Description

Single-cell barcoding and sequencing using droplet microfluidics – a detailed protocol

Juozas Nainys¹, Rapolas Zilionis^1,2, Adrian Veres^2–4, Virginia Savova², David Zemmour⁵, Allon M Klein² & Linas Mazutis¹

¹Institute of Biotechnology, Vilnius University, Vilnius, Lithuania; ²Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA; ³Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA; ⁴Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA; ⁵Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

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.