Organization space omics

Abstract

Provided herein are compositions, kits, and methods for collecting, integrating, and analyzing spatial-based information from tissue at cellular or subcellular levels. Layers of information include, but are not limited to, gene expression, morphology, nucleic acids, and proteins. In some workflows, light-based techniques are incorporated for selective spatial barcoding of regions. In a further step, such barcoded tags are optionally analyzed by high throughput next generation sequencing.

Description

[0001] Cross-references

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

[0003] Sequence Listing

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

[0005] background

[0006] The integration of imaging-based analysis and high-throughput screening of cells allows spatial selection of information from tissue samples. In many published methods, limitations around the number of recorded visual features or the way in which information from such features is gathered result in the loss of certain cellular or subcellular location-specific information (including spatial information relating one feature to another). Therefore, there is a need for improved compositions, devices, methods and systems for collecting multi-channel spatial information at the cellular and subcellular levels, including but not limited to morphological, genomic, transcriptomic and proteomic data.

[0007] Overview

[0008] Provided herein is a method for generating biological cell information, the method comprising: contacting a population of cells with a nucleic acid polymerase and a primer to generate an amplification product; depositing a DNA barcode onto the population of cells, wherein the DNA barcode comprises a region complementary to a region of the amplification product, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase; 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 deposition, selective irradiation, and washing steps, wherein: the DNA barcode in the first deposition step comprises a different sequence from the DNA barcode in the second deposition step, and for the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different locations.

[0009] The present invention provides a method for generating spatial information of biological cells, which method comprises: contacting a population of cells with a capture probe, wherein the capture probe comprises a DNA binding region and a target binding region; depositing a DNA barcode onto the population of cells, wherein the DNA barcode comprises a region bound to the DNA binding region, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase; selectively irradiating the population of cells to form a DNA barcode-amplification product complex; washing the population of cells to remove the DNA barcode that is not complexed with the amplification product; and repeating the deposition, selective irradiation and washing steps, wherein: the DNA barcode in the first deposition step comprises a different sequence from the DNA barcode in the second deposition step, and for the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different positions.

[0010] Provided herein is a method for generating spatial information of biological cells, the method comprising: capturing a three-dimensional image of a tissue sample; contacting the tissue sample with a nucleic acid polymerase and a primer to generate an amplification product; depositing a DNA barcode onto the tissue sample, wherein the DNA barcode comprises a region complementary to a region of the amplification product, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase; selectively irradiating the tissue sample in three dimensions to form a DNA barcode-amplification product complex; washing the tissue sample to remove the DNA barcode that is not complexed with the amplification product; and repeating the deposition, selective irradiation and washing steps, wherein: the DNA barcode in the first deposition step comprises a different sequence from the DNA barcode in the second deposition step, and for the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different locations of the tissue sample.

[0011] Provided herein is a method for generating spatial information of biological cells, comprising: capturing an image of a population of cells, wherein the population of cells is fixed to a substrate; reverse transcribing RNA from the population of cells to generate cDNA; depositing a DNA barcode onto the population of cells; hybridizing the DNA barcode with the cDNA, wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase; selectively irradiating the population of cells to form cross-linked barcodes; washing to remove uncross-linked DNA barcodes; and repeating the steps of deposition, hybridization, selective radiation, and washing, wherein the DNA barcodes in the deposition step are different.

[0012] Provided herein is a method for generating spatial information of biological cells, the method comprising: depositing DNA barcodes onto a population of cells, wherein the DNA barcodes are hybridized to a target region; and wherein the DNA barcodes comprise a photoreactive nucleobase capable of cross-linking with another nucleobase; selectively irradiating the population of cells at a region of interest to form a covalently linked DNA barcode-target complex, wherein the irradiation is 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 deposition, selective irradiation, and washing steps, wherein the DNA barcode in the first deposition step comprises a different sequence from the DNA barcode in the second deposition step.

[0013] Provided herein is a method for generating bioinformatics, the method comprising: depositing primers on a population of nucleic acids, the primers hybridizing to a target sequence of the nucleic acids to form a nucleic acid-primer complex; depositing DNA barcodes on the nucleic acid-primer complexes, wherein the DNA barcodes hybridize to the primers to form nucleic acid concatemers; wherein the DNA barcodes comprise a photoreactive nucleobase capable of cross-linking with another nucleobase; irradiating the nucleic acid concatemers to form covalently linked concatemers; and removing DNA barcodes that are not in the covalently linked concatemers.

[0014] Provided herein is a method for generating biological cell information, the method comprising: depositing a DNA barcode on a population of cells, wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase; forming concatemers via a light-based step, comprising repeating the following steps: a) selectively irradiating the population of cells at a first wavelength to form a covalently linked complex of the DNA barcode hybridized to a substrate nucleic acid sequence; and b) selectively irradiating the population of cells at a second wavelength to delink the DNA barcode hybridized to the substrate nucleic acid sequence; and collecting the concatemers, wherein the collecting step is before or after the selective irradiation of the population of cells at the second wavelength, and wherein the concatemers comprise information about the population of cells.

[0015] Provided herein is a method for 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; depositing a DNA barcode onto the sample, wherein the DNA barcode comprises a region complementary to a region of the amplification product, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase; selectively irradiating the sample to form a covalently linked DNA barcode-amplification product complex, wherein for a first selective irradiation step and a second selective irradiation step, the selective irradiation occurs at different locations; washing the sample to remove DNA barcodes that are not complexed with the amplification product; and determining the spatial organization information based at least in part on the generated amplification product and the DNA barcode.

[0016] Provided herein is a method for generating spatial information of biological cells, the method comprising: depositing a DNA barcode onto a population of cells, wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase, and wherein the DNA barcode is hybridized with a target region; selectively irradiating a population of cells in at least one region of interest (ROI) to form a covalently linked DNA barcode-target complex, wherein the irradiation is 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 deposition, selective irradiation, and washing steps, wherein the DNA barcode in the first deposition step comprises a different sequence from the DNA barcode in the second deposition step, and wherein the second selective irradiation step is applied at the same or different duration or intensity as the first selective irradiation step.

[0017] Provided herein are nucleic acid libraries generated according to the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and the accompanying drawings, which set forth illustrative embodiments utilizing the principles of the present invention, wherein:

[0020] Figure 1A and Figure 1B Diagram shows the process workflow incorporating sample preparation, light-based in situ barcoding of populations of cells, and transcript extraction and sequencing.

[0021] Figure 1C Diagram showing the workflow of fractional spatial barcoding.

[0022] Figure 2A and Figure 2B Illustrated to follow Figure 1C Scheme of fractional barcoding of the workflow described in .

[0023] Figure 3 A photo-directed cross-linking reaction as described herein is illustrated.

[0024] Figure 4 Diagram shows an iterative barcode addition method in which a barcode strand is added to the 5' end of the cDNA.

[0025] Figure 5A Illustration of photo-directed cross-linking used to generate barcoded concatemers.

[0026] Figure 5B Data reconstruction from barcode concatemers is shown.

[0027] Figure 6 The diagram illustrates the process of cross-junction synthesis.

[0028] Figure 7 Schematic representation of the workflow for cross-linking across barcode concatemers to generate continuous nucleic acids.

[0029] Figure 8 The diagram shows the coding combination of four barcode chains using the binary coding method.

[0030] Fig. 9 The diagram shows the encoding combination of two barcode chains using the quaternary encoding method.

[0031] Fig.10 The diagram shows the encoding combination of two barcode chains using the fractional encoding method.

[0032] Fig.11 Representative images of brain, spleen, and liver sections fixed with paraformaldehyde (PFA), paraformaldehyde with Triton X-100 (PFA-Tx), and formalin fixation-paraffin embedding (formalin) after photodirected transcriptome barcoding with fluorescently labeled barcodes are shown, showing that transcriptome barcoding is comparable across tissue types and fixation conditions; the bar in the lower right corner of the image corresponds to 100 μm.

[0033] Fig. 12A is an image of a FFPE liver section showing barcoding of the fluorescent marker in the region of interest marked by the dotted line.

[0034] Fig. 12Bis a heat map showing z-scores of expression detected in 50 genes in PFA-Tx fixed brain sections and PFA-Tx and FFPE fixed liver samples, indicating increased expression of typical organ marker genes and consistent detection levels across preparation methods.

[0035] Fig. 12C It is shown as Fig. 12B Volcano plot of relative expression levels of selected gene sets with typical organ-specific expression in the samples described in .

[0036] Fig.12D is a heat map showing the z-scores of the relative expression levels of 2,653 differentially expressed genes, indicating increased expression detected in typical organ marker genes.

[0037] Fig.12E is a heat map showing Pearson correlations calculated to compare mRNA expression values ​​in FFPE and PFA-Tx fixed tissues, showing high correlations among liver or brain samples and low correlations when comparing liver samples to brain samples.

[0038] Fig.13A and Fig. 13B is an image showing liver sections, including at 70°C ( Fig.13A ) and 95℃( Fig. 13B ) Photo-directed barcoded rectangular regions after heat-induced epitope retrieval (HIER) treatment.

[0039] Fig. 13C Yes Fig.13A and Fig. 13B The bar graph of average barcode fluorescence intensity in the samples shown in FIG. 1 shows that the fluorescence intensity is three times higher in the samples treated at 95° C. compared to the samples treated at 70° C.

[0040] Fig.13D is a bar graph of average Cq values ​​for cDNA libraries generated collected from slices treated with HIER at 70°C and 95°C, with and without UV treatment, showing that samples treated at 95°C amplified in fewer cycles than samples treated at 70°C, and that control samples (without UV treatment) required at least 50% more amplification cycles than the treated samples.

[0041] Fig.14A are images of mouse liver sections of photo-directed barcoded regions after illumination with a DMD using a 380 nm LED (lower rectangle) or a 405 nm laser (upper rectangle).

[0042] Fig. 14B It is shown in Fig.14A Image of autofluorescence in the same liver section depicted in .

[0043] Fig. 14C Is from Fig.14A and Fig. 14B superposition of images.

[0044] Fig.14D yes Fig.14A A magnified view of the dashed rectangle shows the intensity of the barcoding of four regions exposed to different power levels of illumination from a 405 nm laser.

[0045] Fig.14E It is shown in Fig.14D Image of autofluorescence in the same liver section depicted in .

[0046] Fig.14F Is from Fig.14D and Fig.14E superposition of images.

[0047] Fig.15A is an image of a mouse retina showing barcoding of FITC labels in five regions of interest (ROIs) processed from left to right with increasing illumination power levels in each ROI.

[0048] Fig. 15B is with Fig.15A Image of the same retinal section showing barcoding of the Cy3 marker in the same ROI after treatment with illumination at a consistent power level across all ROIs.

[0049] Fig. 15C Is from Fig.15A and Fig. 15B superposition of images.

[0050] Fig.15D yes Fig.15A and Fig. 15B Bar graph of image intensity ratios for the ROIs depicted in Figure 2.

[0051] Fig.16A is an image of a mouse brain section stained with DRAQ5 for use in generating a photomask, with the dotted line indicating the region drawn for generating the photomask.

[0052] Fig. 16B Is from Fig.16A Image of the same mouse brain slice, showing the Fig.16A The image generated in the photomask is then subjected to photo-directed barcoding followed by the binding of fluorescent barcoded regions.

[0053] Fig. 16C Is from Fig.16A and Fig. 16BOverlay of images showing the barcoded regions relative to the nuclear staining features on the sample. DETAILED DESCRIPTION

[0054] Provided herein are compositions, devices, methods and systems for use in accessible and scalable visual selection methods, which can directly associate multidimensional and high-resolution cell phenotypes (including morphology, protein markers and spatial organization) with transcriptomes, genomes or proteome spectra of different sample types. The methods described herein are applicable to cell states in analytical tissues, synthetic tissues or solutions. Methods for sample preparation provided for imaging and regional selection, methods for in situ barcoding and imaging, and recording extraction and sequencing to provide spatially limited data from samples are described herein.

[0055] Provided herein is a method for spatially indexing the transcribed nucleic acid in the region of interest (ROI) selected in cells and tissues. In some embodiments, the transcribed nucleic acid is a reverse transcribed DNA or a transcribed RNA. In some embodiments, the barcode sequence from the sequencing read provides spatial index data. In some embodiments, the spatial index data is combined with the imaging method described earlier herein to provide an association between cell phenotype information and transcriptome information.

[0056] Workflow

[0057] Figure 1A An exemplary workflow for analyzing biological cells is provided in. In short, a step of sample preparation (e.g., a population of tissue samples or cells) is performed. The sample preparation step may include culturing cells or obtaining tissues or organoids, followed by imaging. Dyes and / or probes may be applied to the sample to enhance visualization of the cells and / or structures therein. Subsequent imaging provides morphological and structural information of the sample to assist in region selection, cell segmentation, and mask generation. The sample is then optionally permeabilized. Permeabilization may be performed prior to imaging the sample or simultaneously with fixation of the sample.

[0058] Following the workflow, for in situ analysis of biological cells, reverse transcription of sample RNA is performed, optionally followed by tailing of the resulting target cDNA. Tailing adds non-templated nucleotides to the 3' end of a nucleic acid chain. In some embodiments, tailing includes ligation of nucleic acid fragments. In some embodiments, tailing includes extension of a nucleic acid chain. In some cases, spatially directed barcoding within a selected region of interest is then performed using a photomask, wherein the region for unmasking is selected based on earlier imaging of the sample. Optionally, the biological cell is further imaged to reveal incorporation of the barcode. Following Figure 1AThe process described in, record extraction and sequencing include displacement of barcoded nucleic acids. The displacement is optionally achieved by applying RNase H. The cross-linking of continuous nucleic acids can also achieve displacement of barcoded nucleic acids. Enzymatic displacement and synthesis reactions can be combined or performed sequentially in any order. After the sequence has been extracted from the sample, the sample can be further imaged or otherwise measured (e.g., mass spectrometry, H&E staining). 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.

[0059] Figure 1B Another exemplary workflow is shown in . Briefly, a step of sample preparation (e.g., a tissue sample or a population of cells) is performed. The sample preparation step may include culturing cells or harvesting tissue or organoids, followed by imaging. Dyes and / or probes may be applied to the sample to enhance visualization of the cells and / or structures therein. Subsequent imaging provides morphological and structural information of the sample to aid in region selection, cell segmentation, and mask generation. The sample is then optionally permeabilized. In this exemplary embodiment, a reverse transcription step is not performed. Optionally, a target in the sample is bound to a recognition binding agent, which may be fused to a nucleic acid or otherwise linked thereto, and then barcoded, such as Figure 1A Described in .

[0060] Figure 1C A fractional spatial barcoding workflow is shown in . Sample preparation and optional imaging steps include methods similar to the previous workflow. In this case, a unique ratio of a barcode or barcode arrangement is assigned to a specific cell or region of interest (ROI). The duration or power of the applied radiation energy is controlled to define the fraction of available targets that will be cross-linked with the contacted barcodes. Figure 2A is an exemplary depiction of controllably increasing illumination power or time to induce more crosslinking events in a specific region (in this case, cells). Multiple rounds of fractional barcode crosslinking can be applied across a sample, allowing cells or regions of interest to be distinguished and identified. Figure 2BShown is an exemplary combination of four different barcode sequences applied across cells or regions. Each cell or region of interest can be assigned an individualized segmentation of the barcode sequence in its read segment, so that after subsequent single cell or single region barcoding and sequencing, the original spatial position of the cell can be restored based on the score of the read segment of each barcode measured with next generation sequencing. In some cases, cells or regions are barcoded separately after sample dissociation. In some embodiments, before sequencing, a sorting method for separating different regions is applied, such as separately pooling barcoding, microfluidics, barcoded beads or other single cell barcoding technologies. Each cell or ROI can be identified based on the score of the barcode cross-linked by light or a combination thereof.

[0061] Sample preparation

[0062] Cell culture / sample acquisition

[0063] The spatial barcoding method described herein can be applied to a wide range of materials. This article provides a method for preparing samples for downstream bioanalysis steps. In some embodiments, samples as described herein include a population of cells. In some embodiments, samples described herein include tissue samples. In some embodiments, samples described herein include layered cell cultures. Non-limiting examples of samples that can be used include: complete tissues, dissected tissues, dissociated cells, organoids, engineered tissues, cultured cells, subcellular compositions, suspensions of cells, organs, tissue biopsies, or any combination thereof. In some embodiments, samples described herein include a population of cultured cells. Organoids include tiny, self-organized, three-dimensional tissue cultures. In some embodiments, organoids are derived from stem cells. Cultured cells may include, but are not limited to, primary cell cultures, dissociated cells, dissected cells, cell lines and / or transformed cells of passage. In some embodiments, the population of cells provided herein includes engineered tissues. In some embodiments, engineered tissues include a population of cells differentiated in vitro by humans. In some embodiments, cells differentiated in vitro by humans are derived from human induced pluripotent stem cells (hiPSC), embryonic stem cells, or adult stem cells. In some embodiments, the human in vitro differentiated 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 renal cells. In some embodiments, the colonies of cells provided herein include retinal cells. In some embodiments, the colonies of cells provided herein are obtained from subjects. In some embodiments, subjects are mammals. In some embodiments, mammals are primates, non-human primates, humans, mice, rats, goats, rabbits, dogs, camelids or cats.

[0064] In some embodiments, the colony of cells includes a homogeneous colony of cells (e.g., cells of the same type or the same species). In some embodiments, the colony of cells includes a heterogeneous colony of cells (e.g., cells of different types, different species, or any combination thereof). In some embodiments, the colony of cells includes cells from a species. In some embodiments, the colony of cells includes cells from two or more species. In some embodiments, the colony of cells includes a cell type. In some embodiments, the colony of cells includes cells from two or more different cell types. In some embodiments, the colony of cells includes 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 includes 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.

[0065] In some embodiments, the sample used in the methods described herein is a tissue sample. In some embodiments, the tissue sample provided herein includes connective tissue, epithelial tissue, muscle tissue or neural tissue. In some embodiments, the tissue sample provided herein includes a combination of tissues. Organs include different types of tissues and have specific functions. 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: 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 complete organ or biopsy. In some embodiments, the biopsy includes tissue with or suspected of having abnormal proliferation or growth. In some embodiments, the biopsy includes cancer cells.

[0066] In some embodiments, the colony of cells described herein includes one or more microorganisms. In some embodiments, the colony of cells includes prokaryotic cells or eukaryotic cells. In some embodiments, the colony of cells provided herein includes bacterial cells, fungal cells, archaeal cells (archaic cell), eukaryotic cells or any combination thereof. In some embodiments, the colony of cells includes mammalian cells. In some embodiments, mammalian cells are cells of humans, non-human primates, primates, pigs, horses, sheep, cats, rats, mice, dogs, llamas, rabbits or goats. In some embodiments, the colony of cells also includes viruses. In some embodiments, the virus is a pathogenic virus. In some embodiments, the colony of cells includes viral vectors. In some embodiments, the colony of cells has been genetically modified. Non-limiting examples of genetic modification include base editing, gene silencing (e.g., by CRISPR / Cas9 system), gene insertion and gene deletion.

[0067] In some embodiments, the sample includes cells. In some embodiments, the sample includes cells from a suspension. In some embodiments, the sample includes a complete organism. In some embodiments, the complete organism is a multicellular organism. In some embodiments, the complete organism is a unicellular organism. Exemplary multicellular organisms include but are not limited to roundworms (Caenorhabditis elegans), zebrafish (zebrafish) (Daniorerio) and fruit flies (Drosophila). Exemplary unicellular organisms include but are not limited to bacteria and yeast.

[0068] Imaging

[0069] The imaging of the sample is used to guide the photomask to perform light-directed barcoding. Imaging is also used to visualize the barcoding of the barcode with a fluorophore label. Provided herein is a method for image capture and enhancement thereof, which incorporates the application of imaging agents. Imaging agents can be used to distinguish cells, cell types, and / or subcellular structures. In some embodiments, a tissue sample or a colony of cells provided herein is contacted with an imaging agent. In some embodiments, the imaging agent is a dye or stain. In some embodiments, the dye or stain includes 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 (official name: osmium tetroxide), propidium iodide, resazurin, rhodamine, safranin, trypan blue, tetrazolium salts, or any combination thereof.

