Journal
NATURE PROTOCOLS
Volume 12, Issue 1, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2016.154
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Funding
- Lithuanian-Swiss Research and Development Program [CH-3-SMM-01/03]
- Edward J Mallinckrodt Foundation Grant
- Marie Curie Individual Fellowship [705791]
- Burroughs Wellcome Fund CASI Award
- HSCI Medical Scientist Training Fellowship
- Harvard Presidential Scholars Fund
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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 in Drops, 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 in Drops 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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