# US 12,600,961 B2 · Methods and systems for droplet-based single cell barcoding

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- Number: US 12,600,961 B2
- Document ID: US12600961B2
- Status: Granted
- Kind: B2
- Application number: 17/517,282
- Filed: 2021-11-02
- Published: 2022-06-23
- Granted: 2026-04-14
- Earliest claimed: 2017-01-30
- Assignees: 10x Genomics, Inc.
- Applicants: 10x Genomics, Inc.
- Inventors: Zahra Kamila Belhocine; Rajiv Bharadwaj; Christopher Hindson; Michael Schnall-Levin; Bill Kengli Lin; Anthony Makarewicz; Pranav Patel; Andrew D. Price; Mohammad Rahimi Lenji; Tobias Daniel Wheeler; Yifeng Yin
- Web page: https://patentgrep.com/patent/US12600961B2

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/09: Recombinant DNA-technology
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- B01L 3/502761: {specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules}
- B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics}
- B01L 2200/0652: Sorting or classification of particles or molecules
- B01L 2200/0668: Trapping microscopic beads
- B01L 2200/0673: Handling of plugs of fluid surrounded by immiscible fluid
- B01L 2300/021: Identification, e.g. bar codes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

## Abstract

Methods and systems are provided for sample preparation techniques and sequencing of macromolecular constituents of cells and other biological materials.

## Claims

### Claim 1

A method for processing or analyzing one or more components from a sample, comprising:
(a) subjecting a plurality of cell beads to bulk denaturation, wherein a cell bead of said plurality of cell beads comprises one or more macromolecular constituents, derived from a cell or a virus, encapsulated within a polymer or gel matrix, wherein said one or more macromolecular constituents comprise a nucleic acid molecule, and wherein said bulk denaturation comprises denaturing said nucleic acid molecule to generate a denatured nucleic acid molecule in said cell bead;
(b) partitioning said plurality of cell beads and a plurality of barcode beads into a plurality of partitions, wherein a barcode bead of said plurality of barcode beads comprises a plurality of nucleic acid barcode molecules for barcoding said nucleic acid molecule, and wherein upon partitioning, a partition of said plurality of partitions comprises said cell bead and said barcode bead.

### Claim 2 (depends on 1)

The method of claim 1, further comprising, (c) using a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules and said denatured nucleic acid molecule or derivative thereof to generate a barcoded nucleic acid molecule.

### Claim 3 (depends on 2)

The method of claim 2, further comprising subjecting said barcoded nucleic acid molecule or derivative thereof to sequencing.

### Claim 4 (depends on 2)

The method of claim 2, further comprising coupling a functional sequence to said barcoded nucleic acid molecule or derivative thereof, wherein said functional sequence permits attachment of said barcoded nucleic acid molecule or derivative thereof to a flow cell of a sequencer.

### Claim 5 (depends on 1)

The method of claim 1, wherein, in (a), said one or more macromolecular constituents derived from said cell comprises an organelle, a cell protein, a ribosome, or a cellular enzyme.

### Claim 6 (depends on 1)

The method of claim 1, wherein said cell bead comprises said cell or said virus.

### Claim 7 (depends on 1)

The method of claim 1, wherein said plurality of partitions is a plurality of droplets or a plurality of wells.

### Claim 8 (depends on 1)

The method of claim 1, wherein said barcode bead or said cell bead is degradable upon application of a stimulus selected from the group consisting of a chemical stimulus, a biological stimulus, a temperature change, exposure to light, and a pH change.

### Claim 9 (depends on 1)

The method of claim 1, further comprising: prior to (a), partitioning a plurality of macromolecular constituents and polymer or gel precursors into a plurality of first partitions; and subjecting said plurality of first partitions to conditions sufficient to polymerize or crosslink said polymer or gel precursors in said plurality of first partitions to generate said plurality of cell beads.

### Claim 10 (depends on 1)

The method of claim 1, wherein said plurality of nucleic acid barcode molecules is a plurality of double-stranded nucleic acid barcode molecules.

### Claim 11 (depends on 10)

The method of claim 10, further comprising (i) performing one or more nucleic acid extension reactions on said denatured nucleic acid molecule to generate a double-stranded nucleic acid molecule comprising a sequence of said denatured nucleic acid molecule and (ii) ligating said nucleic acid barcode molecule to said double-stranded nucleic acid molecule to generate a barcoded nucleic acid molecule.

### Claim 12 (depends on 11)

The method of claim 11, wherein said one or more nucleic acid extension reactions comprise:
(i) annealing a first primer to said denatured nucleic acid molecule and performing a first nucleic acid extension reaction in the presence of uracil to generate a first nucleic acid extension product comprising a uracil-containing moiety;
(ii) excising said uracil-containing moiety to generate a nick in said first nucleic acid extension product;
(iii) performing a second nucleic acid extension reaction on said first nucleic acid extension product comprising said nick to generate a plurality of single-stranded nucleic acid fragments;
(iv) annealing a second primer to a single-stranded nucleic acid fragment of said plurality of single-stranded nucleic acid fragments; and
(v) performing a third nucleic acid extension reaction to generate a second nucleic acid extension product.

### Claim 13 (depends on 12)

The method of claim 12, wherein said second nucleic acid extension reaction is performed using a polymerase having strand displacement activity, wherein the polymerase engages the first nucleic acid extension product at the nick, and wherein said plurality of single-stranded nucleic acid fragments are displaced from the first nucleic acid extension product.

### Claim 14 (depends on 1)

The method of claim 1, wherein said bulk denaturation comprises contacting said plurality of cell beads with a chemical agent.

### Claim 15 (depends on 14)

The method of claim 14, wherein said chemical agent is sodium hydroxide.

### Claim 16 (depends on 1)

The method of claim 1, wherein said plurality of cell beads comprises a plurality of magnetic particles.

### Claim 17 (depends on 16)

The method of claim 16, further comprising, using a magnetic source to purify said plurality of cell beads subsequent to said bulk denaturation.

### Claim 18 (depends on 1)

The method of claim 1, wherein said plurality of nucleic acid barcode molecules are releasable from said barcode bead upon application of a stimulus selected from the group consisting of a chemical stimulus, a biological stimulus, a temperature change, exposure to light, and a pH change.

### Claim 19 (depends on 1)

The method of claim 1, wherein a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises one or more functional sequences selected from the group consisting of: a unique molecular index (UMI), a target-specific primer sequence, a random primer sequence, a sequencing primer sequence, and a sequence configured to attach to a flow cell of a sequencer.