IP Library Granted Patent US 12,235,262
Granted Patent B1
US 12,235,262 · App. 17/014,909 · Granted Feb 25, 2025

Methods and systems for single cell protein analysis

Inventor: Paul Giresi (Palo Alto, CA)
Assignee: 10X GENOMICS, INC.
G01N33/54306G01N33/6842
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Quick Facts
Patent No.
US 12,235,262
App. No.
17/014,909
Granted
Feb 25, 2025
Kind
B1
Abstract

Provided herein are methods identifying protein-protein interactions in a single cell context. In some cases, the methods may be used to measure the abundance of protein-protein interactions in a single cell context. In some cases, the methods may be used to map relationships (e.g., spatial relationships) between proteins in a single cell context. Generally, the methods employ the use of proximity probes coupled with splint oligonucleotides to link information about the relationship of proteins within a single cell context, which may then be read in a downstream process (e.g., a sequencing reaction).

Claims (20)

1. A method for processing a labelled cell, comprising:

(a) partitioning a plurality of labelled cells into a plurality of partitions, wherein a partition of the plurality of partitions comprises a cell barcode molecule and a labelled cell of the plurality of labelled cells, wherein the labelled cell comprises (i) a first labelling agent coupled to a first protein of the labelled cell, and (ii) a second labelling agent coupled to a second protein of the labelled cell, wherein the first labelling agent comprises a first proximity probe comprising a first barcode sequence and a first unique molecular identifier (UMI), wherein the second labelling agent comprises a second proximity probe comprising a second barcode sequence and a second UMI, and wherein the cell barcode molecule comprises a third UMI and a cell barcode sequence that is complementary to the first and second barcode sequences; and

(b) generating a barcoded molecule in the partition, wherein the barcoded molecule comprises the first barcode sequence, the first UMI, the second barcode sequence, the second UMI, the third UMI, and the cell barcode sequence, or derivatives thereof.

2. The method of claim 1 , wherein the first protein is a surface protein.

3. The method of claim 1 , wherein the second protein is a surface protein.

4. The method of claim 1 , wherein (a) further comprises partitioning in the partition a support comprising the cell barcode molecule.

5. The method of claim 4 , wherein the support comprises a plurality of cell barcode molecules comprising the cell barcode molecule attached thereto.

6. The method of claim 5 , wherein cell barcode molecules of the plurality of cell barcode molecules comprise the cell barcode sequence.

7. The method of claim 1 , wherein the labelled cell is a single labelled cell and the cell barcode sequence is specific to the single labelled cell.

8. The method of claim 4 , wherein the cell barcode molecule is releasably attached to the support.

9. The method of claim 1 , wherein the partition is a droplet.

10. The method of claim 1 , wherein the partition is a well.

11. The method of claim 1 , wherein the first protein is an internal protein.

12. The method of claim 1 , wherein the second protein is an internal protein.

13. The method of claim 1 , wherein the first barcode sequence comprises a barcode sequence corresponding to a type of the first labelling agent or a type of the first protein.

14. The method of claim 1 , wherein the first labelling agent comprises at least one additional proximity probe, wherein the at least one additional proximity probe comprises a donor probe comprising a donor sequence or an acceptor probe comprising an acceptor sequence.

15. The method of claim 1 , wherein the first proximity probe is:

(a) single stranded; or

(b) single stranded and comprises a donor sequence or an acceptor sequence at an end of the first proximity probe, wherein the cell barcode molecule comprises a first attachment sequence complementary to the donor sequence and a second attachment sequence complementary to the acceptor sequence.

16. The method of claim 1 , wherein the cell barcode molecule comprises a first single stranded portion, a double stranded portion, and a second single stranded portion, wherein the first single stranded portion and the second single stranded portion flank the double stranded portion, wherein the first single stranded portion comprises a first attachment sequence complementary to a donor sequence of the first proximity probe, and wherein the second single stranded portion comprises a second attachment sequence complementary to an acceptor sequence of the second proximity probe.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 18, 2021
From: GIRESI, PAUL
To: 10X GENOMICS, INC.
Reel/Frame 056280/0602 →
Continuity (1)
Provisional Application 62897698 · Sep 9, 2019
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