Granted Patent B2

US 12,735,810 · App. 19/658,124 · Granted Sep 15, 2026

Inventors: Solongo B. Ziraldo (Pleasanton, CA); Geoffrey McDermott (Livermore, CA); Shea Thompson Lance (Livermore, CA)

Assignee: 10x Genomics, Inc.

C40B50/06

Abstract

Claims (33)

1 . A method, comprising:

generating a first emulsion using: (i) a first aqueous fluid comprising a reagent, and (ii) a partitioning fluid comprising a fluorinated oil and a fluorosurfactant comprising a perfluoropolyether (PFPE), wherein the first emulsion comprises a first plurality of droplets dispersed in the partitioning fluid, wherein a first droplet of the first plurality of droplets comprises the reagent at a first concentration;

generating a second emulsion using: (i) a second aqueous fluid comprising a plurality of cells and a plurality of beads, wherein a bead of the plurality of beads comprises a plurality of nucleic acid barcode molecules, and (ii) the partitioning fluid, wherein the second emulsion comprises a second plurality of droplets dispersed in the partitioning fluid, wherein a second droplet of the second plurality of droplets comprises a cell of the plurality of cells and the bead of the plurality of beads, and wherein the reagent is absent from the second droplet or present in the second droplet at a second concentration that is lower than the first concentration;

contacting the first emulsion with the second emulsion; and

transferring the reagent from the first droplet to the second droplet via an aggregate of surfactant molecules comprising the reagent.

2 . The method of claim 1 , wherein the first aqueous fluid further comprises an ammonium salt and wherein the reagent is a surfactant.

3 . The method of claim 2 , wherein the surfactant is SDS.

4 . The method of claim 3 , wherein the method further comprises lysing the cell within the second droplet with the surfactant.

5 . The method of claim 4 , wherein the reagent is absent from the second droplet before contacting the first emulsion with the second emulsion.

6 . The method of claim 3 , wherein the aggregate of surfactant molecules is a micelle.

7 . The method of claim 3 , wherein the aggregate of surfactant molecules comprises the fluorosurfactant.

8 . The method of claim 1 , wherein the fluorosurfactant comprises a carboxylic acid-terminated PFPE.

9 . The method of claim 8 , wherein the fluorosurfactant is a first fluorosurfactant and the partitioning fluid further comprises a second fluorosurfactant.

10 . The method of claim 9 , wherein the second fluorosurfactant is a polyethylene glycol (PEG)-PFPE block copolymer.

11 . The method of claim 10 , wherein the second fluorosurfactant is a PFPE-PEG-PFPE triblock copolymer.

12 . The method of claim 3 , wherein generating the first emulsion comprises combining and agitating the first aqueous fluid and the partitioning fluid.

13 . The method of claim 12 , wherein generating the second emulsion comprises combining and agitating the second aqueous fluid and the partitioning fluid.

14 . The method of claim 4 , wherein the method further comprises:

hybridizing a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to a messenger ribonucleic acid (mRNA) of the cell within the second droplet; and

extending the nucleic acid barcode molecule in a reverse transcription reaction using the mRNA as template to generate a barcoded nucleic acid molecule.

15 . The method of claim 14 , wherein the nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules comprises a bead-specific barcode sequence, and wherein the barcoded nucleic acid molecule comprises the bead-specific barcode sequence or a complement thereof and a sequence of the mRNA or a complement thereof.

16 . The method of claim 15 , wherein the method further comprises sequencing the barcoded nucleic acid molecule or a derivative thereof.

17 . The method of claim 1 , wherein the first aqueous fluid further comprises n-dodecyl-beta-D-maltoside (DBDM), sodium lauroyl sarcosinate, t-Octylphenoxypolyethoxyethanol, or polysorbate 20.

18 . The method of claim 1 , wherein:

the first aqueous fluid further comprises an ammonium salt;

the reagent is sodium dodecyl sulfate (SDS);

the fluorosurfactant is a first fluorosurfactant and the partitioning fluid further comprises a second fluorosurfactant;

the first fluorosurfactant comprises a carboxylic acid-terminated PFPE;

the second fluorosurfactant is a polyethylene glycol (PEG)-PFPE block copolymer;

the aggregate of surfactant molecules is a micelle; and

the reagent is absent from the second droplet before contacting the first emulsion with the second emulsion.

19 . The method of claim 18 , wherein the second fluorosurfactant is a PFPE-PEG-PFPE triblock copolymer.

20 . The method of claim 19 , wherein the method further comprises lysing the cell within the second droplet with the reagent.

Assignments (1)

Continuity (7)

References Cited (400)