US 12,252,730 B2
In situ combinatorial labeling of cellular molecules
Georg Seelig, Seattle, WA (US); Alexander B. Rosenberg, Seattle, WA (US); and Charles Roco, Seattle, WA (US)
Assigned to University of Washington, Seattle, WA (US)
Filed by UNIVERSITY OF WASHINGTON, Seattle, WA (US)
Filed on Apr. 21, 2023, as Appl. No. 18/304,670.
Application 18/304,670 is a continuation of application No. 16/649,601, granted, now 11,680,283, previously published as PCT/US2018/052283, filed on Sep. 21, 2018.
Claims priority of provisional application 62/561,806, filed on Sep. 22, 2017.
Prior Publication US 2023/0304073 A1, Sep. 28, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6806 (2018.01)
CPC C12Q 1/6806 (2013.01) [C12Q 2521/107 (2013.01); C12Q 2525/161 (2013.01); C12Q 2543/101 (2013.01); C12Q 2563/179 (2013.01); C12Q 2563/185 (2013.01); C12Q 2565/514 (2013.01)] 27 Claims
 
1. A method of cell-specifically labeling RNA molecules within cells, the method comprising:
(a) fixing and permeabilizing a plurality of cells, wherein the plurality of cells is fixed and permeabilized at a temperature below about 8° C.;
(b) dividing the plurality of cells into a first plurality of aliquots, wherein each of the first plurality of aliquots comprises more than one cell;
(c) generating complementary DNA (cDNA) molecules within cells of the first plurality of aliquots by reverse transcribing RNA molecules within the cells, wherein
the RNA molecules are reverse transcribed using reverse transcription (RT) primers each comprising: (i) a poly (T) sequence or a random nucleotide sequence, and (ii) an RT primer barcode sequence; and wherein
the RT primer barcode sequences present within the RT primers are specific to each aliquot of the first plurality of aliquots;
(d) pooling the cells from the first plurality of aliquots;
(e) dividing the pooled cells from the first plurality of aliquots into a second plurality of aliquots, wherein each of the second plurality of aliquots comprises more than one cell;
(f) coupling nucleic acid tags to cDNA molecules within cells of the second plurality of aliquots, wherein each of the nucleic acid tags comprises a tag barcode sequence, and wherein the tag barcode sequences present within the nucleic acid tags are specific to each aliquot of the second plurality of aliquots;
(g) pooling the cells from the second plurality of aliquots;
(h) lysing the pooled cells from the second plurality of aliquots to release the cDNA molecules and form a lysate; and
(i) adding a protease inhibitor and a binding agent to the lysate such that the cDNA molecules bind the binding agent.