	US 12,247,248 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 Jun. 25, 2024, as Appl. No. 18/753,319.
Application 18/753,319 is a continuation of application No. 18/455,113, filed on Aug. 24, 2023.
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 2024/0336953 A1, Oct. 10, 2024
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)]

23 Claims

1. A kit for preparing a nucleic acid library for single-cell transcriptome analysis, the kit comprising:

(a) a plurality of single-stranded nucleic acid molecules configured to hybridize to the transcriptome of a cell, each comprising:

(i) a first hybridization sequence, located at a first end of the single-stranded nucleic acid molecule, comprising complementarity to the transcriptome of the cell; and

(ii) 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 the one or more sets comprises a barcode sequence;

wherein multiple distinct barcode sequences are present among the nucleic acid tags of each set;

wherein the nucleic acid tags of at least one of the one or more sets comprise a single-stranded terminal region comprising a third hybridization sequence, and

wherein the third hybridization sequence is complementary to the second hybridization sequence present within the plurality of single-stranded nucleic acid molecules within the cell;

(d) a lysis agent;

(e) a plurality of amplification primers, comprising forward and reverse amplification primers, wherein at least a portion of the forward amplification primers or at least a portion of the reverse amplification primers comprises an index sequence, and wherein multiple distinct index sequences are present among the forward amplification primers or among the reverse amplification primers; and

(f) a deoxyribonucleic acid (DNA) polymerase suitable for polymerase chain reaction (PCR) amplification.