IP Library › Granted Patent US 12,281,357
Granted Patent B1
US 12,281,357 · App. 17/174,961 · Granted Apr 22, 2025

In situ spatial barcoding

Inventors: Augusto Manuel Tentori (Pleasanton, CA); Rajiv Bharadwaj (Pleasanton, CA); Bill Kengli Lin (Pleasanton, CA); Hanyoup Kim (Pleasanton, CA); Denis Pristinski (Pleasanton, CA)
Assignee: 10x Genomics, Inc.
C12Q1/6876C12Q1/6841
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Quick Facts
Patent No.
US 12,281,357
App. No.
17/174,961
Filed
Feb 12, 2021
Granted
Apr 22, 2025
Kind
B1
Art Unit
1635
USPC
435/6.11
Abstract

Provided herein are materials and methods for in situ labeling of a biological sample for use in spatial analysis of one or more analytes in the biological sample. In some aspects, in situ labeling of a biological sample includes labeling the biological sample with one or more spatial barcodes.

Claims (22)

1. A method for identifying location of a nucleic acid analyte in a region of interest within a tissue section, the method comprising:

(a) contacting the tissue section with a substrate, wherein the substrate comprises a plurality of capture probes attached to the substrate on its surface, and 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 tissue section;

(c) before step (d), selectively delivering a permeabilization reagent comprising pepsin or proteinase K to the region of interest, wherein the region of interest is a portion of the tissue section on the substrate;

(d) hybridizing the nucleic acid analyte in the region of interest to the capture probe; and

(e) determining (i) the sequence of the spatial barcode or a complement thereof, and (ii) all or a portion of the sequence of the nucleic acid analyte, or a complement thereof; and using the determined sequences of (i) and (ii) to identify the location of the nucleic acid analyte in the region of interest in the tissue section.

2. The method of claim 1 , wherein identifying the region of interest comprises staining the tissue section using immunofluorescence, immunohistochemistry, hematoxylin, or eosin.

3. The method of claim 1 , wherein identifying the region of interest comprises imaging the tissue section.

4. The method of claim 1 , wherein, prior to (a), the tissue section was previously stained using immunofluorescence, immunohistochemistry, hematoxylin, or eosin.

5. The method of claim 1 , wherein selectively delivering the permeabilization reagent increases diffusion of the nucleic acid analyte to the capture probe at the region of interest as compared to areas of the tissue section outside the region of interest.

6. The method of claim 1 , wherein the permeabilization reagent reduces thickness of a hydrogel at the region of interest as compared to areas of the tissue section outside the region of interest.

7. The method of claim 1 , wherein the permeabilization reagent comprises a phosphate buffer.

8. The method of claim 1 , wherein selectively delivering the permeabilization reagent to the region of interest utilizes a liquid dispensing mechanism.

9. The method of claim 1 , wherein selectively delivering the permeabilization reagent to the region of interest utilizes laser irradiation.

10. The method of claim 1 , further comprising extending the capture probe using the nucleic acid analyte as a template, thereby generating an extended capture probe.

11. The method of claim 10 , further comprising amplifying the extended capture probe.

12. The method of claim 1 , wherein the determining comprises sequencing (i) the sequence of the spatial barcode or the complement thereof, and (ii) all or a portion of the sequence of the nucleic acid analyte, or the complement thereof.

13. The method of claim 1 , wherein the capture probe further comprises a unique molecular identifier, a cleavage domain, a functional domain, or any combination thereof.

14. The method of claim 1 , wherein the tissue section is a fixed tissue section, a fresh tissue section, or a frozen tissue section.

15. The method of claim 14 , wherein the fixed tissue section is a formalin-fixed, paraffin-embedded tissue section.

16. The method of claim 1 , wherein the nucleic acid analyte comprises RNA.

17. The method of claim 16 , wherein the RNA is an mRNA molecule.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 13, 2021
From: TENTORI, AUGUSTO MANUEL; BHARADWAJ, RAJIV; LIN, BILL KENGLI; KIM, HANYOUP; PRISTINSKI, DENIS
To: 10X GENOMICS, INC.
Reel/Frame 058369/0082 →
Continuity (2)
Provisional Application 63013503 · Apr 21, 2020
Provisional Application 62976490 · Feb 14, 2020
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