Highly Multiplexed, Efficient, and Automated Single‐Cell MicroRNA Sequencing with Digital Microfluidics.

Abstract

Single‐cell microRNA (miRNA) sequencing has allowed for comprehensively studying the abundance and complex networks of miRNAs, which provides insights beyond single‐cell heterogeneity into the dynamic regulation of cellular events. Current benchtop‐based technologies for single‐cell miRNA sequencing are low throughput, limited reaction efficiency, tedious manual operations, and high reagent costs. Here, a highly multiplexed, efficient, integrated, and automated sample preparation platform is introduced for single‐cell miRNA sequencing based on digital microfluidics (DMF), named Hiper‐seq. The platform integrates major steps and automates the iterative operations of miRNA sequencing library construction by digital control of addressable droplets on the DMF chip. Based on the design of hydrophilic micro‐structures and the capability of handling droplets of DMF, multiple single cells can be selectively isolated and subject to sample processing in a highly parallel way, thus increasing the throughput and efficiency for single‐cell miRNA measurement. The nanoliter reaction volume of this platform enables a much higher miRNA detection ability and lower reagent cost compared to benchtop methods. It is further applied Hiper‐seq to explore miRNAs involved in the ossification of mouse skeletal stem cells after bone fracture and discovered unreported miRNAs that regulate bone repairing.

DMF-DM-seq: Digital-Microfluidics Enabled Dual-Modality Sequencing of Single-Cell mRNA and microRNA with High Integration, Sensitivity, and Automation

“…As reported in our previous studies, − two hydrophilic microwells were spotted on the hydrophobic surface of the DMF chip for single-cell isolation. With microscopic observation, the wettability-based single-cell isolation strategy features selectivity, high efficiency, and convenience, and the detailed workflow is depicted in Figure S4.…”

“…Here, based on our pioneering work on highly multiplexed single-cell miRNA sequencing, we reported highly integrated, efficient, and automated sequencing library preparation platform for single-cell mRNA and miRNA coanalysis based on DMF, named DMF-enabled dual-modal sequencing (DMF-DM-seq) . With the combination of microscopic observation, an external magnetic field, local heating modules, the procedures of single-cell isolation, lysis, mRNA and miRNA separation, and simultaneous sequencing library preparation of mRNA and miRNA are highly integrated in a single DMF device.…”