# US 10,501,739 B2 · Method, systems and apparatus for single cell analysis

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- Number: US 10,501,739 B2
- Document ID: US10501739B2
- Status: Granted
- Kind: B2
- Application number: 16/169,959
- Filed: 2018-10-24
- Published: 2019-04-18
- Granted: 2019-12-10
- Earliest claimed: 2017-10-18
- Assignees: Mission Bio, Inc.
- Applicants: Mission Bio, Inc.
- Inventors: Dennis Jay Eastburn
- Web page: https://patentgrep.com/patent/US10501739B2

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/1075: {by coupling phenotype to genotype, not provided for in other groups of this subclass}
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- 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

## Abstract

The disclosed embodiments relate to method, apparatus and system for high throughput single-cell DNA sequencing with droplet microfluidic. In an exemplary embodiment, a method for analyzing nucleic acids within a cell includes the steps of: (a) flowing individual cells together with a material capable of forming a polymer or microsphere that retains nucleic acids into a carrier fluid such that droplets are formed; (b) breaking the emulsion and collecting the microsphere hydrogels in an aqueous fluid; and (c) performing combinatorial labeling on the nucleic acids contained within the microspheres/hydrogels.

## Claims

### Claim 1

A method for analyzing nucleic acids within a cell, comprising:
(a) combining a plurality of molecular barcodes on a polymeric support with a plurality of cells in a carrier fluid to form a plurality of microsphere in an emulsion, wherein the carrier fluid defines a continuous phase fluid;
(b) disrupting the polymeric support to capture a plurality of nucleic acids contained within a respective plurality of cells and then re-polymerizing the support material;
(c) breaking the emulsion and collecting the microspheres in an aqueous fluid wherein each of the plurality of nucleic acids is retained within a respective one of the plurality of microspheres, each collected microsphere having a respective nucleic acid and an associated barcode;
(d) re-emulsifying the collected microspheres; and
(e) using the associated barcode to label the associated nucleic acid with a reaction.

### Claim 2 (depends on 1)

The method of claim 1, wherein step (a) is performed under microfluidic control.

### Claim 3 (depends on 1)

The method of claim 1, wherein step (a) is done with agitation or shaking.

### Claim 4 (depends on 1)

The method of claim 1, wherein step (c) is performed under microfluidic control.

### Claim 5 (depends on 1)

The method of claim 1, wherein step (c) is done with agitation or shaking.

### Claim 6 (depends on 1)

The method of claim 1, wherein the polymeric support defines a polymeric solid support.