US 11,739,321 B2
Compositions and methods for genomic DNA and gene expression analysis in single cells
James Eberwine, Philadelphia, PA (US); Jae-Hee Lee, Philadelphia, PA (US); Jifen Li, Philadelphia, PA (US); Stephen Fisher, Philadelphia, PA (US); Youtao Lu, Philadelphia, PA (US); Junhyong Kim, Philadelphia, PA (US); Jai-Yoon Sul, Philadelphia, PA (US); Jinchun Wang, Santa Clara, CA (US); and Mimi Healy, Santa Clara, CA (US)
Assigned to Agilent Technologies, Inc., Santa Clara, CA (US)
Filed by Agilent Technologies, Inc., Santa Clara, CA (US); and The Trustees of the University of Pennsylvania, Philadelphia, PA (US)
Filed on Jan. 7, 2020, as Appl. No. 16/736,085.
Claims priority of provisional application 62/898,824, filed on Sep. 11, 2019.
Claims priority of provisional application 62/789,073, filed on Jan. 7, 2019.
Prior Publication US 2020/0216841 A1, Jul. 9, 2020
Int. Cl. C12Q 1/6816 (2018.01); C12N 15/11 (2006.01); C07H 19/10 (2006.01); C07H 21/02 (2006.01); C07H 21/04 (2006.01)
CPC C12N 15/11 (2013.01) [C07H 19/10 (2013.01); C12Q 1/6816 (2013.01); C07H 21/02 (2013.01); C07H 21/04 (2013.01); C12Y 207/07006 (2013.01); C12Y 207/07007 (2013.01); C12Y 207/07049 (2013.01)] 24 Claims
 
1. A method for identifying regions of open DNA in a cell, the method comprising:
(a) introducing a population of oligonucleotide molecules into the cell, wherein each molecule comprises, from 5′ to 3′, an amplification segment, an index barcode segment, a hybridization segment, and a reversibly terminating nucleotide;
(b) incubating the cell under conditions to allow for the hybridization segments of the population of oligonucleotide molecules to anneal to regions of open DNA;
(c) activating at least a portion of the annealed oligonucleotide molecules to expose an extendable 3′ hydroxy group; and
(d) synthesizing cDNAs from the open DNA by extending the activated oligonucleotide molecules from their extendable 3′ hydroxy groups.