% to about 60%. Further provided herein is a method, wherein the detergent comprises polysorbate 20, polysorbate 80 or Triton X-100. Further provided herein is a method, wherein the detergent is present in an amount between about 0.05% (v / v) and about 5% (v / v). Further provided herein is a method, wherein the DNA barcode further comprises a detectable label. Further provided herein is a method, wherein the step comprising washing the population of cells is omitted.

[0166] Provided herein is a method for generating biological cell spatial information, the method comprising the steps of contacting a population of cells with a capture probe, the capture probe comprising a DNA binding region and a target binding region; disposing a DNA barcode on the population of cells, the DNA barcode comprising a region that binds to the DNA binding region, the DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating the population of cells to form a DNA barcode-amplification product complex; washing the population of cells to remove DNA barcodes that are not complexed with an amplification product; and repeating the disposing, selectively irradiating and washing steps, wherein the DNA barcode in a first disposing step of the disposing steps comprises a different sequence than the DNA barcode in a second disposing step of the disposing steps, and the selectively irradiating step is performed at different locations for the first selectively irradiating step and the second selectively irradiating step. Further provided herein is a method further comprising determining biological cell spatial information based at least in part on the DNA barcode-amplification product complex. Further provided herein is a method, in which the capture probe comprises RNA or DNA. Further provided herein is a method, in which the capture probe comprises a protein. Further provided herein is a method, in which the method further comprises a step of synthesizing a continuous DNA strand, the step of synthesizing comprises contacting the population of cells with a polymerase. Further provided herein is a method, in which the step of synthesizing comprises cross-junction synthesis. Further provided herein is a method, in which the method further comprises a step of releasing the continuous DNA strand from the population of cells. Further provided herein is a method, in which the steps of synthesizing and releasing are performed simultaneously. Further provided herein is a method, in which the released continuous DNA strands are pooled. Further provided herein is a method, in which the pooled continuous DNA strands are amplified. Further provided herein is a method, in which the pooled continuous DNA strands are sequenced.Further provided herein is a method further comprising identifying targets associated with a continuous DNA strand. Further provided herein is a method further comprising mapping targets to different locations. Further provided herein is a method in which the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK). Further provided herein is a method in which the selectively irradiating step is performed using a photomask. Further provided herein is a method in which the photomask is varied in different selectively irradiating steps. Further provided herein is a method in which the photomask is manually generated. Further provided herein is a method in which the photomask is machine generated. Further provided herein is a method in which the selectively irradiating step comprises applying irradiation at a wavelength of about 300 nm to 450 nm. Further provided herein is a method in which the selectively irradiating step comprises applying irradiation at a wavelength of about 350 nm to 420 nm. Further provided herein is a method, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 365 nm to 405 nm. Further provided herein is a method, wherein the selectively irradiating step comprises applying irradiation to a preselected region of interest (ROI). Further provided herein is a method, wherein the ROI is cell-specific or sub-cellular specific. Further provided herein is a method, wherein the ROI is based on morphology. Further provided herein is a method, wherein the positioning step is performed manually, automatically, robotically, or any combination thereof. Further provided herein is a method, wherein the positioning step is performed using a modified inkjet printer. Further provided herein is a method, wherein the positioning step or the washing step is performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handler, inkjet printer, automated liquid handler, or any combination thereof. Further provided herein is a method, wherein the positioning step or the washing step is performed using a pipettor.Further provided herein is a method in which the repeating step is completed at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more times. Further provided herein is a method in which at least 2, 3, 4, 5, 6, 7, 8 or 9 different DNA barcode strands are arranged. Further provided herein is a method in which the method further comprises contacting the population of cells with a cell permeabilizing agent. Further provided herein is a method in which the method further comprises contacting the population of cells with an agent that generates a population of fixed cells. Further provided herein is a method in which the agent that generates a population of fixed cells comprises formalin, formaldehyde, paraformaldehyde, methanol, acetic acid, or any combination thereof. Further provided herein is a method, wherein the fixed cell population is fixed to a substrate. Further provided herein is a method, wherein the fixed cell is a formalin-fixed paraffin-embedded (FFPE) cell, is fixed with paraformaldehyde (PFA), or is fixed with paraformaldehyde with Triton X-100 (PFA-Tx). Further provided herein is a method, wherein the cell population comprises one or more of: cultured cells; tissue obtained from a subject; engineered tissue; or organoid. Further provided herein is a method, wherein the cell population comprises tissue, wherein the tissue is an intact tissue or a dissected tissue. Further provided herein is a method, wherein the cell population comprises cultured cells, wherein the cultured cells are dissociated cells. Further provided herein is a method, wherein the engineered tissue comprises a population of human cells differentiated in vitro. Further provided herein is a method, wherein the human cells differentiated in vitro are derived from human induced pluripotent stem cells (hiPSC), embryonic stem cells, or adult stem cells. Further provided herein is a method, wherein the population of cells comprises retinal cells.Further provided herein is a method further comprising imaging the population of cells after the washing step. Further provided herein is a method wherein the washing step comprises disposing a solution comprising a chemical denaturant and a detergent. Further provided herein is a method wherein the chemical denaturant comprises formamide. Further provided herein is a method wherein the formamide is present in an amount of about 40% (v / v) to about 60% (v / v). Further provided herein is a method wherein the formamide is present in an amount of about 50% to about 60%. Further provided herein is a method wherein the detergent comprises polysorbate 20, polysorbate 80 or Triton X-100. Further provided herein is a method wherein the detergent is present in an amount between about 0.05% (v / v) and about 5% (v / v). Further provided herein is a method wherein the DNA barcode further comprises a detectable label. Further provided herein is a method wherein the target binding region binds to DNA, RNA, protein, or a combination thereof. Further provided herein is a method further comprising a plurality of contacting steps. Further provided herein is a method further comprising a plurality of contacting steps comprising: contacting a population of cells with a first capture probe that recognizes a nucleic acid; and contacting a population of cells with a second capture probe that recognizes a protein. Further provided herein is a method further comprising: the DNA binding region comprises a DNA barcode binding region. Further provided herein is a method further comprising: contacting a population of cells with a DNA polymerase or an RNA polymerase; and a primer to generate an amplification product. Further provided herein is a method further comprising: the RNA polymerase is a reverse transcriptase. Further provided herein is a method further comprising: omitting a step comprising: washing a population of cells.

[0167] Provided herein is a method for generating biological cellular spatial information, the method comprising the steps of capturing an image of a three-dimensional tissue sample; contacting the tissue sample with a nucleic acid polymerase and a primer to generate an amplification product; placing a DNA barcode on the tissue sample, the DNA barcode comprising a region complementary to a region of the amplification product, the DNA barcode comprising a photoreactive nucleic acid base capable of crosslinking to another nucleic acid base; selectively irradiating the three-dimensional tissue sample to form a DNA barcode-amplification product complex; washing the tissue sample to remove DNA barcodes that are not complexed with an amplification product; and repeating the placing, selectively irradiating, and washing steps, wherein the DNA barcode in a first one of the placing steps comprises a different sequence than the DNA barcode in a second one of the placing steps, and the selectively irradiating step is performed at different locations of the tissue sample for the first selectively irradiating step and the second selectively irradiating step. Further provided herein is a method further comprising determining biological cell spatial information based at least in part on the amplification product and the DNA barcode. Further provided herein is a method wherein a tissue sample is obtained from a subject. Further provided herein is a method wherein the tissue sample is an engineered tissue or an organoid. Further provided herein is a method wherein the tissue sample comprises a population of cells, and wherein the population of cells is a heterogeneous population of cells. Further provided herein is a method wherein the tissue sample comprises dissected cells or intact cells. Further provided herein is a method wherein the nucleic acid polymerase is an RNA polymerase or a DNA polymerase. Further provided herein is a method wherein the RNA polymerase is a reverse transcriptase. Further provided herein is a method wherein the primer is an RNA primer or a DNA primer. Further provided herein is a method wherein the primer is conjugated to a protein. Further provided herein is a method wherein the protein is an antibody or an antibody fragment.Further provided herein is a method in which an RNA polymerase transcribes RNA from the tissue sample to generate cDNA. Further provided herein is a method further comprising a step of hybridizing a DNA barcode to the cDNA. Further provided herein is a method further comprising a step of synthesizing a continuous DNA strand, the continuous DNA strand being complementary to the cDNA and at least one of the DNA barcodes. Further provided herein is a method in which the continuous DNA strand is complementary to the cDNA and two or more of the DNA barcodes. Further provided herein is a method in which the synthesizing step is by cross-junction synthesis. Further provided herein is a method further comprising a step of releasing the continuous DNA strand from the tissue sample. Further provided herein is a method in which the synthesizing and releasing steps are performed simultaneously. Further provided herein is a method in which the released continuous DNA strands are pooled. Further provided herein is a method in which the pooled continuous DNA strands are amplified. Further provided herein is a method in which the pooled continuous DNA strands are sequenced. Further provided herein is a method further comprising identifying targets associated with continuous DNA strands. Further provided herein is a method further comprising mapping targets to different locations within a fixed population of cells. Further provided herein is a method wherein the placing step is performed manually, automatically, robotically, or any combination thereof. Further provided herein is a method wherein the placing step is performed using a modified inkjet printer. Further provided herein is a method wherein the placing step or the washing step is performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handler, inkjet printer, automated liquid handler, or any combination thereof. Further provided herein is a method wherein the placing step or the washing step is performed using a pipettor.Further provided herein are methods in which the repeating step is completed at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more times. Further provided herein are methods in which at least 2, 3, 4, 5, 6, 7, 8 or 9 different barcode strands are disposed. Further provided herein are methods further comprising imaging the population of cells after the washing step. Further provided herein are methods in which the washing step comprises disposing a solution comprising a chemical denaturant and a detergent. Further provided herein are methods in which the chemical denaturant comprises formamide. Further provided herein are methods in which the formamide is present in an amount of about 40% (v / v) to about 60% (v / v). Further provided herein is a method, wherein formamide is present in an amount of about 50% to about 60%. Further provided herein is a method, wherein the detergent comprises polysorbate 20, polysorbate 80 or Triton X-100. Further provided herein is a method, wherein the detergent is present in an amount between about 0.05% (v / v) and about 5% (v / v). Further provided herein is a method, wherein the DNA barcode further comprises a detectable label. Further provided herein is a method, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK). Further provided herein is a method, wherein the selectively irradiating step is performed with a photomask. Further provided herein is a method, wherein the photomask is varied in different selectively irradiating steps. Further provided herein is a method, wherein the photomask is manually generated. Further provided herein is a method, wherein the photomask is generated by machine. Further provided herein is a method, wherein the selectively irradiating step includes applying irradiation to a preselected region of interest (ROI). Further provided herein is a method, wherein the ROI is cell-specific or sub-cellular specific.Further provided herein is a method, wherein the ROI is based on morphology. Further provided herein is a method, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 300 nm to 450 nm. Further provided herein is a method, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 350 nm to 420 nm. Further provided herein is a method, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 365 nm to 405 nm. Further provided herein is a method, wherein the step comprising washing the tissue sample is omitted.