[0070] In some embodiments, the sample is visualized with immunofluorescence (IF) staining. In some embodiments, the IF staining reagent includes fluorophore-conjugated streptavidin. In some embodiments, the imaging agent is a fluorophore. Exemplary fluorophores for inclusion in the workflow described herein include, but are not limited to, 1,5IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthylfluorescein (pH 10); 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; Acriflavin; Acriflavin 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 Fluor647 TM Alexa Fluor 660 TM Alexa Fluor 680 TM ; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aniline Blue; Anthrocylstearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAGTM CBQCA; ATTO-TAG TM FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); berberine sulfate; β-lactamase; BFP blue-shifted GFP (Y66H); BG-647; Bimane; bisbenzamide; Blancophor FFG; Blancophor SV; BOBO TM -1;BOBO TM -3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; 576 / 589; Bodipy581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy FI; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X Conjugate; Bodipy TMR-X,SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO TM -1; BO-PRO TM -3; Brilliant Sulphoflavin FF; Calcium Crimson; Calcium Crimson Blue TM ; Calcium Green; Calcium Green-1Ca2+ dye; Calcium Green-2Ca2+; Calcium Green-5NCa2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcium Fluorescent White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue TM ;CascadeYellow;catecholamine;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 TM ; Cy3.1 8; Cy3.5 TM ;Cy3 TM ;Cy5.1 8;Cy5.5 TM ;Cy5 TM;Cy7 TM ; cyan GFP; cyclic AMP fluorescent sensor (FiCRhR); d2; Dabcyl; dansyl; dansylamide; dansylcadaverine; dansyl chloride; dansylDHPE; dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (dihydrofluorescein 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; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1); Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA; Feulgen (para-purpurin); FITC; FL-645; FlazoOrange; Fluo-3; Fluo-4; Fluorescein diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxy stilbamidine); Fluor-Ruby; FluorX; FM 1-43 TM ;FM 4-46; Fura Red TM(high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl Brilliant Red B; Genacryl Brilliant Yellow10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GFP(S65T); GFP red shifted (rsGFP); GFP wild type, no-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbene (FluoroGold); Serotonin; 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; Lissamine B; LOLO-1; LO-PRO-1; Fluorescent Yellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10GFF; Maxilon Brilliant Flavin 8GFF; Merocyanine; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromodiamine; Monobromodiamine (mBBr-GSH); Monochlorodiamine; MPS (Methyl Green Pyronine Stilbene));NBD;NBD amine;Nile Red;Nitrobenzoxadidole;Norepinephrine;Nuclear Fast Red;Nuclear Yellow;Nylosan Brilliant IavinE8G;Oregon Green TM ;Oregon Green 488-X; Oregon GreenTM 488; Oregon Green TM 500; Oregon Green TM 514; Pacific Blue; Feulgen; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); 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; Primuline; Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufm; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine Bextra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycoerythrin (PE); Red-shifted 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 BFP); sgGFP TM ;sgGFP TM(super glow GFP); SITS; SITS (Primula); SITS (Stilbene Isothiosulphonic Acid); SPQ (6-methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine; Texas Red TM ;Texas Red-X TM Conjugates; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazolyl Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (calcium fluorescent white); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC (Tetramethyl Rodamine 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; YOYO-1; YOYO-3; or any combination thereof. Many suitable forms of these fluorescent compounds are available and can be used.

[0071] In some embodiments, the samples described herein are imaged using a microscope. In some embodiments, the microscope used in the methods described herein comprises a compound microscope, a fluorescence microscope, a confocal microscope, a light sheet microscope, a Raman microscope, a digital microscope, or any combination thereof.

[0072] Sample fixation

[0073] Methods described herein are not limited by sample fixation conditions. In some embodiments, the colony of cells is fixed and then frozen. In some embodiments, the colony of cells is frozen and then fixed after thawing and / or sectioning. In some embodiments, the colony of fixed cells is sectioned and then fixed for a second time. In some embodiments, the colony of cells is stored at room temperature after fixation. In some embodiments, the colony of cells is fixed with organic solvents such as alcohol and acetone. In some embodiments, the colony of cells (e.g., tissue) is embedded in paraffin and then fixed. In some embodiments, the colony of cells is fixed with a cross-linking agent. In some embodiments, the cross-linking agent includes formalin, formaldehyde, paraformaldehyde, disulfide-bis (succinimidyl propionate) (DSP) or any combination thereof.

[0074] In some embodiments, provided herein is a sample frozen at -5 ° C to -200 ° C. In some embodiments, provided herein is a sample 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, provided herein is a tissue sample or a colony of cells that are rapidly frozen. In some embodiments, provided herein is a tissue sample or a colony of cells that are frozen by slow or balanced freezing.

[0075] In some embodiments, the sample comprises tissue. In some embodiments, the tissue of the slice is sliced. In some embodiments, the thickness of the slice is up to about 5um, about 10um, about 15um, about 20um, about 25um, about 30um, about 35um, about 40um, about 45um, about 50um or larger.

[0076] In some embodiments, the samples described herein include cells. In some embodiments, the cells are bound to a surface. In some embodiments, the cells are in solution. In some embodiments, the cells are connected to a hydrogel. In some embodiments, the cells are connected to a synthetic scaffold. In some embodiments, the cells are directly or indirectly attached to the synthetic scaffold. In some embodiments, the synthetic scaffold includes beads or semisolid gels. In some embodiments, the synthetic scaffold includes extracellular matrix proteins. In some embodiments, the extracellular matrix proteins include collagen or fibronectin.

[0077] In some embodiments, the sample provided herein is fixed on a microscope slide or a cover glass. In some embodiments, the microscope slide is a chamber slide. In some embodiments, the chamber slide includes one or more chambers. In some embodiments, the slide includes polystyrene. In some embodiments, the slide includes glass.

[0078] In some embodiments, slide includes coating to promote the colony of tissue sample or cell provided herein to adhere to the slide surface. In some embodiments, coating includes poly-L-lysine or poly-D-lysine. In some embodiments, coating includes gelatin. In some embodiments, coating includes 3-aminopropyltriethoxysilane (APES). In some embodiments, adhesive tape is used to adhere the sample to the slide. In some embodiments, 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.

[0079] In some embodiments, the sample is incorporated into the hydrogel. In some embodiments, the hydrogel comprises a synthetic gel or a polyepoxide. In some embodiments, the embedded sample is generated at the tissue-hydrogel matrix. In some embodiments, the embedded sample provides an expansion of the population of tissue samples or 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, (Corning Inc., Corning, NY), genetically engineered proteins, 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 comprises poly(2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate (HEMA), poly(2-hydroxypropyl methacrylate) (PHPMA), poly(N-isopropylacrylamide (PNIPAm), poloxamer ( ThermoFisher Scientific, Inc., Waltham, MA), acrylate-modified PEG, acrylate-modified hyaluronic acid, amine end-functionalized 4-arm star-PEG, poly(ethylene glycol) diacrylate (PEGDA), poly(vinyl alcohol (PVA), polyvinylpyrrolidone (PVP), acrylamide / acrylic acid copolymer, linear cationic polyallylammonium chloride, poly(N-isopropylacrylamide) (PNIPAM), or any combination thereof. In some embodiments, the biodegradable hydrogel includes degradable polyethylene glycol (PEG), polypropylene fumarate-PEG (PPF-PEG), poly(2-hydroxyethyl methacrylate-poly(ε-caprolactone) (PHEMA-PCL), methacrylic acid (MAA), PEG-PEGMA, carboxymethyl cellulose (CMC), synthetic peptides, or any combination thereof. In some embodiments, the bioactive hydrogel comprises a cell-adhesive hydrogel, an enzyme-sensitive hydrogel, a hydrogel carrying a growth factor, 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 peptides / protein, polysaccharides, PVA / DNA, Pluronic / dextran, PHPMA / protein, or any combination thereof.

[0080] In some embodiments, the samples described herein include nucleic acids. In some embodiments, the samples include nucleic acids directly or indirectly bound or tethered to a surface. In some embodiments, the samples include nucleic acids in solution. In some embodiments, the samples include nucleic acids connected to a hydrogel. In some embodiments, the samples include nucleic acids in a scaffold. In some embodiments, nucleic acids are directly or indirectly attached to a synthetic matrix. In some embodiments, the synthetic matrix includes beads or semisolid gels.

[0081] The analysis using the method described herein can be applied to immobilized nucleic acids. In some embodiments, immobilized nucleic acids are fixed to substrates. In some embodiments, fixation is carried out via formalin / formaldehyde treatment. In some embodiments, fixation is carried out via glyoxal or oxalaldehyde treatment. In some embodiments, fixed nucleic acids are present in tissues that have been fixed with formalin and paraffin embedded (FFPE), paraformaldehyde (PFA) or fixed with PFA and Triton X-100 (PFA-Tx). The analysis using the method described herein can be applied to any fixed nucleic acids. The method described can be used to characterize nucleic acids, proteins, or any combination thereof.

[0082] The immobilized nucleic acid can be conjugated to an affinity molecule or immobilized to a support, substrate, surface, bead or column. The methods described herein can be used to profile in situ hybridization (ISH) probes, DNA conjugated to antibodies, DNA encoding libraries, CRISPR libraries, viral libraries and / or genomes, nucleic acids covalently linked to transparent hydrogels, nucleic acid-conjugated proteins, nucleic acids localized on a surface (e.g., a glass slide or microarray), or sequences generated in situ (e.g., by in vitro transcription (IVT)).

[0083] In some embodiments, the fixed nucleic acid is conjugated to an affinity molecule. In some embodiments, the affinity molecule includes a component of a ligand / receptor complex. In some embodiments, the affinity molecule includes 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 as described herein is a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a 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 small peptide molecule.

[0084] In some embodiments, the fixed nucleic acid is conjugated to the support. In some embodiments, the support is biodegradable. In some embodiments, the support is non-biodegradable. In some embodiments, the support is biocompatible. In some embodiments, the support includes a synthetic material. In some embodiments, the synthetic material includes silicone, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylactic acid-co-glycolic acid (PLGA) or any combination thereof. In some embodiments, the support includes natural materials. In some embodiments, natural materials include derivatives of the extracellular matrix, collagen, fibrin, polysaccharides (including chitosan or glycosaminoglycans (GAGs)) or any combination thereof. In some embodiments, glycosaminoglycans (GAGs) include hyaluronic acid.

[0085] In some embodiments, fixed nucleic acid is conjugated to a surface comprising a flexible material. In some embodiments, flexible materials include but are not limited to 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 but is not limited to 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 surface described herein can be made of a material including silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), glass or any combination thereof.

[0086] 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.

[0087] optimization

[0088] Additional steps can be applied to optimize hybridization or barcoding in the analyzed sample. In some embodiments, the sample is subjected to a permeabilization step. In some embodiments, permeabilization is not performed. In some embodiments, permeabilization occurs simultaneously with fixation. In some embodiments, permeabilization is performed after fixation. In some embodiments, the permeabilization agent includes acetone, alcohol, surfactant, or any combination thereof.

[0089] In some cases, fixation may affect protein biochemistry, masking epitopes or binding sites. The methods described herein can provide for the demasking of binding sites, making them more available for binding. In some embodiments, the samples described herein are also treated with antigen retrieval methods. In some embodiments, the antigen retrieval method is protease-induced epitope retrieval (PIER). PIER treatment can utilize enzymes such as proteinase K, trypsin, or pepsin to demask antigens in the sample. In some embodiments, the antigen retrieval method is heat-induced epitope retrieval (HIER). The sample can be heated using a microwave oven, a pressure cooker, a steamer, an autoclave, a heating block, a hot plate, a water bath, or another heat source. HIER-treated samples can be heated from 50°C to 120°C. In some embodiments, the sample is heated from 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 capable of reversing epitope masking. In some embodiments, the antigen retrieval step is combined with a permeabilization step.

[0090] In some embodiments, the antigen retrieval step is performed independently. Additional treatments can be performed in conjunction with epitope retrieval. For example, paraffin-embedded samples can be dewaxed before PIER or HIER. Dewaxing can be achieved by using an organic solvent, such as xylene, cycloalkane solvents, d-limonene or any suitable solvent.

[0091] Barcoding

[0092] Barcoding can be directly applied to a target in a sample, a transcription product of a sample, a reverse transcription product of a sample, or another element (e.g., an antibody) that specifically binds to a target or a transcription product of a sample. As described herein, a target can be any substance to which a specific binding product can be applied. Specific binding can be affinity binding, hybridization, ligand / receptor binding, aptamer binding, immune binding, or any other type of specific binding or a combination thereof. In some embodiments, the nucleic acid in a 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, RNA is messenger RNA (mRNA). In some embodiments, the target nucleic acid is a regulatory RNA. In some embodiments, the RNA is a circular RNA (circRNA), a ribosomal RNA (rRNA), a 16S ribosomal RNA (16S rRNA), a transfer RNA (tRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), a noncoding RNA, a long noncoding RNA (lncRNA), or a micro RNA (miRNA).

[0093] In some embodiments, the target nucleic acid is an amplification product. Nucleic acid amplification can 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, isothermal amplification is performed at ambient temperature and / or room temperature. In some embodiments, ambient temperature and / or room temperature are about 20°C to 25°C. In some embodiments, 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 exchange reaction signal amplification (SABER). In some embodiments, the target nucleic acid is generated by a synthetic reaction. In some embodiments, the target nucleic acid is generated by a primer exchange reaction (PER) or RCA. In some embodiments, PER is a PER assay based on terminal deoxynucleotidyl transferase (Tdt). In some embodiments, the target nucleic acid is a long repeat single-stranded RNA or DNA sequence. In some embodiments, amplification is performed at multiple temperatures (e.g., in one cycle). In some embodiments, amplification is performed using a non-isothermal, linear or exponential amplification method. In some embodiments, amplification is PCR. In some embodiments, PCR is asymmetric PCR. In some embodiments, amplification is isothermal. Exemplary 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).

[0094] 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 in situ detection of protein-protein interactions at a distance of approximately less than 40 nanometers. In some embodiments, the target nucleic acid is a PLA oligonucleotide. In some embodiments, the barcode nucleic acid provided herein also comprises a PLA oligonucleotide. In some embodiments, the target nucleic acid comprises a first PLA oligonucleotide, wherein the first PLA oligonucleotide is conjugated to a first protein of interest. In some embodiments, the first PLA oligonucleotide is hybridized with a second PLA oligonucleotide, wherein 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: antibodies, antibody fragments, cell surface receptors, cell surface proteins and intracellular proteins, mitochondrial proteins, nuclear proteins, transcription factors or scaffold proteins. In some embodiments, the first PLA oligonucleotide and the second PLA oligonucleotide are connected to form circular DNA. In some embodiments, circular DNA is amplified. In some embodiments, circular DNA includes a barcode chain provided herein. In some embodiments, the circular DNA is contacted with a barcode strand provided herein.In a PLA reaction, the barcodes can be used to identify specific protein-protein or nucleic acid and protein interactions within a sample.

[0095] In some embodiments, the target nucleic acid is exposed to a DNA or RNA polymerase. In some embodiments, a polymerase refers to an enzyme that performs template-directed synthesis of a polynucleotide. In some embodiments, a DNA polymerase is isolated from or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or a modified form thereof. In some embodiments, polymerases include, but are not limited to, Klenow fragment (New England Biolabs Inc.), Taq DNA polymerase (QIAGEN), 9°N TM DNA polymerase (NewEngland Biolabs Inc.), Deep Vent TMDNA 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.

[0096] In some embodiments, the primer is attached to an affinity molecule, a scaffold, a substrate, a surface, a bead, or a column. In some embodiments, the primer is in suspension, solution, or a hydrogel.

[0097] Reverse transcription

[0098] The generation of cDNA complementary to the target mRNA can include in situ reverse transcription. In some embodiments, in situ reverse transcription includes the incorporation of primers. In some embodiments, primers describe short nucleic acid sequences. In some embodiments, primers are DNA. In some embodiments, primers are RNA. In some embodiments, primers have regions complementary to DNA or RNA chains. In some embodiments, primers provide free 3'-OH, and DNA polymerase starts synthesizing deoxyribonucleotide chains at this free 3'-OH. In some embodiments, primers contain about 5 to about 50 or more nucleotides. In some embodiments, primers contain about 10 to about 30 nucleotides. In some embodiments, primers as described herein include 3' sequences NNNNNNN (7 N), NNNNGGG (5 N and 3 G), NNNNNCCC (5 N and 3 C), NNNNNAAA (5 N and 3 A) or NNNNNTTT (5 N and 3 T).

[0099] 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, tailing is achieved by using terminal transferase and dXTP (dATP, dCTP, dGTP, dTTP or any combination thereof). Adding ddXTP (ddATP, ddCTP, ddGTP, ddTTP or any combination thereof) or another terminating nucleotide at the 3' end or 5' end prevents subsequent extension during the cross-linking step later. In some embodiments, ddXTP or another terminating nucleotide is included at low concentrations to randomly terminate the 3' end or 5' end. In some embodiments, 3' or 5' overhangs are added using other strategies. In some embodiments, overhangs are added by connection. In some embodiments, overhangs are added by extension.

[0100] Target Binding

[0101] like Figure 1B As described in, the barcoding of the sample includes target binding. In some embodiments, the target binding is affinity binding. Affinity binding may include binding to a ligand, an epitope, an aptamer, a protein, a receptor, a small molecule, or any other specific recognition binding. In some embodiments, the target binding is hybridization. Hybridization may include hybridization of nucleic acid chains, including but not limited to deoxyribonucleic acid (DNA) chains, ribonucleic acid (RNA) chains, peptide nucleic acid (PNA) chains, locked nucleic acid (LNA) chains, and combinations thereof. For example, 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.

[0102] In some embodiments, the target is recognized by an antibody, antibody fragment, protein, peptide, nanobody, small molecule, nucleic acid, probe, aptamer, siRNA, lipid, nanoparticle, lipid nanoparticle, or other affinity agent.

[0103] Barcode

[0104] Provided herein is a method for information labeling with nucleic acid. As described herein, information labeling provides non-genetic information. In some embodiments, 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, non-genetic information includes sample information, such as tube number, well number, sample source, charge, quality or other sample characteristics, or any combination thereof. In some embodiments, non-genetic information includes digital information. The method provided herein allows barcodes to be deposited on surfaces. Such barcodes can convey information about the identity, substrate, timing, position, intensity, charge, affinity, etc. of the target. Therefore, barcodes provide nucleic acid media for digital information storage. In some embodiments, the barcode also includes a unique molecular identifier (UMI).

[0105] In some embodiments, the colony of cells for analysis as provided herein is a living cell. In some embodiments, living cells are directly contacted with a barcode sequence, or indirectly contacted via a connecting molecule. In some embodiments, the connecting molecule binds to a cell surface receptor. In some embodiments, the connecting 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 adhesion point. In some embodiments, the barcode sequence is introduced into a living cell intracellularly. In some embodiments, the barcode sequence is attached to a lipid nanoparticle (LNP) as a carrier for delivery into a cell. In some embodiments, the cell is transfected or transformed to receive the barcode sequence. In some embodiments, the cell is mechanically perturbed so that the cell can take up the barcode sequence.

[0106] 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 cell 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 by in situ transcription. In some embodiments, in situ transcription is reverse transcription. In some embodiments, RNA is generated in situ from a partially or completely double-stranded DNA by transcription.

