Materials and methods for localized detection of nucleic acids in a tissue sample
View Patent ↗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 method of generating a spatial transcriptomics gene expression image having subcellular resolution, comprising:
a. generating, on a flat array surface, high density, bridge-amplified clusters of probes having a distance between centers of said clusters of 1 μm or less, wherein each probe comprises a spatial barcode sequence and a capture domain;
b. identifying, by sequencing said spatial barcode sequence, a location on said flat array surface of each cluster;
c. contacting said flat array surface with a tissue section under conditions such that RNA from cells in said tissue section hybridizes to said capture domain of said probes;
d. reverse transcribing target sequences from said RNA on 3′ ends of said probes;
e. generating second strand copies of said probes and eluting said second strand copies from said array;
f. generating a sequencing library from eluted second strand copies;
g. sequencing said sequencing library to generate sequencing data; and
h. generating a spatial transcriptomics gene expression image having subcellular resolution from said sequencing data.
2. The method of claim 1 , wherein each probe in a given cluster comprises an identical spatial barcode sequence, and wherein said spatial barcode sequence for each cluster is unique.
3. The method of claim 1 , wherein said flat array surface comprises 0.5-2 million clusters per 1 mm 2 of surface.
4. The method of claim 3 , wherein said flat array surface comprises about 1.5 million clusters per 1 mm 2 of surface.
5. The method of claim 1 , wherein each cluster comprises at least 200 probes.
6. The method of claim 1 , wherein each cluster comprises at least 500 probes.
7. The method of claim 1 , wherein each cluster comprises at least 800 probes.
8. The method of claim 1 , wherein each cluster has a diameter of 500-1200 nm.
9. The method of claim 8 , wherein each cluster has an average diameter of 0.6 μm.
10. The method of claim 1 , wherein said flat array 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.
11. The method of claim 1 , wherein said capture domain is identical for each probe.
12. The method of claim 1 , wherein said capture domain comprises a poly-T oligonucleotide comprising at least 10 deoxythymidine residues.
13. The method of claim 1 , wherein each probe further comprises a sequencing barcode.
14. The method of claim 1 , wherein each probe further comprises one or more filler sequences.
15. The method of claim 1 , wherein each probe further comprises a unique molecular identifier (UMI) barcode sequence.
16. The method of claim 1 , wherein each probe further comprises a cleavage domain comprising a binding site for a restriction endonuclease.
17. The method of claim 1 , further comprising imaging the tissue before or after reverse transcribing target sequences from said RNA on 3′ ends of said probes.
18. The method of claim 17 , further comprising correlating the identified location of each cluster on said flat array surface with a corresponding location within said tissue section.