Granted Patent B2

US 12,612,714 · App. 19/283,119 · Granted Apr 28, 2026

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

Assignee: 10x Genomics, Inc.

C40B50/06

Abstract

Claims (30)

1 . A system for transferring a lysis surfactant from a first droplet to a second droplet via micelles, the system comprising:

a partitioning fluid comprising a fluorinated oil and a fluorosurfactant, wherein the fluorosurfactant comprises a perfluoropolyether (PFPE);

a lysis reagent comprising: water, an ammonium salt, and a lysis surfactant, wherein the lysis surfactant is sodium dodecyl sulfate (SDS); and

a plurality of beads, wherein a bead of the plurality of beads is coupled to a plurality of nucleic acid barcode molecules;

wherein the partitioning fluid and the lysis reagent, when combined and vortexed, are capable of forming a first emulsion comprising a first plurality of droplets and a plurality of micelles dispersed in the fluorinated oil;

wherein the first plurality of droplets comprises the first droplet, the first droplet comprising: 1) a first droplet interior comprising the lysis reagent, and 2) a first droplet surface comprising the fluorosurfactant;

wherein a micelle of the plurality of micelles comprises the lysis surfactant; and

wherein the micelle is capable of transferring the lysis surfactant to the second droplet contacted with the first emulsion, wherein the second droplet comprises: 1) a second droplet interior comprising a cell and the bead of the plurality of beads; and 2) a second droplet surface comprising the fluorosurfactant.

2 . The system of claim 1 , wherein the partitioning fluid and the plurality of beads, when combined with a plurality of cells and vortexed, are capable of forming a second emulsion comprising a second plurality of droplets dispersed in the fluorinated oil, and wherein the second plurality of droplets comprises the second droplet.

3 . The system of claim 2 , wherein the micelle is capable of transferring the lysis surfactant to the second droplet in a combined emulsion formed by contacting the first emulsion with the second emulsion.

4 . The system of claim 1 , wherein the lysis surfactant is capable of lysing the cell inside the second droplet.

5 . The system of claim 1 , wherein the fluorosurfactant inhibits droplet coalescence.

6 . The system of claim 1 , wherein the second droplet does not coalesce with any droplet of the first plurality of droplets.

7 . The system of claim 1 , wherein the fluorosurfactant comprises a carboxylic acid-terminated PFPE ID.

8 . The system of claim 7 , wherein the fluorosurfactant is a first fluorosurfactant and the partitioning fluid further comprises a second fluorosurfactant, wherein the first droplet surface and the second droplet surface further comprise the second fluorosurfactant.

9 . The system of claim 8 , wherein the second fluorosurfactant is a block copolymer comprising polyethylene glycol (PEG) and a perfluoropolyether (PFPE).

10 . The system of claim 9 , wherein the second fluorosurfactant is a PFPE-PEG-PFPE triblock copolymer.

11 . The system of claim 9 , wherein the first fluorosurfactant or the second fluorosurfactant inhibits droplet coalescence.

12 . The system of claim 1 , wherein the system further comprises an RNAse inhibitor, and wherein the second droplet further comprises the RNAse inhibitor.

13 . The system of claim 12 , wherein the partitioning fluid, the plurality of beads, and the RNAse inhibitor, when combined with a plurality of cells and vortexed, are capable of forming a second emulsion comprising a second plurality of droplets dispersed in the fluorinated oil, and wherein the second plurality of droplets comprises the second droplet.

14 . The system of claim 1 , wherein the first plurality of droplets does not comprise nucleic acids.

15 . The system of claim 1 , wherein a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules comprises a bead-specific barcode sequence and a sequence capable of hybridizing to a messenger ribonucleic acid (mRNA) of the cell.

16 . The system of claim 15 , wherein the nucleic acid barcode molecule is configured to be extended by a reverse transcriptase using the mRNA as template to generate a barcoded nucleic acid molecule comprising the bead-specific barcode sequence and a sequence complementary to a sequence of the mRNA.

17 . The system of claim 16 , wherein the bead-specific barcode sequence identifies the second droplet and the sequence complementary to the sequence of the mRNA identifies the mRNA.

18 . The system of claim 16 , wherein the system comprises the reverse transcriptase.

19 . The system of claim 18 , wherein the system comprises a template switching oligonucleotide configured to be used as template by the reverse transcriptase to further extend the nucleic acid barcode molecule.

20 . The system of claim 3 , wherein the second plurality of droplets comprises monodisperse droplets.

21 . The system of claim 1 , wherein the bead is a hydrogel bead.

22 . The system of claim 9 , wherein the second fluorosurfactant inhibits droplet coalescence.

23 . The system of claim 1 , wherein the micelle of the plurality of micelles further comprises the fluorosurfactant.

Assignments (1)

Continuity (5)

References Cited (400)