[0107] The barcodes provided herein generally encode non-genetic data. In some embodiments, the barcode chain is a single-stranded nucleic acid. The hybridization domain is introduced into the barcode chain to allow the barcode to be arbitrarily arranged by iterative hybridization, crosslinking and washing steps. In some embodiments, the nucleic acid barcode chain comprises a barcode domain. In some embodiments, the length of the barcode domain is about 5 to about 10 bases. In some embodiments, the barcode domain flanks a group of two alternating hybridization domains. In some embodiments, the barcode domain flanks different hybridization domains. In some embodiments, the barcode domain flanks a group of 3, 4, 5, 6 or more alternating hybridization domains. In some embodiments, the hybridization domain is a crosslinking domain. In some embodiments, the hybridization domain is a complementary strand. In some embodiments, a group of two alternating hybridization domains comprises a crosslinking strand and a complementary strand. In some embodiments, the nucleic acid barcode comprises a barcode domain connected to (i) a crosslinking strand comprising a photoactivated nucleotide and (ii) a complementary strand. In some embodiments, the length of the alternating hybridization domain can be at least 5, at least 10, at least 15, or at least 20 bases. In some embodiments, the alternating hybridization domain comprises bases that hybridize to the hybridization domain of the second nucleic acid barcode. In some embodiments, the cross-linked strand of one nucleic acid barcode hybridizes to the complementary strand of another nucleic acid barcode.

[0108] Methods for barcode deposition provided herein include, but are not limited to, applying a force to transfer the barcode from a reservoir to a sample. Exemplary forces for applying include laminar pressure, capillary pressure, slipstream pressure, magnetic force, electrostatic force, creeping force, acoustic force, vibration force, centripetal force, centrifugal force, or any combination thereof.

[0109] In some embodiments, a buffer exchange process is used for barcode deposition. In some embodiments, an electrophoresis process is used for barcode deposition. In such a method, the migration of charged particles (e.g., nucleic acid barcode molecules) in an electric field is due to electrophoresis, and the electrode is close to the substrate containing the cell. In some embodiments, electrophoresis is applied to remove nucleic acids from the surface. In some embodiments, electrophoresis is applied to remove concatemers described herein from the surface. The electrode can generate a uniform field across the surface, or a specific electrode can be used for each region of interest. In some embodiments, temperature changes are used for barcode deposition. In other embodiments, the barcode sequence includes a temperature-sensitive feature, and an increase in temperature can make hybridization areas that are unavailable at lower temperatures available. In some embodiments, the ROI includes a heating unit or electrode adjacent to the ROI that is sufficient to increase the temperature at the ROI. In some embodiments, the barcode includes a melting temperature, and the ROI is heated to reach the barcode melting temperature, thereby allowing selective hybridization of one or more barcodes.

[0110] The exemplary methods described herein can also be combined to incorporate any combination of the described methods. For example, a sample barcoded with iterative construction of barcodes can also be tagged using the fractional barcoding methods described herein.

[0111] Nucleic acids described herein can be non-specifically or specifically conjugated to another molecule (e.g., protein, epitope or another nucleic acid) in the sample. In some embodiments, barcodes are conjugated to molecules. In some embodiments, barcodes include nucleic acids conjugated to antibodies, antibody fragments, proteins, peptides, nanobodies, small molecules, nucleic acid therapeutic agents, probes, aptamers, siRNA, lipids, nanoparticles, lipid nanoparticles or other affinity reagents. In some embodiments, antibody fragments as described herein are F (ab') 2 fragments, Fab' fragments, Fab fragments, Fv fragments, IgG fragments, Fc fragments or any combination thereof. In some embodiments, lipids are emulsions, liposomes, particles or any combination thereof. In some embodiments, antibodies are IgA, IgG or IgM antibodies or their functional fragments. In some embodiments, tissue samples provided herein or cell colonies are imaged after barcoding.

[0112] In some embodiments, the barcodes provided herein are deposited onto the sample at a concentration of about 20 nM to about 500 nM. In some embodiments, the barcodes described herein are deposited onto 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. The binding of the barcode to the target region can be stable or transient.

[0113] In some embodiments, the length of the nucleic acid barcode domain is about 5-10 bases. In some embodiments, the length of the nucleic acid barcode domain is 4, 5, 6, 7, 8, 9, 10 or more bases. In some embodiments, the length of the nucleic acid barcode domain is about 10 or about 10-15 bases. Each barcode domain can be assigned a position value of "0" or "1". In some embodiments, the series of nucleic acid barcodes is equivalent to a string of 0 and 1.

[0114] Photo-directed cross-linking

[0115] The nucleic acid barcode chains can be connected and covalently fixed together by photochemical cross-linking reactions. 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 can be carried out in an aqueous solution. In some embodiments, a photoreactive cross-linking agent is used in the reaction. In some embodiments, a photoreactive nucleobase is used in the reaction. In such embodiments, the cross-linked chain of the nucleic acid barcode comprises at least one photoreactive nucleobase. In some embodiments, the photoreactive nucleobase can be any modified nucleobase that can form a crosslink 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-threonine alcohol (CNVD). In some embodiments, the photoreactive nucleobase includes vinyl, acrylate, N-hydroxysuccinimide, amine, carboxylate or thiol chemical groups. In some embodiments, the photoreactive nucleobase comprises bromodeoxyuridine.In some embodiments, the CNVK crosslinking base exhibits the highest crosslinking efficiency with thymine (T) bases positioned adjacent to bases on the complementary strand and can be directly incorporated into the DNA hybridization domain itself as a base substitution.

[0116] The nucleic acid barcode chains can be connected and covalently fixed together by photochemical cross-linking reactions. 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 can be carried out in an aqueous solution. In some embodiments, a photoreactive cross-linking agent is used in the reaction. In some embodiments, a photoreactive nucleobase is used in the reaction. In such embodiments, the cross-linked chain of the nucleic acid barcode comprises at least one photoreactive nucleobase. In some embodiments, the photoreactive nucleobase can be any modified nucleobase that can form a crosslink 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-threonine alcohol (CNVD). In some embodiments, the photoreactive nucleobase includes vinyl, acrylate, N-hydroxysuccinimide, amine, carboxylate or thiol chemical groups. In some embodiments, the photoreactive nucleobase includes bromodeoxyuridine. In some embodiments, the CNVK crosslinking base exhibits the highest crosslinking efficiency with a thymine (T) base located adjacent to a base on the complementary strand and can be directly incorporated into the DNA hybridization domain itself as a base substitution. In some embodiments, the crosslinking reaction is performed using light of 365 nm wavelength and is completed within 1 second, such as Figure 3 In some embodiments, more than one photocrosslinker molecule is incorporated so that different barcodes contain different photocrosslinker molecules that can crosslink at different wavelengths. Such changes in the barcodes allow methods to change crosslinking events at one or more regions of interest. In some embodiments, a reverse crosslinking reaction is performed. For example, applying a wavelength of 312nm for a few minutes can provide crosslinking reversal. In some embodiments, a series of crosslink-then-decrosslinking reactions are performed to form concatemers. In some embodiments, molecules are deposited on a substrate provided herein to locally generate light wavelengths to support photosensitive crosslinking reactions.

[0117] In some embodiments, radiation is provided at a specific wavelength or wavelength range. The cross-linking reaction can be designed to use visible light or ultraviolet light of any wavelength. In some embodiments, the radiation is ultraviolet (UV) radiation. In some embodiments, radiation is provided at a wavelength within the range of about 300 nanometers (nm) to about 450nm. In some embodiments, the radiation 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 radiation is provided at a wavelength of about 365 nm. In some embodiments, the radiation is provided at a wavelength of about 405 nm.

[0118] In some embodiments, the radiation is provided for a specific amount of time. In some embodiments, the radiation is provided for about 1 second to up to 10 seconds. In some embodiments, the radiation is provided for about 100 milliseconds (ms) to up to 10 seconds. In some embodiments, radiation 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 cross-linking reaction can be completed within 0.01, 0.1, 0.25, 0.5, 1, 5, or 10 seconds. In some embodiments, the cross-linking reaction can be completed within 0.5, 1, 5, 10, 20, 30, 40, 50, or 60 minutes.

[0119] In some embodiments, the cross-linking reaction is performed using light of 365 nm wavelength and is completed within 1 second, such as Figure 3 In some embodiments, more than one photocrosslinker molecule is incorporated so that different barcodes contain different photocrosslinker molecules that can crosslink at different wavelengths. Such changes in the barcodes allow methods to change crosslinking events at one or more regions of interest. In some embodiments, a reverse crosslinking reaction is performed. For example, applying a wavelength of 312nm for a few minutes can provide crosslinking reversal. In some embodiments, a series of crosslink-then-decrosslinking reactions are performed to form concatemers. In some embodiments, molecules are deposited on a substrate provided herein to locally generate light wavelengths to support photosensitive crosslinking reactions.

[0120] In some embodiments, the cross-linking reaction has a negligible effect on bases adjacent to the photoreactive nucleobase.In some embodiments, other photochemical nucleic acid cross-linkers, including psoralens, are used in combination with nucleic acid barcodes.

[0121] In some embodiments, a cross-linking reaction is applied to a nucleic acid barcode strand that is hybridized to the 5' end of a cDNA. In some embodiments, a cross-linking reaction is applied to a nucleic acid barcode strand that is hybridized to the 3' end of a cDNA. In some embodiments, additional barcode strands are iteratively added to the 5' or 3' end of a cDNA-nucleic acid barcode structure using binding and cross-linking as previously described. Figure 4 A process comprising iterative addition of a barcode strand to the 5' end of a cDNA according to methods described herein is shown. In some embodiments, the cross-linking reaction occurs simultaneously with hybridization.

[0122] The cycle of hybridization and cross-linking can be repeated without a wash step to remove uncross-linked barcode strands. In such an embodiment, the barcode strands are designed with different or alternating hybridization domains so that only subsequently added barcode strands will hybridize and cross-link to existing target / barcode concatemers.

[0123] In some embodiments, the cross-linked chain is a single-stranded nucleic acid having a length of 200 nucleotides or less. In some embodiments, the length of the cross-linked chain may be 5-100, 5-75, 5-50, 5-25, 5-15 or 5-10 nucleotides. In some embodiments, the length of the cross-linked chain may be 10-100, 10-50, 10-25, 10-20, 15-25 or 15-25 nucleotides. In some embodiments, the length of the cross-linked chain may be 5, 6, 7, 8, 9 or 10 nucleotides. In some embodiments, the cross-linked chain comprises at least one photoreactive nucleotide. In some embodiments, the cross-linked chain comprises at least one 3-cyanovinylcarbazole nucleoside (CNVK) or 3-cyanovinylcarbazole modified d-threonine alcohol (CNVD) nucleotide. In some embodiments, the cross-linked chain is complementary to the complementary chain, wherein the cross-linked chain is a component of the first nucleic acid barcode and the complementary chain is a component of the second nucleic acid barcode. In some embodiments, the cross-linked chain is DNA or RNA. In some embodiments, the cross-links are also functionalized with moieties such as fluorophores, quantum dots, biotin, streptavidin, functional chemical groups, acrylamide, maleimide, lipids, metal isotopes, other tags (e.g., antibodies), or nanoparticles.

[0124] In some embodiments, the length of the nucleic acid barcode chain is at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides. In some embodiments, the length of the nucleic acid barcode chain is 20 nucleotides. In some embodiments, the nucleic acid barcode chain comprises a barcode domain having a length of 2 to 10 nucleotides, a cross-linked chain having a length of 2 to 12 nucleotides, and a complementary chain having a length of 2 to 12 nucleotides. In some embodiments, the nucleic acid barcode chain comprises the following structure in the 5' to 3' direction: 5'-cross-linked chain to barcode domain to complementary chain-3'. In some embodiments, the nucleic acid barcode chain comprises the following structure in the 5' to 3' direction: 5'-complementary chain to barcode domain to cross-linked chain-3'.

[0125] The cross-linked strands can be designed to stably bind or transiently bind to the complementary strand. In some embodiments, the cross-linked strands can be covalently or non-covalently bound to the complementary strand. In some embodiments, the cross-linked strands can be complementary to the complementary strand. The cross-linked strands can 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. In addition, the cross-linked strands can bind to complementary domains of different nucleic acid barcodes. In some embodiments, the cross-linked strands can bind to complementary domains of nucleic acids that contain their own cross-linked strands but do not contain barcode strands.

[0126] Region Selection

[0127] Provided herein are methods in which cells (or subcellular compartments such as nuclei) of a sample are sorted by defined features before or after barcoding or labeling. In some embodiments, cells are sorted before each iteration of barcoding. In some embodiments, cells are sorted before any barcoding step. In some embodiments, cells are sorted after all barcoding steps. In some embodiments, cells are sorted in one step based on features (e.g., surface receptors). In other 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 cells are barcoded using methods described herein. In other embodiments, barcoded cells are further sorted based on a second feature, and are optionally further barcoded.

[0128] Provided herein are methods for nucleic acid barcoding, the methods comprising 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, cell nuclei or multi-cellular arrangements are sorted rather than single cells.

[0129] In some embodiments, sample imaging provides a basis for identifying a cell region of interest (ROI). In some embodiments, the ROI includes a barcoded region targeted for spatial definition of cDNA in the sample. In some embodiments, the ROI is based on the morphology of the sample, such as cells, cell structures, tissue layers, or subcellular structures. In some embodiments, the ROI has regular or irregular boundaries. In some embodiments, the ROI describes the boundaries of the barcoded space defined 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).

[0130] In some embodiments, barcode is deposited to the ROI across the surface provided herein with a preselected pattern. In some embodiments, pattern formation spans the grid of the repeated shape of the region, or an alternating shape is preselected for barcode deposition. In some embodiments, pattern formation spans the grid of the repeated shape of the region, and less than all shapes are preselected for barcode deposition. In some embodiments, the shape is a square, rectangle, triangle, ellipse, diamond or sphere. In other embodiments, the shape can be a complex geometric or non-geometric pattern (e.g., the outline of tissue or cell features, alphanumeric characters).

[0131] In some embodiments, light-directed cross-linking is selectively applied to cell samples. Cross-linking is selectively performed by shielding areas from radiation, allowing cross-linking only in unshielded areas. A photomask provides a template to shield the area receiving radiated light in the sample. In some embodiments, a photomask is generated to allow radiation of one or more regions of interest (ROI). In some embodiments, the photomask is manually or machine generated. In some embodiments, one or more photomasks are generated. In some embodiments, a photomask associated with each ROI is generated. In some embodiments, a photomask is generated according to sample imaging as previously described herein. In some embodiments, a photomask is applied to the sample before radiation. In some embodiments, each cross-linking step includes different photomasks. In some embodiments, multiple cross-linking steps use the same photomask.

[0132] ROIs define regions within a sample where one or more target crosslinks occur. In some embodiments, one or more ROIs define different regions. In some embodiments, one or more ROIs are congruent. In some embodiments, one or more ROIs overlap. In some embodiments, one or more ROIs are separate. In some embodiments, one or more ROIs are not separate.

[0133] In some embodiments, in addition to or in place of a photomask, as described herein, light is focused on one or more ROIs. In some embodiments, a digital micromirror device (DMD) is used to focus light. In some embodiments, a confocal microscope is used to focus light. In some embodiments, the confocal microscope is a laser scanning confocal microscope. In some embodiments, a DVD recorder is used to focus light. In some embodiments, a Blu-ray disc recorder is used to focus light. In some embodiments, the light source is an entity independent of another device (such as a microscope). In some embodiments, the light source is a laser. In some embodiments, the laser has a dominant wavelength of about 180nm to about 1mm. In some embodiments, the laser has a dominant wavelength of about 180nm to about 400nm. In some embodiments, the laser has a dominant wavelength of about 400nm to about 700nm. In some embodiments, the laser has a dominant wavelength of about 700nm to about 1mm.

[0134] The optical system used with the method described herein can provide selective z-plane illumination. In some embodiments, the optical system can provide light with about 2nm to about 10μm resolution, about 5nm to about 9μm resolution, about 10nm to about 8μm resolution, about 15nm to about 7μm resolution, about 20nm to about 6μm resolution, about 25nm to about 5μm resolution, about 30nm to about 4μm resolution, about 35nm to about 3μm resolution, about 40nm to about 2μm resolution, about 45nm to about 1μm resolution, about 50nm to about 800nm ​​resolution, about 100nm to about 600nm resolution, about 200nm to about 400nm resolution or any range therebetween in the z-plane. In some embodiments, the light in the z-plane has a resolution of 10μm or more. In some embodiments, the light is focused on the surface of a tissue sample or a cell colony provided herein. In some embodiments, the light is focused on a plane comprising at least a portion of a tissue sample or a cell colony provided herein. In some embodiments, the light is focused on one or more planar surfaces on or within a tissue sample or a cell colony provided herein. In some embodiments, the light-directed crosslinking described herein is applied to the surface of a tissue sample or a colony of cells provided herein. In some embodiments, the light-directed crosslinking described herein is applied across multiple planes within a tissue sample or a colony of cells. In some embodiments, the light-directed crosslinking described herein is applied to multiple planes of a three-dimensional tissue sample or a colony of cells provided herein. Exemplary devices for illumination limited in the z plane include two-photon microscopes and light sheet microscopes.

[0135] 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 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 the xy plane. In some embodiments, the light in the xy plane has a resolution of 10 μm or more.

[0136] In some embodiments, a nucleic acid described herein is covalently linked to at least one other nucleic acid through a single photoresponsive element of a hybridization domain.

[0137] Fractional barcoding

[0138] By irradiating with longer duration or higher intensity, the number of crosslinks formed in a 1D, 2D or 3D region can be controllably increased on a specific region, sample or strand in a cell or test tube. Intensity is measured in Watts per square centimeter (W / cm 2 ) measured by the amount of radiation. Because the radiation exposure time and power level can be precisely controlled, the fraction of crosslinks formed in a particular area or volume can be precisely controlled (e.g., on a microscope, using an LED or other light source on a sample or test tube). Such regulation allows for region-specific photosensitive crosslinking events of the barcodes, thereby allowing for the preselected gradation ("fractionation") of any given barcode (or tag) to attach it to a target within a particular region of interest (tissue region, cell, subcellular region, microwell, tube, etc.). In some embodiments, the light intensity is about 1 to about 5000 W / cm 2 In 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 1 W / cm 2 In some embodiments, the light intensity exceeds 5000 W / cm 2 The above intensity is the intensity when the intensity is focused on a flat light. Similar intensity can be applied in 3D, in which case the quantity is W / cm 3 When irradiating a solution, the volume of the sample is irradiated. In such a case, the intensity per volume unit is applied, such as with a UV gun.

[0139] The duration or power of the radiation exposure allows the fraction of crosslinks formed in a specific region of interest (ROI) to be controllably adjusted. In other words, the fraction of sequences crosslinked to the barcode in a specific ROI can be controlled by the radiation exposure time or intensity level. In some embodiments, up to about 100% of the barcodes are crosslinked after radiation. 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 with one or more target sequences after radiation. In some embodiments, about 10% of the barcodes in the ROI are photocrosslinked with one or more target sequences after radiation. In some embodiments, about 20% of the barcodes in the ROI are photocrosslinked with one or more target sequences after radiation. 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.

[0140] The duration or power of the radiation, or both, can be adjusted in a gradient to provide a series of increasing or decreasing barcoded reaction fractions across one or more ROIs. The gradient range of duration or power can be 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 thereof or intervals therein.

[0141] Barcoding can also be adjusted by introducing a certain amount of interfering nucleic acid chains in the solution during the hybridization step, the radiation step, or both steps. In some embodiments, the interfering nucleic acid is bound to the target nucleic acid to prevent the barcoding reaction. In some embodiments, the interfering nucleic acid is bound to the barcode chain to prevent the barcoding reaction. The regulation of the barcoding reaction can be achieved by changing the amount of the interfering nucleic acid chain added. In some embodiments, the amount of the interfering nucleic acid chain is the percentage of the target nucleic acid calculated in the sample. In some embodiments, the amount of the interfering nucleic acid chain is the percentage of the barcode chain added. In some embodiments, the interfering nucleic acid chain is added in an amount of about 1% to about 1000% of the target nucleic acid or barcode chain. 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%, about 90% to about 100% of the target nucleic acid or barcode strand.

