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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This work was supported by the Lithuanian-Swiss Research and Development Program (grant no. CH-3-SMM-01/03) and an Edward J Mallinckrodt Foundation Grant (to A.M.K.). L.M. holds a Marie Curie Individual Fellowship (705791). A.M.K. holds a Burroughs Wellcome Fund CASI Award. A.V. is supported by the HSCI Medical Scientist Training Fellowship and the Harvard Presidential Scholars Fund. Liquid-handling robotics was carried out at the ICCB-Longwood Screening Facility at Harvard Medical School (HMS); microfabrication was carried out at the HMS Microfabrication/Microfluidics core facility.
L.M. and A.M.K. are inventors on a patent application (PCT/US2015/026443) that includes some of the ideas described in this article. A.M.K. is a cofounder and science advisory board member of 1CellBio. L.M. is affiliated with Droplet Genomics. The rest of the authors declare no competing financial interests.
Integrated supplementary information
Supplementary Methods 1 and 2, Supplementary Table 1, and Supplementary Figure 1 (PDF 280 kb)
Supplementary Tables 2 (XLSX 41 kb)
Supplementary Tables 3 (XLSX 45 kb)
Supplementary Tables 4 (XLSX 44 kb)
Designs for microfluidic chips. (ZIP 442 kb)
Hamilton Microlab STAR-let liquid handler method files. (ZIP 38 kb)
Python script for sequencing data processing. (ZIP 27 kb)
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Zilionis, R., Nainys, J., Veres, A. et al. Single-cell barcoding and sequencing using droplet microfluidics. Nat Protoc 12, 44–73 (2017). https://doi.org/10.1038/nprot.2016.154
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