[0168] Provided herein is a method for generating biological cell spatial information, comprising the steps of capturing an image of a population of cells, the population of cells being immobilized on a substrate; reverse transcribing RNA from the population of cells to generate cDNA; disposing a DNA barcode on the population of cells; hybridizing the DNA barcode to the cDNA, the DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating the population of cells to form a crosslinked barcode; washing to remove non-crosslinked DNA barcodes; and repeating the disposing, hybridizing, selectively irradiating and washing steps, where the DNA barcode in the disposing step is varied. Further provided herein is a method further comprising determining biological cell spatial information based at least in part on the cDNA and the DNA barcode. Further provided herein is a method where the crosslinked barcode forms a linkage of barcodes. A population of cells is 1 micrometer squared (μm 2 Further provided herein is a method in which up to about 55 unique cDNA molecules are generated per square micrometer (μm 2 Further provided herein is a method in which up to about 10 unique cDNA molecules are generated per square micrometer (μm 2Further provided herein is a method in which up to about 5 unique cDNA molecules are generated per cubic micrometer (μm 3 Further provided herein is a method in which up to about 5000 unique cDNA molecules are generated per cubic micrometer (μm 3 Further provided herein is a method in which up to about 1000 unique cDNA molecules are generated per cubic micrometer (μm 3Further provided herein is a method, in which up to about 500 unique cDNA molecules are generated per 1000 cells / ml. Further provided herein is a method, further comprising synthesizing a continuous DNA strand complementary to the cDNA and the cross-linked barcode. Further provided herein is a method, in which the synthesizing is by cross-junction synthesis. Further provided herein is a method, in which the synthesizing and releasing are performed simultaneously. Further provided herein is a method, in which the released continuous DNA strands are pooled. Further provided herein is a method, in which the pooled continuous DNA strands are amplified. Further provided herein is a method, in which the pooled continuous DNA strands are sequenced. Further provided herein is a method, in which the further comprises identifying targets associated with the continuous DNA strands. Further provided herein is a method, in which the further comprises mapping the targets to different locations within the fixed population of cells. Further provided herein is a method, in which the population of cells is contacted with a permeabilizing agent. Further provided herein is a method, wherein the population of cells is contacted with one or more agents to generate a population of fixed cells. Further provided herein is a method, wherein the agent is formalin, formaldehyde, paraformaldehyde, methanol, acetic acid, or any combination thereof. Further provided herein is a method, wherein the fixed cells are formalin-fixed paraffin-embedded (FFPE) cells, fixed with paraformaldehyde (PFA), or fixed with paraformaldehyde with Triton X-100 (PFA-Tx). Further provided herein is a method, wherein the population of cells includes one or more of: cultured cells; tissue obtained from a subject; or engineered tissue. Further provided herein is a method, wherein the population of cells is retinal cells. Further provided herein is a method, wherein the placing or washing step is performed manually, automatically, robotically, or any combination thereof.Further provided herein is a method in which the placing or washing step is performed using a modified inkjet printer. Further provided herein is a method in which the placing or washing step is performed using microfluidics, digital microfluidics, acoustic liquid handlers, inkjet printers, automated liquid handlers, or any combination thereof. Further provided herein is a method in which the placing or washing step is performed using a pipettor. Further provided herein is a method in which the repeating step is completed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, or more. Further provided herein is a method in which at least 2, 3, 4, 5, 6, 7, 8, or 9 different barcode strands are placed. Further provided herein is a method further comprising imaging the permeabilized cells after the washing step. Further provided herein is a method wherein the washing step comprises disposing a solution comprising a chemical denaturant and a detergent. Further provided herein is a method wherein the chemical denaturant comprises formamide. Further provided herein is a method wherein the formamide is present in an amount of about 40% (v / v) to about 60% (v / v). Further provided herein is a method wherein the formamide is present in an amount of about 50% to about 60%. Further provided herein is a method wherein the detergent comprises polysorbate 20, polysorbate 80 or Triton X-100. Further provided herein is a method wherein the detergent is present in an amount between about 0.05% (v / v) and about 5% (v / v). Further provided herein is a method wherein the DNA barcode further comprises a detectable label. Further provided herein is a method wherein the hybridizing step is at the 5' end of the transcribed cDNA.Further provided herein is a method in which the hybridizing step is at the 3' end of the transcribed cDNA. Further provided herein is a method in which the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK). Further provided herein is a method in which the selectively irradiating step is performed with a photomask. Further provided herein is a method in which the photomask is varied in different selectively irradiating steps. Further provided herein is a method in which the photomask is manually generated. Further provided herein is a method in which the photomask is generated by machine. Further provided herein is a method in which the selectively irradiating step includes applying irradiation to a preselected region of interest (ROI). Further provided herein is a method in which the ROI is cell-specific or sub-cellular specific. Further provided herein is a method in which the ROI is based on morphology. Further provided herein is a method in which the population of cells includes retinal cells. Further provided herein is a method in which the selectively irradiating step comprises applying irradiation at a wavelength of about 300 nm to 450 nm. Further provided herein is a method in which the selectively irradiating step comprises applying irradiation at a wavelength of about 350 nm to 420 nm. Further provided herein is a method in which the selectively irradiating step comprises applying irradiation at a wavelength of about 365 nm to 405 nm. Further provided herein is a method in which the washing step is omitted.

[0169] Provided herein is a method for generating biological cell spatial information, comprising the steps of: arranging DNA barcodes on a population of cells, the DNA barcodes hybridizing to a target region, the DNA barcodes comprising photoreactive nucleobases capable of crosslinking to another nucleobase; selectively irradiating the population of cells in the region of interest to form covalently linked DNA barcode-target complexes, the irradiating step being selectively varied by intensity or duration; washing the population of cells to remove DNA barcodes that are not complexed with the target region; and repeating the arranging, selectively irradiating and washing steps, wherein the DNA barcodes in the first of the arranging steps comprise a different sequence than the DNA barcodes in the second of the arranging steps. Provided herein is a method further comprising the step of determining biological cell spatial information based at least in part on the target region and the DNA barcodes. Provided herein is a method further comprising the step of synthesizing a continuous DNA strand complementary to the covalently linked barcodes. Further provided herein is a method further comprising the step of collecting the continuous DNA strand or an amplification product thereof. Further provided herein is a method further comprising the step of sequencing the continuous DNA strand or an amplification product thereof. Further provided herein is a method in which the step comprising washing the population of cells is omitted.

[0170] Methods are added to the methods described herein, where the population of cells is present on a synthetic substrate. Methods are further provided herein, where the synthetic substrate comprises a hydrogel. Methods are further provided herein, where the synthetic substrate comprises an extracellular matrix protein. Methods are further provided herein, where the synthetic substrate comprises collagen or fibronectin.

[0171] Provided herein is a method for generating biological information, comprising the steps of: arranging a primer that hybridizes to a target sequence of the nucleic acid on a population of nucleic acids to form a nucleic acid primer-complex; arranging a DNA barcode on the nucleic acid-primer complex, the DNA barcode hybridizing to the primer to form a nucleic acid concatemer, the DNA barcode comprising a photoreactive nucleic acid base capable of crosslinking to another nucleic acid base; irradiating the nucleic acid concatemer to form a covalently linked concatemer; and removing the DNA barcode that is not in the covalently linked concatemer. Provided herein further is a method, in which the steps of arranging the DNA barcode, irradiating and removing are repeated, and the DNA barcode in the first of the arranging steps comprises a different sequence from the DNA barcode in the second of the arranging steps. Provided herein further is a method, in which the primer is bound to an affinity molecule, a scaffold, a substrate, a surface, a bead or a column.

[0172] Methods are added to the methods described herein in which the selective irradiating step is varied in intensity or duration to provide for the generation of a preselected fraction of DNA barcode-amplification product complexes or crosslinked barcodes.

[0173] Methods are added to the methods described herein that further include the step of comparing information collected from the disease state sample to a non-disease state sample.

[0174] Methods are added to the methods described herein that further include the steps of contacting the sample with a reagent for intervention, the reagent for intervention comprising a compound, a nucleic acid or a cell; and comparing information collected from the contacted sample area with information collected from the uncontacted sample area.

[0175] Provided herein is a method for generating biological cell information, the method comprising: disposing DNA barcodes on a population of cells, where the DNA barcodes comprise photoreactive nucleobases capable of crosslinking to another nucleobase; forming concatemers via a light-based process comprising repeating the following steps: selectively irradiating the population of cells with a first wavelength to form covalently linked complexes of DNA barcodes hybridized to a substrate nucleic acid sequence; and selectively irradiating the population of cells with a second wavelength to cleave the DNA barcodes hybridized to the substrate nucleic acid sequence; and collecting the concatemers, where the collecting step is before or after the step of selectively irradiating the population of cells with the second wavelength, and the concatemers comprise information about the population of cells.

[0176] Provided herein is a method of generating spatial organization information, the method comprising at least partially removing paraffin from a sample comprising paraffin-embedded tissue; heating the sample to a temperature of at least about 50° C.; contacting the sample with a nucleic acid polymerase and a primer to generate an amplification product; disposing a DNA barcode on the sample, the DNA barcode comprising a region complementary to a region of the amplification product, the DNA barcode comprising a photoreactive nucleic acid base capable of crosslinking to another nucleic acid base; selectively irradiating the sample to form a covalently linked DNA barcode-amplification product complex, the selectively irradiating step being performed at different locations for the first selectively irradiating step and the second selectively irradiating step; washing the sample to remove DNA barcodes not complexed with the amplification product; and determining the spatial organization information based at least in part on the generated amplification products and DNA barcodes. Further provided herein is a method, wherein the heating the sample is at a temperature between 50° C. and 120° C. Further provided herein is a method further comprising determining biological cell spatial information based at least in part on the generated amplification products and DNA barcodes. Further provided herein is a method further comprising synthesizing a continuous DNA strand, the synthesizing comprising contacting the DNA barcode-amplification product complex with a polymerase. Further provided herein is a method further comprising releasing the continuous DNA strand from the population of cells. Further provided herein is a method in which the synthesizing and releasing steps are performed simultaneously. Further provided herein is a method in which the synthesizing is performed before the releasing step. Further provided herein is a method in which the released continuous DNA strands are pooled. Further provided herein is a method in which the pooled continuous DNA strands are amplified.Further provided herein is a method, in which the pooled consecutive DNA strands are sequenced. Further provided herein is a method, further comprising identifying the target associated with the consecutive DNA strand. Further provided herein is a method, further comprising mapping the target to different positions.

[0177] Provided herein is a method of generating biological cellular spatial information, the method comprising the steps of: placing a DNA barcode on a population of cells, where the DNA barcode comprises a photoreactive nucleobase capable of crosslinking to another nucleobase, and where the DNA barcode hybridizes to a target region; selectively irradiating the population of cells in at least one region of interest (ROI) to form covalently linked DNA barcode-target complexes, where the irradiating step is selectively varied by intensity or duration; washing the population of cells to remove DNA barcodes that are not complexed to the target region; and repeating the placing, selectively irradiating, and washing steps, where the DNA barcode in a first one of the placing steps comprises a different sequence than the DNA barcode in a second one of the placing steps, and where the second selectively irradiating step is applied at the same or different duration or intensity as the first selectively irradiating step. Further provided herein is a method, wherein the first selectively illuminating step, the second selectively illuminating step, or both, are applied as an intensity gradient across at least one ROI. Further provided herein is a method, wherein the second intensity gradient is applied parallel, orthogonal, perpendicular, or at another angle to the first intensity gradient.

[0178] Provided herein are nucleic acid libraries produced according to the methods described herein. EXAMPLES

[0179] The following examples further illustrate the described embodiments without limiting the scope of the disclosure.

[0180] Example 1 Fixation of samples Samples are fixed according to the following method: MRC-5 (human, ATCC CCL-171) and HEK293T cells (human, ATCC CRL-1573) are grown in Dulbecco's modified Eagle's medium (Gibco No. 10564) supplemented with 10% (vol / vol) serum (Gibco No. 10437 for MRC-5, Peak Serum PS-FB2 for HEK293T), 50 U / mL penicillin and 50 μg / mL streptomycin (Gibco No. 15070). EY.T4 embryonic fibroblasts (mouse)49 are grown in Dulbecco's modified Eagle's medium supplemented with 15% (vol / vol) serum, 50 U / mL penicillin and 50 μg / mL streptomycin. Cells are cultured at 37° C. in the presence of 5% CO2.

