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

Single-cell barcoding and sequencing using droplet microfluidics.

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Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
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.
Division 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.

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