Droplet-based method and apparatus for composite single-cell nucleic acid analysis
The present invention generally relates to a combination of molecular barcoding and emulsion-based microfluidics to isolate, lyse, barcode, and prepare nucleic acids from individual cells in a high-throughput manner.
1. A method for creating a composite single-cell cDNA library, wherein the RNAs from different cells are tagged individually, allowing the cell identity of each RNA to be retained in a single library, said method comprising:
(a) loading an aqueous input comprising a plurality of single cells and an aqueous input comprising a plurality of at least a thousand RNA capture microbeads into a microfluidic device configured for joining the two aqueous inputs into a plurality of emulsion droplets, wherein each RNA capture microbead comprises a plurality of capture oligonucleotides attached to the microbead surface, each capture oligonucleotide comprising (i) a cell-of-origin barcode sequence that is the same for all capture oligonucleotides on the same bead but differs from the barcode sequence of capture oligonucleotides on other beads, (ii) a unique molecular identifier (UMI) sequence that is different for each capture oligonucleotide on the same microbead and (iii) a capture sequence that binds to cellular RNA, and wherein the maximum complexity of cell-of-origin barcodes for the plurality of RNA capture microbeads is 4 n where n is the length of the cell-of-origin barcode sequence and n is at least 6, and wherein the cell-of-origin barcode sequence is contiguous with the UMI sequence;
(b) co-encapsulating single cells and single RNA capture microbeads in emulsion droplets by co-flowing the two aqueous inputs across an oil channel in the microfluidic device, and wherein each emulsion droplet has a diameter from 50 μm to 210 μm;
(c) lysing the cells in the emulsion droplets, thereby capturing the cellular RNA on the RNA capture oligonucleotides; and
(d) performing a reverse transcription reaction to generate cDNA copies of the captured RNA that incorporate the barcode sequence and a UMI, thereby recording the cell-of-origin for the captured RNA and identifying individual transcripts.
2. The method of claim 1 , further comprising amplifying the cDNA, wherein the method of amplifying the cDNA is template switch amplification; T7 linear application; exponential isothermal amplification; or PCR.
3. The method of claim 1 , further comprising preparing a massively parallel single cell RNA sequencing library from the cDNA and sequencing the sequencing library.
4. The method of claim 1 , further comprising collecting cDNA-attached beads.
5. The method of claim 1 , wherein the capture oligonucleotides are attached to the beads via a cleavable linker and the method comprises lysing the cells and releasing the capture oligonucleotides from the beads.
6. The method of claim 1 , wherein the emulsion droplet has a diameter of 125 μm.
7. The method of claim 1 , wherein the diameter of the RNA capture microbeads is from 10 μm to 95 μm.
8. The method of claim 1 , wherein the capture sequence is a poly-T (dT) sequence.
9. The method of claim 1 , wherein the capture oligonucleotide further comprises a common priming sequence which comprises the same sequence across all beads in the plurality of beads.
10. The method of claim 1 , wherein the capture oligonucleotide further comprises a second barcode sequence that comprises the same sequence across all capture oligonucleotides on a bead but differs from the first barcode sequence.
11. The method of claim 1 , wherein the capture oligonucleotide is a single-stranded oligonucleotide.
12. The method of claim 1 , wherein the plurality of capture oligonucleotides are attached to the surface of the microbead via one or more linkers.
13. The method of claim 12 wherein the linker is a non-cleavable straight-chain polymer or substituted hydrocarbon polymer.
14. The method of claim 12 , wherein the linker is a chemically-cleavable or photolabile linker.
15. The method of claim 1 , wherein the reverse transcription reaction is performed in the droplets.
16. The method of claim 1 , wherein the reverse transcription reaction is performed after releasing the microbeads from the droplets.
17. The method of claim 1 , wherein the bead material is porous.
18. The method of claim 17 , wherein the bead material is methacrylate resin.
19. The method of claim 1 , wherein the plurality of RNA capture microbeads comprise 100,000 to 10 million uniquely barcoded RNA capture microbeads.
20. The method of claim 1 , wherein the cell-of-origin barcode is 6-12 nucleotides.
21. The method of claim 1 , wherein the capture oligonucleotide comprises at least one chemically modified nucleotide.