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 cells, the method comprising:
(a) providing a plurality of permeabilized, fixed cells;
(b) generating complementary DNA (cDNA) molecules within the plurality of cells by reverse transcribing RNA molecules within the cells, wherein the RNA molecules are reverse transcribed using reverse transcription (RT) primers each comprising a poly(T) sequence or a random nucleotide sequence;
(c) dividing the plurality of cells comprising the cDNA molecules into a plurality of aliquots, wherein each aliquot comprises more than one cell;
(d) 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;
(e) combining the cells from the plurality of aliquots;
(f) dividing the combined cells from the plurality of aliquots into a plurality of samples;
(g) lysing the cells in the plurality of samples to release the cDNA molecules; and
(h) amplifying the released cDNA molecules in the plurality of samples using one or more amplification primers, wherein at least one of the amplification primers used in each sample comprises an index sequence, and wherein the index sequences present within the amplification primers are specific to each sample.
2. The method of claim 1 , wherein steps (c) through (e) are repeated one or more times.
3. The method of claim 2 , wherein steps (c) through (e) are repeated 1, 2, 3, 4, or 5 times.
4. The method of claim 2 , wherein steps (c) through (e) are repeated a number of times sufficient to generate at least as many unique barcode sequence combinations as the number of cells in the plurality of cells.
5. The method of claim 2 , wherein steps (c) through (e) are repeated a number of times sufficient to provide a greater than 50%, 90%, 95%, or 99% probability that the cDNAs in each cell are bound to a unique barcode sequence combination.
6. The method of claim 1 , wherein the RT primers each further comprise a 5′ overhang comprising a 5′ overhang sequence.
7. The method of claim 1 , wherein the nucleic acid tags each comprise a 3′ region and a 5′ region flanking the barcode sequence.
8. The method of claim 1 , wherein the coupling comprises ligating 3′ ends of the nucleic acid tags to 5′ ends of the cDNA molecules.
9. The method of claim 8 , wherein the 3′ ends of the nucleic acid tags are brought into proximity to the 5′ ends of the cDNA molecules by prehybridizing the nucleic acid tags with linker nucleic acid strands that are complementary to the 3′ terminal sequence of the nucleic acid tags and to the 5′ terminal sequence of the cDNA molecules.
10. The method of claim 9 , wherein the RT primers each further comprise a 5′ overhang comprising a 5′ overhang sequence, and wherein the 5′ terminal sequence of the cDNA molecules comprises the 5′ overhang sequence from an RT primer.
11. The method of claim 9 , wherein steps (c) to (e) are repeated one or more times, wherein the nucleic acid tags each comprise a 5′ region located 5′ to the barcode sequence, and wherein the 5′ terminal sequence of the cDNA molecules comprises the 5′ region from a previously coupled nucleic acid tag.
12. The method of claim 9 , wherein the coupling is stopped by introducing a plurality of ligation stop oligos that are complementary to all or part of the linker nucleic acid strand.
13. The method of claim 1 , wherein the nucleic acid tags are DNA tags.
14. The method of claim 1 , further comprising, prior to step (a), fixing and/or permeabilizing the plurality of cells.
15. The method of claim 1 , wherein steps (c) to (e) are performed one or more times, and wherein the nucleic acid tags that are coupled to the cDNA molecules during the last of the one or more times comprise a UMI, a capture agent, a flow-cell binding site, and/or a primer-binding site.
16. The method of claim 15 , wherein the capture agent comprises biotin.
17. The method of claim 1 , wherein the barcode sequences comprise at least 8 nucleotides.
18. 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.
19. The method of claim 1 , wherein all of the RT primers comprise a poly(T) sequence.
20. The method of claim 1 , wherein all of the RT primers comprise a random nucleotide sequence.
21. The method of claim 1 , wherein a portion of the RT primers comprise a poly(T) sequence and the remainder of the RT primers comprise a random nucleotide sequence.
22. The method of claim 1 , wherein at least a portion of the poly(T) sequences are anchored poly(T) sequences.
23. The method of claim 1 , wherein at least a portion of the poly(T) sequences comprise 15 dTs.
24. The method of claim 1 , wherein at least a portion of the random nucleotide sequences are random hexamer sequences.
25. The method of claim 1 , further comprising:
(i) sequencing the cDNA molecules amplified in step (h).
26. The method of claim 25 , further comprising:
(j) grouping the sequencing reads obtained in (i) by barcode sequence and/or index sequence.
27. The method of claim 26 , wherein the sequencing reads are grouped by a combination of barcode sequence and index sequence.
28. The method of claim 26 , wherein steps (c) to (e) have been repeated one or more times, and wherein grouping by barcode sequence comprises grouping by barcode sequence combination.
29. The method of claim 26 , wherein the RT primers further comprise a barcode sequence, and wherein the grouping is further performed based on the barcode sequence in the RT primer.
30. The method of claim 1 , wherein the plurality of aliquots comprises 96 aliquots distributed in a 96-well plate.