Single-cell barcoding and sequencing using droplet microfluidics View Full Text


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Article Info

DATE

2017-01

AUTHORS

Rapolas Zilionis, Juozas Nainys, Adrian Veres, Virginia Savova, David Zemmour, Allon M Klein, Linas Mazutis

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. More... »

PAGES

44-73

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nprot.2016.154

    DOI

    http://dx.doi.org/10.1038/nprot.2016.154

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1019900861

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/27929523


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