title: Single-cell barcoding and sequencing using droplet microfluidics.
abstract: 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.
authors.affiliation: Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.|Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Rapolas
authors.initials: R
authors.lastname: Zilionis
authors.affiliation: Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Juozas
authors.initials: J
authors.lastname: Nainys
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.|Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.|Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Adrian
authors.initials: A
authors.lastname: Veres
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Virginia
authors.initials: V
authors.lastname: Savova
authors.affiliation: Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: David
authors.initials: D
authors.lastname: Zemmour
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Allon M
authors.initials: AM
authors.lastname: Klein
authors.affiliation: Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Linas
authors.initials: L
authors.lastname: Mazutis
mesh_terms: D004867:Equipment Design; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation*; D056656:Lab-On-A-Chip Devices; D017423:Sequence Analysis, RNA / Q000295:instrumentation*; D059010:Single-Cell Analysis / Q000295:instrumentation*