[0142] The population of targets as described herein within a sample can be encoded for identification using fractional applications of one or more barcodes. Binary, ternary, quaternary or more base encodings can be used. In some embodiments, any combination of fractional applications of different barcodes that allow for differentiation of barcode fractions can be used. The fractional barcodes can be read using sequencing technology, fluorescence imaging, spectrophotometry or any other detection method. In an exemplary embodiment, one ROI in a sample contains 40% of barcode 1+20% of barcode 2+20% of barcode 3, while another ROI contains 20% of barcode 1+40% of barcode 2+20% of barcode 3. In some embodiments, the fractions do not add up to 100%. In some embodiments, the fractions add up to 100%. In some embodiments, the fractions are distinguishable and deconvoluted.

[0143] Barcode processing

[0144] Methods as described herein include applying reagents (including barcode strands, wash sequences, etc.) to a sample. In some embodiments, the application method includes traditional microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handlers for depositing new solutions (e.g., Echo Liquid Beckman Coulter, Indianapolis, IN), an inkjet printer with improved content, an automated liquid handler, optionally incorporating a pipette, a robotic arm for handling slides between solution baths, or any combination thereof. In some embodiments, the method of application is performed on the stage of a device (e.g., a microscope). In some embodiments, the method of application is performed off the stage of a device (e.g., a microscope).

[0145] In some embodiments, the diffusion of nucleic acids is enhanced by adding molecular crowding agents. In some embodiments, crowding agents include polymers, proteins, or any combination thereof. In some embodiments, polymers include polyethylene glycol. In some embodiments, proteins include serum albumin, such as bovine serum albumin. In some embodiments, the diffusion of nucleic acids is enhanced by heating a tissue sample or a population of cells provided herein. In some embodiments, the diffusion is enhanced by heating to more than 30°C, more than 35°C, more than 40°C, more than 45°C, more 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, about 50°C.

[0146] The sample can be treated to prevent or reduce non-specific binding of the barcode strand. In some embodiments, before applying the barcode strand, a blocking agent is added to a population of tissue samples or cells provided herein. In some embodiments, while applying the barcode strand, a blocking agent is added to a population of tissue samples or cells provided herein. In some embodiments, the blocking agent includes non-specific nucleic acid sequences, sheared salmon sperm DNA, tRNA, yeast tRNA, single-stranded oligonucleotides, double-stranded oligonucleotides, polysaccharides, charged polysaccharides, negatively charged polysaccharides, negatively charged molecules, charged molecules, bovine serum albumin (BSA), skim milk powder, surfactants, non-ionic polymer surfactants (e.g., poly(ethylene oxide) / poly(propylene oxide) triblock copolymers-PLURONIC TM F-127, F-68), crowding agent or any combination thereof. In some embodiments, ribosome depletion or targeted depletion of high-abundance sequences occurs before any of the following: reverse transcription, barcoding, sequence extraction, cross-binding synthesis or PCR amplification. In some embodiments, ribosome depletion or targeted depletion of high-abundance sequences occurs after PCR amplification. In some embodiments, an in situ hybridization (ISH) probe targeting a high-abundance sequence is included before or during reverse transcription to prevent specific high-abundance sequences from acting as binding sites for reverse transcription primers. In some embodiments, ISH probes are combined with nuclease activity (e.g., RNase H) to specifically digest high-abundance sequences in situ before reverse transcription. In some embodiments, double-stranded specific nucleases (e.g., dsDNA enzymes) are applied after PCR to specifically enrich high-abundance sequences.

[0147] After the radiation and cross-linking reaction, in some embodiments, the uncross-linked barcodes are removed. In some embodiments, the uncross-linked barcodes are removed by washing. In some embodiments, the uncross-linked barcodes are not removed before additional cross-linking cycles. In some embodiments, the sample is washed with a chemical denaturant solution. In some embodiments, the chemical denaturant solution comprises 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 comprises a surfactant. In some embodiments, the concentration of the surfactant 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 surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 ( 20, Merck KGaA, Darmstadt, Germany) or polysorbate 80 ( 80, Merck KGaA, Darmstadt, Germany). In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the nonionic surfactant is a nonionic polyoxyethylene surfactant. In some embodiments, the surfactant is polyethylene glycol (PEG), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (CAS No.: 9002-93-1), N,N-dimethyltetradecylamine N-oxide (TDAO, CAS No.: 3332-27-2). In some embodiments, the surfactant is Triton X-100, Nereid, TDAO or a combination thereof. In some embodiments, washing is performed at room temperature. In some embodiments, washing is performed at a temperature above or below room temperature. In some embodiments, washing is performed at 4°C, 16°C, 25°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C or higher.

[0148] In situ hybridization

[0149] In some embodiments, in situ hybridization (ISH) is applied. The ISH step generally involves depositing small nucleic acids (e.g., oligomers of 15-40 nucleotides). In some embodiments, a nucleic acid probe identifies one or more regions of a genome. In some embodiments, the nucleic acid probe of ISH identifies RNA. In the case of single-molecule in situ hybridization (smISH), many small nucleic acids identify RNA together, wherein the combination of a single probe results in a weak signal, but the ability to obtain a signal from the collection of all probes (directly or via the connection of a label to the probe) results in a strong signal compared with the combination of a single probe. In some embodiments, before or after antigen repair (e.g., amplification reaction), ISH or improved ISH reactions are applied to samples described herein. In other embodiments, the probe for ISH is also used as a barcode or for the formation of concatemers for barcode binding, followed by methods described herein.

[0150] Iterative addition of nucleic acid barcodes to cDNA can produce nucleic acid concatemers. In some embodiments, the nucleic acid concatemer includes at least three nucleic acid barcode chains. The nucleic acid concatemer may include nucleic acid barcode chains covalently linked to each other via photoreactive nucleotides. In some embodiments, the nucleic acid concatemer may include 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 or at least 10 or more nucleic acid barcode chains. In some embodiments, the nucleic acid concatemer may include 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 or at least 10 or more barcode domains, each barcode domain containing data (e.g., each barcode domain can be uniquely / independently assigned a bit value). Figure 5A-Figure 5B One embodiment of digital data storage using light-directed concatenation of barcodes is shown in FIG. The concatemer of nucleic acid barcodes can comprise at least two, at least three, at least four, at least five, at least ten, at least 15, at least 20, at least 25, at least 30, at least 50 nucleic acid barcodes, or any number therebetween.

[0151] 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.

[0152] In some embodiments, the CNVK-labeled chain can hybridize 1 to 10 bases or 10 to 20 bases with the docking chain. In some embodiments, salt concentration, temperature, denaturant concentration or chain concentration affect the stability of chain binding. In some embodiments, the stability of chain binding is adjusted so that the average binding of the CNVK-labeled chain is less than 1 second, between 1 and 10 seconds or less than 1 minute. In some embodiments, the CNVK-labeled chain can have an increased homology domain with the docking chain. In some embodiments, the CNVK-labeled chain has a homology spanning 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, salt is increased or the temperature is lowered, so as to adjust the chain binding to be longer than one minute on average. In some embodiments, conditions can be adjusted to determine the average binding time of the CNVK-labeled chain and the docking chain.

[0153] The concatemers of nucleic acid barcodes can be in various positions on a substrate as described herein. For example, the concatemers can be in a linear relationship relative to each other, have two different orientations relative to the target, or have multiple orientations in different directions. The barcodes can be positioned along x, y, and z coordinates in space.

[0154] In some embodiments, the concatemers described herein are linked to proteins. In other embodiments, proteases are applied to release concatemers from proteins. In some embodiments, the barcodes described herein are linked to proteins. In other embodiments, proteases are applied to release barcodes from proteins. Exemplary proteases for inclusion include, but are not limited to, proteinase K. In some embodiments, proteases are applied prior to antigen repair reactions (e.g., reverse transcription), prior to barcoding reactions, prior to concatemerization reactions, and / or prior to concatemer repair reactions.

[0155] Imaging

[0156] Nucleic acid barcodes optionally comprise additional detectable labels, such as fluorophores, luminescent and bioluminescent labels (e.g., biotin, luciferases (e.g., bacteria, fireflies, click beetles, etc.), luciferin, and aequorin), radioactive labels (e.g., 3H, 125I, 35S, 14C, 33P, or 32P), enzymes (e.g., β-galactosidases, glucuronidases, phosphatases (e.g., alkaline phosphatases), peroxidases (e.g., horseradish peroxidase), and cholinesterases), 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 comprise the addition of a detectable label as described herein.

[0157] The detection resolution using the barcoding methods described herein is not limited by light diffraction. Such barcoding methods allow for the detection of samples with very high density as well as very low density targets. Additionally, the barcoding methods described herein can be applied in two or three dimensions without losing resolution or requiring significant adjustments to the method.

[0158] In some embodiments, fluorescent in situ hybridization (FISH) is applied. The FISH step generally involves depositing small nucleic acids (e.g., oligomers of 15-40 nucleotides) with labels (e.g., fluorescent labels). In some embodiments, the nucleic acid fluorescent probes of FISH identify one or more regions of the genome. In some embodiments, the nucleic acid fluorescent probes of FISH identify RNA. In some embodiments, FISH or modified FISH reactions are applied to samples described herein before or after antigen repair (e.g., amplification reactions). In other embodiments, the probes for FISH are also used as barcodes or for the formation of concatemers for barcode binding, followed by methods described herein.

[0159] After washing to remove uncrosslinked DNA barcodes, the bound barcodes can be visualized or detected. In some embodiments, visualization or detection is by colorimetric, fluorescent, ultraviolet, radiation or other means.

[0160] The barcoding methods described herein may include single cell or single cell nucleus labeling methods. In some embodiments, split pool barcoding is used. In such embodiments, samples are repeatedly distributed into wells for barcoding and pooling. The final library contains discrete combinations of barcodes.

[0161] Transcript extraction and sequencing

[0162] 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 a single ROI are collected in a region-specific manner. In some embodiments, DNA strands from a single cell are pooled individually. In some embodiments, DNA from a single cell or other defined ROI is individually barcoded and sequenced.

[0163] In some embodiments, the displacement of barcoded nucleic acid includes applying reagents to release the nucleic acid containing barcodes from the surface. The reagents for releasing the nucleic acid containing barcodes can include RNA enzymes, DNA enzymes and / or proteases. In some embodiments, record extraction and sequencing include the displacement of barcoded nucleic acid. Optionally, the displacement is achieved by applying RNA hydrolases. In some embodiments, the RNA hydrolases are RNA enzymes H. The cross-linking of continuous nucleic acids can also achieve the displacement of barcoded nucleic acids. Enzymatic displacement and synthesis reactions can be combined or carried out in any order. After the sequence has been extracted from the sample, the sample can be further imaged or other determinations (e.g., mass spectrometry, H&E staining). Subsequent polymerase chain reaction (PCR) amplification and optional purification are followed by optional library preparation and next generation sequencing. The resulting sequence analysis can be merged with the captured imaging data to provide integrated spatial and sequence-based information output.

[0164] The generated barcode concatemers are displaced and synthesized into continuous DNA strands from the tissue samples or cell populations provided herein. These steps can occur sequentially, in any order or simultaneously. Continuous DNA strands are optionally collected. The nucleic acids are then optionally amplified and sequenced. Sequencing data is optionally combined with earlier imaging data to provide spatially defined sequencing information.

[0165] Provided herein are methods for concatemer replacement. In some embodiments, concatemers bound to RNA are replaced from a population of tissue samples or cells provided herein. In some embodiments, concatemers are chemically replaced. In some embodiments, concatemers are enzymatically replaced. In some embodiments, concatemers are replaced using enzymes that specifically cleave RNA (e.g., RNase H). In some embodiments, concatemers are replaced using enzymes that specifically cleave DNA, DNA enzymes (e.g., DNA enzyme I). In some embodiments, concatemers are physically replaced. In some embodiments, concatemers are replaced by applying energy. In some embodiments, energy is chemical energy, electrical energy, radiation energy, mechanical energy, thermal energy, nuclear energy, or any combination thereof. In some embodiments, concatemers are replaced by applying heat. In some embodiments, concatemers are replaced by electrophoresis. In some embodiments, concatemers are replaced by a process of cross-binding. In some embodiments, cross-binding is performed before replacing barcoded DNA. In some embodiments, cross-binding is performed after replacing barcoded DNA. In some embodiments, replacement and cross-binding occur simultaneously.

[0166] Cross-linking

[0167] Provided herein are methods for crossover synthesis, wherein a nucleic acid combined with one or more barcode sequences is copied to a new single strand of DNA. The crossover synthesis is performed directly in situ, or during / after the displacement. Figure 6 The basic strategy for crossover formation is depicted in Figure 1. Two nucleic acids that have hybridized together to form a knot are shown. A primer binds to the first nucleic acid in front of the knot (left). A strand displacement polymerase is used to copy the x domain (also referred to herein as the knot domain) until it reaches the terminator (shown in black and also referred to herein as the closure domain). The new x domain competes with the old x domain in a random walk branch migration process. Eventually, the new x domain can mate with an exposed x on the second template strand (right, also referred to herein as the synthesis region). * The domains bind. Polymerization can then continue on the second template strand, making copies along the new backbone.

[0168] In some embodiments, cross-linking is performed to produce DNA, RNA, PNA or LNA. In some embodiments, the method of cross-linking includes contacting the target nucleic acid or barcode provided herein with a strand displacement polymerase. In some embodiments, cross-linking is performed at room temperature (e.g., at least about 20°C to about 25°C). In some embodiments, cross-linking is performed at a temperature below room temperature (e.g., about 15°C, about 10°C, about 4°C). In some embodiments, cross-linking is performed at a temperature above room temperature (e.g., about 30°C, about 37°C). In some embodiments, nucleic acid amplification or cross-linking is performed at a constant temperature. In some embodiments, nucleic acid amplification or cross-linking is performed at different or varying temperatures.

[0169] Provided herein are methods in which a polymerase is prevented or stopped from moving along the backbone of a template strand and / or copying a template strand by a blocking domain. In some embodiments, the blocking domain comprises a T domain known to increase the number of double-stranded nucleic acids. m In some embodiments, the modified nucleotides include, but are not limited to, locked nucleic acids (LNA), 2'-O-methoxy-ethyl (2'-MOE) nucleotides, 2,6-diaminopurine, G-clamps (analogs of C with 4 hydrogen bonds), and guanidino G-clamp nucleotides, etc.

[0170] The compositions and methods described herein can be used to synthesize a specified sequence. Figure 7 An exemplary embodiment is shown in FIG. Figure 7As shown in Figure 1, by hybridizing multiple barcode strands together, crossover reactions can be cascaded to form longer sequences. Each junction between barcode strands displays the same domain motif, so that the strand domain copied on the 3' end of the growing strand before reaching the terminator (b, c, d, e) can cross over the crossover junction and interact with the complementary sequence exposed on the next barcode strand (b * 、c * d * 、e * Any nucleic acid sequence can be added in the template region between the motif domains (shown in grey) to enable the assembly of longer sequences.

[0171] In some embodiments, at least one closed domain comprises a poly monomer stretch. For example, the closed domain may comprise a stretch of poly-A, poly-T, poly-C or poly-G nucleotides.

[0172] In some embodiments, the closed domain or terminator may include covalent crosslinking of two barcode chains. In some embodiments, the closed domain or terminator includes one or more proteins. In some embodiments, one or more proteins are combined with one or more of the barcode chains. In some embodiments, one or more proteins are crosslinked with one or more of the barcode chains. In some embodiments, covalent crosslinking is photocrosslinking or chemical crosslinking. In some embodiments, covalent crosslinking includes crosslinking the nucleotides at the 5' end of the first barcode chain with the nucleotides at the 3' end of the second template chain. In some embodiments, crosslinking is at an oligonucleotide or a single nucleic acid backbone bond (backbone linkage). In some embodiments, crosslinking is by a phosphodiester bond. In some embodiments, the crosslinking reaction is between CNVK nucleotides and complementary nucleotides. In some embodiments, the crosslinked nucleotides serve as terminators for polymerases.

[0173] In some embodiments, the closed region comprises any desired nucleotide sequence or number of nucleotides. In some embodiments, each closed domain can have any length. In some embodiments, the length of each closed domain can be 1 nucleotide to 100 nucleotides. In some embodiments, the length of each closed domain is independently one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides. Note that the closed domain can be a single nucleotide.

[0174] 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 may 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 may have complementary sequences. Thus, the nucleic acids may be alternately arranged and cross-linked to generate concatemers described herein.

[0175] Note that the nucleic acids described herein optionally comprise a barcode domain. In an exemplary embodiment, the second nucleic acid of the junction comprises a barcode domain at the 5' end. In some embodiments, one of the first synthesis region or the second synthesis region of the nucleic acid comprises a barcode domain.

[0176] purification

[0177] The methods and systems described herein can include purification of samples. In some embodiments, the barcoded samples are purified by chemical means, such as precipitating the nucleic acids. In some embodiments, the barcoded samples are purified using column chromatography (e.g., ion exchange, reverse phase, or size exclusion chromatography). In some embodiments, the barcoded samples are purified using on-bead binding. In some embodiments, the barcoded samples are purified using electrophoretic properties.

[0178] PCR amplification

[0179] Amplification methods are provided herein. PCR amplification methods are 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 (Long-range PCR), single-cell PCR, rapid cycle PCR, methylation-specific PCR (MSP), hot start PCR, high-fidelity PCR, in situ PCR, variable number of tandem repeats (VNTR) PCR, asymmetric PCR, PCR based on repetitive sequences, overlap extension PCR, assembly PCR, sequence-specific PCR (Intersequence-specific PCR, ISSR), connection-mediated PCR, methylation-specific PCR, miniprimer PCR (Miniprimer PCR), solid phase PCR, touchdown PCR (Touch down PCR) or any combination thereof. In some embodiments, reverse transcriptase is applied to amplification described herein. In some embodiments, reverse transcriptase is MuLV reverse transcriptase or avian myeloblastosis virus (Avian myeloblastosis virus, AMV) reverse transcriptase. In some embodiments, DNA polymerase is applied to amplification described herein. In some embodiments, DNA polymerase is T7 DNA polymerase, thermophilic eubacterial microorganism Thermus aquaticus DNA polymerase, Pfu DNA polymerase or Bst DNA polymerase. In some embodiments, DNA ligase is applied to the connection chain. In some embodiments, DNA ligase is EC 6.5.1.1. In some embodiments, DNA ligase is T4 DNA ligase.

[0180] 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, but is not limited to, 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 an SP6 RNA polymerase promoter region (SEQ ID NO: 3: 5'ATTTAGGTGACACTATAG 3').

[0181] Provided herein are methods for preparing amplicons. In some embodiments, the amplicons are purified. In some embodiments, the amplicons are analyzed by gel electrophoresis. In some embodiments, the amplicons are separated. 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.

[0182] Samples can also be prepared for sequencing using any combination of one or more of tagmentation, fragmentation, adapter ligation, rRNA reduction, or other library preparation steps.

[0183] Sequencing

[0184] Provided herein is a method for sequencing. In some embodiments, sequencing includes next generation sequencing (NGS) technology. In some embodiments, sequencing includes parallel sequencing. Sequencing may include paired end, unpaired end or long read sequencing. In some embodiments, sequencing includes high throughput sequencing, such as single molecule real-time sequencing, ion semiconductor sequencing (Ion Torrent), pyrophosphate sequencing, synthetic sequencing, combined probe anchor synthesis, connection sequencing (SOLiD), nanopore sequencing, genapsys sequencing or chain termination (Sanger) sequencing. In some embodiments, sequencing includes large-scale parallel sequencing.

[0185] Image and sequence analysis

[0186] The density of the barcoded cDNA target can be measured by the number of unique sequences detected. In some embodiments, the raw sequencing reads are filtered to obtain those that map only once to the transcriptome. The reads are "de-duplicated", that is, the same molecules that have been sequenced as reads more than once according to reads with the same UMI sequence and mapped to the same transcript in the transcriptome are identified. All identical sequencing reads are collapsed into a single "de-duplicated" read. A gene expression matrix is ​​generated, compiling the unique counts of each gene sequenced in the transcriptome. Therefore, the difference in the number of counts across all genes can be compared across samples.

