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Single-cell barcoding and sequencing using droplet microfluidics

Nature Protocols volume 12, pages 4473 (2017) | Download Citation

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.

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Acknowledgements

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.

Author information

Affiliations

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

    • Rapolas Zilionis
    • , Juozas Nainys
    •  & Linas Mazutis
  2. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Rapolas Zilionis
    • , Adrian Veres
    • , Virginia Savova
    •  & Allon M Klein
  3. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Adrian Veres
  4. Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.

    • Adrian Veres
  5. Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.

    • David Zemmour

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Contributions

A.M.K. and L.M. developed the original inDrops method. A.M.K., A.V., and V.S. developed the bioinformatic pipeline for raw sequencing data analysis. R.Z., A.M.K., and L.M. analyzed data provided in the Anticipated Results. D.Z. designed library PCR primers and custom sequencing primers. R.Z., J.N., A.M.K., and L.M. wrote the manuscript.

Competing interests

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.

Corresponding authors

Correspondence to Allon M Klein or Linas Mazutis.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Methods 1 and 2, Supplementary Table 1, and Supplementary Figure 1

Excel files

  1. 1.

    Supplementary Table 2

    Supplementary Tables 2

  2. 2.

    Supplementary Table 3

    Supplementary Tables 3

  3. 3.

    Supplementary Table 4

    Supplementary Tables 4

Zip files

  1. 1.

    Supplementary Data 1

    Designs for microfluidic chips.

  2. 2.

    Supplementary Data 2

    Hamilton Microlab STAR-let liquid handler method files.

  3. 3.

    Supplementary Software

    Python script for sequencing data processing.

Videos

  1. 1.

    Coencapsulation of cells, RT/lysis reagents, and barcoding hydrogel beads (slowed down 23×).

  2. 2.

    Transfer of reagents to a syringe for injection into the microfluidic device.

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    Hydrogel droplet production (slowed down 166×).

  4. 4.

    Preparation of the syringe with hydrogel beads.

  5. 5.

    Exposure of emulsion to UV light to release photocleavable primers.

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DOI

https://doi.org/10.1038/nprot.2016.154

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