	US 12,227,793 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 Dec. 12, 2023, as Appl. No. 18/536,721.
Application 18/536,721 is a continuation of application No. 18/455,152, filed on Aug. 24, 2023.
Application 18/455,152 is a continuation of application No. 18/188,504, filed on Mar. 23, 2023.
Application 18/188,504 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 2024/0110226 A1, Apr. 4, 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)]

26 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) reverse transcribing the transcriptome of each of the plurality of cells within the cells, thereby generating a plurality of cDNA molecules in each cell corresponding to the cell's transcriptome;

(c) coupling nucleic acid tags to the plurality of cDNA molecules within each cell, thereby generating a plurality of tagged cDNA molecules;

(d) lysing the plurality of cells and releasing the tagged cDNA molecules, thereby forming a lysate comprising the released tagged cDNA molecules;

(e) isolating the released tagged cDNA molecules from the lysate;

(f) amplifying the isolated tagged cDNA molecules using amplification primers; and

(g) sequencing the amplified tagged cDNA molecules.