| 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)] | 30 Claims |
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1. A method of labeling RNA molecules with nucleus-specific tags, the method comprising:
(a) fixing and permeabilizing a plurality of nuclei, wherein each of the plurality of fixed, permeabilized nuclei comprises RNA, and wherein the plurality of nuclei is fixed and permeabilized at a temperature below about 8° C.;
(b) dividing the plurality of nuclei into a first plurality of aliquots, wherein each aliquot of the first plurality of aliquots comprises more than one nucleus;
(c) reverse transcribing RNA molecules within the nuclei of the first plurality of aliquots to generate complementary DNA (cDNA) molecules, wherein the RNA molecules are reverse transcribed using reverse transcription (RT) primers each comprising:
(i) a poly(T) sequence or a random nucleotide sequence,
(ii) an RT primer barcode sequence,
wherein multiple distinct RT primer barcode sequences are present among the RT primers used in the first plurality of aliquots; and
wherein the RT primer barcode sequences present in each individual aliquot of the first plurality of aliquots are specific to the individual aliquot; and
(iii) a 5′ overhang comprising a 5′ overhang sequence located 5′ of the poly(T) or the random nucleotide sequence,
wherein the 5′ overhang sequence is the same in all of the RT primers used in the first plurality of aliquots, and
wherein following reverse transcription of the RNA, the 5′ overhang sequence is present at the 5′ end of each of the cDNA molecules;
(d) combining the nuclei from the first plurality of aliquots;
(e) dividing the combined nuclei from the first plurality of aliquots into a second plurality of aliquots, wherein each of the second plurality of aliquots comprises more than one nucleus;
(f) coupling nucleic acid tags to the cDNA molecules within nuclei of the second plurality of aliquots, thereby generating tagged cDNA molecules, wherein each of the nucleic acid tags comprises:
i) a tag 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 tag barcode sequences are present among the nucleic acid tags used in the second plurality of aliquots, and
wherein the tag barcode sequences present in each individual aliquot of the second plurality of aliquots are specific to the individual aliquot; and
(g) combining the nuclei from the second plurality of aliquots;
(h) dividing the combined nuclei from the second plurality of aliquots into a plurality of samples, wherein each sample of the plurality of samples comprises more than one nucleus; and
(i) lysing the nuclei in the plurality of samples, thereby releasing the tagged cDNA molecules and forming a lysate in each sample of the plurality of samples.
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