Granted Patent B2

US 12,344,892 · App. 18/988,119 · Granted Jul 1, 2025

Inventors: Michael Schnall-Levin (San Francisco, CA); Michael Ybarra Lucero (South San Francisco, CA); Tarjei Sigurd Mikkelsen (Hayward, CA); Patrik Stahl (Stockholm, SE); Jonas Frisen (Stockholm, SE); Maja Marklund (Stockholm, SE); Enric Llorens Bobadilla (Stockholm, SE)

Assignee: 10x Genomics, Inc.

C12Q1/6881C12Q1/48C12Q1/6806C12Q1/6841C12Q1/6874C12Y207/07

Abstract

Claims (36)

1. A method for spatially tagging a biological sample, the method comprising:

(a) providing:

(i) a first substrate comprising the biological sample, wherein the biological sample comprises a nucleic acid analyte; and

(ii) a second substrate comprising a hydrogel array, wherein the hydrogel 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;

(b) aligning the biological sample on the first substrate with the hydrogel array on the second substrate;

(c) releasing the capture probe from the hydrogel array such that the capture probe migrates to the biological sample and the capture domain of the capture probe hybridizes to the nucleic acid analyte of the biological sample; and

(d) extending the capture probe using the nucleic acid analyte as a template, thereby generating a spatially tagged nucleic acid product.

2. The method of claim 1 , wherein the releasing in (c) comprises cleaving the plurality of capture probes from the hydrogel array.

3. The method of claim 1 , wherein the releasing in (c) comprises degrading the hydrogel array.

4. The method of claim 3 , wherein the releasing in (c) comprises treating with a chemical degrading agent.

5. The method of claim 4 , wherein the chemical degrading agent is DTT.

6. The method of claim 4 , wherein the chemical degrading agent is sodium periodate.

7. The method of claim 1 , wherein the releasing in (c) comprises exposure to light.

8. The method of claim 1 , wherein the releasing in (c) comprises heating.

9. The method of claim 1 , wherein each capture probe of the plurality of capture probes is coupled to a feature.

10. The method of claim 9 , wherein the feature is a bead, a spot, a well, or a hydrogel pad.

11. The method of claim 9 , wherein the plurality of capture probes is coupled to the feature by a cleavage domain and wherein the releasing in (c) comprises cleavage of the cleavage domain.

12. The method of claim 11 , wherein the cleavage domain comprises a labile chemical bond.

13. The method of claim 12 , wherein the labile chemical bond is a disulfide bond.

14. The method of claim 12 , wherein the labile chemical bond is a photo-sensitive bond.

15. The method of claim 12 , wherein the labile chemical bond is a vicinal diol linkage.

16. The method of claim 1 , wherein the plurality of capture probes is encapsulated within, embedded within, or layered on the hydrogel array.

17. The method of claim 1 , wherein the hydrogel array comprises polyacrylamide.

18. The method of claim 1 , wherein the hydrogel array comprises N,N′-bis-(acryloyl) cystamine (BAC).

19. The method of claim 1 , wherein the hydrogel array comprises agarose.

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

21. The method of claim 1 , wherein the capture domain comprises a random N-mer sequence.

22. The method of claim 1 , wherein the nucleic acid analyte is a RNA molecule.

23. The method of claim 1 , wherein the nucleic acid analyte is a DNA molecule.

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

25. The method of claim 1 , further comprising determining: (i) a sequence of the spatial barcode or a complement thereof and (ii) all or a portion of a sequence of the spatially tagged nucleic acid product or a complement thereof.

26. The method of claim 1 , further comprising, after the extending in (d), amplifying the spatially tagged nucleic acid product.

27. The method of claim 26 , further comprising sequencing the spatially tagged nucleic acid product or an amplicon thereof.

28. The method of claim 1 , further comprising, prior to the releasing in (c), permeabilizing the biological sample.

29. The method of claim 28 , further comprising, prior to the permeabilizing, embedding the biological sample in a hydrogel matrix.

30. The method of claim 29 , further comprising clearing biological macromolecules from the embedded biological sample such that the nucleic acid analyte remains in the hydrogel matrix.

Assignments (3)

Continuity (23)

References Cited (400)

Cited By (12)

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