Methods for generating barcoded nucleic acid molecules using fixed cells

Patent metadata

Field	Value
Publication number	US-12241890-B2
Application number	US-202017131174-A
Country	US
Kind code	B2
Filing date	Dec 22, 2020
Priority date	Dec 23, 2019
Publication date	Mar 4, 2025
Grant date	Mar 4, 2025

Abstract

The present disclosure provides compositions and methods for using fixed biological samples in partition-based assays. In at least one embodiment, the disclosure provides a composition comprising a fixed biological sample and an un-fixing agent contained in a partition, such as a discrete droplet. In some embodiments, the disclosure provides un-fixing agent compounds capable of catalytically cleaving crosslinks in fixed biological samples, particularly crosslinked nucleic acids, such as RNA.

First claim

What is claimed is:

1. A method for generating a plurality of barcoded nucleic acid molecules using fixed cells, the method comprising:
(a) providing a plurality of fixed cells, wherein a fixed cell of said plurality of fixed cells comprises a plurality of crosslinked nucleic acid molecules;
(b) un-fixing said fixed cell with an un-fixing agent to provide an un-fixed cell comprising a plurality of un-crosslinked nucleic acid molecules from said plurality of crosslinked nucleic acid molecules; and
(c) generating the plurality of barcoded nucleic acid molecules from said plurality of un-crosslinked nucleic acid molecules, wherein a barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises i) a sequence corresponding to an un-crosslinked nucleic acid molecule of said plurality of said un-crosslinked nucleic acid molecules or a complement thereof, and ii) a barcode sequence or a complement thereof, wherein said un-fixing agent comprises a compound selected from the group consisting of (4-aminopyridin-3-yl)phosphonic acid, (3-aminopyridin-2-yl)phosphonic acid, (5-aminopyrimidin-4-yl)phosphonic acid, and a combination thereof.

2. The method of claim 1 , wherein (c) is performed in a plurality of partitions.

3. The method of claim 2 , wherein said plurality of partitions is a plurality of droplets or a plurality of wells.

4. The method of claim 2 , wherein a partition of said plurality of partitions comprises said un-fixed cell and a support comprising said plurality of nucleic acid barcode molecules.

5. The method of claim 4 , wherein said support is a bead.

6. The method of claim 1 , wherein said barcode sequence is a partition-specific barcode sequence.

7. The method of claim 1 , wherein said plurality of fixed cells is a plurality of paraformaldehyde fixed cells.

8. The method of claim 1 , wherein said un-fixing agent further comprises a protease.

9. The method of claim 8 , wherein said protease is a thermolabile protease.

10. The method of claim 8 , wherein said protease is a cold-active protease.

11. The method of claim 1 , wherein said sequence corresponding to an un-crosslinked nucleic acid molecule is a sequence corresponding to an un-crosslinked RNA molecule.

12. The method of claim 1 , wherein said fixed cell comprises a labeling agent.

13. The method of claim 12 , wherein said labeling agent is selected from the group consisting of protein, a peptide, an antibody, a lipophilic moiety, a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, a protein scaffold, and any combination thereof.

14. The method of claim 12 , wherein said labeling agent comprises a reporter oligonucleotide comprising a sequence that identifies the labeling agent.

15. The method of claim 14 , further comprising generating an additional barcoded nucleic acid molecule from said reporter oligonucleotide and said plurality of nucleic acid barcode molecules, wherein said additional barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises i) said reporter sequence or a complement thereof and ii) said barcode sequence or a complement thereof.

Assignees

• 10X Genomics Inc

Inventors

• Delaney Joshua
• Gohil Shalini
• Herschleb Jill
• Lowe Adam
• Kim Albert
• Tjandra Meiliana

Classifications

• G01N2001/307

  non-toxic, no Hg, no formaldehyde · CPC title

• G01N33/5047

  Cells of the immune system · CPC title

• G01N2333/948

  acting on peptide bonds (3.4) · CPC title

• C12N9/48

  acting on peptide bonds (3.4) · CPC title

• C07D239/24

  having three or more double bonds between ring members or between ring members and non-ring members · CPC title