Spatial analysis utilizing degradable hydrogels

Patent metadata

Field	Value
Publication number	US-11981960-B1
Application number	US-202117368259-A
Country	US
Kind code	B1
Filing date	Jul 6, 2021
Priority date	Jul 6, 2020
Publication date	May 14, 2024
Grant date	May 14, 2024

Abstract

Provided are methods of capturing an analyte from a biological sample using a hydrogel that includes capture probes to capture the analyte, identifying a region of interest of the biological sample, and isolating the region of interest from the biological sample by removing a portion of the hydrogel that corresponds to the region of interest. Compositions and kits for performing the methods are also provided.

First claim

What is claimed is:

1. A method of determining a location and/or an abundance of an analyte at a region of interest from a biological sample, the method comprising:
(a) aligning the biological sample with a substrate comprising a hydrogel layered over a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain;
(b) identifying the region of interest in the biological sample;
(c) degrading a portion of the hydrogel corresponding to the region of interest of the biological sample, thereby exposing the capture probe;
(d) contacting the biological sample with the substrate;
(e) hybridizing the analyte at the region of interest to the capture domain; and
(f) determining (i) a sequence of the spatial barcode, or a complement thereof, and (ii) all or a portion of a sequence of the analyte, or a complement thereof, and using the sequences of (i) and (ii) to determine the location and/or the abundance of the analyte in the region of interest in the biological sample.

2. The method of claim 1 , wherein degrading the portion of the hydrogel comprises exposing the region of interest to UV light, laser light, natural light, a heating device, a radiation device, a plasma device, a microwave device or a degradation agent, wherein the degradation agent degrades the portion of the hydrogel underneath the biological sample.

3. The method of claim 1 , further comprising dissolving the hydrogel at regions outside of the region of interest.

4. The method of claim 3 , further comprising, at the regions outside of the region of interest:
hybridizing an analyte outside of the region of interest to a capture domain outside of the region of interest; and
determining (i) a sequence of a spatial barcode of the capture domain outside of the region of interest, or a complement thereof, and (ii) all or a portion of a sequence of the analyte outside of the region of interest, or a complement thereof, and using the sequences of (i) and (ii) to determine the location and/or the abundance of the analyte outside of the region of interest in the biological sample.

5. The method of claim 1 , wherein degrading the portion of the hydrogel corresponding to the region of interest comprises treating the hydrogel at the region of interest with a reducing agent.

6. The method of claim 5 , wherein the reducing agent comprises dithiothreitol.

7. The method of claim 5 , wherein the treating the hydrogel at the region of interest with a reducing agent is performed before step (d).

8. The method of claim 1 , wherein step (d) is performed before steps (b) and (c).

9. The method of claim 5 , wherein the treating the hydrogel at the region of interest with a reducing agent is performed after step (d).

10. The method of claim 1 , wherein the biological sample is stained using hematoxylin and eosin (H&E), immunofluorescence, or immunohistochemistry.

11. The method of claim 1 , wherein the hydrogel comprises hydrogel subunits selected from the group consisting of: acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, poly(ethylene glycol) and derivatives thereof, gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, or any combination thereof.

12. The method of claim 1 , wherein the hydrogel comprises one or more permeabilization reagents, wherein the one or more permeabilization reagents comprises pepsin or proteinase K.

13. The method of claim 1 , wherein the capture probe further comprises one or more functional domains, a unique molecular identifier, a cleavage domain, or any combination thereof, wherein the cleavage domain is a cleavable linker selected from the group consisting of a photocleavable linker, a UV-cleavable linker, an enzymatic cleavable linker, and a pH-sensitive cleavable linker.

14. The method of claim 1 , wherein the biological sample is a fresh frozen tissue sample or formalin fixed paraffin embedded (FFPE) tissue sample.

15. The method of claim 1 , wherein the analyte is an RNA molecule.

Assignees

• 10X Genomics Inc

Inventors

• Lin Bill Kengli
• Herschleb Jill
• Uytingco Cedric

Classifications

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title