Materials and methods for localized detection of nucleic acids in a tissue sample

Abstract

The present disclosure relates to materials and methods for spatial detection of nucleic acid in a tissue sample or a portion thereof. In particular, provided herein are materials and methods for detecting RNA so as to obtain spatial information about the localization, distribution or expression of genes in a tissue sample. In some embodiments, the materials and methods provided herein enable detection of gene expression in a single cell.

1 . A substrate for spatial detection of nucleic acid in a tissue sample, the substrate comprising a plurality of capture probes immobilized on a surface of the substrate, wherein:
 a. each capture probe comprises a capture domain and a spatial barcode;   b. the plurality of capture probes is arranged in clusters, each cluster comprising multiple capture probes;   c. each capture probe in a cluster comprises the same spatial barcode; and   d. the spatial barcode for each cluster is unique,   wherein the substrate comprises 0.5-2 million clusters per 1 mm 2  of surface.   
     
     
         2 . The substrate of  claim 1 , wherein:
 (i) each cluster comprises at least 200 capture probes, at least 500 capture probes, or at least 800 capture probes;   (ii) each cluster has a diameter of 500-1200 nm; and/or   (iii) the substrate comprises about 1.5 million clusters per 1 mm 2  of surface.   
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . The substrate of  claim 1 , wherein the surface comprises a material selected from glass, silicon, poly-L-lysine coated materials, nitrocellulose, polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polyacrylamide, polypropylene, polyethylene and polycarbonate. 
     
     
         9 . The substrate of  claim 1 , wherein the capture domain for each capture probe is the same, and/or wherein the capture domain comprises a poly-T oligonucleotide comprising at least 10 deoxythymidine residues or a DNA sequence complementary to a nucleotide sequence of a target nucleic acid. 
     
     
         10 . (canceled) 
     
     
         11 . The substrate of  claim 1 , wherein each capture probe further comprises a sequencing barcode, each capture probe further comprises one or more filler sequences, each capture probe in a cluster comprises a unique molecular identifier (UMI) barcode, and/or each capture probe further comprises a cleavage domain. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The substrate of  claim 11 , wherein the cleavage domain comprises a binding site for a restriction endonuclease. 
     
     
         16 . The substrate of  claim 1 , wherein the nucleic acid is RNA. 
     
     
         17 . A method comprising replicating the substrate of  claim 1  to a second media to produce a second substrate. 
     
     
         18 . A method for spatial detection of RNA in a tissue sample, comprising:
 a. providing the substrate of  claim 1 ;   b. contacting the substrate with a tissue sample and allowing RNA molecules of the tissue sample to bind to the capture domain of the capture probes;   c. generating cDNA molecules from the bound RNA molecules; and   d. sequencing the cDNA molecules.   
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 18 , further comprising determining the location of each cluster of capture probes on the substrate prior to contacting the substrate with the tissue sample, wherein determining the location of each cluster comprises determining the sequence of the spatial barcode for at least one capture probe in each cluster, and assigning the sequence to a location on the substrate. 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 20 , further comprising correlating the sequence of the spatial barcode for each sequenced cDNA molecule with the location of the cluster of capture probes on the substrate having a corresponding spatial barcode. 
     
     
         24 . The method of  claim 23 , further comprising imaging the tissue before or after generating the cDNA molecules, and determining the spatial location of the RNA molecules within the tissue sample by correlating the location of the cluster of capture probes on the substrate with a corresponding location within the tissue sample. 
     
     
         25 . (canceled) 
     
     
         26 . A method for spatial detection of nucleic acid in a tissue sample, comprising:
 a. providing the substrate of  claim 1 ;   b. contacting the substrate with a tissue sample and allowing nucleic acid molecules of the tissue sample to bind to the capture domain of the capture probes; and   c. sequencing the bound nucleic acid molecules.   
     
     
         27 . The method of  claim 26 , further comprising determining the location of each cluster of capture probes on the substrate prior to contacting the substrate with the tissue sample, wherein determining the location of each cluster comprises determining the sequence of the spatial barcode for at least one capture probe in each cluster, and assigning the sequence to a location on the substrate. 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 27 , further comprising correlating the sequence of the spatial barcode for each sequenced nucleic acid molecule with the location of the cluster of capture probes on the substrate having a corresponding spatial barcode. 
     
     
         31 . The method of  claim 30 , further comprising imaging the tissue before or after sequencing the nucleic acid molecules, and determining the spatial location of the nucleic acid molecules within the tissue sample by correlating the location of the cluster of capture probes on the substrate with a corresponding location within the tissue sample. 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 18 , wherein the method determines RNA expression in a single cell within the tissue sample. 
     
     
         35 . The method of  claim 34 , wherein the method determines RNA expression in subcellular components within the single cell. 
     
     
         36 . The method of  claim 35 , wherein the subcellular components comprise the nucleus, the cytoplasm and/or the mitochondria of the cell. 
     
     
         37 . A kit comprising the substrate of  claim 1 . 
     
     
         38 . (canceled) 
     
     
         39 . A method of determining RNA expression in a single cell in a tissue sample, comprising contacting the tissue sample with the substrate of  claim 1 . 
     
     
         40 . A method of determining RNA expression in subcellular components of a single cell in a tissue sample, comprising contacting the tissue sample with the substrate of  claim 1 . 
     
     
         41 . The method of  claim 40 , wherein the subcellular components comprise the nucleus, the cytoplasm, and/or the mitochondria.