METHODS AND KITS FOR LABELING CELLULAR MOLECULES
Methods of uniquely labeling or barcoding molecules within a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs, cDNAs, DNAs, proteins, peptides, and/or antigens.
1 . A method of labeling ribonucleic acid (RNA) molecules within cells, the method comprising:
(a) providing a plurality of fixed and permeabilized cells, wherein each of the plurality of cells comprises RNA molecules;
(b) generating complementary deoxyribonucleic acid (cDNA) molecules within the plurality of cells by reverse transcribing the RNA molecules using reverse transcription (RT) primers;
(c) tagging the cDNA molecules within the plurality of cells with one or more barcode sequences, thereby generating a plurality of tagged cDNA molecules;
(d) lysing the plurality of cells to release the tagged cDNA molecules; and
(e) isolating tagged cDNA molecules released from the lysed plurality of cells.
2 . The method of claim 1 , further comprising:
(f) amplifying the tagged cDNA molecules isolated from the lysed plurality of cells.
3 . The method of claim 2 , further comprising:
(g) sequencing the isolated amplified tagged cDNA molecules.
4 . The method of claim 1 , wherein at least a portion of the RT primers comprise a poly(T) sequence or a random sequence.
5 . The method of claim 1 , wherein at least a portion of the RT primers comprise a gene-specific sequence.
6 . The method of claim 1 , wherein the tagging of the cDNA molecules within the plurality of cells in step (c) is cell-specific.
7 . The method of claim 6 , wherein the cell-specific tagging of the cDNA molecules within the plurality of cells comprises performing steps (i) through (iii) one or more times:
(i) dividing the plurality of cells comprising the cDNA molecules into a plurality of aliquots, wherein each aliquot comprises more than one cell;
(ii) coupling nucleic acid tags to cDNA molecules within cells of the aliquots, wherein each nucleic acid tag comprises a barcode sequence, and wherein the barcode sequences present within the nucleic acid tags are specific to each aliquot; and
(iii) combining the cells from the plurality of aliquots.
8 . The method of claim 1 , wherein the cells are lysed using a lysis agent comprising a proteinase.
9 . The method of claim 8 , wherein the proteinase is proteinase K.
10 . The method of claim 7 , wherein at least a portion of the nucleic acid tags coupled to the cDNA molecules comprise a capture agent, and wherein the capture agent is used to isolate the tagged cDNA molecules released from the lysed plurality of cells in step (e).
11 . The method of claim 10 , wherein the capture agent is biotin.
12 . The method of claim 3 , wherein the sequencing is performed using a next generation sequencing (NGS) method.
13 . The method of claim 7 , wherein the nucleic acid tags are coupled to the cDNA molecules within the cells by ligation.
14 . The method of claim 1 , wherein the plurality of cells are mammalian cells.
15 . The method of claim 4 , wherein at least a portion of the random sequences are random hexamer sequences.
16 . The method of claim 4 , wherein at least a portion of the poly(T) sequences comprise 15 dTs.
17 . The method of claim 4 , wherein at least a portion of the poly(T) sequences are anchored poly(T) sequences.
18 . The method of claim 1 , wherein the cells are isolated cells.
19 . The method of claim 1 , wherein the cells are present within a tissue.
20 . The method of claim 1 , wherein the cells are adherent cells.