Granted Patent B2

US 12,371,688 · App. 18/409,174 · Granted Jul 29, 2025

Inventors: Hanyoup Kim (Foster City, CA); David Sukovich (Oakland, CA); Layla Katiraee (Castro Valley, CA); Augusto Manuel Tentori (Dublin, CA); Lauren Gutgesell (Pleasanton, CA); Janine Hensel (Menlo Park, CA); Seayar Mohabbat (Manteca, CA)

Assignee: 10x Genomics, Inc.

C12N15/1065

Abstract

Claims (26)

1. A method for processing a nucleic acid analyte in a biological sample mounted on a first substrate, the method comprising:

(a) providing the biological sample, wherein the biological sample is a tissue sample that was previously treated with a fixative comprising an aldehyde and embedded in an optimal cutting temperature (OCT) compound;

(b) hybridizing a first probe and a second probe to the nucleic acid analyte, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to sequences that are not adjacent to each other of the nucleic acid analyte, and wherein the second probe comprises a capture probe binding domain;

(c) extending the first probe, thereby (i) filling in a gap between the first probe and the second probe and (ii) generating an extended first probe;

(d) coupling the extended first probe and the second probe, thereby generating a connected probe;

(e) aligning the first substrate with a second substrate comprising an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain;

(f) releasing the connected probe from the nucleic acid analyte when at least a portion of the biological sample is aligned with at least a portion of the array; and

(g) hybridizing the connected probe to the capture domain of the capture probe.

2. The method of claim 1 , wherein the fixative comprises paraformaldehyde.

3. The method of claim 1 , wherein the fixative comprises formaldehyde.

4. The method of claim 1 , wherein the biological sample was treated with a sucrose gradient.

5. The method of claim 4 , wherein the biological sample was embedded in the OCT compound after treatment with the sucrose gradient.

6. The method of claim 4 , wherein the biological sample was treated with ethanol.

7. The method of claim 1 , wherein the first probe is extended using a polymerase.

8. The method of claim 1 , wherein the first probe is extended via a Mu polymerase, a DNA polymerase, an RNA polymerase, a reverse transcriptase, a VENT polymerase, or a Taq polymerase.

9. The method of claim 1 , wherein the first probe is extended via a DNA polymerase.

10. The method of claim 1 , wherein generating the connected probe comprises ligating the extended first probe to the second probe using chemical ligation.

11. The method of claim 1 , wherein generating the connected probe comprises ligating the extended first probe to the second probe using a ligase, optionally wherein the ligase is selected from a T4 RNA ligase (Rnl2), a PBCV-1 ligase, a single stranded DNA ligase, or a T4 DNA ligase.

12. The method of claim 1 , wherein the first probe, the second probe, or both the first probe and the second probe comprises a primer sequence.

13. The method of claim 1 , wherein the first probe and/or the second probe is a DNA probe.

14. The method of claim 1 , wherein releasing the connected probe comprises contacting the biological sample with an endoribonuclease, wherein the endoribonuclease is an RNase H enzyme.

15. The method of claim 1 , wherein the biological sample was previously stained using immunofluorescence, immunohistochemistry, or hematoxylin and eosin.

16. The method of claim 1 , further comprising contacting the biological sample with a permeabilization agent, wherein the permeabilization agent comprises proteinase K or pepsin.

17. The method of claim 1 , wherein the nucleic acid analyte comprises mRNA.

18. The method of claim 1 , further comprising extending the connected probe using the capture probe as a template, thereby generating an extended connected probe.

19. The method of claim 1 , further comprising determining (i) all or a part of the sequence of the nucleic acid analyte, or a complement thereof, and (ii) the spatial barcode, or a complement thereof, from the extended connected probe or a complement thereof, and using the determined sequence of (i) and (ii) to determine a location of the nucleic acid analyte in the biological sample.

Assignments (1)

Continuity (8)

References Cited (400)

Cited By (11)

US 12,497,654 US 12,508,590 US 12,529,094 US 12,553,805 US 12,553,898 US 12,566,113 US 12,566,114 US 12,571,029 US 12,624,475 US 12,637,706 US 12,692,492