[0181] Cells are pre-washed with phosphate buffered saline (PBS), 0.1% TWEEN® 80 in PBS, 0.1% polysorbate 20 (TWEEN® 20) in PBS, or Hank's Balanced Salt Solution (HBSS). Samples are fixed in 1-10% formaldehyde and then washed with PBS or HBSS. Cells are permeabilized with 0%-2.5% Triton X-100 + 0.1% Tween® 20, as appropriate. Fixed samples can be maintained at a temperature of 4-45°C. Samples are fixed at room temperature (20-25°C) for approximately 5-20 minutes prior to barcode hybridization. Alternatively, samples are fixed at a lower temperature of 4°C and stored for up to 2 weeks prior to barcoding. After fixation, samples are washed with PBS, or 0.1% TWEEN® 20 in PBS. Bovine serum albumin (BSA) is used as a blocking agent to reduce non-specific detection of target nucleic acids and proteins. Other blocking or detergent agents can be used. When blocking, cells are incubated in BSA or detergent at room temperature.

[0182] Example 2 Barcode hybridization and imaging Fixed tissue samples, such as those produced in Example 1, are hybridized with barcodes to target nucleic acids using the reagents listed in Table 1. Reagent conditions include exemplary ranges of concentrations that allow DNA barcode hybridization to the target. [Table 1]

[0183] A barcoding solution consisting of 250 nM barcode 1 strand mixed in PBS, 2 mg / ml sheared salmon sperm DNA (Invitrogen AM9680), 10% dextran sulfate (wt / vol, Sigma Aldrich S4030) and 500 mM NaCl in TWEEN® 20 (PBST) is applied to the fixed tissue sample for 20 min to 1 h. The sample is washed three times with PBST containing 1 M NaCl for approximately 1 min each. The fixed sample can be kept at a temperature of approximately 20-60 °C in the barcoding solution prior to irradiation.

[0184] Using microscopy, the desired area on the tissue sample is visually identified in the brightfield image. A hand-drawn mask is set as the photostimulation area. A high-power liquid light guide-coupled LED source is applied at 365 nm to the selected area of ​​interest on the sample per barcoding round. The microscope focus is set to the desired layer on the sample to manually select the photomask area. The sample is photostimulated, imaged, and then washed with approximately 0.1x-1x PBS; 25-80% formamide; and 0%-1% Tween® 20.

[0185] Additional barcode strands can be added to the barcoding solution after finding the same cell area in the fixed sample. After barcoding, the sample is optionally treated with a cross-junction synthesis solution consisting of Rnase H and reverse transcription buffer. cDNA can be extracted by washing the cells with PBST and collecting the supernatant in a tube for amplification and / or next generation sequencing. cDNA can be pooled together or separately in sequential barcoding steps for sequencing analysis.

[0186] Example 3 Binary Encoding The combination of four barcodes can define 16 distinct ROIs using on / off binary encoding. The sample is spatially defined with 16 regions of interest (ROIs). Four unique barcodes are applied in four rounds of crosslinking iterations. In each of the four crosslinking rounds, light is applied to half of the ROI. Every fraction illuminated in each round contains approximately ¼ (25%) or less saturation, and in each region, 2 of the four barcode strands are crosslinked or not crosslinked. 4 = 16 possible combinations. This is shown in Figure 8, where a filled square indicates some fraction of reads in the area containing the barcode corresponding to that row, and an open square indicates that there was no illumination during that barcoding round. As shown on the right side of the figure, a binary encoding is assigned to the four barcodes, allowing for 16 possible combinations.

[0187] Example 4 Quaternary Encoding The combination of the two barcodes can define 16 distinct ROIs using quaternary encoding. The sample is spatially defined with 16 regions of interest (ROIs). Two unique barcodes are applied in two rounds of crosslinking iterations. In each of the two crosslinking rounds, light is applied to 75% of the ROI at one of three different power levels, such that the most illuminated fraction in each round contains approximately ½ (50%) or less sites of crosslink saturation. This results in a 40% crosslinked fraction of the two barcode strands. 2 = 16 distinct combinations. The combinations are shown in Figure 9, where the gradation of filled squares indicates different fractions of reads in the area containing the barcode corresponding to that row, and open squares indicate that there was no illumination during that barcoding round. On the right, the quaternary encoding for the two barcodes can be assigned to each of these cases.

[0188] Example 5 Fraction Encoding Fractional barcoding provides a much larger number of possible distinct identification encodings. For example, illumination at 11 different power levels using only two barcodes can define 121 distinct ROIs. The sample is spatially defined with 11 regions of interest (ROIs). Two unique barcodes are applied in two rounds of crosslinking iterations. In each of the crosslinking rounds, light is applied to each ROI in 10 different gradients so that there is a unique ratio of barcode fractions. Light is applied in gradients that provide barcoded fractions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%. These 11 ratios of the two barcode strands are shown in FIG. 10, where the gradient of filled squares indicates different fractions of reads in the region containing the barcode corresponding to that row, and the open squares indicate that there was no illumination during that barcoding round. This is the cross-linked fraction of the two barcode strands of 121 (=11 *11). On the right, a ratio-based encoding for the two barcodes can be assigned to each of these cases. Figure 10 shows only a small portion of the 121 possible combinations of barcode fractions.

[0189] Example 6 Spatial barcoding across multiple tissues and tissue fixation protocols The robustness of the method to different tissue fixation protocols is an important feature of the spatial sequencing method described herein: it was found that consistent sequencing results were successfully generated from tissues prepared under different fixation conditions.

[0190] Fixed Brain, spleen and liver samples were collected from mice using three different fixation conditions. Fixation condition 1 (PFA) included transferring freshly dissected organs into 4% PFA in 1x PBS at room temperature for 30 minutes, followed by transfer to PBS and cryopreservation at -80°C. Fixation condition 2 (PFA-Tx) included transferring freshly dissected organs into 4% PFA with 0.25% Triton X-100 in 1x PBS for 30 minutes at RT, followed by three 5-minute washes in PBS, transfer to 7% sucrose in 1x PBS for 10 minutes, and then a final transfer to a 1:1 mixture of optimal cutting temperature (OCT) compound + 30% sucrose in 1x PBS. Samples were then cryopreserved at -80°C. Fixation condition 3 (formalin or FFPE) involved transfer of freshly dissected organs into 10% neutral buffered formalin overnight at room temperature for fixation, followed by transfer into PBS and frozen storage at -80°C.

[0191] Frozen tissue blocks were sectioned at 15 μm onto microscope cover slips and separated using well chambers for exchange of individually contained fluids.

[0192] Cleaning and barcoding Samples were washed three times with 60% formamide in 1x PBS, 0.1% Tween-20, then twice with 1x PBS, 0.1% Tween-20, 1M NaCl, then twice with 1x PBS, 0.1% Tween-20. A-tailing of the cDNA was then performed in situ using 1x ThermoPol buffer, 1 mM dATP and 1000 U / mL terminal transferase enzyme. Sections were incubated with this solution for 45 min at 37°C, then washed three times with 1x PBS, 0.1% Tween-20.

[0193] Light-induced barcoding of cDNA was performed using a Nikon Ti2-E microscope with a Mightex Polygon 1000 DMD connected to a 380 nm LED. Samples were incubated with a barcoding solution containing 250 nM fluorophore (Cy5)-labeled UMI-containing CNVK-containing barcode strand, 500 mM NaCl, and 10% dextran sulfate in 1×PBS, 0.1% Tween-20, at room temperature for 30 min. The barcoding solution was then removed and samples were washed with 1×PBS, 0.1% Tween-20, 1 M NaCl.

[0194] Photo Masking Square regions of interest for illumination were hand-drawn in Nikon Elements Software, and light was selectively directed to these regions using a DMD. Brain tissue was illuminated using a single large rectangular mask, whereas other tissues were illuminated using three individual square regions. Illumination was performed with 20% 380 nm LED power for 10 seconds. Samples were then washed eight times with 60% formamide in 1×PBS, 0.1% Tween-20, followed by two washes in 1×PBS, 0.1% Tween-20 with NaCl. Barcode signals were then imaged after washing, and visible fluorescently labeled barcode strands in the regions of interest (ROIs) indicate successful light-induced barcoding. Figure 11 shows representative images of brain, spleen and liver sections after light-induced transcriptome barcoding. Samples were prepared according to fixation conditions 1 (PFA), 2 (PFA-Tx) and 3 (formalin) as described herein. In the brain, barcode placement was largely restricted to cell bodies, with only faint signals observed in axon-dense regions, consistent with the majority of in situ transcribed cDNAs localized to cell bodies. Successful transcriptome barcoding was observed in all tissue types and all fixation conditions.

[0195] Extraction and sequencing Barcoded cDNA was then extracted from the samples by treatment with 250 U / mL RNase H in 1× ThermoPol reaction buffer and incubated for 45 minutes at 37° C. The barcoded cDNA was then transferred to a PCR tube for copying and amplification for next generation sequencing.

[0196] The extracted cDNA was copied using a poly-T primer hybridized to the A-tail of the cDNA sequence. Copies of the concatenated cDNA-barcode sequence were created by mixing the extracted cDNA with the primer sequence and adding strand-displacing Bst polymerase at 800 U / mL, 1× ThermoPol buffer, dNTPs at 100 uM, and poly-T primers at 20 nM for 45 minutes at 37° C. Samples were heat inactivated at 80° C. for 20 minutes.

[0197] The concentration of the cDNA library was quantified and the library was amplified in a qPCR machine using a 1:1 ratio of Sybr Green I, Kapa HiFi buffer, 600 nM each of forward and reverse primers, 600 μM dNTPs and Kapa HiFi Hot Start Polymerase PCR mix: 8° C. for 3 min, followed by 30 cycles of 98° C. for 20 s and 72° C. for 2 min. The amplified library was then tagmented and prepared for next generation sequencing using standard library preparation. Sequencing was performed on an Illumina MiniSeq machine using custom R1 and index 2 primers.

[0198] Data analysis To analyze next-generation sequencing data, sequences were parsed using a unique molecular identifier (UMI)-tool. cDNA sequences were mapped to the mouse genome (vM27) using the STAR aligner. All reads that were multiply mapped to the genome were discarded, and then single-mapped reads were mapped to annotated transcripts using the FeatureCounts tool. Unique cDNA sequences in the library were identified by de-duplication based on the UMIs that were attached to the cDNA via barcode oligos during the light-induced barcoding step. Only single UMI / mapped cDNA pairs were kept for all cases, resulting in a matrix of unique cDNA molecule counts across all genes. This was further referred to as the RNA count vector for each sample, and the RNA count matrix refers to a matrix that contains multiple RNA count vectors.

[0199] Next generation sequencing data from brain and liver samples fixed in formalin and PFA were analyzed for differential expression of genes to evaluate the consistency of transcriptome sequencing data across fixation conditions.Briefly, DESeq2 was used for differential expression analysis across samples, and significantly differentially expressed genes were called as genes with an adjusted p-value (after Benjamini-Hochberg correction for multiple hypothesis testing) of <0.05.Counting matrices were generated using the number of unique barcoded cDNAs detected in each spatially targeted region per gene.Table 2 shows the generated matrix.

[0200] As shown in Table 2, when comparing expression levels detected in PFA-fixed brains to formalin-fixed brains, there was only a single significantly differentially expressed gene. Similarly, there were zero significantly differentially expressed genes across formalin-fixed versus PFA-fixed liver samples. In contrast, when cross-organ comparisons were performed for all pairs of fixation conditions, these same samples showed hundreds to >1000 significantly differentially expressed genes. These data support that spatial transcriptomic methods produce consistent results from the same organ across fixation conditions and reproducible organ-specific results across organs. [Table 2]

[0201] Figure 11 demonstrates successful barcode placement across tissue samples from brain, spleen and liver tissues prepared using three different fixation conditions. Gene expression data shows that sequencing libraries from the same tissue were highly similar to each other, independent of the different fixation conditions. Differential expression analysis demonstrated hundreds to thousands of statistically differentially expressed genes when compared across organ types. At most, a single differentially expressed gene was found when compared across fixation conditions for the same organ.

