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 cell-specifically labeling RNA molecules within a plurality of cells, the method comprising:
(a) providing a plurality of fixed, permeabilized cells in admixture in a well or compartment, wherein each of the plurality of cells comprises ribonucleic acid (RNA) molecules;
(b) without lysing the cells, reverse transcribing the RNA molecules within the plurality of cells, thereby generating complementary deoxyribonucleic acid (cDNA) molecules within the plurality of cells, wherein primers used to reverse transcribe the RNA molecules comprise a poly(T) sequence, a mix of random sequences, or both a poly(T) sequence and a mix of random sequences, wherein each primer further comprises a first barcode sequence; wherein the first barcode sequence is specific to each well or compartment;
(c) dividing the plurality of cells comprising the cDNA molecules into a plurality of primary aliquots, wherein the plurality of primary aliquots comprises a first primary aliquot and a second primary aliquot;
(d) providing primary nucleic acid tags comprising a second barcode sequence to the plurality of primary aliquots, wherein the primary nucleic acid tags comprising a second barcode sequence provided to the first primary aliquot are different in sequence from the primary nucleic acid tags provided to the second primary aliquot;
(e) coupling the provided primary nucleic acid tags comprising a second barcode sequence of (d) to the cDNA molecules from each of the plurality of primary aliquots, thereby tagging the cDNA molecules with the primary nucleic acid tags comprising a second barcode sequence and producing primary nucleic acid-tagged cDNA molecules, whereby the primary nucleic acid-tagged cDNA molecules of the first primary aliquot are tagged with a different primary nucleic acid tag than the primary nucleic acid-tagged cDNA molecules of the second primary aliquot;
(f) combining the plurality of primary aliquots;
(g) dividing the combined primary aliquots of (f) into a plurality of secondary aliquots, wherein the plurality of secondary aliquots comprises a first secondary aliquot and a second secondary aliquot;
(h) providing secondary nucleic acid tags comprising a third barcode sequence to the plurality of secondary aliquots, wherein the secondary nucleic acid tags comprising a third barcode sequence provided to the first secondary aliquot are different in sequence from the secondary nucleic acid tags provided to the second secondary aliquot; and
(i) coupling the provided secondary nucleic acid tags comprising a third barcode sequence of (h) to the primary nucleic acid-tagged cDNA molecules of (e) thereby tagging the primary nucleic acid-tagged cDNA molecules with the secondary nucleic acid tags comprising a third barcode sequence and producing secondary nucleic acid-tagged cDNA molecules, whereby the secondary nucleic acid-tagged cDNA molecules of the first secondary aliquot are tagged with a different secondary nucleic acid tag than the secondary nucleic acid-tagged cDNA molecules of the second secondary aliquot.
2. The method of claim 1 , further comprising (j) repeating (f), (g), (h), and (i) with subsequent aliquots.
3. The method of claim 2 , wherein (j) is repeated a number of times sufficient to generate a unique series of nucleic acid tags for the cDNA molecules in a single cell of the plurality of cells.
4. The method of claim 3 , wherein the number of times is 1, 2, 3, 4, or 5.
5. The method of claim 1 , wherein each of the primary nucleic acid tags comprises a first strand comprising the second barcode sequence.
6. The method of claim 5 , wherein each of the primary nucleic acid tags further comprises a 3′ hybridization sequence flanking the 3′ end of the second barcode sequence, or a 5′ hybridization sequence flanking the 5′ end of the second barcode sequence, or both.
7. The method of claim 1 , wherein each of the secondary nucleic acid tags comprises a first strand comprising the third barcode sequence.
8. The method of claim 7 , wherein each of the secondary nucleic acid tags further comprises a 3′ hybridization sequence flanking the 3′ end of the third barcode sequence, or a 5′ hybridization sequence flanking the 5′ end of the third barcode sequence, or both.
9. The method of claim 1 , further comprising, prior to (a), hybridizing a single-stranded adapter sequence to each of the RNA molecules.
10. The method of claim 1 , further comprising, subsequent to (b), ligating an adapter sequence to each of the cDNA molecules.
11. The method of claim 1 , further comprising, subsequent to (b), coupling an adapter sequence to each of the cDNA molecules by integrating the adapter sequence into the cDNA molecules using a transposase and releasing the transposase to expose the adapter sequence.
12. The method of claim 1 , further comprising ligating the primary nucleic acid tag of a primary nucleic acid-tagged cDNA molecule to the secondary nucleic acid tag.
13. The method of claim 10 , wherein the ligation is performed within the plurality of cells.
14. The method of claim 1 , further comprising removing primary nucleic acid tags or secondary nucleic acid tags that are unbound to the cDNA molecules.
15. The method of claim 2 , wherein a final nucleic acid tag coupled to the cDNA molecules comprises a capture agent.
16. The method of claim 1 , further comprising lysing the plurality of cells to release the cDNA molecules from within the plurality of cells wherein the lysing is subsequent to (i).
17. The method of claim 12 , further comprising lysing the plurality of cells to release the cDNA molecules from within the plurality of cells prior to the ligation.
18. The method of claim 16 , wherein the lysing is subsequent to (i), and wherein a majority of the nucleic acid-tagged cDNA molecules from a single cell comprises the same plurality of nucleic acid tags coupled thereto.
19. The method of claim 1 , wherein the plurality of cells is selected from the group consisting of mammalian cells, yeast cells, bacterial cells, and combinations thereof.
20. The method of claim 1 , further comprising, prior to (a), ligating a single-stranded adapter sequence to each of the RNA molecules.
21. The method of claim 20 , wherein a 5′ end of the single-stranded adapter sequence is ligated to a 3′ end of an RNA molecule or a 3′ end of the single-stranded adapter sequence is ligated to a 5′ end of an RNA molecule.
22. The method of claim 1 , wherein the primers comprise a poly(T) sequence.
23. The method of claim 1 , wherein the primers comprise a random hexamer sequence.
24. The method of claim 10 , further comprising providing the cDNA molecules with a plurality of ligation stop oligos complementary to the primary nucleic acid tags after ligating the primary nucleic acid tag.
25. The method of claim 1 , wherein the plurality of cells is fixed using formaldehyde.
26. The method of claim 1 , wherein combining the plurality of primary aliquots includes combining cells of the plurality of primary aliquots; and wherein dividing the combined primary aliquots of (f) into a plurality of secondary aliquots includes dividing cells of the combined primary aliquots in the plurality of secondary aliquots.
27. The method of claim 8 , wherein each of the secondary nucleic acid tags further comprises a second strand comprising: a first portion complementary to at least one of the 5′ hybridization sequence, and a second portion complementary to the 3′ hybridization sequence.