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

Abstract

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. Here we 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. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We 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. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

inDrops-2: a flexible, versatile and cost-efficient droplet microfluidics approach for high-throughput scRNA-seq of fresh and preserved clinical samples

“…Being an open and flexible platform inDrops-2 can accommodate diverse workflows matching user-specific needs and has a potential to further democratize single cell technologies. Indeed, it has recently been shown that inDrops-based platforms such as VASA-Seq [35] outperforms commercial and plate-based methods in terms of gene and transcript capture, while spinDrops [73] benefits applications based on target cell enrichment by on-chip sorting. Beyond transcriptomics, inDrops-based platforms have been successfully tailored to probe other –omic modalities in individual cells such as open chromatin by HyDrop [33] or genome by Microbe-Seq [74], to name a few.…”

Advanced sequencing-based high-throughput and long-read single-cell transcriptome analysis

“…2d). 31 The first microfluidic device was utilized for the generation and sorting of droplets using in-line fluorescence-activated droplet sorting. The second microfluidic device was exploited for droplet pico-injection where RT mix can be injected into droplets after cell lysis.…”

Development and future of droplet microfluidics

“…The single-cell screening can be further combined with genome sequencing to map the relationships between their genotypes and phenotypes. 191 Through sorting and sequencing of a specific set of IgG-secreting primary cells, the immunoglobulin G (IgG) secretion and the sequences of paired V H – V L genes can be coupled. 192 Single glial cells can also be sorted and collected by nucleic acid cytometry, thus enabling interrogation of the signaling pathways that regulate the pathogenic activities.…”