[0187] In some embodiments, the density of barcoded cDNA targets is in the range of 1 μm2. 2 ) of a tissue sample, a population of cells, an organoid, or an organ. In some embodiments, the density of barcoded cDNA targets is in a cubic micrometer (μm 3 ) of a tissue sample, a population of cells, an organoid, or an organ. In some embodiments, the methods provided herein generate barcodes per square micrometer (μm 2) about 55 tags or barcodes. In some embodiments, the methods provided herein generate 2 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, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000, about 2250, about 2500, about 2750, about 3000, about 3500, about 4000, about 4500, about 5000 tags.

[0188] In some embodiments, the methods provided herein generate 3 ) up to about 5 tags per cubic micron (μm 3 ), up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, up to about 50, up to about 55, up to about 60, up to about 65, up to about 70, up to about 80, up to about 90, up to about 100, up to about 150, up to about 200, up to about 250, up to about 300, up to about, up to about 350, up to about 400, up to about 450, up to about 500, up to about 550, up to about 600, up to about 650, Up to about 700, up to about 800, up to about 900, up to about 1000, up to about 1200, up to about 1400, up to about 1600, up to about 1800, up to about 2000, up to about 2250, up to about 2500, up to about 2750, up to about 3000, up to about 3500, up to about 4000, up to about 4500, up to about 5000, up to about 6000, up to about 70000, up to about 8000, up to about 9000, up to about 10,000 tags.

[0189] Additional analysis

[0190] Provided herein are methods for protein analysis. In some embodiments, proteins recovered from samples described herein are analyzed by mass spectrometry, which is an analytical tool for measuring 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, amount and / or identity of a component in a sample.

[0191] Also provided herein is a method for screening a region of interest affected by intervention compared to an unaffected region of interest. In some embodiments, the method includes screening one or more regions of interest in a sample. In some embodiments, the method includes screening one or more regions of interest across one or more samples. In some embodiments, one or more regions of interest are affected by intervention before screening, after screening, or between multiple screenings. In some embodiments, the impact includes applying small molecules, peptides, antibodies, cells, proteins, energy, or any combination thereof. In some embodiments, the impact includes thermal energy, radiation energy, chemical energy, nuclear energy, electrical energy, kinetic energy, acoustic energy, or any combination thereof.

[0192] Applications of the methods described herein include, but are not limited to, identification of drug targets, identification of biomarkers, profiling, characterization of cell and disease models, characterization of differentiation states and cell states, tissue mapping, and multidimensional analysis.

[0193] Exemplary embodiments

[0194] Provided herein is a method for generating biological cell information, the method comprising: contacting a population of cells with a nucleic acid polymerase and a primer to generate an amplification product; depositing a DNA barcode onto the population of cells, wherein the DNA barcode comprises a region complementary to a region of the amplification product, and wherein the DNA barcode comprises a photoreactive nucleobase capable of crosslinking with another nucleobase; selectively irradiating a population of cells to form a covalently linked DNA barcode-amplification product complex; washing a population of cells to remove DNA barcodes that are not complexed with the amplification product; and repeating the deposition, selective irradiation and washing steps, wherein: the DNA barcode in the first deposition step comprises a different sequence from the DNA barcode in the second deposition step, and for the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different locations. Also provided herein is a method, the method further comprising determining biological cell spatial information based at least in part on the amplification product and the DNA barcode. Also provided herein is a method, the method further comprising synthesizing a continuous DNA chain, wherein the synthesis comprises contacting the DNA barcode-amplification product complex with a polymerase. Also provided herein is a method, wherein the synthesis comprises cross-linking. Also provided herein is a method, the method further comprising replacing a continuous DNA chain from a population of cells. Also provided herein is a method, wherein synthesis and displacement occur simultaneously. Also provided herein is a method, wherein the continuous DNA chains displaced are collected (e.g., combined for batch processing and / or analysis). Also provided herein is a method, wherein the continuous DNA chains collected are amplified. Also provided herein is a method, wherein the continuous DNA chains collected are sequenced. Also provided herein is a method, the method also includes identifying a target associated with the continuous DNA chain. Also provided herein is a method, the method also includes mapping the target to different positions. Also provided herein is a method, wherein the primer is a random primer. Also provided herein is a method, wherein the primer is a non-random primer. Also provided herein is a method, wherein the photoreactive nucleobase is 3-cyano vinyl carbazole phosphoramidite (CNVK). Also provided herein is a method, wherein selective radiation is performed using a photomask. Also provided herein is a method, wherein the photomask is different in different selective radiation steps. Also provided herein is a method, wherein the photomask is manually generated. Also provided herein is a method, wherein the photomask is generated by a machine. Also provided herein is a method, wherein the selective radiation step includes applying radiation at a wavelength of about 300nm to up to 450nm. Also provided herein are methods, wherein the selective radiation step comprises applying radiation at a wavelength of about 350 nm to up to 420 nm. Also provided herein are methods, wherein the selective radiation step comprises applying radiation at a wavelength of about 365 nm to up to 405 nm. Also provided herein are methods, wherein the selective radiation step comprises applying radiation to a preselected region of interest (ROI). Also provided herein are methods, wherein the ROI is cell-specific or subcellular-specific. Also provided herein are methods, wherein the ROI is based on morphology.Also provided herein is a method, wherein the deposition is performed manually, automatically, robotically, or any combination thereof. Also provided herein is a method, wherein the deposition is performed with a modified inkjet printer. Also provided herein is a method, wherein the deposition or washing is performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handlers, inkjet printers, automatic liquid handlers, or any combination thereof. Also provided herein is a method, wherein the deposition or washing is performed with a pipette. Also provided herein is a method, wherein the repetition 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. Also provided herein is a method, wherein at least 2, 3, 4, 5, 6, 7, 8 or 9 different DNA barcode chains are deposited. Also provided herein is a method, the method also includes contacting a population of cells with a cell permeabilizing agent. Also provided herein is a method, which also includes contacting a colony of cells with an agent that generates a colony of fixed cells. Also provided herein is a method, wherein the agent that generates a colony of fixed cells includes formalin, formaldehyde, paraformaldehyde, methanol, acetic acid, or any combination thereof. Also provided herein is a method, wherein a colony of fixed cells is fixed to a substrate. Also provided herein is a method, wherein the fixed cells are cells of formalin-fixed paraffin embedding (FFPE), cells of paraformaldehyde fixation (PFA), or cells fixed with paraformaldehyde and Triton X-100 (PFA-Tx). Also provided herein is a method, wherein the colony of cells includes one or more of the following: cultured cells; tissues obtained from a subject; engineered tissues; or organoids. Also provided herein is a method, wherein the tissue is a complete tissue or anatomic tissue. Also provided herein is a method, wherein the cultured cells are dissociated cells. Also provided herein is a method, wherein the engineered tissue includes a colony of cells differentiated in vitro by humans. Also provided herein is a method, wherein the cells differentiated in vitro by humans are derived from human induced pluripotent stem cells (hiPSC), embryonic stem cells, or adult stem cells. Also provided herein is a method, wherein the population of cells is retinal cells. Also provided herein is a method, the method further comprising imaging the population of cells after a washing step. Also provided herein is a method, wherein washing comprises depositing a solution comprising a chemical denaturant and a surfactant. Also provided herein is a method, wherein the chemical denaturant comprises formamide. Also provided herein is a method, wherein formamide is present in an amount of about 40% (v / v) to about 60% (v / v). Also provided herein is a method, wherein formamide is present in an amount of about 50% to about 60%. Also provided herein is a method, wherein the surfactant comprises polysorbate 20, polysorbate 80 or Triton X-100. Also provided herein is a method, wherein the surfactant is present in an amount between about 0.05% and about 5% (v / v).Also provided herein are methods wherein the DNA barcode further comprises a detectable label. Also provided herein are methods wherein the step comprising washing the population of cells is omitted.

[0195] Provided herein is a method for generating biological cell spatial information, the method comprising: contacting a population of cells with a capture probe, wherein the capture probe comprises a DNA binding region and a target binding region; depositing a DNA barcode onto a population of cells, wherein the DNA barcode comprises an area bound to the DNA binding region, and wherein the DNA barcode comprises a photoreactive nucleobase capable of crosslinking with another nucleobase; selectively irradiating a population of cells to form a DNA barcode-amplification product complex; washing a population of cells to remove DNA barcodes that are not complexed with amplification products; and repeating the deposition, selective irradiation and washing steps, wherein: the DNA barcode in the first deposition step comprises a different sequence from the DNA barcode in the second deposition step, and for the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different locations. Also provided herein is a method, the method further comprising determining biological cell spatial information based at least in part on the DNA barcode-amplification product complex. Also provided herein is a method, wherein the capture probe comprises RNA or DNA. Also provided herein is a method, wherein the capture probe comprises a protein. Also provided herein is a method, the method further comprising synthesizing a continuous DNA chain, wherein the synthesis comprises contacting a population of cells with a polymerase. Also provided herein is a method, wherein the synthesis comprises cross-linking into. Methods are also provided herein, and the methods also include replacing continuous DNA chains from a population of cells. Methods are also provided herein, wherein synthesis and replacement occur simultaneously. Methods are also provided herein, wherein the replaced continuous DNA chains are collected. Methods are also provided herein, wherein the collected continuous DNA chains are amplified. Methods are also provided herein, wherein the collected continuous DNA chains are sequenced. Methods are also provided herein, and the methods also include identifying targets associated with continuous DNA chains. Methods are also provided herein, and the methods also include mapping targets to different positions. Methods are also provided herein, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK). Methods are also provided herein, wherein selective radiation is performed using a photomask. Methods are also provided herein, wherein the photomask is different in different selective radiation steps. Methods are also provided herein, wherein the photomask is manually generated. Methods are also provided herein, wherein the photomask is generated by a machine. Methods are also provided herein, wherein the selective radiation step includes applying radiation at a wavelength of about 300nm to up to 450nm. Methods are also provided herein, wherein the selective radiation step includes applying radiation at a wavelength of about 350nm to up to 420nm. Also provided herein are methods, wherein the selective irradiation step comprises applying radiation at a wavelength of about 365 nm to up to 405 nm. Also provided herein are methods, wherein the selective irradiation comprises applying radiation to a preselected region of interest (ROI). Also provided herein are methods, wherein the ROI is cell-specific or subcellular-specific. Also provided herein are methods, wherein the ROI is based on morphology. Also provided herein are methods, wherein the deposition is performed manually, automatically, robotically, or any combination thereof.Also provided herein is a method, wherein the deposition is performed with a modified inkjet printer. Also provided herein is a method, wherein deposition or washing is performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid processors, inkjet printers, automatic liquid processors or any combination thereof. Also provided herein is a method, wherein deposition or washing is performed with a pipette. Also provided herein is a method, wherein repetition is performed 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. Also provided herein is a method, wherein at least 2, 3, 4, 5, 6, 7, 8 or 9 different DNA barcode chains are deposited. Also provided herein is a method, wherein the method also includes contacting a colony of cells with a cell permeabilizing agent. Also provided herein is a method, wherein the method also includes contacting a colony of cells with an agent that generates a colony of fixed cells. Also provided herein is a method, wherein the agent for generating a fixed cell population includes formalin, formaldehyde, paraformaldehyde, methanol, acetic acid or any combination thereof. Also provided herein is a method, wherein the fixed cell population is fixed to a substrate. Also provided herein is a method, wherein the fixed cells are cells fixed with formalin paraffin embedding (FFPE), cells fixed with paraformaldehyde (PFA) or cells fixed with paraformaldehyde and Triton X-100 (PFA-Tx). Also provided herein is a method, wherein the cell population includes one or more of the following: cultured cells; tissue obtained from a subject; engineered tissue; or organoid. Also provided herein is a method, wherein the cell population includes tissue, and the tissue is a complete tissue or anatomic tissue. Also provided herein is a method, wherein the cell population includes cultured cells, and the cultured cells are dissociated cells. Also provided herein is a method, wherein the engineered tissue includes a population of cells differentiated in vitro by humans. Also provided herein is a method, wherein the cells differentiated in vitro by humans are derived from human induced pluripotent stem cells (hiPSC), embryonic stem cells or adult stem cells. Also provided herein is a method, wherein the cell population includes retinal cells. Also provided herein is a method, the method also comprising imaging the population of cells after the washing step. Also provided herein is a method, wherein the washing comprises depositing a solution comprising a chemical denaturant and a surfactant. Also provided herein is a method, wherein the chemical denaturant comprises formamide. Also provided herein is a method, wherein formamide is present in an amount of about 40% (v / v) to about 60% (v / v). Also provided herein is a method, wherein formamide is present in an amount of about 50% to about 60%. Also provided herein is a method, wherein the surfactant comprises polysorbate 20, polysorbate 80 or Triton X-100. Also provided herein is a method, wherein the surfactant is present in an amount between about 0.05% and about 5% (v / v).Also provided herein is a method, wherein the DNA barcode further comprises a detectable label. Also provided herein is a method, wherein the target binding region is bound to DNA, RNA, protein, or a combination thereof. Also provided herein is a method, the method further comprising more than one contacting step. Also provided herein is a method, wherein the more than one contacting step comprises: contacting a population of cells with a first capture probe that recognizes nucleic acid; and contacting a population of cells with a second capture probe that recognizes protein. Also provided herein is a method, wherein the DNA binding region comprises a DNA barcode binding region. Also provided herein is a method, wherein the DNA binding region is RNA or DNA. Also provided herein is a method, the method further comprising contacting a population of cells with a DNA polymerase or an RNA polymerase; and a primer to generate an amplification product. Also provided herein is a method, wherein the RNA polymerase is a reverse transcriptase. Also provided herein is a method, wherein the step of washing the population of cells is omitted.

[0196] Provided herein is a method for generating biological cell spatial information, the method comprising: capturing a three-dimensional image of a tissue sample; contacting the tissue sample with a nucleic acid polymerase and a primer to generate an amplification product; depositing a DNA barcode onto the tissue sample, wherein the DNA barcode comprises a region complementary to a region of the amplification product, and wherein the DNA barcode comprises a photoreactive nucleobase capable of crosslinking with another nucleobase; selectively irradiating the tissue sample in three dimensions to form a DNA barcode-amplification product complex; washing the tissue sample to remove the DNA barcode that is not complexed with the amplification product; and repeating the deposition, selective irradiation and washing steps, wherein: the DNA barcode in the first deposition step comprises a sequence different from the DNA barcode in the second deposition step, and for the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different locations of the tissue sample. Also provided herein is a method, the method further comprising determining biological cell spatial information based at least in part on the amplification product and the DNA barcode. Also provided herein is a method, wherein the tissue sample is obtained from a subject. Also provided herein is a method, wherein the tissue sample is an engineered tissue or an organoid. Also provided herein is a method, wherein the tissue sample comprises a population of cells, wherein the population of cells is a heterogeneous population of cells. Also provided herein is a method, wherein the tissue sample comprises dissected cells or intact cells. Also provided herein is a method, wherein the nucleic acid polymerase is an RNA polymerase or a DNA polymerase. Also provided herein is a method, wherein the RNA polymerase is a reverse transcriptase. Also provided herein is a method, wherein the primer is an RNA primer or a DNA primer. Also provided herein is a method, wherein the primer is conjugated to a protein. Also provided herein is a method, wherein the protein is an antibody or an antibody fragment. Also provided herein is a method, wherein the RNA polymerase transcribes RNA from a tissue sample to generate cDNA. Also provided herein is a method, the method also includes hybridizing a DNA barcode with the cDNA. Also provided herein is a method, the method also includes synthesizing a continuous DNA chain, wherein the continuous DNA chain is complementary to the cDNA and at least one DNA barcode. Also provided herein is a method, wherein the continuous DNA chain is complementary to the cDNA and two or more DNA barcodes. Also provided herein is a method, wherein the synthesis is performed by cross-linking. Also provided herein is a method, the method also includes replacing a continuous DNA chain from a tissue sample. Also provided herein is a method, wherein synthesis and replacement occur simultaneously. Also provided herein is a method, wherein the replaced continuous DNA chain is pooled. Also provided herein is a method, wherein the collected continuous DNA strands are amplified. Also provided herein is a method, wherein the collected continuous DNA strands are sequenced. Also provided herein is a method, the method also includes identifying a target associated with the continuous DNA strand. Also provided herein is a method, the method also includes mapping the target to different locations within a population of fixed cells. Also provided herein is a method, wherein the deposition is performed manually, automatically, robotically, or any combination thereof.Also provided herein is a method, wherein the deposition is performed with a modified inkjet printer. Also provided herein is a method, wherein the deposition or washing is performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handlers, inkjet printers, automatic liquid handlers, or any combination thereof. Also provided herein is a method, wherein the deposition or washing is performed with a pipette. Also provided herein is a method, wherein the repetition 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 times. Also provided herein is a method, wherein at least 2, 3, 4, 5, 6, 7, 8 or 9 different barcode chains are deposited. Also provided herein is a method, the method also includes imaging the population of cells after the washing step. Also provided herein is a method, wherein the washing includes depositing a solution comprising a chemical denaturant and a surfactant. Also provided herein is a method, wherein the chemical denaturant includes formamide. Also provided herein is a method, wherein formamide is present in an amount of about 40% (v / v) to about 60% (v / v). Also provided herein is a method, wherein formamide is present in an amount of about 50% to about 60%. Also provided herein is a method, wherein the surfactant comprises polysorbate 20, polysorbate 80 or Triton X-100. Also provided herein is a method, wherein the surfactant is present in an amount between about 0.05% and about 5% (v / v). Also provided herein is a method, wherein the DNA barcode further comprises a detectable label. Also provided herein is a method, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK). Also provided herein is a method, wherein selective radiation is performed with a photomask. Also provided herein is a method, wherein the photomask is different in different selective radiation steps. Also provided herein is a method, wherein the photomask is manually generated. Also provided herein is a method, wherein the photomask is generated by a machine. Also provided herein is a method, wherein selective radiation comprises applying radiation to a preselected region of interest (ROI). Also provided herein are methods, wherein the ROI is cell-specific or subcellular-specific. Also provided herein are methods, wherein the ROI is based on morphology. Also provided herein are methods, wherein the selective radiation step comprises applying radiation at a wavelength of about 300nm to up to 450nm. Also provided herein are methods, wherein the selective radiation step comprises applying radiation at a wavelength of about 350nm to up to 420nm. Also provided herein are methods, wherein the selective radiation step comprises applying radiation at a wavelength of about 365nm to up to 405nm. Also provided herein are methods, wherein the step of washing the tissue sample is omitted.

