US 12,227,741 B2
Method to perform high-throughput single cell genomic and phenotypic analyses
Polly Fordyce, Menlo Park, CA (US); Kara Brower, Palo Alto, CA (US); Sandy Klemm, Palo Alto, CA (US); and William Greenleaf, Menlo Park, CA (US)
Assigned to CZ Biohub SF, LLC, San Francisco, CA (US); and The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
Appl. No. 17/257,509
Filed by CZ Biohub SF LLC, San Francisco, CA (US); and The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US)
PCT Filed Jul. 1, 2019, PCT No. PCT/US2019/040139
§ 371(c)(1), (2) Date Dec. 31, 2020,
PCT Pub. No. WO2020/009998, PCT Pub. Date Jan. 9, 2020.
Claims priority of provisional application 62/693,800, filed on Jul. 3, 2018.
Prior Publication US 2021/0261953 A1, Aug. 26, 2021
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6806 (2018.01)
CPC C12N 15/1093 (2013.01) [C12N 15/1075 (2013.01); C12Q 1/6806 (2013.01)] 20 Claims
OG exemplary drawing
 
1. A method of producing a library of single cells for multi-parameter analysis, comprising:
(a) generating a population of double emulsion droplets at least some of which comprise single cells encapsulated therein, wherein the double emulsion droplets further comprise reagents for a first single cell assay to analyze a first parameter;
(b) incubating the droplets for a time sufficient to generate a reaction product for the first single cell assay;
(c) sorting the population of droplets by flow cytometry to select for single droplets comprising one or more cells, or the contents of one or more cells;
(d) distributing single droplets that comprise single cells into separate dry compartments;
(e) breaking individual droplets in each separate dry compartment by shear or osmotic force to produce a plurality of individual compartments each with the content of a single droplet;
(f) diluting the contents of individual compartments by at least 500-fold to provide a diluted reaction volume; and
(g) measuring a second parameter in a second single cell assay in the diluted reaction volume.