Methods and kits for labeling cellular molecules

A method of labeling RNA molecules 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 RNA;
• (b) reverse transcribing RNA molecules within the cells 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, and
• ii) 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, and
• wherein following reverse transcription of the RNA, the 5′ overhang sequence is present at the 5′ end of each of the cDNA molecules;
• (c) dividing the plurality of cells comprising the cDNA molecules 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 cDNA molecules within cells of the plurality of aliquots, thereby generating tagged cDNA molecules, 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 cDNA molecules;
• and
• (h) amplifying the released tagged cDNA molecules in the plurality of samples using amplification primers,
• wherein at least a portion of the amplification primers used in each of the plurality of samples 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.