Probe-based analysis of nucleic acids and proteins

Patent metadata

Field	Value
Publication number	US-2024254552-A1
Application number	US-202418412111-A
Country	US
Kind code	A1
Filing date	Jan 12, 2024
Priority date	Jun 29, 2022
Publication date	Aug 1, 2024
Grant date	—

Abstract

Provided herein are systems and methods for processing biomolecules (e.g., nucleic acid molecules, proteins) from a sample. A method for processing biomolecules may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule) and barcoding the probe-nucleic acid molecule complex or derivatives thereof. Such a method can comprise performing a nucleic acid reaction, e.g., extension, denaturation, and amplification. A method for processing a sample may comprise hybridizing probes to (i) target regions of a nucleic acid molecule (e.g., RNA molecule) and (ii) a reporter oligonucleotide of a feature binding group, and barcoding the probe-associated molecules. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.

First claim

What is claimed is:

1 . A method for analyzing a cell, comprising:
(a) providing a sample comprising a cell comprising a nucleic acid molecule, wherein the sample is an embedded, fixed tissue embedded in a solid medium; (b) removing at least a portion of the solid medium from the embedded, fixed tissue, thereby obtaining a fixed tissue comprising the cell comprising the nucleic acid molecule; (c) dissociating the fixed tissue into a plurality of cells, wherein the plurality of cells comprises the cell comprising the nucleic acid molecule; and (d) in the cell comprising the nucleic acid molecule, using the nucleic acid molecule and a barcode molecule comprising a barcode sequence to generate a barcoded nucleic acid molecule comprising (i) a sequence corresponding to the nucleic acid molecule; and (ii) the barcode sequence or reverse complement thereof.

2 . The method of claim 1 , wherein (d) comprises hybridizing the barcode molecule to the nucleic acid molecule.

3 . The method of claim 2 , further comprising extending the barcode molecule to form the barcoded nucleic acid molecule.

4 . The method of claim 1 , wherein, in (a), the cell comprising the nucleic acid molecule further comprises a feature, wherein the feature comprises a protein, a peptide, a lipid, or a carbohydrate.

5 . The method of claim 4 , further comprising, prior to or during (d), contacting the cell comprising the nucleic acid molecule with a feature binding group and coupling the feature binding group to the feature, wherein the feature binding group comprises an oligonucleotide comprising a feature nucleic acid sequence that identifies the feature binding group.

6 . The method of claim 5 , further comprising, using the feature nucleic acid sequence and an additional barcode molecule comprising an additional barcode sequence to generate an additional barcoded nucleic acid molecule comprising (i) the feature nucleic acid sequence or reverse complement thereof, and (ii) the additional barcode sequence or reverse complement thereof.

7 . The method of claim 1 , wherein the fixed tissue is fixed at least about 1 year prior to (a).

8 . The method of claim 7 , wherein the fixed tissue is fixed at least about 5 years prior to (a).

9 . The method of claim 1 , further comprising, in (b), adding a first solvent to the embedded, fixed tissue, thereby dissolving the solid medium, thereby obtaining a tissue.

10 . The method of claim 9 , wherein the first solvent is a non-polar solvent.

11 . The method of claim 10 , wherein the non-polar solvent comprises xylene.

12 . The method of claim 10 , wherein the non-polar solvent comprises neo-Clear® xylene alternative.

13 . The method of claim 9 , further comprising removing the first solvent from the tissue.

14 . The method of claim 13 , further comprising adding a second solvent to the tissue.

15 . The method of claim 14 , wherein the second solvent is a polar solvent.

16 . The method of claim 15 , wherein the polar solvent comprises ethanol.

17 . The method of claim 14 , further comprising removing the second solvent from the tissue.

18 . The method of claim 17 , further comprising adding a rehydration agent to the tissue.

19 . The method of claim 18 , wherein the rehydration agent comprises water.

20 . The method of claim 18 , further comprising removing the rehydration agent from the tissue.

21 . The method of claim 20 , further comprising adding buffer to the tissue.

22 . The method of claim 21 , further comprising removing the buffer from the tissue.

23 . The method of claim 22 , further comprising, in (c), dissociating the tissue.

24 . The method of claim 23 , further comprising resuspending the tissue in a supernatant.

25 . The method of claim 24 , further comprising filtering the tissue.

26 . The method of claim 25 , further comprising washing the tissue.

27 . The method of claim 26 , further comprising resuspending the tissue.

28 . The method of claim 23 , wherein the dissociating the tissue is performed using an automated dissociator.

29 . The method of claim 23 , wherein the dissociating the tissue is performed using a manual pulverizer.

30 . The method of claim 1 , wherein (b) and (c) provide a yield of cells of at least about 1×10 5 cells per two 25 micrometer sections of the fixed tissue.

Assignees

• 10X Genomics Inc

Inventors

• Pfeiffer Katherine
• Kohlway Andrew Scott
• Hill Andrew John
• Lund Paul Eugene
• Abousoud Jawad N
• Bhattacharjee Sharmila Chatterjee
• Stott Ryan

Classifications

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title