[0202] Example 7 Spatial barcoding and transcriptome sequencing can be applied to FFPE and PFA-Tx fixed tissues Historically, it has been extremely difficult to sequence RNA from formalin-fixed paraffin-embedded (FFPE) tissues. However, many samples are routinely preserved using FFPE, and being able to generate data from FFPE samples as described herein provides a great advantage. Light-induced transcriptome barcoding in FFPE liver tissue was demonstrated using image-based readout. When FFPE liver sequencing results were compared (in the form of gene counting matrices), it was further demonstrated that the detected expression of genes was consistent between FFPE-fixed and PFA-Tx-fixed livers. FFPE liver gene counts also correlated well with PFA-Tx gene counts, but not with brain counts.

[0203] For PFA-Tx samples, mouse liver was fixed with 4% PFA, 0.25% Triton-X-100 in 1×PBS solution by intracardiac perfusion, then dissected and incubated in fixative overnight at 4°C. Tissue was then washed with 1×PBS and immersed in 7.5% sucrose in 1×PBS. Tissue was then immersed in a 1:1 solution of 30% sucrose and OCT cryoprotectant solution in 1×PBS. Tissue was then stored at -80°C until use. For FFPE liver tissue, tissue was submerged in fresh 10% neutral buffered formalin solution for between 4 and 72 hours, long enough to ensure proper fixation. Tissue was then embedded in paraffin and stored at 4°C until use. For PFA brain tissue samples, brains were dissected from animals after death and dropped into 4% PFA, 0.25% Triton-X-100 in 1xPBS solution at room temperature for 30 minutes, then immersed in 7.5% sucrose in 1xPBS. Tissues were then immersed in a 1:1 solution of 30% sucrose and OCT cryoprotectant solution in 1xPBS. Tissues were then stored at -80°C until spatial sequencing.

[0204] 15 micron sections were sliced, placed on microscope slides and baked at 60°C for 1 hour. For FFPE samples, sections were deparaffinized and antigen retrieval was performed in decloaking buffer at 90°C for 15 minutes, then at 70°C for 30 seconds, then cooled to room temperature. Sections were then washed twice with water, slides were air-dried, then re-wetted with 1x PBS, 0.1% Tween, before proceeding to the reverse transcription step below. For non-FFPE brain and liver tissues, sections were washed three times with 1x PBS, 0.1% Tween-20 and incubated with antigen retrieval buffer (Tris-EDTA, pH 8) at 70°C for 30 minutes. Samples were then returned to 1x PBS, 0.1% Tween, before proceeding to the reverse transcription step below.

[0205] All tissue sections underwent the same processing steps for cDNA library generation, barcoding and sequence library preparation: tissue sections were washed three times with 1x PBS, 0.1% Tween-20 and then incubated with reverse transcription mix (300 μM dNTPs, 0.5% Triton-X-100, 6 mM RNaseOUT, 1 μM RT primer and 8 U / μl Maxima RT enzyme in RT buffer) at 22°C for 30 min, followed by 12 cycles of 8°C for 30 s, 15°C for 30 s, 25°C for 30 s, 30°C for 1 min, 37°C for 1 min and 42°C for 2 min, followed by 42°C for 30 min.

[0206] Sample cleaning and barcoding was performed as described in Example 6.

[0207] Regions of interest for illumination were hand-drawn in Nikon Elements Software, and light was selectively directed to these regions using a DMD. Illumination was performed for 10 seconds at 20% LED power. Three section replicates of each HIER condition (both 95°C and 70°C) were treated as "no UV controls" that did not receive illumination with the 380 nm LED, but were otherwise treated identically. Samples were then washed eight times with 60% formamide in 1x PBS and 0.1% Tween-20, followed by two washes in 1x PBS, 0.1% Tween-20, and NaCl. Barcode signals were then imaged after washing, and visible fluorescently labeled barcode strands in the ROI indicate successful light-induced barcoding. Figure 12A shows a representative image of light-induced spatial transcriptome barcoding in FFPE liver sections, with circular and square regions of interest for illumination noted. This figure shows successful light-induced transcriptome barcoding in FFPE samples.

[0208] Sample extraction and sequencing were performed as described in Example 6.

[0209] Sequencing data analysis was performed as described in Example 6. The top 50 differentially expressed genes from the full transcriptome sequencing data derived from spatially barcoded brain samples (n=6 replicates) fixed with PFA-Tx versus liver samples (n=3, two of which were fixed with PFA-Tx perfusion and drop fixation, and one was fixed with standard FFPE fixation procedure) were summarized in a heatmap in FIG. 12B from the calculated z-scores. Differential expression analysis revealed gene expression patterns across the two organs, and consistent expression patterns of the top differentially expressed genes were found across liver samples with different fixation conditions. Region 1 of FIG. 12B shows the z-scores of expression levels of canonically brain-expressed genes detected in fixed brain samples that show high expression levels. Region 2 of FIG. 12B shows the z-scores of expression levels of canonically brain-expressed genes detected in fixed liver samples that show low expression levels. Region 3 of Figure 12B shows the z-scores of expression levels of canonically liver-expressed genes detected in fixed brain samples that exhibit low expression levels, and Region 4 of Figure 12B shows the z-scores of expression levels of canonically liver-expressed genes detected in fixed liver samples that exhibit high expression levels.

[0210] The differential expression results in liver and brain samples are summarized in a volcano plot in Figure 12C. Liver data are from two PFA-Tx fixed sections and one FFPE fixed section. Brain data are from six PFA-TX fixed sections. Data are derived from the same differential expression data used in Figure 12B. Regardless of fixation conditions in liver samples, differential expression was powered enough to recover thousands of differentially expressed genes between organs, with a corrected p-value << 0.05 for multiple hypothesis testing (y-axis) and a log2 fold change (x-axis) with an absolute value of up to 10. As expected, selected genes composed of gene sets with canonical organ-specific expression are displayed.

[0211] A heatmap generated from the z-scores of the DESeq2 analysis of the full cohort of 2,653 differentially expressed genes is shown in FIG. 12D. Rows indicate individual genes and columns indicate individual samples (the same samples analyzed in FIG. 12B and FIG. 12C). Colors reflect the z-scores of normalized transcript counts. Region 1 of FIG. 12D shows the z-scores of expression levels of canonically brain-expressed genes detected in fixed brain samples that generally show higher expression levels compared to the average across all samples. A higher z-score (darker color) indicates enrichment of that gene in that sample compared to the average across all samples. Specifically, the z-score is: ((gene expression value in sample of interest)-(average expression across all samples)) / standard deviation across all samples. Region 2 of FIG. 12D shows the z-scores of expression levels of canonically brain-expressed genes detected in fixed liver samples that generally show lower expression levels compared to the average. Region 3 of Figure 12D shows the z-score of expression levels of canonically liver-expressed genes detected in fixed brain samples, which generally show lower expression levels compared to the average. Region 4 of Figure 12D shows the z-score of expression levels of canonically liver-expressed genes detected in fixed liver samples, which show higher expression levels compared to the average. The high number of significantly differential genes across brain samples compared to liver samples, and the consistency of gene expression (shown as color, z-score) compared to brain samples further supports the consistency of FFPE-fixed liver sample transcriptomes with PFA-Tx-fixed liver transcriptomes.

[0212] To directly assess the consistency of the spatially barcoded and sequenced transcriptomes for FFPE-fixed versus PFA-Tx-fixed tissues, pairwise Pearson correlations were calculated to compare the unique mRNA gene expression vectors, alternatively referred to as mRNA counting vectors, for each pair of samples. The resulting Pearson correlation numbers are summarized in a heatmap shown in Figure 12E. This metric directly assesses the concordance of gene expression across all genes in the genome. Colors indicate the Pearson correlation coefficient between paired samples. For FFPE versus PFA-Tx livers, the Pearson correlation coefficients were >0.7 in all cases, indicating a strong correlation between liver samples despite the different fixations. The Pearson correlation coefficients for all inter-organ comparisons were <0.3.

[0213] Example 8 Heat-induced epitope retrieval (HIER) to increase recovery of barcoded cDNA Protocol optimization for the tissue pre-treatment step was performed to increase the amount of barcoded cDNA that could be recovered from a given tissue area. This example tested heat-induced epitope retrieval conditions at two different temperatures and found that higher temperatures resulted in higher amounts of barcoded cDNA. Quantification of fluorescent barcode images and qPCR Cq values ​​indicate that the library was amplified. When samples were treated with a 95°C heat step, the intensity of barcoding increased the image data and decreased the Cq value. Both of these measurements indicated that more cDNA was barcoded when this tissue pre-treatment step was added.

[0214] Mouse liver was fixed with 4% PFA, 0.25% Triton-X-100 in 1xPBS solution by intracardiac perfusion, then dissected and incubated in fixative overnight at 4°C. Tissue was then washed with 1xPBS and immersed in 7.5% sucrose in 1xPBS. Tissue was then immersed in a 1:1 solution of 30% sucrose and OCT cryoprotectant solution in 1xPBS. Tissue was then stored at -80°C until use. Tissue was then immersed in a 1:1 solution of 30% sucrose and OCT cryoprotectant solution in 1xPBS. Tissue was then stored at -80°C until spatial sequencing was performed.

[0215] Frozen tissue blocks were then sectioned onto microscope coverslips at 15 μm and separated using a well chamber for exchange of individually contained liquid. Slides were baked at 60° C. for 1 hour. Sections were then washed three times with 1× PBS, 0.1% Tween-20 and incubated for 30 minutes at either 70° C. or 95° C. with antigen retrieval buffer (Tris-EDTA, pH 8) for heat-induced epitope retrieval (HIER) to directly compare the two conditions. All tissue sections then underwent the same processing steps for cDNA library generation, barcoding and sequence library preparation. Tissue sections were washed three times with 1x PBS containing 0.1% Tween-20 and then incubated with reverse transcription mix (300 μM dNTPs, 0.5% Triton-X-100, 6 mM RNaseOUT, 1 μM RT primer and 8 U / μl Maxima RT enzyme in RT buffer) at 22°C for 30 min, followed by 12 cycles of 8°C for 30 s, 15°C for 30 s, 25°C for 30 s, 30°C for 1 min, 37°C for 1 min and 42°C for 2 min, followed by incubation at 42°C for 30 min.

[0216] Samples were washed three times with 1x PBS, 0.1% Tween-20 containing 60% formamide, then twice with 1x PBS, 0.1% Tween-20 and 1M NaCl, then twice with 1x PBS, 0.1% Tween-20. A-tailing of cDNA was then performed in situ using 1x A-ThermoPol buffer, 1 mM dATP and 1000 U / mL terminal transferase enzyme. Sections were incubated with this solution for 45 min at 37°C, then washed three times with 1x PBS, 0.1% Tween-20.

[0217] Light-induced barcoding of cDNA was then performed using a Nikon Ti2-E microscope with a Mightex Polygon 1000 DMD connected to a 380 nm LED. Samples were incubated with a barcoding solution containing 250 nM FITC-labeled UMI-containing CNVK-containing barcode strands, 500 mM NaCl and 10% dextran sulfate in 1×PBS, 0.1% Tween-20 for 30 minutes at room temperature. The barcoding solution was then removed and samples were washed with 1×PBS, 0.1% Tween-20 and 1 M NaCl.

