Single-cell barcoding and sequencing using droplet microfluidics

Nat Protoc. 2017 Jan;12(1):44-73. doi: 10.1038/nprot.2016.154. Epub 2016 Dec 8.


Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile >75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.

MeSH terms

  • Equipment Design
  • High-Throughput Nucleotide Sequencing / instrumentation*
  • Lab-On-A-Chip Devices*
  • Sequence Analysis, RNA / instrumentation*
  • Single-Cell Analysis / instrumentation*