US 12,305,219 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. 18/887,080.
Application 18/887,080 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 2025/0011835 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)] 25 Claims
 
1. A method of preparing a nucleic acid library for single-cell transcriptome analysis, the method comprising:
(a) fixing and permeabilizing a plurality of cells;
(b) generating a plurality of tagged cDNA molecules within each of the plurality of fixed and permeabilized cells;
wherein the plurality of tagged cDNA molecules generated within each of the plurality of cells correspond to the transcriptome of the cell;
wherein the plurality of fixed and permeabilized cells are present within a plurality of aliquots during the generating, with each aliquot of the plurality of aliquots comprising more than one cell; and
wherein each of the plurality of tagged cDNA molecules comprises one or more aliquot-specific barcode sequences;
(c) lysing the plurality of cells, thereby releasing the plurality of tagged cDNA molecules from each of the plurality of cells;
(d) isolating the tagged cDNA molecules released from each of the plurality of cells;
(e) amplifying the isolated tagged cDNA molecules using a plurality of amplification primers;
wherein at least a portion of the plurality of amplification primers comprise an index sequence; and
wherein multiple index sequences are present among the plurality of amplification primers; and
(f) sequencing the amplified tagged cDNA molecules.