Granted Patent B2

US 12,385,083 · App. 17/312,069 · Granted Aug 12, 2025

Inventor: Preyas Shah (Pleasanton, CA)

Assignee: 10x Genomics, Inc.

C12Q1/6844B01L3/50853B01L3/545B01L9/523C12N15/1065C12Q1/6837C12Q1/6841C12Q1/6874C12Q1/6876C12Q1/6881G01N21/6458G01N33/4833G01N35/00732G02B21/16G02B21/26G02B21/34G02B21/365B01L2300/0829C12Q1/6855C12Q1/6869C12Q2525/161C12Q2525/179C12Q2527/156C12Q2543/101C12Q2563/149C12Q2565/537C12Q2565/60C12Q2600/158G01N2021/6439G01N2035/00752

Abstract

Claims (26)

1. A method of identifying a location of a biological analyte in a biological sample comprising:

(a) providing a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain;

(b) contacting the substrate with a plurality of analyte capture agents, wherein an analyte capture agent of the plurality of analyte capture agents comprises an analyte binding moiety and a capture agent barcode domain, and the capture probe binds to the analyte capture agent via the capture domain of the capture probe;

(c) producing a spatially-tagged analyte capture agent, by extending the capture agent barcode domain to include a sequence that is complementary to the spatial barcode of the capture probe;

(d) labelling a cell of the biological sample with one or more spatially-tagged analyte capture agents, by contacting the one or more spatially-tagged analyte capture agents with the biological sample, wherein at least one spatially-tagged analyte capture agent interacts with the biological analyte within the biological sample, and denaturing the spatially-tagged analyte capture agent from the capture probe;

(e) dissociating the biological sample into a plurality of cells, wherein the at least one spatially-tagged analyte capture agent is associated with a dissociated cell; and

(f) using the at least one spatially-tagged analyte capture agent to identify the location of the target biological analyte in the biological sample.

2. The method of claim 1 , wherein the capture agent barcode domain comprises a poly(A) 3′ tail.

3. The method of claim 1 , wherein the capture agent barcode domain comprises an analyte binding moiety barcode and a poly(A) 3′ tail.

4. The method of claim 1 , wherein the target biological analyte is a protein.

5. The method of claim 1 , wherein the capture domain is configured to hybridize to a poly(A) tail.

6. The method of claim 1 , wherein the capture domain comprises a poly(T) sequence.

7. The method of claim 1 , wherein the target biological analyte comprises at least one of RNA, DNA, a small molecule, and a metabolite.

8. The method of claim 1 , wherein the analyte capture agent comprises an analyte binding moiety linked to the capture agent barcode domain by a disulfide bond.

9. The method of claim 1 , wherein the capture agent barcode domain further comprises a PCR handle.

10. The method of claim 1 , wherein the at least one spatially-tagged analyte capture agent interacts with at least one target biological analyte within the biological sample before denaturing the spatially-tagged analyte capture agent from the capture probe.

11. The method in claim 1 , wherein the substrate can be reused after releasing the spatially-tagged analyte capture agent from the capture probe.

12. The method in claim 11 , wherein the substrate is washed after the one or more spatially-tagged analyte capture agents are released from the one or more capture probes.

13. The method of claim 1 , wherein after the one or more spatially-tagged analyte capture agents are released from the one or more capture probes, the method further comprises repeating the steps (b) through (f).

14. The method of claim 1 , wherein the biological sample comprises a tissue, an organ, an organism, or a cell culture sample.

15. The method of claim 1 , further comprising imaging the biological sample.

16. The method of claim 15 , wherein imaging is used to identify the location of the target biological analyte within the biological sample.

17. The method of claim 16 , wherein identifying the location of the target biological analyte occurs at single cell resolution.

18. The method of claim 1 , wherein the biological sample is a tissue sample.

19. The method of claim 18 , wherein the tissue sample is a tissue section.

20. The method of claim 19 , wherein the tissue section is a formalin-fixed paraffin-embedded (FFPE) tissue section or a fresh frozen tissue section.

Assignments (1)

Continuity (69)

References Cited (400)

Cited By (9)

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