	US 11,987,838 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. 18/536,654.
Application 18/536,654 is a continuation of application No. 17/814,771, filed on Jul. 25, 2022.
Application 17/814,771 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 2024/0102080 A1, Mar. 28, 2024
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)]

30 Claims

1. A method of labeling genomic DNA with cell-specific tags, the method comprising:

(a) providing a plurality of fixed and permeabilized cells, wherein each of the plurality of fixed, permeabilized cells comprises genomic DNA;

(b) fragmenting the genomic DNA within the cells to produce a plurality of genomic DNA fragments, and appending a nucleic acid adapter in a non-target-specific manner to the ends of the plurality of genomic DNA fragments within the cells, thereby producing a plurality of adapter-coupled genomic DNA fragments;

(c) dividing the plurality of cells comprising the adapter-coupled genomic DNA fragments into a plurality of aliquots, wherein each aliquot of the plurality of aliquots comprises more than one cell;

(d) coupling nucleic acid tags to the adapter-coupled genomic DNA fragments within cells of the plurality of aliquots, thereby generating tagged genomic DNA fragments, wherein each of the nucleic acid tags comprises:

i) a barcode sequence, and

ii) a 3′ hybridization sequence located 3′ of the barcode sequence and/or a 5′ hybridization sequence located 5′ of the barcode sequence,

wherein multiple distinct barcode sequences are present among the nucleic acid tags used in the plurality of aliquots, and

wherein the barcode sequences present in each individual aliquot of the plurality of aliquots are specific to the individual aliquot;

(e) combining the cells from the plurality of aliquots;

(f) dividing the combined cells from the plurality of aliquots into a plurality of samples, wherein each sample of the plurality of samples comprises more than one cell;

(g) lysing the cells in the plurality of samples to release the tagged genomic DNA fragments; and

(h) amplifying the released tagged genomic DNA fragments in the plurality of samples using amplification primers,

wherein at least a portion of the amplification primers used in each sample comprise an index sequence,

wherein multiple distinct index sequences are present among the amplification primers used in the plurality of samples, and

wherein the index sequences present in each individual sample of the plurality of samples are specific to the individual sample.