[0218] Regions of interest for illumination were hand-drawn in Nikon Elements Software, and the light was selectively directed to these regions using the DMD. Illumination was performed with 20% 385 nm LED power for 10 seconds. The samples were then washed 8 times with 60% formamide in 1×PBS, 0.1% Tween-20, followed by two washes in 1×PBS, 0.1% Tween-20 with NaCl. Barcode signals were then imaged after washing, and visible fluorescently labeled barcode strands in the ROI indicate successful light-induced barcoding. Images of barcoded liver sections after HIER treatment at 70° C. and 95° C. are shown in FIG. 13A and FIG. 13B, respectively. The images show stronger barcoding signals in the higher temperature conditions.

[0219] The amount of barcode placement was quantified based on fluorescence imaging of tissue sections after transcriptome barcoding with FITC-labeled barcode strands. The average intensity of fluorescence within a rectangular barcoded area acquired across n=3 replicates for each HIER condition was calculated. Figure 13C shows a bar plot of the calculated means. Error bars indicate standard deviation. These data demonstrate that the efficiency of barcode placement is increased after HIER at 95°C compared to 70°C.

[0220] Sample extraction and sequencing was performed as described in Example 6. After thermocycling, the efficiency of light-induced barcoding was compared across + / -UV conditions by comparing Cq qPCR values ​​(a lower signal is expected from the -UV condition compared to +UV).

[0221] Quantification of the number of barcoded molecules in the barcoded cDNA libraries prepared from the different HIER and + / -UV conditions was assessed using qPCR designed to use primers that only amplify barcoded cDNA. The bar plot in Figure 13D shows the average Cq values ​​across n=3 replicates with standard deviation error bars. Samples +UV received spatially patterned UV illumination with a 380 nm LED for barcoding, whereas -UV samples did not. The 95°C HIER condition +UV amplified approximately 2.5 cycles faster than the 70°C HIER +UV condition, consistent with the higher number of barcoded cDNA molecules in the 95°C HIER condition. Combined with the increased image intensity of the barcoded region after light-induced barcoding, these data support that 95°C HIER allows for more light-induced barcoding. Example 9 Spatial Light Guided Bar Coding Using Separate Optical Systems - Patent application The method for light-induced spatial barcoding was demonstrated over a wide range of optical parameters, including wavelength, light delivery modality (LED vs. laser), illumination time and illumination power. This was shown by representative image data when fluorescent DNA barcodes were placed on tissue sections that underwent reverse transcription in situ to introduce barcode docking sites on the 5' end of cDNA. A Cy5-labeled barcode strand containing CNVK was then hybridized to the sample, and a rectangular area was illuminated with different optical parameters within the same tissue section. Light-induced barcoding was successful for a wide range of parameters, with some conditions producing more efficient barcoding than others. These data highlight the robustness of the method for light-induced spatial barcoding.

[0222] Mouse liver was fixed with 4% PFA, 0.25% Triton-X-100 in 1xPBS solution by intracardiac perfusion, then dissected and incubated in fixative overnight at 4°C. Tissue was then washed with 1xPBS and immersed in 7.5% sucrose in 1xPBS. Tissue was then immersed in a 1:1 solution of 30% sucrose and OCT cryoprotectant solution in 1xPBS. Tissue was then stored at -80°C until spatial sequencing.

[0223] 15 micron sections were sliced, placed on microscope slides and baked for 1 hour at 60° C. Sections were then washed three times with 1× PBS, 0.1% Tween-20 and incubated with antigen retrieval buffer (Tris-EDTA, pH 8) at 95° C. for 30 minutes.

[0224] The tissue sections underwent the following processing steps for cDNA library generation: the tissue sections were washed three times with 1x PBS and 0.1% Tween-20, then incubated with reverse transcription mix (300 μM dNTPs, 0.5% Triton-X-100, 6 mM RNaseOUT, 1 μM RT primer and 8 U / μl Maxima RT enzyme in RT buffer) at 22°C for 30 min, followed by 12 cycles of 8°C for 30 s, 15°C for 30 s, 25°C for 30 s, 30°C for 1 min, 37°C for 1 min and 42°C for 2 min, followed by incubation at 42°C for 30 min.

[0225] Samples were washed three times with 60% formamide in 1x PBS, 0.1% Tween-20, then twice with 1x PBS, 0.1% Tween-20, 1M NaCl, then twice with 1x PBS, 0.1% Tween-20. A-tailing of cDNA was then performed in situ using 1x A-ThermoPol buffer, 1 mM dATP and 1000 U / mL terminal transferase enzyme. Sections were incubated with this solution for 45 min at 37°C, then washed three times with 1x PBS, 0.1% Tween-20.

[0226] Light-induced barcoding of cDNA was then performed using a Nikon Ti2-E microscope with two separate optics. Optic 1 was a Mightex Polygon 1000 DMD connected to a 380 nm LED. Optic 2 was a 405 nm laser connected to an optimicroscanner. All illumination was performed through a 20× air objective. For barcoding, samples were incubated for 30 min at room temperature with a barcoding solution consisting of 250 nM fluorophore (Cy5)-labeled UMI-containing CNVK-containing barcode strands in 1× PBS, 0.1% Tween-20, 500 mM NaCl, and 10% dextran sulfate. The barcoding solution was then removed and samples were washed with 1× PBS, 0.1% Tween-20, 1 M NaCl, and then barcoded.

[0227] A rectangular region of interest was drawn within Nikon Elements Software to guide the lighting. For optic 1, lighting power was sampled between 1-10% and time was sampled between 1-20 seconds. Conditions using 5% power and using 1, 5, 10 and 20 seconds for a single lighting round versus two lighting rounds were also tested. For optic 2, lighting power was adjusted between 0.1%, 0.5%, 1%, 2%, 5%, 10%, crossed with dwell time variables of 10 μs, 100 μs and 1000 μs. Together, this provided a broad sampling of the parameter space for lighting conditions.

[0228] After barcoding, the samples were then washed 8 times with 60% formamide in 1×PBS, 0.1% Tween-20, followed by 2 washes in 1×PBS, 0.1% Tween-20 with NaCl. The barcode signal was then imaged after washing (FIG. 14A). Image capture of autofluorescence of the same field of view (FIG. 14B) was merged with the barcode imaging (FIG. 14C) to show the location and correlation of barcoding within the sample. The imaging shows visible fluorescently labeled barcode strands in the ROI, indicating successful light-induced barcoding under both conditions.

[0229] FIG. 14A shows light-induced spatial barcoding using two optical modalities for UV light delivery: an OptiMicro Scanner (Galvo) with a 405 nm laser light source (top rectangle), and a DMD with a 380 nm LED light source (bottom rectangle). The use of the OptiMicro Scanner resulted in a higher precision of light delivery, allowing for higher resolution. However, it was significantly more time-consuming for the OptiMicro Scanner, as it illuminated every individual pixel in the area of ​​interest sequentially. The 405 nm laser is more ubiquitous on most confocal systems, offering the advantage of accessibility. The DMD / 380 nm LED setup was advantageous, as it allowed for collimated light delivery to any spatial pattern within the field of view (FOV). This provides for shorter processing times for large areas of interest.

[0230] Figure 14D shows the relative intensity of four barcoded regions from the dashed rectangular region in Figure 14A, labeled using a 405 nm laser at the indicated power and duration. Autofluorescence of the same area is shown in Figure 14E. Figure 14F shows a combined image of both barcoding and autofluorescence.

[0231] Example 10 Fractional barcoding of cellular transcriptomes The ability to adjust the fraction of barcoded cDNA sequences in a cell or tissue region allows encoding of information.As described herein, if the local fraction of barcoded transcripts can be read out, fractional barcoding can be used to allow regional identification.These data support that more than one spatial barcode can be placed on the same cell / tissue region, and that adjustable fraction of barcoded cDNA can be achieved in a deterministic manner.

[0232] Retinal tissue was collected from mice and transferred to 4% PFA and 0.25% Triton X-100 in 1x PBS for 30 min at RT, followed by three 5 min washes in PBS, transferred to 7% sucrose in 1x PBS for 10 min, and then finally transferred to a 1:1 mixture of OCT + 30% sucrose in 1x PBS. Samples were then stored frozen at -80°C.

[0233] The frozen tissue blocks were then sectioned at 15 μm onto microscope cover slips and separated using a well chamber for exchange of the individually contained liquid. The tissue sections then underwent the following processing steps for cDNA library generation: the tissue sections were washed three times with 1×PBS 0.1% Tween-20, then incubated with reverse transcription mix (300 μM dNTPs, 0.5% Triton-X-100, 6 mM RNaseOUT, 1 μM RT primer and 8 U / μl Maxima RT enzyme in RT buffer) and incubated at 22°C for 30 minutes, followed by 12 cycles of 8°C for 30 seconds, 15°C for 30 seconds, 25°C for 30 seconds, 30°C for 1 minute, 37°C for 1 minute and 42°C for 2 minutes, followed by 42°C for 30 minutes.

[0234] Samples were washed three times with 60% formamide in 1x PBS, 0.1% Tween-20, then twice with 1x PBS, 0.1% Tween-20, 1M NaCl, then twice with 1x PBS, 0.1% Tween-20. A-tailing of cDNA was then performed in situ using 1x A-ThermoPol buffer, 1 mM dATP and 1000 U / mL terminal transferase enzyme. Sections were incubated with this solution for 45 min at 37°C, then washed three times with 1x PBS, 0.1% Tween-20.

[0235] Light-induced barcoding of cDNA was then performed using a Nikon Ti2-E microscope with a Mightex Polygon 1000 DMD connected to a 380 nm LED. Samples were incubated with the first barcode in barcoding solution 1 consisting of 250 nM FITC-labeled UMI-containing CNVK-containing barcode strands, 500 mM NaCl and 10% dextran sulfate in 1×PBS, 0.1% Tween-20 for 30 minutes at room temperature. Barcoding solution 1 was then removed and samples were washed with 1×PBS, 0.1% Tween-20, 1 M NaCl.

[0236] For the bicolor barcoding shown in Figures 15A-C, square regions of interest for illumination were hand-drawn in Nikon Elements Software, and UV light was selectively directed to these regions using a DMD. From left to right, different amounts of barcode 1 were placed in separate square regions using each of the following illumination conditions (n=3 replicates per condition): 0% power / 0 seconds, 1% power / 5 seconds, 5% power / 5 seconds, 10% power / 5 seconds, 20% power / 5 seconds. Imaging of the ROI with barcode 1 is shown in Figure 15A, where power in the area increases from left to right.

[0237] Barcode 2 was then incubated with the sections in a solution consisting of 250 nM fluorophore (Cy3)-labeled UMI-containing CNVK-containing barcode strands in 1×PBS, 0.1% Tween-20, 500 mM NaCl, and 10% dextran sulfate at room temperature for 30 minutes. The barcoding solution was then removed and the samples were washed with 1×PBS, 0.1% Tween-20, 1 M NaCl. Illumination was performed for 10 seconds with 20% 380 nm LED power in all previously barcoded squares.

[0238] The samples were then washed 8 times with 60% formamide in 1×PBS, 0.1% Tween-20, followed by 2 washes in 1×PBS, 0.1% Tween-20 with NaCl. The barcode signal was then imaged after washing, as shown in FIG. 15B. The imaging shows that visible fluorescently labeled barcode strands in the ROI indicate successful light-induced barcoding. FIG. 15C shows an overlay of the two imaged ROIs.

[0239] Light-induced barcoding was quantified through the analysis of image data by calculating the average intensity of fluorescence in the barcoded region of interest in the channel corresponding to the barcode.Since the DNA barcodes were conjugated to separate fluorophores (FITC and Cy3), the fluorescence in each channel could be quantified to evaluate the placement of each barcode.Figure 15D shows a bar graph showing the ratio of the average image intensity value of the FITC / Cy3 channel in each condition after barcoding.