[0197] Provided herein is a method for generating biological cell spatial information, the method comprising: capturing an image of a population of cells, wherein the population of cells is fixed to a substrate; reverse transcribing RNA from the population of cells to generate cDNA; depositing a DNA barcode onto the population of cells; hybridizing the DNA barcode with the cDNA, wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase; selectively irradiating the population of cells to form cross-linked barcodes; washing to remove uncross-linked DNA barcodes; and repeating the deposition, hybridization, selective radiation, and washing steps, wherein the DNA barcodes in the deposition step are different. Also provided herein is a method, the method further comprising determining biological cell spatial information based at least in part on the cDNA and the DNA barcode. Also provided herein is a method, wherein the cross-linked barcodes form a tandem of barcodes. Also provided herein is a method, wherein a barcode per square micrometer (μm) is generated. 2 ) of cells up to about 55 unique cDNA molecules. Also provided herein are methods wherein each square micrometer (μm) is generated 2 ) of cells up to about 10 unique cDNA molecules. Also provided herein are methods wherein each square micrometer (μm) is generated 2 ) of cells up to about 5 unique cDNA molecules. Also provided herein are methods wherein each cubic micrometer (μm) is generated 3 ) of cells up to about 5000 unique cDNA molecules. Also provided herein are methods wherein each cubic micrometer (μm) is generated 3 ) of cells up to about 1000 unique cDNA molecules. Also provided herein are methods wherein each cubic micrometer (μm) is generated 3) of a population of cells up to about 500 unique cDNA molecules. Methods are also provided herein, the method also comprising synthesizing a continuous DNA chain complementary to the cDNA and the cross-linked barcode. Methods are also provided herein, wherein the synthesis is performed by cross-linking. Methods are also provided herein, the method also comprising replacing the continuous DNA chain from the population of cells. Methods are also provided herein, wherein synthesis and replacement occur simultaneously. Methods are also provided herein, wherein the replaced continuous DNA chains are collected. Methods are also provided herein, wherein the collected continuous DNA chains are amplified. Methods are also provided herein, wherein the collected continuous DNA chains are sequenced. Methods are also provided herein, the method also comprises identifying a target associated with the continuous DNA chain. Methods are also provided herein, the method also comprises mapping the target to different positions within the population of fixed cells. Methods are also provided herein, wherein the population of cells is contacted with a permeabilizing agent. Methods are also provided herein, wherein the population of cells is contacted with one or more agents that generate a population of fixed cells. Methods are also provided herein, wherein the agent is formalin, formaldehyde, paraformaldehyde, methanol, acetic acid or any combination thereof. Also provided herein is a method, wherein the fixed cells are formalin-fixed paraffin-embedded (FFPE) cells, paraformaldehyde-fixed (PFA) cells, or paraformaldehyde and Triton X-100 (PFA-Tx) fixed cells. Also provided herein is a method, wherein the population of cells includes one or more of the following: cultured cells; tissue obtained from a subject; or engineered tissue. Also provided herein is a method, wherein the population of cells is retinal cells. Also provided herein is a method, wherein deposition or washing is performed manually, automatically, robotically, or any combination thereof. Also provided herein is a method, wherein deposition or washing is performed with an improved inkjet printer. Also provided herein is a method, wherein deposition or washing is performed using microfluidics, digital microfluidics, acoustic liquid processors, inkjet printers, automatic liquid processors, or any combination thereof. Also provided herein is a method, wherein deposition or washing is performed with a pipette. Also provided herein are methods, wherein the repetitions are performed 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. Also provided herein are methods, wherein at least 2, 3, 4, 5, 6, 7, 8 or 9 different barcode chains are deposited. Also provided herein are methods, the methods further comprising imaging the permeabilized cells after the washing step. Also provided herein are methods, wherein the washing comprises depositing a solution comprising a chemical denaturant and a surfactant. Also provided herein are methods, wherein the chemical denaturant comprises formamide. Also provided herein are methods, wherein formamide is present in an amount of about 40% (v / v) to about 60% (v / v). Also provided herein are methods, wherein formamide is present in an amount of about 50% to about 60%.Also provided herein is a method, wherein the surfactant comprises polysorbate 20, polysorbate 80 or Triton X-100. Also provided herein is a method, wherein the surfactant is present in an amount between about 0.05% and about 5% (v / v). Also provided herein is a method, wherein the DNA barcode further comprises a detectable label. Also provided herein is a method, wherein hybridization is at the 5' end of the transcribed cDNA. Also provided herein is a method, wherein hybridization is at the 3' end of the transcribed cDNA. Also provided herein is a method, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK). Also provided herein is a method, wherein selective radiation is performed with a photomask. Also provided herein is a method, wherein the photomask is different in different selective radiation steps. Also provided herein is a method, wherein the photomask is manually generated. Also provided herein is a method, wherein the photomask is generated by a machine. Also provided herein is a method, wherein selective radiation comprises applying radiation to a preselected region of interest (ROI). Also provided herein is a method, wherein the ROI is cell-specific or subcellular-specific. Also provided herein are methods, wherein the ROI is based on morphology. Also provided herein are methods, wherein the population of cells comprises retinal cells. Also provided herein are methods, wherein the selective irradiation step comprises applying radiation at a wavelength of about 300nm to up to 450nm. Also provided herein are methods, wherein the selective irradiation step comprises applying radiation at a wavelength of about 350nm to up to 420nm. Also provided herein are methods, wherein the selective irradiation step comprises applying radiation at a wavelength of about 365nm to up to 405nm. Also provided herein are methods, wherein washing is omitted.

[0198] Provided herein is a method for generating biological cell spatial information, the method comprising: depositing a DNA barcode onto a population of cells, wherein the DNA barcode is hybridized to a target region, and wherein the DNA barcode comprises a photoreactive nucleobase capable of crosslinking with another nucleobase; selectively irradiating a population of cells at an area of ​​interest to form a covalently linked DNA barcode-target complex, wherein the irradiation is selectively varied by intensity or duration; washing a population of cells to remove DNA barcodes that are not complexed with the target region; and repeating the deposition, selective irradiation, and washing steps, wherein the DNA barcode in the first deposition step comprises a sequence different from that in the second deposition step. Also provided herein is a method, the method further comprising determining biological cell spatial information based at least in part on the target region and the DNA barcode. Also provided herein is a method, the method further comprising synthesizing a continuous DNA chain complementary to the covalently linked barcode. Also provided herein is a method, the method further comprising collecting a continuous DNA chain or an amplified product thereof. Also provided herein is a method, the method further comprising sequencing a continuous DNA chain or an amplified product thereof. Also provided herein is a method, wherein the step of washing a population of cells is omitted.

[0199] Further to the methods described herein are methods wherein the colony of cells is present on a synthetic substrate. Methods are also provided herein wherein the synthetic substrate comprises a hydrogel. Methods are also provided herein wherein the synthetic substrate comprises an extracellular matrix protein. Methods are also provided herein wherein the synthetic substrate comprises collagen or fibronectin.

[0200] Provided herein is a method for bioinformatics generation, the method comprising: depositing a primer on a population of nucleic acids, the primer hybridizing to a target sequence of the nucleic acid to form a nucleic acid primer-complex; depositing a DNA barcode on the nucleic acid-primer complex, wherein the DNA barcode hybridizes with the primer to form a nucleic acid concatemer, and wherein the DNA barcode comprises a photoreactive nucleobase capable of crosslinking with another nucleobase; irradiating the nucleic acid concatemer to form a covalently linked concatemer; and removing the DNA barcode that is not in the covalently linked concatemer. Also provided herein is a method, wherein the steps of depositing the DNA barcode, irradiating, and removing are repeated, wherein the DNA barcode in the first deposition step comprises a sequence different from the DNA barcode in the second deposition step. Also provided herein is a method, wherein the primer is attached to an affinity molecule, a scaffold, a substrate, a surface, a bead, or a column.

[0201] Further of the methods described herein are methods wherein the selective irradiation is varied in intensity or duration to provide for the generation of a preselected fraction of DNA barcode-amplification product complexes or cross-linked barcodes.

[0202]

[0013] Further aspects of the methods described herein include comparing the information collected from the disease state sample to a non-disease state sample.

[0203] Further aspects of the methods described herein include contacting the sample with an agent for intervention, wherein the agent for intervention includes a compound, a nucleic acid, or a cell; and comparing information collected from the contacted sample area with the non-contacted sample area.

[0204] Provided herein is a method for generating biological cell information, the method comprising: depositing a DNA barcode on a population of cells, wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase; forming concatemers via a light-based step, comprising repeating the following steps: selectively irradiating the population of cells at a first wavelength to form a covalently linked complex of the DNA barcode hybridized to a substrate nucleic acid sequence; and selectively irradiating the population of cells at a second wavelength to delink the DNA barcode hybridized to the substrate nucleic acid sequence; and collecting the concatemers, wherein the collecting step is before or after the selective irradiation of the population of cells at the second wavelength, and wherein the concatemers comprise information about the population of cells.

[0205] Provided herein is a method for 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; depositing a DNA barcode onto the sample, wherein the DNA barcode comprises a region complementary to a region of the amplification product, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase; selectively irradiating the sample to form a covalently linked DNA barcode-amplification product complex, wherein for the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different locations; washing the sample to remove the DNA barcode that is not complexed with the amplification product; and determining the spatial organization information based at least in part on the generated amplification product and the DNA barcode. Also provided herein is a method, wherein the temperature at which the sample is heated is 50°C to 120°C. Also provided herein is a method, the method further comprising determining biological cell spatial information based at least in part on the generated amplification product and the DNA barcode. Also provided herein is a method, the method further comprising synthesizing a continuous DNA chain, wherein the synthesis comprises contacting the DNA barcode-amplification product complex with a polymerase. Also provided herein is a method, wherein synthesis includes cross-linking. Also provided herein is a method, the method also includes replacing a continuous DNA chain from a population of cells. Also provided herein is a method, wherein synthesis and replacement occur simultaneously. Also provided herein is a method, wherein synthesis occurs before replacement. Also provided herein is a method, wherein synthesis occurs after replacement. Also provided herein is a method, wherein the replaced continuous DNA chains are collected. Also provided herein is a method, wherein the collected continuous DNA chains are amplified. Also provided herein is a method, wherein the collected continuous DNA chains are sequenced. Also provided herein is a method, the method also includes identifying a target associated with the continuous DNA chain. Also provided herein is a method, the method also includes mapping the target to a different position.

[0206] Provided herein is a method for generating spatial information of biological cells, the method comprising: depositing a DNA barcode onto a population of cells, wherein the DNA barcode comprises a photoreactive nucleobase capable of crosslinking with another nucleobase, and wherein the DNA barcode is hybridized with a target region; selectively irradiating a population of cells at at least one region of interest (ROI) to form a covalently linked DNA barcode-target complex, wherein the radiation is 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 deposition, selective irradiation and washing steps, wherein the DNA barcode in the first deposition step comprises a sequence different from that in the second deposition step, and wherein the second selective irradiation step is applied with the same or different duration or intensity as the first selective irradiation step. Also provided herein is a method, wherein the first selective irradiation step, the second selective irradiation step, or both are applied as an intensity gradient across at least one ROI. Also provided herein is a method, wherein the second intensity gradient is applied parallel, orthogonal, perpendicular, or at another angle relative to the first intensity gradient.

[0207] Provided herein are nucleic acid libraries generated according to the methods described herein.

[0208] Example

[0209] The following examples further illustrate the described embodiments but are not intended to limit the scope of the disclosure.

[0210] Example 1: Fixation of samples

[0211] Samples were fixed according to the following method. MRC-5 (human, ATCC CCL-171) and HEK293T cells (human, ATCC CRL-1573) were grown in Dulbecco's Modified Eagle Medium (Gibco #10564) supplemented with 10% (vol / vol) serum (Gibco #10437 for MRC-5 and Peak Serum PS-FB2 for HEK293T), 50 U / mL penicillin and 50 μg / mL streptomycin (Gibco #15070). EY.T 4 embryonic fibroblasts (mouse) 49 were grown in Dulbecco's Modified Eagle Medium supplemented with 15% (vol / vol) serum, 50 U / mL penicillin and 50 μg / mL streptomycin. Cells were cultured at 37°C in the presence of 5% CO2.

[0212] The cells were washed with phosphate buffered saline (PBS), 0.1% 80. In PBS solution with 0.1% polysorbate 20 ( 20) or Hank's balanced salt solution (HBSS). The samples were fixed in 1%-10% formaldehyde and then washed with PBS or HBSS. Optionally, 0%-2.5% Triton X-100 + 0.1% 20 to permeabilize the cells. Fixed samples can be maintained at a temperature of 4°C to 45°C. Prior to barcode hybridization, samples are fixed at room temperature (20°C to 25°C) for approximately 5 to 20 minutes. Alternatively, samples are fixed at a lower temperature of 4°C and stored for up to two weeks prior to barcoding. After fixation, samples are washed with PBS or 0.1% 20 washes. Bovine serum albumin (BSA) is used as a blocking agent to reduce nonspecific detection of target nucleic acids and proteins. Other blocking agents or surfactants can be used. When blocking, cells are incubated in BSA or surfactant at room temperature.

[0213] Example 2: Barcode hybridization and imaging.

[0214] For fixed tissue samples, such as those generated in Example 1, barcodes are hybridized to target nucleic acids using the reagents listed in Table 1. Reagent conditions include exemplary concentration ranges that allow DNA barcodes to hybridize to targets.

[0215] Table 1. Barcode hybridization reagents

[0216]

[0217] The barcoding solution consisting of 250 nM barcode 1 strand was mixed in PBS and 20 (PBST) mixed with 2mg / ml sheared salmon sperm DNA (Invitrogen AM9680), 10% dextran sulfate (wt / vol, Sigma Aldrich S4030) and 500mM NaCl, applied to fixed tissue samples for 20min to 1 hour. The samples were washed 3 times with PBST containing 1M NaCl, each for about 1min. Prior to irradiation, the fixed samples can be maintained in the barcoding solution at a temperature of about 20°C to up to 60°C.

[0218] Using microscopy, the desired area on the tissue sample is visually identified in the bright field image. A hand-drawn mask is set to the photostimulation area. In each barcode wheel, a high-power liquid light guide-coupled LED source is applied at 365 nm to the selected area of ​​interest on the sample. The microscope focus is set to the desired layer on the sample and the photomask area is manually selected. The sample is photostimulated, imaged, and then treated with approximately 0.1×-1× PBS; 25% to 80% formamide; and 0% to up to 1% 20 Washing.

[0219] After finding the same cell region in the fixed sample, additional barcode strands can be added to the barcoding solution. After barcoding, the sample is optionally treated with a cross-linking solution consisting of RNase H and reverse transcription buffer. cDNA can be extracted for amplification and / or next generation sequencing by washing the cells with PBST and collecting the supernatant in a tube. cDNA can be pooled together or separately pooled for sequencing analysis in a continuous barcoding step.

[0220] Example 3: Binary encoding.

[0221] The combination of four barcodes can be used to define 16 different ROIs using on / off binary coding. The sample is spatially defined with 16 regions of interest (ROIs). The 4 unique barcodes are applied in 4 rounds of crosslinking iterations. In each of the four rounds of crosslinking, light is applied to half of the ROIs. The fraction of any illumination in each round contains about 1 / 4 (25%) saturation or less, providing 2 of the 4 barcode strands in each region that are crosslinked or not crosslinked. 4 = 16 possible combinations. Figure 8 , where filled squares represent some fraction of reads in the region containing a barcode corresponding to that column, and empty white squares represent no illumination during that barcoding round. As shown on the right side of the figure, four barcodes are assigned binary codes, allowing 16 possible combinations.

[0222] Example 4: quaternary encoding.

[0223] The combination of the two barcodes can define 16 different 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 ROIs at one of three different power levels so that the fraction of maximum illumination in each round includes crosslink saturation of approximately 1 / 2 (50%) or less of the sites. This provides a 4-fold increase in the crosslink fraction of the two barcode strands. 2 = 16 different combinations. Fig. 9 The combinations are illustrated in , where the hierarchy of filled squares represents the different fractions of reads in the region containing the barcode corresponding to that column, and the empty white squares represent the absence of illumination during that barcoding round. On the right, the quaternary encodings of the two barcodes can be assigned to each of these cases.

[0224] Example 5: Fractional encoding.

[0225] Fractional barcoding provides a very large number of possible different identification codes. For example, illumination at 11 different power levels using only two barcodes can define 121 different ROIs. The sample is spatially defined with 121 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 at 10 different levels so that there is a unique ratio of barcode scores. Light is applied at levels that provide 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% barcode scores. These 11 ratios of the two barcode chains are Fig.10 , where the hierarchy of filled squares represents the different fractions of reads in the region containing the barcode corresponding to that column, and the empty white squares represent the absence of illumination during that barcoding round. This allows a cross-linking fraction of 121 (=11 * 11) different combinations. On the right, the ratio-based encoding of the two barcodes can be assigned to each of these cases. Fig.10 Only a portion of the 121 possible combinations of barcode scores are shown.

[0226] Example 6: Spatial barcoding methods across multiple tissues and tissue fixation protocols

[0227] The robustness of the method to different tissue fixation protocols is an important feature of the spatial sequencing method described here. We found that tissues prepared under different fixation conditions successfully generated consistent sequencing results.

[0228] fixed

[0229] Brain, spleen and liver samples were collected from mice using three different fixation conditions.Fixation condition 1 (PFA) includes transferring freshly dissected organs to 4% PFA in 1 × PBS for 30 min at room temperature, then transferring to PBS and freezing at -80 ° C.Fixation condition 2 (PFA-Tx) includes transferring freshly dissected organs to 4% PFA with 0.25% Triton X-100 in 1 × PBS for 30 min at RT, then washing for three 5 min in PBS, transferring to 7% sucrose in 1 × PBS for 10 min, and then finally transferring to 30% sucrose in 1:1 mixture of 30% sucrose in optimal cutting temperature (OCT) compound+1 × PBS.Then samples were frozen at -80 ° C.Fixation condition 3 (formalin or FFPE) includes transferring freshly dissected organs to 10% neutral buffered formalin, fixing overnight at room temperature, then transferring to PBS and freezing at -80 ° C.

[0230] Frozen tissue blocks were sectioned at 15 μm onto microscope coverslips and separated using well chambers for individually contained liquid exchange.

[0231] Washing and barcoding

[0232] The samples were washed three times with 1×PBS, 0.1% Tween-20 and 60% formamide, followed by two washes with 1×PBS, 0.1% Tween-20, 1M NaCl, and then two washes with 1×PBS, 0.1% Tween-20. A-tailing of the cDNA was then performed in situ using 1×ThermoPol buffer, 1 mM dATP and 1000 U / mL terminal transferase. The sections were incubated with the solution at 37°C for 45 minutes and then washed three times with 1×PBS, 0.1% Tween-20.

[0233] Photo-directed barcoding of cDNA was performed using a Nikon Ti2-E microscope with a Mightex Polygon 1000DMD connected to a 380 nm LED. The samples were incubated with a barcoding solution (including 250 nM of a 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 minutes. The barcoding solution was then removed and the samples were washed with 1× PBS, 0.1% Tween-20, 1 M NaCl.

[0234] Light masking

[0235] 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 with a single large rectangular mask, while other tissues were illuminated with three separate square regions. Illumination was performed for 10 seconds at 20% 380nm LED power. The samples were then washed eight times in 60% formamide, 0.1% Tween-20 in 1×PBS, followed by two washes in 1×PBS, 0.1% Tween-20 and NaCl. The barcode signal was then imaged after washing, with the fluorescently labeled barcode strands visualized in the region of interest (ROI), indicating successful light-directed barcodes. Fig.11Representative images of brain, spleen and liver sections after photo-directed transcriptome barcoding are shown. Samples were prepared according to fixation conditions 1 (PFA), 2 (PFA-Tx) and 3 (formalin) as described herein. In the brain, barcode deposition was primarily limited to cell bodies, and almost no signal was observed in axon-dense areas, which is consistent with the localization of most in situ transcribed cDNAs to cell bodies. Successful transcriptome barcoding was observed in all tissue types and all fixation conditions.

[0236] Extraction and sequencing

[0237] The barcoded cDNA was then extracted from the samples by treatment with 250 U / mL of RNase H in 1× ThermoPol reaction buffer and incubated for 45 minutes at 37° C. The barcoded cDNA was then transferred to PCR tubes for copying and amplification for next generation sequencing.

[0238] The extracted cDNA was copied using a poly-T primer that hybridized to the A-tail of the cDNA sequence. Copies of the ligated cDNA-barcode sequence were generated by mixing the extracted cDNA with the primer sequence and adding strand displacement Bst polymerase to 800 U / mL, 1× ThermoPol buffer, dNTPs to 100 uM, and poly-T primer to 20 nM for 45 min at 37°C. The samples were heat inactivated at 80°C for 20 minutes.

[0239] The concentration of the cDNA library was quantified and the library was amplified in PCR in a qPCR machine with a 1:1 ratio PCR mix (SybrGreen I, Kapa HiFi buffer, 600 nM each of forward and reverse primers, 600 μM of dNTPs and Kapa HiFi hot start polymerase): 8°C for 3 min, followed by 30 cycles of 98°C for 20 seconds and 72°C for 2 min. The amplified library was then tagged 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.

[0240] Data analysis

[0241] In order to analyze the next generation sequencing data, the sequence was parsed using a unique molecular identifier (UMI) tool. The cDNA sequence was mapped to the mouse genome (vM27) using the STAR aligner. All reads that were multiplexed to the genome were discarded, and the single mapped reads were then mapped to the annotated transcripts using the FeatureCounts tool. Based on the UMI attached to the cDNA via the barcode oligonucleotide during the light-directed barcoding step, the unique cDNA sequences in the library were identified by deduplication. For all cases, only a single UMI / mapped cDNA pair was retained, generating a unique cDNA molecule count matrix across all genes. This is also referred to as the RNA count vector for each sample, and the RNA count matrix refers to a matrix containing multiple RNA count vectors.

