Single-cell barcoding and sequencing using droplet microfluidics

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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Figure 1: Single-cell transcriptome barcoding in drops.
Figure 2: Synthesis of barcoding hydrogel beads.
Figure 3: Library preparation.
Figure 4: The inDrops platform.
Figure 5: Design of the microfluidic devices.
Figure 6: Quality control of barcoded hydrogel bead synthesis.
Figure 7: Setting the qPCR threshold.
Figure 8: BioAnalyzer electropherograms of amplified RNA (Step 145) and sequencing-ready DNA libraries (Step 164).
Figure 9: Results of sequencing a library of 2,000 immune 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.

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Affiliations

Authors

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.

Corresponding authors

Correspondence to Allon M Klein or Linas Mazutis.

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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.

Integrated supplementary information

Supplementary information

Supplementary Text and Figures

Supplementary Methods 1 and 2, Supplementary Table 1, and Supplementary Figure 1 (PDF 280 kb)

Supplementary Table 2

Supplementary Tables 2 (XLSX 41 kb)

Supplementary Table 3

Supplementary Tables 3 (XLSX 45 kb)

Supplementary Table 4

Supplementary Tables 4 (XLSX 44 kb)

Supplementary Data 1

Designs for microfluidic chips. (ZIP 442 kb)

Supplementary Data 2

Hamilton Microlab STAR-let liquid handler method files. (ZIP 38 kb)

Supplementary Software

Python script for sequencing data processing. (ZIP 27 kb)

Coencapsulation of cells, RT/lysis reagents, and barcoding hydrogel beads (slowed down 23×). (MOV 1264 kb)

Transfer of reagents to a syringe for injection into the microfluidic device. (MOV 10824 kb)

Hydrogel droplet production (slowed down 166×). (MP4 2832 kb)

Preparation of the syringe with hydrogel beads. (MOV 10829 kb)

Exposure of emulsion to UV light to release photocleavable primers. (MOV 5549 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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