Granted Patent B2

US 12,742,199 · App. 19/315,008 · Granted Sep 22, 2026

Inventors: Yifeng Yin (Elk Grove, CA); Michael Schnall-Levin (San Francisco, CA); Eswar Prasad Ramachandran Iyer (Oakland, CA)

Assignee: 10x Genomics, Inc.

C12Q1/6837

Abstract

Claims (36)

1 . A method comprising:

(a) providing a substrate comprising a plurality of features attached thereto, wherein a feature of the plurality of features comprises (i) a primer comprising a poly(T) sequence; and (ii) a capture probe comprising a spatial barcode sequence and a unique molecular identifier (UMI);

(b) hybridizing the primer comprising the poly(T) sequence to an mRNA molecule of a tissue section;

(c) extending the 3′ end of the primer by reverse transcription of the mRNA molecule to generate cDNA, wherein the reverse transcription adds a poly(C) sequence to the 3′ end of the cDNA;

(d) denaturing the mRNA molecule from the cDNA;

(e) hybridizing the capture probe to the cDNA;

(f) barcoding the cDNA using the capture probe as a template to generate a first strand cDNA; and

(g) generating a second strand cDNA that is a complement of the first strand cDNA.

2 . The method of claim 1 , wherein the first strand cDNA or the second strand cDNA is released from the substrate.

3 . The method of claim 2 , further comprising amplifying the released second strand cDNA, thereby generating a plurality of amplicons.

4 . The method of claim 1 , wherein a sequencing library is constructed from the second strand cDNA.

5 . The method of claim 4 , wherein the sequencing library is sequenced.

6 . The method of claim 1 , wherein the capture probe further comprises a cleavage domain.

7 . The method of claim 1 , wherein the primer and/or the capture probe further comprises an adapter sequence.

8 . The method of claim 1 , wherein the substrate comprises at least one million features.

9 . The method of claim 1 , wherein the feature in the plurality of features comprises an area of less than about 5 μm 2 .

10 . The method of claim 1 , wherein the tissue section is stained and imaged prior to the reverse transcription.

11 . The method of claim 1 , wherein the substrate comprises a slide or a flow cell.

12 . The method of claim 2 , wherein the first strand cDNA or the second strand cDNA is released from the substrate by nucleic acid cleavage or denaturation.

13 . The method of claim 3 , wherein an amplicon of the plurality of amplicons is analyzed to obtain sequencing data.

14 . The method of claim 13 , wherein the sequencing data is associated with a location on the substrate using the spatial barcode sequence.

15 . The method of claim 14 , wherein the location on the substrate is correlated with a location of the mRNA molecule in the tissue section.

16 . The method of claim 1 , wherein the capture probe is attached to the substrate by a 5′ end.

17 . The method of claim 16 , further comprising blocking the free 3′ end of the capture probe.

18 . The method of claim 1 , wherein the tissue section is overlaid with the substrate prior to the reverse transcription.

19 . The method of claim 18 , wherein the tissue section is permeabilized prior to the reverse transcription.

20 . The method of claim 19 , wherein the mRNA molecule is released from the permeabilized tissue section.

21 . The method of claim 1 , further comprising hybridizing a template switching oligonucleotide to the poly(C) sequence in (c).

22 . The method of claim 21 , further comprising extending the 3′ end of the cDNA using the template switching oligonucleotide as a template.

23 . The method of claim 22 , further comprising removing the template switching oligonucleotide from the extended cDNA.

24 . The method of claim 2 , further comprising amplifying the released first strand cDNA, thereby generating a plurality of amplicons.

25 . The method of claim 1 , wherein the primer and/or the capture probe further comprises a sequencing primer sequence.

26 . The method of claim 10 , wherein the staining comprises hematoxylin and/or eosin staining.

27 . The method of claim 10 , wherein the staining comprises immunostaining or immunohistochemical staining.

28 . The method of claim 1 , wherein the tissue section is a fresh-frozen tissue section.

29 . The method of claim 22 , wherein in (f) the extended cDNA is further extended using the capture probe as a template to generate the first strand cDNA.

Assignments (1)

Continuity (39)

References Cited (400)