[0240] Example 11 Small molecule dyes guide spatial barcoding Barcoding of specific cells, subcellular features and tissue regions can enable highly selective transcriptome sequencing. Our light-induced barcoding allows for high-resolution barcode placement and can be guided based on small molecule stains. Herein, we demonstrate pre-staining of tissue sections with a small molecule stain (Deep Red Anthraquinone 5 (DRAQ5™)) that stains cell nuclei by intercalating into double-stranded DNA with high affinity to guide photomasking for light-induced transcriptome barcoding into individual cells. Results demonstrate that barcoding can be guided by the pre-stain and restricted to regions of interest guided by the stain.

[0241] Mouse brains were dissected into 10% neutral buffered formalin for overnight fixation at room temperature, then transferred into PBS and stored frozen at -80°C. Frozen tissue blocks were then sectioned onto microscope coverslips at 15 μm and separated using a well chamber for exchange of individually contained liquid. Slides were baked at 60°C for 1 hour. Sections were then washed three times with 1×PBS, 0.1% Tween-20 and incubated with antigen retrieval buffer (Tris-EDTA, pH 8) at 70°C for 30 minutes. Samples were then returned to 1×PBS, 0.1% Tween before proceeding to the following reverse transcription step.

[0242] The tissue sections underwent steps for cDNA library generation: They were washed three times with 1x PBS containing 0.1% Tween-20, then incubated with reverse transcription mix (300 μM dNTPs, 0.5% Triton-X-100, 6 mM RNaseOUT, 1 μM RT primer and 8 U / μl Maxima RT enzyme in RT buffer) at 22°C for 30 min, followed by 12 cycles of 8°C for 30 s, 15°C for 30 s, 25°C for 30 s, 30°C for 1 min, 37°C for 1 min and 42°C for 2 min, followed by incubation at 42°C for 30 min.

[0243] Samples were washed three times with 60% formamide in 1x PBS, 0.1% Tween-20, then twice with 1x PBS, 0.1% Tween-20, 1M NaCl, then twice with 1x PBS, 0.1% Tween-20. A-tailing of the cDNA was then performed in situ using 1x ThermoPol buffer, 1 mM dATP and 1000 U / mL terminal transferase enzyme. Sections were incubated with this solution for 45 min at 37°C, then washed three times with 1x PBS, 0.1% Tween-20.

[0244] Light-induced barcoding of cDNA was performed using a Nikon Ti2-E microscope with a Mightex Polygon 1000 DMD connected to a 380 nm LED and a 40× water immersion objective. Samples were incubated with barcoding solution 1 consisting of 250 nM fluorophore (FITC)-labeled UMI-containing CNVK-containing barcode strands in 1× PBS, 0.1% Tween-20, 500 mM NaCl, and 10% dextran sulfate at room temperature for 30 min. The barcoding solution was then removed and the samples were washed with 1× PBS, 0.1% Tween-20, 1 M NaCl. DRAQ5 nuclear stain was included in this wash solution and kept on the samples during light-induced barcoding to label cell nuclei. Imaging of nuclear stained cells is shown in FIG. 16A.

[0245] A photomask was generated based on the nuclear stain to delineate three specific cells, shown as dashed lines in FIG. 16A. Illumination was performed for 10 seconds with 10% 380 nm LED power. Samples were then washed eight times with 60% formamide in 1×PBS, 0.1% Tween-20, followed by two washes in 1×PBS, 0.1% Tween-20 with NaCl.

[0246] The barcode signal was then imaged after washing, as shown in Figure 16B. The appearance of fluorescently labeled barcode strands in the ROI indicates successful light-induced barcoding. Figure 16C is a representative overlay of small molecule nuclear stain and fluorescent DNA barcode images.

[0247] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in carrying out the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for generating biological cell information, comprising: contacting the population of cells with a nucleic acid polymerase and a primer to generate an amplification product; placing a DNA barcode on said population of cells, said DNA barcode comprising a region complementary to a region of said amplification product, said DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating said population of cells to form covalently linked DNA barcode-amplified product complexes; washing the population of cells to remove the DNA barcodes that are not complexed with an amplification product; and repeating the steps of placing, selectively irradiating, and cleaning; the DNA barcode in a first placing step of the placing steps comprises a different sequence than the DNA barcode in a second placing step of the placing steps; the selectively illuminating step is performed at different locations for a first selectively illuminating step and a second selectively illuminating step. A method comprising:

2. 10. The method of claim 1, further comprising determining biological cell spatial information based at least in part on the amplification products and the DNA barcodes.

3. 3. The method of claim 1 or 2, further comprising a step of synthesizing a continuous DNA strand, said synthesizing step comprising contacting said DNA barcode-amplification product complex with a polymerase.

4. The method of claim 3 , wherein the step of combining comprises cross-junction combination.

5. 5. The method of claim 4, further comprising releasing the continuous DNA strand from the population of cells.

6. The method of claim 3 , wherein the synthesizing and releasing steps are performed simultaneously.

7. The method of claim 6, wherein the released continuous DNA strands are pooled.

8. The method of claim 7, wherein the pooled contiguous DNA strands are amplified.

9. 8. The method of claim 7, wherein the pooled contiguous DNA strands are sequenced.

10. 10. The method of claim 9, further comprising identifying a target associated with said continuous DNA strand.

11. The method of claim 10 further comprising mapping the targets to the different locations.

12. The method of any one of claims 1 to 11, wherein the primer is a random primer.

13. The method of any one of claims 1 to 12, wherein the primer is a non-random primer.

14. 14. The method of any one of claims 1 to 13, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK).

15. 15. The method of claim 1, wherein the selectively irradiating step is performed using a photomask.

16. The method of claim 15 , wherein the photomask is varied at different selectively irradiating steps.

17. The method of claim 15 , wherein the photomask is manually generated.

18. The method of claim 15 , wherein the photomask is machine generated.

19. 19. The method of any one of claims 1 to 18, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 300 nm to 450 nm.

20. 20. The method of claim 1, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 350 nm to 420 nm.

21. 21. The method of any one of claims 1 to 20, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 365 nm to 405 nm.

22. 22. The method of any one of claims 1 to 21, wherein the selectively irradiating step comprises applying irradiation to a preselected region of interest (ROI).

23. The method of claim 22, wherein the ROI is cell-specific or sub-cellular-specific.

24. The method of claim 22 , wherein the ROI is based on morphology.

25. 25. The method of any one of claims 1 to 24, wherein the placing step is performed manually, automatically, robotically, or any combination thereof.

26. 26. The method of any one of claims 1 to 25, wherein the placing step is performed using a modified inkjet printer.

27. 27. The method of any one of claims 1 to 26, wherein the placing or washing steps are performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, an acoustic liquid handler, an inkjet printer, an automated liquid handler, or any combination thereof.

28. 28. The method of any one of claims 1 to 27, wherein the placing or washing step is performed using a pipettor.

29. 29. The method of any one of claims 1 to 28, wherein the repeating step is completed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times or more.

30. 30. The method of any one of claims 1 to 29, wherein at least 2, 3, 4, 5, 6, 7, 8 or 9 different DNA barcode strands are arranged.

31. 31. The method of any one of claims 1 to 30, further comprising contacting the population of cells with a cell permeabilizing agent.

32. 32. The method of any one of claims 1 to 31, further comprising contacting the population of cells with an agent that produces a fixed population of cells.

33. 33. The method of claim 32, wherein the agent that produces the population of fixed cells comprises formalin, formaldehyde, paraformaldehyde, methanol, acetic acid, or any combination thereof.

34. 33. The method of claim 32, wherein the population of fixed cells is fixed to a substrate.

35. 35. The method of any one of claims 32 to 34, wherein the fixed cells are formalin-fixed paraffin-embedded (FFPE) cells, fixed with paraformaldehyde (PFA), or fixed with paraformaldehyde with Triton X-100 (PFA-Tx).

36. wherein said population of cells Cultured cells; Tissue obtained from the subject; Manipulated organization; or Organoids 36. The method of any one of claims 1 to 35, comprising one or more of:

37. 37. The method of claim 36, wherein the tissue is an intact tissue or a dissected tissue.

38. 37. The method of claim 36, wherein the cultured cells are dissociated cells.

39. 37. The method of claim 36, wherein the engineered tissue comprises a population of human cells differentiated in vitro.

40. 40. The method of claim 39, wherein the in vitro differentiated human cells are derived from human induced pluripotent stem cells (hiPSCs), embryonic stem cells or adult stem cells.

41. 41. The method of any one of claims 1 to 40, wherein the population of cells is retinal cells.

42. 42. The method of any one of claims 1 to 41, further comprising imaging the population of cells after the washing step.

43. 43. The method of any one of claims 1 to 42, wherein the cleaning step comprises depositing a solution comprising a chemical denaturant and a cleaning agent.

44. 44. The method of claim 43, wherein the chemical denaturant comprises formamide.

45. 45. The method of claim 44, wherein the formamide is present in an amount of about 40% (v / v) to about 60% (v / v).

46. 45. The method of claim 44, wherein the formamide is present in an amount of about 50% to about 60%.

47. 44. The method of claim 43, wherein the detergent comprises polysorbate 20, polysorbate 80, or Triton X-100.

48. 44. The method of claim 43, wherein the detergent is present in an amount between about 0.05% (v / v) and about 5% (v / v).

49. 49. The method of any one of claims 1 to 48, wherein the DNA barcode further comprises a detectable label.

50. 50. The method of any one of claims 1 to 49, wherein the step involving washing the population of cells is omitted.

51. 1. A method for generating biological cell spatial information, comprising: contacting a population of cells with a capture probe, said capture probe comprising a DNA binding region and a target binding region; placing a DNA barcode on said population of cells, said DNA barcode comprising a region that binds to said DNA binding region, said DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating said population of cells to form DNA barcode-amplified product complexes; washing the population of cells to remove the DNA barcodes that are not complexed with an amplification product; and repeating the steps of placing, selectively irradiating, and cleaning; the DNA barcode in a first placing step of the placing steps comprises a different sequence than the DNA barcode in a second placing step of the placing steps; the selectively illuminating step is performed at different locations for a first selectively illuminating step and a second selectively illuminating step. A method comprising:

52. 52. The method of Claim 51, further comprising determining biological cell spatial information based at least in part on said DNA barcode-amplification product complexes.

53. 52. The method of claim 51 , wherein the capture probe comprises RNA or DNA.

54. 52. The method of claim 51 , wherein the capture probe comprises a protein.

55. 55. The method of any one of claims 51 to 54, further comprising the step of synthesizing a continuous strand of DNA, said synthesizing step comprising contacting said population of cells with a polymerase.

56. 56. The method of claim 55, wherein the combining step comprises cross-junction combination.

57. 57. The method of claim 56, further comprising releasing said continuous DNA strand from said population of cells.

58. 58. The method of claim 57, wherein the synthesizing and releasing steps are performed simultaneously.

59. 59. The method of claim 58, wherein the released continuous DNA strands are pooled.

60. 60. The method of claim 59, wherein the pooled contiguous DNA strands are amplified.

61. 60. The method of claim 59, wherein the pooled contiguous DNA strands are sequenced.

62. 56. The method of claim 55, further comprising identifying a target associated with said continuous DNA strand.

63. 63. The method of claim 62, further comprising mapping the targets to the different locations.

64. 64. The method of any one of claims 51 to 63, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK).

65. 65. The method of any one of claims 51 to 64, wherein the selectively irradiating step is performed using a photomask.

66. 66. The method of claim 65, wherein the photomask is varied at different selectively irradiating steps.

67. 66. The method of claim 65, wherein the photomask is manually generated.

68. 66. The method of claim 65, wherein the photomask is generated by machine.

69. 69. The method of any one of claims 51 to 68, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 300 nm to 450 nm.

70. 70. The method of any one of claims 51 to 69, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 350 nm to 420 nm.