[0242] Analyze the differential expression of genes from the next generation sequencing data of brain and liver samples fixed in formalin and PFA to evaluate the consistency of transcriptome sequencing data across fixed conditions. In short, DESeq2 is used for differential expression analysis across samples, and genes with significant differential expression are called as genes with adjusted p value <0.05 (after Benjamini-Hochberg correction for multiple hypothesis testing). Count matrices are generated with the number of unique barcoded cDNAs detected in each spatial targeting region of each gene. Table 2 shows the generated matrix.

[0243] Comparing the expression levels detected in PFA-fixed brains and formalin-fixed brains, as shown in Table 2, there is only a single significantly differentially expressed gene. Similarly, across liver samples fixed with formalin and PFA, there are no significantly differentially expressed genes. In contrast, for all paired fixation conditions, when compared across organs, these same samples show hundreds to >1000 significantly differentially expressed genes. These data support that the spatial transcriptomics approach can produce consistent results across the fixation conditions of the same organ, as well as reproducible organ-specific results across organs.

[0244] Table 2: Differential gene expression across tissues and fixation conditions

[0245] Brain, formalin Brain, PFA Liver, formalin Liver, PFA Brain, formalin 0 1 1008 673 Brain, PFA 1 0 511 816 Liver, formalin 1008 511 0 0 Liver, PFA 673 816 0 0

[0246] Fig.11 Successful barcode deposition across tissue samples from brain, spleen, and liver tissues prepared with three different fixation conditions is shown. Gene expression data show that sequencing libraries from the same tissue are very similar to each other, independent of the different fixation conditions. Differential expression analysis shows hundreds to thousands of statistically differentially expressed genes when compared across organ types. When comparing different fixation conditions for the same organ, at most a single differentially expressed gene was found.

[0247] Example 7: Spatial barcoding and transcriptome sequencing can be applied to FFPE and PFA-TX fixed tissues

[0248] Historically, it is extremely difficult to carry out RNA sequencing from the tissue of formalin-fixed paraffin embedding (FFPE). However, many samples have been preserved using FFPE routine, and it is possible to provide major advantages as described herein from FFPE sample generation data. The light-directed transcriptome barcoding in FFPE liver tissue was shown with image-based readout. It is also shown that when comparing FFPE liver sequencing results (in the form of gene count matrix), the gene expression detected in the liver fixed by FFPE and PFA-Tx is consistent. FFPE liver gene count is also well correlated with PFA-Tx gene count, and is not correlated with brain count.

[0249] For PFA-Tx samples, mouse liver was fixed with 4% PFA, 0.25% Triton-X-100 in 1×PBS solution by intracardiac perfusion, followed by dissection and incubation in fixative at 4°C overnight. The tissue was then washed with 1×PBS and immersed in 7.5% sucrose in 1×PBS. The tissue was then immersed in a 1:1 solution of 30% sucrose and OCT cryoprotectant solution in 1×PBS. The tissue was then stored at -80°C until use. For FFPE liver tissue, the tissue was immersed in a fresh 10% neutral buffered formalin solution for a long enough time (between 4 and 72 hours) to ensure adequate fixation. The tissue was then embedded in paraffin and stored at 4°C until use. For PFA brain tissue samples, brains were dissected from postmortem animals and dripped into 4% PFA, 0.25% Triton-X-100 in 1×PBS solution for 30 minutes at room temperature, and then immersed in 7.5% sucrose in 1×PBS. The tissue was then immersed in a 1:1 solution of 30% sucrose and OCT cryoprotectant solution in 1× PBS. The tissue was then stored at -80°C until spatial sequencing.

[0250] Cut into 15 micron slices and place the slices on microscope slides and bake at 60°C for 1 hour. For FFPE samples, the slices are dewaxed and antigen retrieval is performed at 90°C for 15 minutes in decloaking buffer, followed by 30 seconds at 70°C, and then cooled to room temperature. The slices are then washed twice with water, and the slides are air-dried, then rehydrated with 1×PBS, 0.1% Tween, and then the reverse transcription step below is performed. For non-FFPE brain and liver tissues, the slices are washed three times with 1×PBS, 0.1% Tween-20 and incubated at 70°C for 30 minutes with antigen retrieval buffer (Tris-EDTA, pH 8). The samples are then returned to 1×PBS, 0.1% Tween, and the reverse transcription step below is performed.

[0251] All tissue sections were subjected to the same processing steps for cDNA library generation, barcoding, and sequence library preparation. Tissue sections were washed three times with 1×PBS, 0.1% Tween-20, then incubated with reverse transcription mixture (300 μM dNTPs, 0.5% Triton-X-100, 6 mM RNase OUT, 1 μM RT primer, and 8 U / μL Maxima RT enzyme in RT buffer) and incubated at 22°C for 30 min, followed by 12 cycles of 8°C for 30 sec, 15°C for 30 sec, 25°C for 30 sec, 30°C for 1 min, 37°C for 1 min, and 42°C for 2 min, followed by 42°C for 30 min.

[0252] Sample washing and barcoding were performed as described in Example 6.

[0253] Regions of interest for illumination were hand-drawn in Nikon Elements software, and light was selectively directed to these regions using DMD. Illumination was performed for 10 seconds at 20% LED power. Three slices of each HIER condition (both 95°C and 70°C) were repeated as "no UV controls" and were not illuminated with 380nm LEDs, but were otherwise treated identically. The samples were then washed 8 times with 60% formamide and 0.1% Tween-20 in 1×PBS, followed by 2 washes in 1×PBS, Tween-20, and NaCl. The barcode signal was then imaged after washing, with the fluorescently labeled barcode chain visualized in the ROI, indicating successful light-directed barcoding. Fig. 12A Representative images of photo-directed spatial transcriptome barcoding in FFPE liver sections are shown, with circular and square regions of interest used for illumination as described. This figure depicts successful photo-directed transcriptome barcoding in FFPE samples.

[0254] Sample extraction and sequencing were performed as described in Example 6.

[0255] Sequencing data analysis was performed as described in Example 6. The top 50 differentially expressed genes from the whole transcriptome sequencing data were compiled according to the calculated z-scores in Fig. 12B In the heat map of the whole transcriptome sequencing data from spatially barcoded brain samples fixed with PFA-Tx (n=6 replicates) and liver samples (n=3, two of which were fixed with PFA-Tx perfusion and drop fixation, and one fixed with standard FFPE fixation procedures). 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. Fig. 12B Region 1 shows the z-scores of the expression levels of typical brain expressed genes as detected in fixed brain samples, indicating high expression levels. Fig. 12B Panel 2 shows the z-scores of the expression levels of typical brain expressed genes as detected in fixed liver samples, indicating low expression levels. Fig. 12B Panel 3 shows the z-scores of the expression levels of typical liver-expressed genes as detected in fixed brain samples, indicating low expression levels. Fig. 12B Region 4 shows the z-scores of the expression levels of typical liver-expressed genes as detected in fixed liver samples, indicating high expression levels.

[0256] The differential expression results between liver and brain samples were compiled in Fig. 12C The volcano plot in Figure 2 shows the liver data from two PFA-Tx-fixed sections and one FFPE-fixed section. The brain data from six PFA-TX-fixed sections. Fig. 12B Differential Expression was well-powered to recover thousands of differentially expressed genes between organs regardless of fixation conditions in liver samples, with p-values ​​<< 0.05 after correction for multiple hypothesis testing (y-axis) and log2FoldChanges of up to 10 (x-axis). As expected, selected genes including gene sets with typical organ-specific expression are shown.

[0257] 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 The rows show individual genes, and the columns represent individual samples (with Fig. 12B and Fig. 12C Colors reflect the z-score of normalized transcript counts. Fig.12DRegion 1 shows the z-score of the expression level of typical brain expression genes as detected in fixed brain samples, indicating generally higher expression levels relative to the average across all samples. Relative to the average across all samples, a higher z-score (darker color) shows the enrichment of the gene in the sample. Specifically, the z-score is: ((gene expression value in the sample of interest)-(average expression across all samples)) / standard deviation across all samples. Fig.12D Panel 2 shows the z-scores of expression levels of typical brain-expressed genes as detected in fixed liver samples, indicating generally low expression levels relative to the mean. Fig.12D Panel 3 shows z-scores for expression levels of typical liver-expressed genes as detected in fixed brain samples, indicating generally low expression levels relative to the mean. Fig.12D Region 4 shows z-scores for expression levels of typical liver-expressed genes as detected in fixed liver samples, indicating higher expression levels relative to the mean. The high number of genes that are significantly different across brain and liver samples and the consistency of gene expression relative to brain samples (represented as color, z-score) further supports the consistency of the FFPE-fixed liver sample transcriptome with the PFA-Tx-fixed liver transcriptome.

[0258] To directly evaluate the consistency of spatially barcoded and sequenced transcriptomes of FFPE versus PFA-Tx fixed tissues, pairwise Pearson correlations were calculated to compare the unique mRNA gene expression vectors, also called mRNA count vectors, for each pair of samples. The resulting Pearson correlation numbers were compiled into a heat map as shown in Fig.12E . This metric directly evaluates the consistency of gene expression across all genes in the genome. The colors represent the Pearson correlation coefficient between paired samples. For FFPE liver and PFA-Tx liver, the Pearson correlation coefficient was >0.7 in all cases, indicating that the correlation between liver samples was strong despite the different fixations used. The Pearson correlation coefficients for all inter-organ comparisons were <0.3.

[0259] Example 8: Heat-induced epitope retrieval (HIER) to increase barcoded cDNA recovery.

[0260] Protocol optimization of the tissue pretreatment step was performed to increase the number of barcoded cDNAs 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 the fluorescent barcode images and Cq values ​​from qPCR shows where the library is amplified. When the sample was treated with a 95°C heating step, the intensity of the barcoding increased in the image data and decreased the Cq values. Both measurements indicate that more cDNA is barcoded when this tissue pretreatment step is added.

[0261] Mouse livers were fixed with 4% PFA, 0.25% Triton-X-100 in 1×PBS solution by intracardiac perfusion, followed by dissection and incubation in fixative at 4°C overnight. The tissue was then washed with 1×PBS and immersed in 7.5% sucrose in 1×PBS. The tissue was then immersed in a 1:1 solution of 30% sucrose and OCT cryoprotectant solution in 1×PBS. The tissue was then stored at -80°C until use. The tissue was then immersed in a 1:1 solution of 30% sucrose and OCT cryoprotectant solution in 1×PBS. The tissue was then stored at -80°C until spatial sequencing was performed.

[0262] The frozen tissue blocks were then sliced ​​onto microscope coverslips at 15 μm and separated using well chambers for individually contained liquid exchange. The slides were baked at 60°C for 1 hour. The 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 or 95°C for 30 minutes 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 1× PBS with 0.1% Tween-20, then incubated with reverse transcription mix (300 μM dNTPs, 0.5% Triton-X-100, 6 mM RNase OUT, 1 μM RT primer, and 8 U / μL Maxima RT enzyme in RT buffer) and incubated 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.

[0263] The samples were washed three times with 1×PBS, 0.1% Tween-20 and 60% formamide, followed by two washes with 1×PBS, 0.1% Tween-20 and 1M NaCl, followed by two washes with 1×PBS, 0.1% Tween-20. A-tailing of the cDNA was then performed in situ using 1×A-ThermoPol buffer, 1 mM dATP and 1000 U / mL terminal transferase. The sections were incubated with the solution at 37°C for 45 minutes and then washed three times with 1×PBS, 0.1% Tween-20.

[0264] Then the photo-directed barcoding of cDNA was performed using a Nikon Ti2-E microscope with a Mightex Polygon1000DMD connected to a 380nm LED. The samples were incubated with a barcoding solution (including 250nM FITC-labeled, UMI-containing, CNVK-containing barcode strands, 500mM NaCl, and 10% dextran sulfate in 1×PBS, 0.1% Tween-20) at room temperature for 30 minutes. The barcoding solution was then removed and the samples were washed with 1×PBS, 0.1% Tween-20, and 1M NaCl.

[0265] Regions of interest for illumination were hand-drawn within Nikon Elements software, and light was selectively directed to these areas using the DMD. Illumination was performed at 20% 385nm LED power for 10 seconds. The samples were then washed eight times with 60% formamide, 0.1% Tween-20 in 1× PBS, followed by two washes in 1× PBS, 0.1% Tween-20 with NaCl. The barcode signal was then imaged after washing, where fluorescently labeled barcode chains were visualized in the ROI, indicating successful photo-directed barcoding. Images of barcoded liver sections after 70°C and 95°C HIER treatment are depicted in Figure 2, respectively. Fig.13A and Fig. 13B Medium. Image shows stronger barcode signal at higher temperature.

[0266] After transcriptome barcoding with FITC-labeled barcode strands, the amount of barcode deposition was quantified based on fluorescence imaging of tissue sections. The average fluorescence intensity within the rectangular barcoded area obtained across n=3 replicates for each HIER condition was calculated. Fig. 13C Bar graphs of calculated averages are shown. Error bars represent standard deviations. These data show that barcode deposition efficiency increases after HIER at 95°C relative to 70°C.

[0267] Extraction and sequencing of samples were performed as described in Example 6. After thermal cycling, the efficiency of photo-directed barcoding was compared across + / -UV conditions by comparing Cq qPCR values ​​(-UV conditions are expected to have lower relative to +UV signals).

[0268] Quantification of the number of barcoded molecules in barcoded cDNA libraries prepared from different HIER and + / - UV conditions was evaluated using qPCR designed to use primers that amplify only barcoded cDNA. Fig.13D The bar graph in shows the average Cq values ​​across n=3 replicates, with standard deviation error bars. The +UV samples received spatially patterned UV illumination with a 380nm LED for barcoding, while the -UV samples did not. The 95°C HIER condition +UV amplified approximately 2.5 cycles earlier than the 70°C HIER +UV condition, consistent with the higher number of barcoded cDNA molecules under the 95°C HIER condition. Combined with the increased image intensity of the barcoded region after photo-directed barcoding, these data support that 95°C HIER is able to achieve more photo-directed barcoding.

[0269] Example 9: Barcoding using spatial light orientation using different optical systems

[0270] Across a wide range of optical parameters, including wavelength, light transmission mode (LED vs. laser), illumination time, and illumination power, a method for light-directed spatial barcoding was demonstrated. This is shown by representative image data, where fluorescent DNA barcodes are deposited on tissue sections undergoing in situ reverse transcription to introduce barcode docking sites on the 5' end of the cDNA. A Cy5-labeled barcode strand containing CNVK is then hybridized to the sample, and a rectangular area within the same tissue section is illuminated with different optical parameters. Light-directed barcoding is successful for a wide range of parameters, with some conditions producing more effective barcoding than other conditions. These data emphasize the robustness of the method for light-directed spatial barcoding.

[0271] Mouse livers were fixed with 4% PFA, 0.25% Triton-X-100 in 1×PBS solution by intracardiac perfusion, followed by dissection and incubation in fixative at 4°C overnight. The tissues were then washed with 1×PBS and immersed in 7.5% sucrose in 1×PBS. The tissues were then immersed in a 1:1 solution of 30% sucrose and OCT cryoprotectant solution in 1×PBS. The tissues were then stored at -80°C until spatial sequencing.

[0272] 15 μm sections were cut, placed on microscope slides, and baked for 1 hour at 60° C. The 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.

[0273] Tissue sections were subjected to the following processing steps for cDNA library generation. Tissue sections were washed three times with 1×PBS and 0.1% Tween-20, then incubated with reverse transcription mixture (300 μM dNTPs, 0.5% Triton-X-100, 6 mM RNase OUT, 1 μM RT primer, and 8 U / μL Maxima RT enzyme in RT buffer) and incubated at 22° C. for 30 min, followed by 12 cycles of 8° C. for 30 sec, 15° C. for 30 sec, 25° C. for 30 sec, 30° C. for 1 min, 37° C. for 1 min, and 42° C. for 2 min, followed by 42° C. for 30 min.

[0274] The samples were washed three times with 1×PBS, 0.1% Tween-20 with 60% formamide, followed by two washes with 1×PBS, 0.1% Tween-20, 1M NaCl, and then two washes with 1×PBS, 0.1% Tween-20. A-tailing of the cDNA was then performed in situ using 1×A-ThermoPol buffer, 1 mM dATP, and 1000 U / mL terminal transferase. The sections were incubated with the solution at 37°C for 45 minutes, and then washed three times with 1×PBS, 0.1% Tween-20.

[0275] Photo-directed barcoding of cDNA was then performed using a Nikon Ti2-E microscope with two different optical systems. Optical system 1 was a Mightex Polygon 1000DMD connected to a 380nm LED. Optical system 2 was a 405nm laser connected to an optical microscanner. All illumination was performed through a 20× air objective. For barcoding, the samples were incubated with a barcoding solution (consisting of 250nM of a fluorophore (Cy5)-labeled, UMI-containing, CNVK-containing barcode strand, 500mM NaCl, and 10% dextran sulfate, 0.1% Tween-20 in 1× PBS) at room temperature for 30 minutes. The barcoding solution was then removed and the samples were washed with 1× PBS, 0.1% Tween-20, 1M NaCl before barcoding.

[0276] Rectangular regions of interest were drawn within the Nikon Elements software to guide the illumination. For optical system 1, illumination power was sampled between 1%-10% and time was sampled between 1-20 seconds. Conditions using 5% power and using 1s, 5s, 10s, and 20s for a single illumination round instead of two were also tested. For optical system 2, illumination power was adjusted between 0.1%, 0.5%, 1%, 2%, 5%, 10% with dwell time variables of 10μs, 100μs, and 1000μs. Overall, this provides a broad sampling of the parameter space for illumination conditions.

[0277] After barcoding, samples were then washed eight times with 60% formamide, 0.1% Tween-20 in 1× PBS, followed by two washes in 1× PBS, 0.1% Tween-20 with NaCl. The barcode signal was then imaged after washing ( Fig.14A ). Image capture of autofluorescence in the same field of view ( Fig. 14B ) and barcode imaging ( Fig. 14C ) to show the location and relevance of barcoding within the sample. Imaging showed fluorescently labeled barcode strands visible in the ROI, indicating successful photo-directed barcoding under both conditions.

[0278] Fig.14A Spatial barcoding with light orientation with two optical modes for UV light transmission is shown: an optical microscanner (Galvo) with a 405nm laser light source (upper rectangle) and a DMD with a 380nm LED light source (lower rectangle). The use of an optical microscanner provides higher light transmission accuracy, thereby achieving higher resolution. However, for an optical microscanner, the time is much slower because each individual pixel in the region of interest is illuminated sequentially. The 405nm laser is more common on most confocal systems, presenting an accessibility advantage. The DMD / 380nm LED setup is advantageous because it allows parallel light transmission to arbitrary spatial patterns within the field of view (FOV). This provides shorter processing times for large regions of interest.

[0279] Fig.14D Shown from Fig.14A Relative intensity of the four barcoded regions in the dashed rectangle, labeled with a 405 nm laser, with power and duration as indicated. Autofluorescence of the same region is shown in Fig.14E middle. Fig.14F A combined image of both barcoding and autofluorescence is shown.

[0280] Example 10: Fractional barcoding of cellular transcriptomes

[0281] The ability to adjust the fraction of barcoded cDNA sequences within a cell or tissue region enables encoding information. If the local fraction of barcoded transcripts can be read out, as described herein, fractional barcoding can be used to achieve regional identification. These data support that more than one spatial barcode can be deposited on the same cell / tissue region and that adjustable fractions of barcoded cDNA can be obtained in a deterministic manner.

[0282] Retinal tissues were collected from mice and transferred to 4% PFA with 0.25% Triton X-100 in 1× PBS for 30 min at room temperature, followed by three 5 min washes in PBS, transfer to 7% sucrose in 1× PBS for 10 min, and then finally transferred to a 1:1 mixture of OCT + 30% sucrose in 1× PBS. The samples were then stored frozen at -80°C.

