US 11,965,877 B2
Compositions and methods for molecular labeling
Michael Samuels, Windham, NH (US); Jeffrey Charles Olson, Chelmsford, MA (US); Andrew Watson, Bedford, MA (US); Keith Brown, Carlsbad, CA (US); and Darren R. Link, Lexington, MA (US)
Assigned to Bio-Rad Laboratories, Inc., Hercules, CA (US)
Filed by Bio-Rad Laboratories, Inc., Hercules, CA (US)
Filed on Oct. 5, 2015, as Appl. No. 14/874,553.
Application 14/874,553 is a continuation of application No. 13/398,677, filed on Feb. 16, 2012, granted, now 9,150,852.
Claims priority of provisional application 61/476,714, filed on Apr. 18, 2011.
Claims priority of provisional application 61/444,612, filed on Feb. 18, 2011.
Prior Publication US 2016/0289670 A1, Oct. 6, 2016
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/53 (2006.01); C12N 15/10 (2006.01); C12Q 1/6804 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6874 (2018.01); C40B 50/08 (2006.01); G01N 33/532 (2006.01); G01N 33/543 (2006.01); G01N 33/58 (2006.01)
CPC G01N 33/53 (2013.01) [C12N 15/1075 (2013.01); C12Q 1/6804 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6874 (2013.01); C40B 50/08 (2013.01); G01N 33/532 (2013.01); G01N 33/5436 (2013.01); G01N 33/58 (2013.01); C12Q 2521/10 (2013.01); C12Q 2563/179 (2013.01); C12Q 2565/30 (2013.01); G01N 2458/10 (2013.01)] 13 Claims
 
1. A method for barcoding RNA from a single cell, the method comprising:
forming a plurality of aqueous droplets in oil, each droplet comprising a plurality of different RNA molecules from a single cell and a plurality of nucleic acid constructs not attached to microbeads, wherein each of the nucleic acid constructs comprises a universal priming site, a barcode sequence and a poly(dT) sequence, wherein the barcode sequence is a unique barcode that allows sequence reads to be assigned to one molecule from a sample;
hybridizing at least one nucleic acid construct to at least one RNA molecule in at least one of the droplets to form at least one hybridized nucleic acid construct that is not attached to a microbead;
extending the at least one hybridized nucleic acid construct that is not attached to the microbead to form an extension product that is not attached to a microbead; and
amplifying, using the universal priming site, the extension product to yield an amplified product.