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bioRxiv · 10.1101/803460

Optimized double emulsion flow cytometry with high-throughput single droplet isolation

Brower, K. K.·Carswell-Crumpton, C.·Klemm, S.·Cruz, B.·Kim, G.·Calhoun, S. G.·Nichols, L.·Fordyce, P. M.
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Abstract

Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate variants-of-interest remains a field-wide bottleneck. Here, we present an optimized double emulsion workflow, sdDE-FACS, that enables high-throughput phenotyping, selection, and sorting of droplets using standard flow cytometers. Using a 130 m nozzle, we demonstrate robust post-sort recovery of intact droplets, with little to no shear-induced droplet breakage, at high sort frequency (12-14 kHz) across two industry-standard FACS instruments. We report the first quantitative plate statistics for double emulsion droplet isolation and demonstrate single droplet recovery with >70% efficiency. In addition, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets, an advance in droplet sorting comparable with the capabilities of single-cell FACS. This work resolves several hurdles in the field of high-throughput droplet analysis and paves the way for a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis, marrying the full power of flow cytometry with droplet microfluidics.

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Brower, K. K., Carswell-Crumpton, C., Klemm, S., Cruz, B., Kim, G., Calhoun, S. G., Nichols, L., Fordyce, P. M.. 2019-10-15. Optimized double emulsion flow cytometry with high-throughput single droplet isolation. https://doi.org/10.1101/803460

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