71. 71. The method of any one of claims 51 to 70, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 365 nm to 405 nm.

72. 72. The method of any one of claims 51 to 71, wherein the selectively irradiating step comprises applying irradiation to a preselected region of interest (ROI).

73. 73. The method of claim 72, wherein the ROI is cell-specific or subcellular-specific.

74. 73. The method of claim 72, wherein the ROI is based on morphology.

75. 75. The method of any one of claims 51 to 74, wherein the placing step is performed manually, automatically, robotically, or any combination thereof.

76. 76. The method of any one of claims 51 to 75, wherein the placing step is performed using a modified inkjet printer.

77. 76. The method of any one of claims 51 to 75, wherein the placing or washing steps are performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, an acoustic liquid handler, an inkjet printer, an automated liquid handler, or any combination thereof.

78. 78. The method of any one of claims 51 to 77, wherein the placing or washing step is performed using a pipettor.

79. 79. The method of any one of claims 51 to 78, wherein the repeating step is completed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times or more.

80. 80. The method of any one of claims 51 to 79, wherein at least 2, 3, 4, 5, 6, 7, 8 or 9 different DNA barcode strands are arranged.

81. 81. The method of any one of claims 51 to 80, further comprising contacting said population of cells with a cell permeabilizing agent.

82. 82. The method of any one of claims 51-81, further comprising contacting the population of cells with an agent that produces a fixed population of cells.

83. 83. The method of claim 82, wherein the agent that produces a population of fixed cells comprises formalin, formaldehyde, paraformaldehyde, methanol, acetic acid, or any combination thereof.

84. 83. The method of claim 82, wherein said population of fixed cells is fixed to a substrate.

85. 85. The method of any one of claims 82 to 84, wherein the fixed cells are formalin-fixed paraffin-embedded (FFPE) cells, fixed with paraformaldehyde (PFA), or fixed with paraformaldehyde with Triton X-100 (PFA-Tx).

86. wherein said population of cells Cultured cells; Tissue obtained from the subject; Manipulated organization; or Organoids 86. The method of any one of claims 51 to 85, comprising one or more of:

87. 87. The method of claim 86, wherein said population of cells comprises a tissue, said tissue being an intact tissue or a dissected tissue.

88. 87. The method of claim 86, wherein said population of cells comprises cultured cells, and said cultured cells are dissociated cells.

89. 87. The method of claim 86, wherein the engineered tissue comprises a population of human cells differentiated in vitro.

90. 90. The method of claim 89, wherein the in vitro differentiated human cells are derived from human induced pluripotent stem cells (hiPSCs), embryonic stem cells or adult stem cells.

91. 91. The method of any one of claims 51 to 90, wherein the population of cells comprises retinal cells.

92. 92. The method of any one of claims 51 to 91, further comprising imaging the population of cells after said washing step.

93. 93. The method of any one of claims 51 to 92, wherein the cleaning step comprises depositing a solution comprising a chemical denaturant and a cleaning agent.

94. 94. The method of claim 93, wherein the chemical denaturing agent comprises formamide.

95. 95. The method of claim 94, wherein the formamide is present in an amount of about 40% (v / v) to about 60% (v / v).

96. 95. The method of claim 94, wherein the formamide is present in an amount of about 50% to about 60%.

97. 94. The method of claim 93, wherein the detergent comprises polysorbate 20, polysorbate 80, or Triton X-100.

98. 94. The method of claim 93, wherein the detergent is present in an amount between about 0.05% (v / v) and about 5% (v / v).

99. 99. The method of any one of claims 51 to 98, wherein the DNA barcode further comprises a detectable label.

100. 100. The method of any one of claims 51 to 99, wherein the target binding region binds to DNA, RNA, protein, or a combination thereof.

101. 101. The method of any one of claims 51 to 100, further comprising multiple contacting steps.

102. The plurality of contacting steps comprises: contacting said population of cells with a first capture probe that recognizes a nucleic acid; and contacting said population of cells with a second capture probe that recognizes a protein; 102. The method of claim 101, comprising:

103. 103. The method of any one of claims 51 to 102, wherein the DNA binding region comprises a DNA barcode binding region.

104. 104. The method of any one of claims 51 to 103, wherein the DNA binding region is RNA or DNA.

105. 105. The method of any one of claims 51 to 104, further comprising contacting the population of cells with a DNA polymerase or an RNA polymerase; and a primer to generate an amplification product.

106. 106. The method of claim 105, wherein the RNA polymerase is a reverse transcriptase.

107. 107. The method of any one of claims 51 to 106, wherein the step involving washing the population of cells is omitted.

108. 1. A method for generating biological cell spatial information, comprising: Capturing an image of the three-dimensional tissue sample; contacting the tissue sample with a nucleic acid polymerase and a primer to generate an amplification product; placing a DNA barcode on the tissue sample, the DNA barcode comprising a region complementary to a region of the amplification product, the DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating the tissue sample in three dimensions to form DNA barcode-amplified product complexes; washing the tissue sample to remove the DNA barcodes that are not complexed with an amplification product; and repeating the steps of placing, selectively irradiating, and cleaning; the DNA barcode in a first placing step of the placing steps comprises a different sequence than the DNA barcode in a second placing step of the placing steps; the selectively irradiating step is performed at different locations of the tissue sample for a first selectively irradiating step and a second selectively irradiating step. A method comprising:

109. 109. The method of Claim 108, further comprising determining biological cell spatial information based at least in part on said amplification products and said DNA barcodes.

110. The method of claim 108, wherein the tissue sample is obtained from a subject.

111. The method of claim 108 or claim 110, wherein the tissue sample is an engineered tissue or organoid.

112. 112. The method of any one of claims 108 to 111, wherein the tissue sample comprises a population of cells, and the population of cells is a heterogeneous population of cells.

113. 113. The method of any one of claims 108 to 112, wherein the tissue sample comprises dissected or intact cells.

114. 114. The method of any one of claims 108 to 113, wherein the nucleic acid polymerase is an RNA polymerase or a DNA polymerase.

115. 115. The method of claim 114, wherein the RNA polymerase is a reverse transcriptase.

116. 116. The method of any one of claims 108 to 115, wherein the primer is an RNA primer or a DNA primer.

117. 117. The method of any one of claims 108 to 116, wherein the primer is conjugated to a protein.

118. 118. The method of claim 117, wherein the protein is an antibody or an antibody fragment.

119. 119. The method of any one of claims 108 to 118, wherein the RNA polymerase transcribes RNA from the tissue sample to produce cDNA.

120. 120. The method of any one of claims 108-119, further comprising the step of hybridizing said DNA barcode to said cDNA.

121. 121. The method of any one of claims 108-120, further comprising the step of synthesizing a continuous DNA strand, said continuous DNA strand being complementary to said cDNA and at least one of said DNA barcodes.

122. 122. The method of Claim 121, wherein the continuous DNA strand is complementary to the cDNA and two or more of the DNA barcodes.

123. 122. The method of claim 121, wherein said combining step is by cross-junction synthesis.

124. 124. The method of claim 123, further comprising releasing the continuous DNA strand from the tissue sample.

125. 125. The method of claim 124, wherein the synthesizing and releasing steps are performed simultaneously.

126. 125. The method of claim 124, wherein the released continuous DNA strands are pooled.

127. 127. The method of claim 126, wherein the pooled contiguous DNA strands are amplified.

128. 127. The method of claim 126, wherein the pooled contiguous DNA strands are sequenced.

129. 122. The method of claim 121, further comprising identifying a target associated with said continuous DNA strand.

130. 130. The method of claim 129, further comprising mapping the targets to different locations within a fixed population of cells.

131. 131. The method of any one of claims 108 to 130, wherein the placing step is performed manually, automatically, robotically, or any combination thereof.

132. 132. The method of any one of claims 108 to 131, wherein the placing step is performed using a modified inkjet printer.

133. 133. The method of any one of claims 108 to 132, wherein the placing or washing steps are performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, an acoustic liquid handler, an inkjet printer, an automated liquid handler, or any combination thereof.

134. 134. The method of any one of claims 108 to 133, wherein the placing or washing step is performed using a pipettor.

135. 135. The method of any one of claims 108-134, wherein the repeating step is completed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times or more.

136. 136. The method of any one of claims 108 to 135, wherein at least 2, 3, 4, 5, 6, 7, 8 or 9 different barcode strands are arranged.

137. 137. The method of any one of claims 108 to 136, further comprising imaging the population of cells after said washing step.

138. 138. The method of any one of claims 108 to 137, wherein the cleaning step comprises depositing a solution comprising a chemical denaturant and a cleaning agent.

139. 139. The method of claim 138, wherein the chemical denaturing agent comprises formamide.

140. 140. The method of claim 139, wherein the formamide is present in an amount of about 40% (v / v) to about 60% (v / v).

141. 140. The method of claim 139, wherein the formamide is present in an amount of about 50% to about 60%.

142. 139. The method of claim 138, wherein the detergent comprises polysorbate 20, polysorbate 80, or Triton X-100.

143. 139. The method of claim 138, wherein the detergent is present in an amount between about 0.05% (v / v) and about 5% (v / v).

144. 144. The method of any one of claims 108 to 143, wherein the DNA barcode further comprises a detectable label.

145. 145. The method of any one of claims 108 to 144, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK).

146. 146. The method of any one of claims 108 to 145, wherein the selectively irradiating step is performed with a photomask.

147. 147. The method of claim 146, wherein the photomask is varied at different selectively irradiating steps.

148. 147. The method of claim 146, wherein the photomask is generated manually.

149. 147. The method of claim 146, wherein the photomask is generated by a machine.

150. 150. The method of any one of claims 108 to 149, wherein the selectively irradiating step comprises applying irradiation to a preselected region of interest (ROI).

151. The method of claim 150, wherein the ROI is cell-specific or subcellular-specific.

152. 151. The method of claim 150, wherein the ROI is based on morphology.

153. 153. The method of any one of claims 108 to 152, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 300 nm to 450 nm.

154. 154. The method of any one of claims 108 to 153, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 350 nm to 420 nm.

155. 155. The method of any one of claims 108 to 154, wherein the selectively irradiating step comprises applying irradiation at a wavelength of from about 365 nm to 405 nm.

156. 156. The method of any one of claims 108 to 155, wherein the step involving washing the tissue sample is omitted.

157. 1. A method for generating biological cell spatial information, comprising: Capturing an image of a population of cells, said population of cells being fixed onto a substrate; reverse transcribing RNA from said population of cells to generate cDNA; placing DNA barcodes on said population of cells; hybridizing a DNA barcode to said cDNA, said DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating said population of cells to form crosslinked barcodes; washing to remove uncrosslinked DNA barcodes; and repeating the steps of placing, hybridizing, selectively irradiating and washing, wherein the DNA barcode in the placing step is varied. A method comprising:

158. 158. The method of Claim 157, further comprising determining biological cell spatial information based at least in part on said cDNA and said DNA barcode.

159. 158. The method of claim 157, wherein the cross-linked barcodes form a linkage of barcodes.

160. The population of cells is divided into 1 square micrometer (μm ２ 158. The method of claim 157, wherein a maximum of about 55 unique cDNA molecules are generated per cDNA sequence.

161. The population of cells is divided into 1 square micrometer (μm ２ 158. The method of claim 157, wherein a maximum of about 10 unique cDNA molecules are generated per cDNA fragment.

162. The population of cells is divided into 1 square micrometer (μm ２ 158. The method of claim 157, wherein a maximum of about 5 unique cDNA molecules are generated per cDNA sequence.

163. The population of cells is divided into 1 cubic micrometer (μm ３ 158. The method of claim 157, wherein up to about 5,000 unique cDNA molecules are generated per cDNA sequence.

164. The population of cells is divided into 1 cubic micrometer (μm ３ 158. The method of claim 157, wherein up to about 1000 unique cDNA molecules are generated per cDNA sequence.

