Droplet-based method and apparatus for composite single-cell nucleic acid analysis

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 4n 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.