US 12,252,733 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. 18/455,080.
Application 18/455,080 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 2023/0392192 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)] 25 Claims
 
1. A kit for labeling ribonucleic acid (RNA) molecules in situ, the kit comprising:
(a) a plurality of reverse transcription (RT) primers, each comprising:
(i) a poly(T) sequence or a random sequence, and
(ii) a 5′ RT overhang sequence located 5′ of the poly(T) sequence or random sequence, wherein the 5′ RT overhang sequence comprises a 5′ RT terminal sequence that is the same in all of the plurality of RT primers;
(b) a reverse transcriptase;
(c) one or more sets of nucleic acid tags, wherein each of the nucleic acid tags within the one or more sets comprises:
a barcode sequence, wherein multiple distinct barcode sequences are present among the nucleic acid tags of each set; and
a hybridization sequence, wherein the hybridization sequence is the same among the nucleic acid tags of each set, and wherein the hybridization sequence present in the nucleic acid tags of at least one set is complementary to the 5′ RT terminal sequence;
(d) a ligation agent;
(e) a lysis agent;
(f) a plurality of amplification primers, wherein at least a portion of the amplification primers comprise an index sequence, and wherein multiple distinct index sequences are present among the plurality of amplification primers; and
(g) a deoxyribonucleic acid (DNA) polymerase suitable for polymerase chain reaction (PCR) amplification.