US 12,247,247 B2
Methods and kits for labeling cellular molecules
Georg Seelig, Seattle, WA (US); Richard Muscat, London (GB); and Alexander B. Rosenberg, Seattle, WA (US)
Assigned to University of Washington, Seattle, WA (US)
Filed by University of Washington, Seattle, WA (US)
Filed on Aug. 24, 2023, as Appl. No. 18/455,113.
Application 18/455,113 is a continuation of application No. 18/158,487, filed on Jan. 24, 2023, granted, now 12,043,864.
Application 18/158,487 is a continuation of application No. 17/814,712, filed on Jul. 25, 2022, granted, now 11,639,519, issued on May 2, 2023.
Application 17/814,712 is a continuation of application No. 17/695,671, filed on Mar. 15, 2022, granted, now 11,555,216, issued on Jan. 17, 2023.
Application 17/695,671 is a continuation of application No. 17/521,263, filed on Nov. 8, 2021, granted, now 11,427,856, issued on Aug. 30, 2022.
Application 17/521,263 is a continuation of application No. 17/249,257, filed on Feb. 25, 2021, granted, now 11,168,355, issued on Nov. 9, 2021.
Application 17/249,257 is a continuation of application No. 17/122,321, filed on Dec. 15, 2020, granted, now 11,634,751, issued on Apr. 25, 2023.
Application 17/122,321 is a continuation of application No. 14/941,433, filed on Nov. 13, 2015, granted, now 10,900,065, issued on Jan. 26, 2021.
Claims priority of provisional application 62/080,055, filed on Nov. 14, 2014.
Prior Publication US 2023/0392193 A1, Dec. 7, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6806 (2018.01); C12N 15/10 (2006.01); C12Q 1/6855 (2018.01)
CPC C12Q 1/6806 (2013.01) [C12N 15/1065 (2013.01); C12Q 1/6855 (2013.01)] 20 Claims
 
1. A kit for labeling ribonucleic acid (RNA) molecules for single-cell RNA profiling, the kit comprising:
(a) a plurality of single-stranded nucleic acid molecules, each comprising:
(i) a first hybridization sequence, located at a first end of the single-stranded nucleic acid molecule, comprising complementarity to an RNA transcript;
(ii) a first barcode sequence, wherein multiple distinct first barcode sequences are present among the plurality of single-stranded nucleic acid molecules; and
(iii) a second hybridization sequence, located at a second end of the single-stranded nucleic acid molecule, wherein the second hybridization sequence is the same in each of the plurality of single-stranded nucleic acid molecules;
(b) one or more sets of nucleic acid tags, wherein each of the nucleic acid tags within each of the one or more sets comprises a second barcode sequence,
wherein multiple distinct second barcode sequences are present among each of the one or more sets of nucleic acid tags,
wherein the nucleic acid tags of at least one of the one or more sets of nucleic acid tags comprise a third hybridization sequence that is complementary to the second hybridization sequence present in the plurality of single-stranded nucleic acid molecules, and
wherein at least a portion of the nucleic acid tags further comprise one or more sequence elements selected from the group consisting of a unique molecular identifier (UMI), a capture agent, a next-generation sequencing (NGS) adapter sequence, and an NGS primer binding sequence;
(c) a ligation agent;
(d) a lysis agent, wherein the lysis agent comprises a protease;
(e) a plurality of amplification primers, wherein each of the amplification primers comprises one or more sequence elements selected from the group consisting of an index sequence, a flow cell binding sequence, an NGS primer binding sequence, and an NGS adapter sequence; and
(f) a deoxyribonucleic acid (DNA) polymerase suitable for polymerase chain reaction (PCR) amplification.