165. The population of cells is divided into 1 cubic micrometer (μm ３ 158. The method of claim 157, wherein up to about 500 unique cDNA molecules are generated per cDNA sequence.

166. 166. The method of any one of claims 157 to 165, further comprising the step of synthesizing a contiguous DNA strand complementary to said cDNA and said cross-linked barcode.

167. 167. The method of claim 166, wherein said combining step is by cross-junction synthesis.

168. 168. The method of claim 167, further comprising releasing said continuous DNA strand from said population of cells.

169. 169. The method of claim 168, wherein the synthesizing and releasing steps are performed simultaneously.

170. 169. The method of claim 168, wherein the released continuous DNA strands are pooled.

171. 171. The method of claim 170, wherein the pooled contiguous DNA strands are amplified.

172. 171. The method of claim 170, wherein the pooled contiguous DNA strands are sequenced.

173. 167. The method of claim 166, further comprising identifying a target associated with said continuous DNA strand.

174. 174. The method of claim 173, further comprising the step of mapping said targets to different locations within said fixed population of cells.

175. 175. The method of any one of claims 157 to 174, wherein the population of cells is contacted with a permeabilizing agent.

176. 176. The method of any one of claims 157 to 175, wherein the population of cells is contacted with one or more agents that produce a fixed population of cells.

177. 177. The method of claim 176, wherein the agent is formalin, formaldehyde, paraformaldehyde, methanol, acetic acid, or any combination thereof.

178. 178. The method of any one of claims 176 to 177, wherein the fixed cells are formalin-fixed paraffin-embedded (FFPE) cells, fixed with paraformaldehyde (PFA), or fixed with paraformaldehyde with Triton X-100 (PFA-Tx).

179. wherein said population of cells Cultured cells; Tissue obtained from a subject; or Manipulated tissue 179. The method of any one of claims 157 to 178, comprising one or more of:

180. 180. The method of any one of claims 157 to 179, wherein the population of cells is retinal cells.

181. 181. The method of any one of claims 157 to 180, wherein the placing or washing steps are performed manually, automatically, robotically, or any combination thereof.

182. 182. The method of any one of claims 157 to 181, wherein the placing or washing step is performed using a modified inkjet printer.

183. 183. The method of any one of claims 157 to 182, wherein the placing or washing step is performed using microfluidics, digital microfluidics, an acoustic liquid handler, an inkjet printer, an automated liquid handler, or any combination thereof.

184. 184. The method of any one of claims 157 to 183, wherein the placing or washing step is performed using a pipettor.

185. 185. The method of any one of claims 157 to 184, wherein the repeating step is completed at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times or more.

186. 186. The method of any one of claims 157 to 185, wherein at least 2, 3, 4, 5, 6, 7, 8 or 9 different barcode strands are arranged.

187. 187. The method of any one of claims 157 to 186, further comprising imaging the permeabilized cells after the washing step.

188. 185. The method of any one of claims 157 to 184, wherein the cleaning step comprises depositing a solution comprising a chemical denaturant and a cleaning agent.

189. 189. The method of claim 188, wherein the chemical denaturing agent comprises formamide.

190. 190. The method of claim 189, wherein the formamide is present in an amount of about 40% (v / v) to about 60% (v / v).

191. 190. The method of claim 189, wherein the formamide is present in an amount of about 50% to about 60%.

192. 189. The method of claim 188, wherein the detergent comprises polysorbate 20, polysorbate 80, or Triton X-100.

193. 189. The method of claim 188, wherein the detergent is present in an amount between about 0.05% (v / v) and about 5% (v / v).

194. 194. The method of any one of claims 157 to 193, wherein the DNA barcode further comprises a detectable label.

195. 195. The method of any one of claims 157 to 194, wherein the hybridizing step is at the 5' end of the transcribed cDNA.

196. 195. The method of any one of claims 157 to 194, wherein the hybridizing step is at the 3' end of the transcribed cDNA.

197. 197. The method of any one of claims 157 to 196, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK).

198. 202. The method of any one of claims 157 to 197, wherein the selectively irradiating step is performed using a photomask.

199. 200. The method of claim 198, wherein the photomask is varied at different selectively irradiating steps.

200. 200. The method of claim 199, wherein the photomask is generated manually.

201. 200. The method of claim 199, wherein the photomask is generated by a machine.

202. 202. The method of any one of claims 157 to 201, wherein the selectively irradiating step comprises applying irradiation to a preselected region of interest (ROI).

203. The method of claim 202, wherein the ROI is cell-specific or subcellular-specific.

204. 203. The method of claim 202, wherein the ROI is based on morphology.

205. 205. The method of any one of claims 157 to 204, wherein the population of cells comprises retinal cells.

206. 206. The method of any one of claims 157 to 205, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 300 nm to 450 nm.

207. 207. The method of any one of claims 157 to 206, wherein the selectively irradiating step comprises applying irradiation at a wavelength of about 350 nm to 420 nm.

208. 208. The method of any one of claims 157 to 207, wherein the selectively irradiating step comprises applying irradiation at a wavelength of from about 365 nm to 405 nm.

209. 209. The method of any one of claims 157 to 208, wherein the washing step is omitted.

210. 1. A method for generating biological cell spatial information, comprising: placing a DNA barcode on a population of cells, said DNA barcode hybridizing to a target region, said DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating said population of cells in a region of interest to form covalently linked DNA barcode-target complexes, said irradiating being selectively varied by intensity or duration; washing the population of cells to remove the DNA barcodes that are not complexed with the target region; and repeating the placing, selectively irradiating, and washing steps, wherein the DNA barcode in a first placing step of the placing steps comprises a different sequence than the DNA barcode in a second placing step of the placing steps. A method comprising:

211. 211. The method of claim 210, further comprising determining biological cell spatial information based at least in part on said target region and said DNA barcode.

212. 212. The method of claim 210 or 211, further comprising the step of synthesizing a contiguous DNA strand complementary to the covalently linked barcode.

213. 211. The method of claim 210, further comprising the step of collecting said continuous DNA strand or an amplification product thereof.

214. 214. The method of claim 213, further comprising the step of sequencing said continuous DNA strand or an amplification product thereof.

215. 215. The method of any one of claims 210 to 214, wherein the step involving washing the population of cells is omitted.

216. 216. The method of any one of claims 1-107 and 157-215, wherein the population of cells is present on a synthetic substrate.

217. 217. The method of claim 216, wherein the synthetic substrate comprises a hydrogel.

218. 217. The method of claim 216, wherein the synthetic substrate comprises an extracellular matrix protein.

219. 217. The method of claim 216, wherein the synthetic substrate comprises collagen or fibronectin.

220. 1. A method for generating biological information, comprising: disposing, on the population of nucleic acids, a primer that hybridizes to a target sequence of said nucleic acid to form a nucleic acid primer-complex; disposing a DNA barcode on said nucleic acid-primer complex, said DNA barcode hybridizing to said primer to form a nucleic acid concatemer, said DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; irradiating the nucleic acid concatemers to form covalently linked concatemers; and removing said DNA barcodes that are not in covalently linked concatemers. A method comprising:

221. 221. The method of claim 220, wherein the steps of placing, irradiating, and removing the DNA barcode are repeated, and the DNA barcode in a first one of the placing steps comprises a different sequence than the DNA barcode in a second one of the placing steps.

222. 222. The method of any one of claims 220 or 221, wherein the primer is bound to an affinity molecule, a scaffold, a substrate, a surface, a bead or a column.

223. 223. The method of any one of claims 1 to 222, wherein the selectively irradiating step is varied in intensity or duration to provide for production of a preselected fraction of DNA barcode-amplification product complexes or crosslinked barcodes.

224. 223. The method of any one of claims 1-222, further comprising the step of comparing information collected from the disease state sample with a non-disease state sample.

225. 223. The method of any one of claims 1 to 222, further comprising the steps of contacting the sample with a reagent for intervention, the reagent for intervention comprising a compound, a nucleic acid or a cell; and comparing information collected from contacted sample areas with information collected from uncontacted sample areas.

226. 1. A method for generating biological cell information, comprising: disposing a DNA barcode onto a population of cells, said DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; Steps below: selectively irradiating said population of cells with a first wavelength to form covalently linked complexes of DNA barcodes hybridized to substrate nucleic acid sequences; and Selectively irradiating the population of cells with a second wavelength to release the DNA barcodes hybridized to substrate nucleic acid sequences. forming concatemers via a light-based step comprising repeating collecting the concatemers, said collecting being before or after said selectively irradiating said population of cells with a second wavelength, said concatemers containing information about said population of cells. A method comprising:

227. 1. A method for generating spatial organization information, comprising: At least partially removing paraffin from a sample containing paraffin-embedded tissue; heating the sample to a temperature of at least about 50° C.; contacting the sample with a nucleic acid polymerase and a primer to generate an amplification product; placing a DNA barcode on the sample, the DNA barcode comprising a region complementary to a region of the amplification product, the DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase; selectively irradiating the sample to form covalently linked DNA barcode-amplification product complexes, wherein the selectively irradiating step is performed at different locations for a first selectively irradiating step and a second selectively irradiating step; washing the sample to remove the DNA barcodes that are not complexed with an amplification product; and determining spatial organization information based at least in part on the generated amplification products and the DNA barcodes. A method comprising:

228. 228. The method of claim 227, wherein the step of heating the sample is at a temperature between 50°C and 120°C.

229. 228. The method of claim 227, further comprising determining biological cell spatial information based at least in part on the generated amplification products and the DNA barcodes.

230. 228. The method of Claim 227, further comprising the step of synthesizing a continuous DNA strand, said synthesizing step comprising contacting said DNA barcode-amplification product complex with a polymerase.

231. 228. The method of claim 227, wherein the step of synthesizing comprises cross-junction synthesis.

232. 231. The method of claim 230, further comprising the step of releasing said continuous DNA strand from said population of cells.

233. 233. The method of any one of claims 230 to 232, wherein the synthesizing and releasing steps are performed simultaneously.

234. 233. The method of any one of claims 230 to 232, wherein the synthesizing step is performed before the releasing step.

235. 233. The method of any one of claims 230 to 232, wherein the synthesizing step is performed after the releasing step.

236. 236. The method of any one of claims 232 to 235, wherein the released continuous DNA strands are pooled.

237. 237. The method of claim 236, wherein the pooled contiguous DNA strands are amplified.

238. 237. The method of claim 236, wherein the pooled contiguous DNA strands are sequenced.

239. 231. The method of claim 230, further comprising identifying a target associated with said continuous DNA strand.

240. 240. The method of claim 239, further comprising the step of mapping the targets to the different locations.

241. 1. A method for generating biological cell spatial information, comprising: placing a DNA barcode on a population of cells, said DNA barcode comprising a photoreactive nucleobase capable of crosslinking to another nucleobase, said DNA barcode hybridizing to a target region; selectively irradiating said population of cells in at least one region of interest (ROI) to form covalently linked DNA barcode-target complexes, said irradiating being selectively varied by intensity or duration; washing the population of cells to remove the DNA barcodes that are not complexed with the target region; and repeating the placing, selectively irradiating and washing steps, wherein the DNA barcode in a first placing step of the placing steps comprises a different sequence than the DNA barcode in a second placing step of the placing steps, and the second selectively irradiating step is applied at the same or different duration or intensity as the first selectively irradiating step. A method comprising:

242. 242. The method of claim 241, wherein the first selectively irradiating step, the second selectively irradiating step, or both, are applied as an intensity gradient across the at least one ROI.

243. 243. The method of claim 242, wherein the second intensity gradient is applied parallel, orthogonal, perpendicular, or at another angle to the first intensity gradient.

244. 244. A nucleic acid library produced by the method of any one of claims 1 to 243.

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