[0283] The frozen tissue block is then sliced ​​onto a microscope cover glass with 15 μm, and separated using a well chamber for the liquid exchange contained alone. Then, tissue section experience following treatment steps for cDNA library generation. Tissue section 1 × PBS 0.1% Tween-20 is washed three times, subsequently incubated with reverse transcription mixture (300 μM dNTPs, 0.5% Triton-X-100, 6mM RNAse OUT, 1 μM RT primers and 8U / μL Maxima RT enzyme in RT buffer), and incubated at 22 ° C for 30 min, followed by 12 cycles: 8 ° C continues for 30 seconds, 15 ° C continues for 30 seconds, 25 ° C continues for 30 seconds, 30 ° C continues for 1 min, 37 ° C continues for 1 min and 42 ° C continues for 2 min, followed by 42 ° C continues for 30 min.

[0284] The samples were washed three times with 1×PBS, 0.1% Tween-20 and 60% formamide, followed by two washes with 1×PBS, 0.1% Tween-20, 1M NaCl, and then two washes with 1×PBS, 0.1% Tween-20. A-tailing of the cDNA was then performed in situ using 1×A-ThermoPol buffer, 1 mM dATP and 1000 U / mL terminal transferase. The sections were incubated with the solution at 37°C for 45 minutes and then washed three times with 1×PBS, 0.1% Tween-20.

[0285] Photo-directed barcoding of cDNA was then performed using a Nikon Ti2-E microscope with a Mightex Polygon 1000DMD connected to a 380 nm LED. The samples were incubated with the first barcode in barcoding solution 1 (composed of 250 nM FITC-labeled, UMI-containing, CNVK-containing barcode strand, 500 mM NaCl, and 10% dextran sulfate, 0.1% Tween-20 in 1× PBS) at room temperature for 30 minutes. Barcoding solution 1 was then removed and the samples were washed with 1× PBS, 0.1% Tween-20, 1 M NaCl.

[0286] for Figure 15A-Figure 15C Two-color barcoding shown in , 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. Different amounts of Barcode 1 were deposited in different square regions from left to right using the following illumination conditions (n=3 replicates per condition): 0% power / 0 sec, 1% power / 5 sec, 5% power / 5 sec, 10% power / 5 sec, 20% power / 5 sec. ROIs with Barcode 1 were imaged in Fig.15A It is shown in FIG. 1 that the power increases in the region from left to right.

[0287] Barcode 2 was then incubated with the sections in a solution consisting of 250 nM of a fluorophore (Cy3) labeled, UMI-containing, CNVK-containing barcode strand, 500 mM NaCl, and 10% dextran sulfate, 0.1% Tween-20 in 1× PBS for 30 minutes at room temperature. The barcoding solution was then removed and the samples washed with 1× PBS, 0.1% Tween-20, 1 M NaCl. Illumination was performed for 10 seconds at 20% 380 nm LED power in all previously barcoded squares.

[0288] The samples were then washed eight times with 60% formamide, 0.1% Tween-20 in 1× PBS, followed by two washes in 1× PBS, 0.1% Tween-20 and NaCl. The barcode signal was then imaged after washing, as shown in Figure 2. Fig. 15B Imaging shows fluorescently labeled barcode strands visible in the ROI indicating successful photo-directed barcoding. Fig. 15C An overlay of two imaging ROIs is shown.

[0289] By analyzing the image data, photo-directed barcoding was quantified by calculating the average intensity of fluorescence within the barcoded region of interest in the channel corresponding to the barcode. Since the DNA barcodes were conjugated to different fluorophores (FITC and Cy3), the fluorescence in each channel could be quantified to evaluate the deposition of the respective barcode. Fig.15D Depicted are bar graphs showing the ratio of the mean image intensity values ​​of the FITC / Cy3 channels for each condition after barcoding.

[0290] Example 11: Small molecule dyes direct spatial barcoding

[0291] Barcoding of specific cells, subcellular features, and tissue regions can enable highly selective transcriptome sequencing. Our light-directed barcoding enables high-resolution barcode deposition and can be guided by small molecule stains. Here, we show the ability to barcode specific cells, subcellular features, and tissue regions using a small molecule stain (deep red anthraquinone 5 (DRAQ5)) that stains cell nuclei by intercalating into double-stranded DNA with high affinity. TM ) Tissue sections were prestained to guide light masking to barcode light-directed transcriptomes to single cells. The results show that barcoding can be directed by the prestain and can be restricted to regions of interest directed by the stain.

[0292] Mouse brains were dissected into 10% neutral buffered formalin, fixed overnight at room temperature, then transferred to PBS and stored frozen at -80°C. The frozen tissue blocks were then sliced ​​onto microscope coverslips at 15 μm and separated using well chambers for individually contained liquid exchange. The slides were baked at 60°C for 1 hour. The sections were then washed three times with 1×PBS, 0.1% Tween-20 and incubated at 70°C for 30 minutes with antigen retrieval buffer (Tris-EDTA, pH 8). The samples were then returned to 1×PBS, 0.1% Tween, and the reverse transcription step below was performed.

[0293] Tissue sections were subjected to the steps for cDNA library generation. Tissue sections were washed three times with 1×PBS with 0.1% Tween-20, then incubated with reverse transcription mix (300 μM dNTPs, 0.5% Triton-X-100, 6 mM RNase OUT, 1 μM RT primer, and 8 U / μL Maxima RT enzyme in RT buffer) and incubated at 22° C. for 30 min, followed by 12 cycles of 8° C. for 30 sec, 15° C. for 30 sec, 25° C. for 30 sec, 30° C. for 1 min, 37° C. for 1 min, and 42° C. for 2 min, followed by 42° C. for 30 min.

[0294] The samples were washed three times with 1×PBS, 0.1% Tween-20 with 60% formamide, followed by two washes with 1×PBS, 0.1% Tween-20, 1M NaCl, and then two washes with 1×PBS, 0.1% Tween-20. A-tailing of the cDNA was then performed in situ using 1×ThermoPol buffer, 1 mM dATP, and 1000 U / mL terminal transferase. The sections were incubated with the solution at 37°C for 45 minutes, and then washed three times with 1×PBS, 0.1% Tween-20.

[0295] Photo-directed 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 40X water immersion objective. The samples were incubated with a barcoding solution (consisting of 250 nM of a fluorophore (FITC)-labeled, UMI-containing, CNVK-containing barcode strand, 500 mM NaCl, and 10% dextran sulfate, 0.1% Tween-20 in 1× PBS) 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. DRAQ5 nuclear stain was included in the wash solution, which remained on the sample during photo-directed barcoding to label the nuclei. Imaging of nuclear stained cells was performed at Fig.16A Shown in.

[0296] Photomasks were generated based on nuclear staining to outline three specific cells, e.g. Fig.16A The dashed line in FIG. 1 is indicated. Illumination was performed for 10 seconds at 10% 380 nm LED power. The samples were then washed 8 times with 60% formamide, 0.1% Tween-20 in 1×PBS, followed by two washes in 1×PBS, 0.1% Tween-20 and NaCl.

[0297] The barcode signal is then imaged after washing, e.g. Fig. 16B The appearance of fluorescently labeled barcode strands in the ROI indicates successful photo-directed barcoding. Fig. 16C Representative overlays of small molecule nuclear stain and fluorescent DNA barcode images.

[0298] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will now expect multiple variations, changes and substitutions without departing from the present invention. It should be understood that the various alternatives of the embodiments of the present invention described herein can be used to implement the present invention. The following claims are intended to define the scope of the present invention, and thus encompass methods and structures and their equivalents within the scope of these claims.

Claims

1. A method for generating biological cell information, the method comprising: contacting the population of cells with a nucleic acid polymerase and primers to generate an amplification product; depositing a DNA barcode onto the population of cells, wherein the DNA barcode comprises a region complementary to a region of the amplification product, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking to another nucleobase; selectively irradiating a population of the cells to form covalently linked DNA barcode-amplification product complexes; washing the population of cells to remove DNA barcodes that are not complexed with amplification products; and The steps of deposition, selective irradiation and washing are repeated, wherein: The DNA barcode in the first deposition step comprises a different sequence than the DNA barcode in the second deposition step, and For the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different positions.

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

3. The method according to claim 1 or 2, further comprising synthesizing a continuous DNA chain, wherein the synthesis comprises contacting the DNA barcode-amplification product complex with a polymerase. The method of claim 3 , wherein the synthesizing comprises cross-linking synthesis.

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

6. The method according to any one of claims 3 to 5, wherein the synthesis and replacement occur simultaneously. The method according to claim 6 , wherein the displaced continuous DNA strands are pooled. The method of claim 7 , wherein the pooled continuous DNA strands are amplified.

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

10. The method of claim 9, further comprising identifying a target associated with the continuous DNA strand. The method of claim 10 , further comprising mapping the targets to the different locations.

12. The method according to any one of claims 1 to 11, wherein the primers are random primers.

13. The method according to any one of claims 1 to 12, wherein the primers are non-random primers.

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

15. The method according to any one of claims 1 to 14, wherein the selective irradiation is performed using a photomask.

16. The method of claim 15, wherein the photomask is different in different selective irradiation steps. The method of claim 15 , wherein the photomask is generated manually. The method of claim 15 , wherein the photomask is generated by a machine.

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

20. The method of any one of claims 1 to 19, wherein the selectively irradiating step comprises applying radiation at a wavelength of about 350 nm up to 420 nm.

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

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

23. The method of claim 22, wherein the ROI is cell-specific or subcellular-specific. The method of claim 22 , wherein the ROI is based on morphology.

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

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

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

28. The method according to any one of claims 1 to 27, wherein the depositing or washing is performed with a pipette.

29. according to the method described in any one of claims 1 to 28, wherein said repetition 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. 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 deposited.

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

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

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

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

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

36. The method of any one of claims 1 to 35, wherein the population of cells comprises one or more of: Cultured cells; tissue obtained from a subject; Engineered tissue; or Organoids.

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

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

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

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

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

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

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

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

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

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

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

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

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

51. A method for generating biological cell spatial information, the method comprising: contacting the population of cells with a capture probe, wherein the capture probe comprises a DNA binding region and a target binding region; depositing a DNA barcode onto the population of cells, wherein the DNA barcode comprises a region that binds to the DNA binding region, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking 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 amplification products; and The steps of deposition, selective irradiation and washing are repeated, wherein: The DNA barcode in the first deposition step comprises a different sequence than the DNA barcode in the second deposition step, and For the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different locations.

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

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

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

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

56. The method of claim 55, wherein the synthesizing comprises cross-linking synthesis.

57. The method of claim 56, further comprising replacing the continuous DNA strands from the population of cells.

58. The method of claim 57, wherein the synthesizing and replacing occur simultaneously.

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

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

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

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

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

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

65. The method of any one of claims 51 to 64, wherein the selective irradiation is performed using a photomask.

66. The method of claim 65, wherein the photomask is different in different selective irradiation steps.

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

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

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

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

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

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

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

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

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

76. A method according to any one of claims 51 to 75, wherein the depositing is performed using a modified inkjet printer.

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

78. The method of any one of claims 51 to 77, wherein the depositing or washing is performed with a pipette.

79. The method of any one of claims 51 to 78, wherein the repetitions are performed 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. 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 deposited.

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

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

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

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

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

86. The method of any one of claims 51 to 85, wherein the population of cells comprises one or more of: Cultured cells; tissue obtained from a subject; Engineered tissue; or Organoids.

87. The method of claim 86, wherein the population of cells comprises a tissue, and the tissue is a whole tissue or a dissected tissue.

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

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

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

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

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

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

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

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

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

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

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

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. The method of any one of claims 51 to 100, further comprising more than one contacting step.

102. The method of claim 101, wherein the more than one contacting steps comprise: contacting the population of cells with a first capture probe that recognizes nucleic acid; and The population of cells is contacted with a second capture probe that recognizes the protein.

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

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

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

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

108. A method for generating biological cell spatial information, the method comprising: capturing three-dimensional images of tissue samples; contacting the tissue sample with a nucleic acid polymerase and primers to generate an amplification product; depositing a DNA barcode onto the tissue sample, wherein the DNA barcode comprises a region complementary to a region of the amplification product, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking to another nucleobase; selectively subjecting the tissue sample to three-dimensional radiation to form a DNA barcode-amplification product complex; washing the tissue sample to remove DNA barcodes that are not complexed with the amplification product; and The steps of deposition, selective irradiation and washing are repeated, wherein: The DNA barcode in the first deposition step comprises a different sequence than the DNA barcode in the second deposition step, and For the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different locations of the tissue sample.

109. The method of claim 108, further comprising determining biological cell spatial information based at least in part on the amplification products and the 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 an organoid.

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

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

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

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

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

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

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

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

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

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. The method of claim 121, wherein the synthesis is performed by cross-linking synthesis.

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

125. The method of claim 124, wherein the synthesizing and replacing occur simultaneously.

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

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

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

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

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

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

132. A method according to any one of claims 108 to 131, wherein the depositing is performed using a modified inkjet printer.

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

134. The method of any one of claims 108 to 133, wherein the depositing or washing is performed with a pipette.

135. The method of any one of claims 108 to 134, wherein the repetitions are performed 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. 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 deposited.

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

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

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

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

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

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

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

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

146. The method of any one of claims 108 to 145, wherein the selective irradiation is performed using a photomask.

147. The method of claim 146, wherein the photomask is different in different selective irradiation steps.

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

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

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

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

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

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

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

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

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

157. A method for generating biological cell spatial information, the method comprising: capturing an image of a population of cells, wherein the population of cells is immobilized on a substrate; reverse transcribing RNA from the population of cells to generate cDNA; depositing DNA barcodes onto the population of cells; hybridizing a DNA barcode to the cDNA, wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking to another nucleobase; selectively irradiating a population of said cells to form a cross-linked barcode; Washing to remove uncrosslinked DNA barcodes; and Repeating the deposition, hybridization, selective irradiation and washing steps, Wherein the DNA barcodes in the deposition step are different.

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

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

160. The method of claim 157, wherein the 2 ) of the cell population with up to about 55 unique cDNA molecules.

161. The method of claim 157, wherein each square micrometer (μm) is generated 2 ) of the cell population with up to about 10 unique cDNA molecules.

162. The method of claim 157, wherein each square micrometer (μm) is generated 2 ) of the cell population with up to about 5 unique cDNA molecules.

163. The method of claim 157, wherein each cubic micrometer (μm) is generated 3 ) of the cell population with up to approximately 5,000 unique cDNA molecules.

164. The method of claim 157, wherein each cubic micrometer (μm) is generated 3 ) of the cell population with up to about 1000 unique cDNA molecules.

165. The method of claim 157, wherein each cubic micrometer (μm) is generated 3 ) of the cell population with up to about 500 unique cDNA molecules.

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

167. The method of claim 166, wherein the synthesis is performed by cross-linking synthesis.

168. The method of claim 167, further comprising replacing the continuous DNA strands from the population of cells.

169. The method of claim 168, wherein the synthesizing and replacing occur simultaneously.

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

171. The method of claim 170, wherein the pooled continuous DNA chains are amplified.

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

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

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

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

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

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

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

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

181. The method of any one of claims 157 to 180, wherein the depositing or cleaning is performed manually, automatically, robotically, or any combination thereof.

182. A method according to any one of claims 157 to 181, wherein the depositing or cleaning is performed using a modified inkjet printer.

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

184. The method of any one of claims 157 to 183, wherein the depositing or washing is performed with a pipette.

185. The method of any one of claims 157 to 184, wherein the repetitions are performed 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. 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 deposited.

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

188. The method of any one of claims 157 to 184, wherein the washing comprises depositing a solution comprising a chemical denaturant and a surfactant.

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

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

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

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

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

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

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

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

198. A method according to any one of claims 157 to 197, wherein the selective irradiation is performed using a photomask.

199. The method of claim 198, wherein the photomask is different in different selective irradiation steps.

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

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

202. The method of any one of claims 157 to 201, wherein the selective radiation comprises applying radiation to a preselected region of interest (ROI).

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

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

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

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

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

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

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

210. A method for generating biological cell spatial information, the method comprising: depositing a DNA barcode onto the population of cells, wherein the DNA barcode is hybridized to a target region, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking to another nucleobase; selectively irradiating a population of said cells at a region of interest to form covalently linked DNA barcode-target complexes, wherein said irradiation is selectively varied by intensity or duration; washing the population of cells to remove DNA barcodes that are not complexed with the target region; and The deposition, selective irradiation, and washing steps are repeated, wherein the DNA barcode in the first deposition step comprises a different sequence than the DNA barcode in the second deposition step.

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

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

213. The method according to claim 210 also includes collecting the continuous DNA chain or its amplified product.

214. The method according to claim 213 also includes sequencing the continuous DNA chain or its amplified product.

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

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

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

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

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

220. A method for generating biological information, the method comprising: depositing primers on the population of nucleic acids, the primers hybridizing to target sequences of the nucleic acids to form nucleic acid-primer complexes; depositing a DNA barcode on the nucleic acid-primer complex, wherein the DNA barcode hybridizes to the primer to form a nucleic acid concatemer, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking with another nucleobase; irradiating the nucleic acid concatemers to form covalently linked concatemers; as well as DNA barcodes not in covalently linked concatemers are removed.

221. A method according to claim 220, wherein the steps of depositing DNA barcodes, irradiating and removing are repeated, wherein the DNA barcodes in the first deposition step contain a different sequence than the DNA barcodes in the second deposition step.

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

223. The method of any one of claims 1 to 222, wherein the selective irradiation is varied in intensity or duration to provide for the generation of a preselected fraction of DNA barcode-amplification product complexes or cross-linked barcodes.

224. The method of any one of claims 1 to 222, further comprising comparing information collected from disease state samples with non-disease state samples.

225. The method according to any one of claims 1 to 222 further comprises contacting the sample with an agent for intervention, wherein the agent for intervention comprises a compound, a nucleic acid or a cell; and comparing information collected from the contacted sample area with the non-contacted sample area.

226. A method for generating biological cell information, the method comprising: depositing a DNA barcode on the population of cells, wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking to another nucleobase; The concatemers are formed via a light-based procedure comprising repeating the following steps: selectively irradiating the population of cells at a first wavelength to form a covalently linked complex of the DNA barcode hybridized to the substrate nucleic acid sequence; as well as selectively irradiating the population of cells at a second wavelength to delink the DNA barcodes hybridized to the substrate nucleic acid sequence; and The concatemers are collected, wherein the collecting step is before or after selectively irradiating the population of cells at a second wavelength, and wherein the concatemers contain information about the population of cells.

227. A method for 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 primers to generate an amplification product; depositing a DNA barcode onto the sample, wherein the DNA barcode comprises a region complementary to a region of the amplification product, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking to another nucleobase; selectively irradiating the sample to form a covalently linked DNA barcode-amplification product complex, wherein the selective irradiation occurs at different locations for the first selective irradiation step and the second selective irradiation step; washing the sample to remove DNA barcodes that are not complexed with the amplification product; and Spatial organization information is determined based at least in part on the generated amplification products and the DNA barcodes.

228. The method of claim 227, wherein the sample is heated to a temperature of 50°C to 120°C.

229. 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. The method of claim 227, further comprising synthesizing a continuous DNA strand, wherein the synthesis comprises contacting the DNA barcode-amplification product complex with a polymerase.

231. The method of claim 227, wherein the synthesizing comprises cross-linking synthesis.

232. The method of claim 230, further comprising replacing the continuous DNA strands from the population of cells.

233. A method according to any one of claims 230 to 232, wherein the synthesizing and replacing occur simultaneously.

234. The method of any one of claims 230 to 232, wherein said synthesizing occurs before said replacing.

235. The method of any one of claims 230 to 232, wherein the synthesizing occurs after the replacing.

236. A method according to any one of claims 232 to 235, wherein the displaced continuous DNA strands are pooled.

237. A method according to claim 236, wherein the pooled continuous DNA chains are amplified.

238. A method according to claim 236, wherein the pooled continuous DNA chains are sequenced.

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

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

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

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

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

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

Images