Granted Patent B2

US 12,258,624 · App. 18/499,129 · Granted Mar 25, 2025

Inventors: Joshua Delaney (Oakland, CA); Shalini Gohil (Castro Valley, CA); Veronica Emelina Gonzalez Muñoz (Palo Alto, CA); Joshua Gu (Dublin, CA); Albert Dale Kim (Oakland, CA); Yi Luo (Dublin, CA); Tathagata Mukherjee (San Jose, CA); Monica Nagendran (Pleasanton, CA); James Francis Perna, III (Hayward, CA); Kristen Nguyen Pham (San Jose, CA); Meiliana Tjandra (Dublin, CA)

Assignee: 10x Genomics, Inc.

C12Q1/6841C12Q1/6806C12Q1/6816C12Q1/6823C12Q1/6876

Abstract

Claims (45)

1. A method for sample analysis, comprising:

a) providing a biological sample immobilized on a substrate, wherein the biological sample is fixed;

b) contacting the biological sample with a catalyst that catalyzes de-crosslinking of molecular crosslinks in the biological sample, wherein the catalyst is a compound of formula (I),

or a salt, zwitterion, or solvate thereof, wherein:

A is selected from the group consisting of —COOH, —P(═O)(OH) 2 , and S(═O) 2 OH;

X 1 , X 2 , X 3 , and X 4 are each independently selected from the group consisting of: CH, CR a , and N;

each occurrence of Ra is independently selected from the group consisting of C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, —NO 2 , —NR′R″, and —C(═O)NR′R″; and

each occurrence of R′ and R″ is independently selected from the group consisting of H and C 1-6 alkyl which is optionally substituted with

 wherein n1 is an integer from 12 to 16;

c) contacting the biological sample with a nucleic acid probe that directly or indirectly binds to an RNA analyte at a location in the biological sample;

d) circularizing the nucleic acid probe to form a circularized nucleic acid probe;

e) using a polymerase to perform rolling circle amplification (RCA) using the circularized nucleic acid probe as template to generate an RCA product, optionally wherein the polymerase is a phi29 polymerase; and

f) detecting an optical signal associated with the RCA product, thereby detecting the RNA analyte at the location in the biological sample.

2. The method of claim 1 , wherein the molecular crosslinks are products of one or more crosslinking agents.

3. The method of claim 2 , wherein the one or more crosslinking agents comprise an aldehyde, optionally wherein the crosslinking agent comprises formaldehyde.

4. The method of claim 1 , wherein the catalyst is a transamination catalyst.

5. The method of claim 1 , wherein the catalyst catalyzes de-crosslinking of aminal crosslinks in the biological sample.

6. The method of claim 1 , wherein the catalyst catalyzes breakdown of hemi-aminal adducts and/or aminal adducts in the biological sample.

7. The method of claim 1 , wherein the catalyst comprises one or more compounds selected from the group consisting of

or a salt, zwitterion, or solvate thereof.

8. The method of claim 1 , wherein the catalyst comprises

or a salt, zwitterion, or solvate thereof.

9. The method of claim 1 , wherein the catalyst comprises

or a salt, zwitterion, or solvate thereof.

10. The method of claim 1 , wherein the catalyst comprises

or a salt, zwitterion, or solvate thereof.

11. The method of claim 1 , wherein the catalyst comprises

or a combination thereof, or a salt, zwitterion, or solvate thereof.

12. The method of claim 1 , wherein the substrate is transparent.

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

14. The method of claim 1 , wherein the biological sample is a fresh frozen biological sample that has been crosslinked or is a formaldehyde-fixed paraffin-embedded (FFPE) biological sample.

15. The method of claim 1 , wherein the catalyst is contacted with the biological sample at 80° C. for 30 minutes.

16. The method of claim 1 , wherein a solution or a suspension comprising the catalyst and a buffer is contacted with the biological sample.

17. The method of claim 16 , wherein the buffer comprises citrate, tris(hydroxymethyl)aminomethane (Tris), phosphate-buffered saline (PBS), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), or a combination thereof.

18. The method of claim 16 , wherein the buffer comprises dimethyl sulfoxide (DMSO).

19. The method of claim 16 , wherein the solution or suspension comprises sodium dodecyl sulfate (SDS), urea, and/or a proteinase.

20. The method of claim 19 , wherein the solution or suspension comprises sodium dodecyl sulfate (SDS) and proteinase K.

21. The method of claim 19 , wherein the solution or suspension comprises urea and proteinase K.

22. The method of claim 1 , wherein the RNA analyte is an mRNA and the nucleic acid probe hybridizes to the mRNA.

23. The method of claim 1 , comprising contacting the biological sample with a detectable probe, wherein the detectable probe hybridizes to a barcode sequence in the RCA product.

24. The method of claim 23 , wherein the detectable probe comprises a barcode sequence in a region that does not hybridize to the RCA product.

25. The method of claim 23 , wherein the detectable probe comprises a fluorescent label or a region for binding to a fluorescently labelled probe.

26. The method of claim 1 , wherein the optical signal is detected in situ in the biological sample.

27. The method of claim 1 , wherein the optical signal is detected by imaging the biological sample.

28. The method of claim 27 , wherein the imaging comprises fluorescent microscopy.

Assignments (1)

Continuity (3)

References Cited (400)

Cited By (1)

US 12,516,369