spinDrop – a droplet microfluidic platform to maximise single-cell sequencing information content

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture.

Researchers at the University of Cambridge alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. This methodology increases gene detection rates fivefold, while reducing background noise by up to half. The researchers harness these properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. This method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

Overview of the modular droplet microfluidic workflow
for spinDrop (‘sorting and picoinjection inDrop‘)

A Schematic of the different steps to generate sequencing-ready libraries. First, intact whole cells are made detectable with a Calcein-AM viability stain, cell-surface marker stain, and intact nuclei using a DNA staining dye (such as the Vybrant DNA stain). Then the cells or nuclei are co-encapsulated with barcoded polyacrylamide beads, and droplets with viable cells, intact nuclei or specific cell types are enriched after encapsulation using in-line fluorescence-activated droplet sorting (FADS), hereby discarding empty droplets or droplets containing damaged material. The droplets containing the viable cells or intact nuclei are then lysed and heat-treated, and further re-injected in a second microfluidic device, which will inject the reverse transcriptase mix using coalescence-activated picoinjection. B Schematic of the cell/nuclei and polyacrylamide bead droplet co-encapsulation process in the first microfluidic device (top). Brightfield image of the co-encapsulation process (bottom). C Schematic of fluorescence-activated sorting of droplets containing viable cells or intact nuclei from the pool of empty droplets or droplets containing damaged or unwanted material (top). Brightfield image of the in-line sorting process (bottom). D Schematic of the coalescence-activated droplet picoinjection of a reverse transcriptase mix (top). Brightfield image of the picoinjection process (bottom).