US 12,305,220 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 Sep. 17, 2024, as Appl. No. 18/887,084.
Application 18/887,084 is a continuation of application No. 18/784,019, filed on Jul. 25, 2024, granted, now 12,252,736.
Application 18/784,019 is a continuation of application No. 18/753,319, filed on Jun. 25, 2024, granted, now 12,247,248.
Application 18/753,319 is a continuation of application No. 18/455,113, filed on Aug. 24, 2023, granted, now 12,247,247.
Application 18/455,113 is a continuation of application No. 18/158,487, filed on Jan. 24, 2023, granted, now 12,043,864, issued on Jul. 23, 2024.
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 2025/0011836 A1, Jan. 9, 2025
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)] 16 Claims
 
1. A method of preparing a nucleic acid library for single-cell transcriptome analysis, the method comprising:
(a) fixing a plurality of cells;
(b) generating a plurality of tagged cDNA molecules from the transcriptome of each cell of the plurality of fixed cells,
wherein the plurality of tagged cDNA molecules generated from the transcriptome of each cell corresponds to the transcriptome of the cell; and
wherein each tagged cDNA molecule of the plurality of tagged cDNA molecules comprises a barcode sequence corresponding to the cell of origin of the tagged cDNA molecule;
(c) amplifying the plurality of tagged cDNA molecules generated from the transcriptome of each cell using a plurality of amplification primers,
wherein the plurality of amplification primers comprises 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 comprise an index sequence, and
wherein multiple index sequences are present among the forward amplification primers or among the reverse amplification primers;
(d) sequencing the amplified tagged cDNA molecules; and
(e) mapping the sequencing reads obtained in (d) to a reference genome.