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

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. miRNA sequencing of single cells provides insights from single-cell heterogeneity into underlying regulatory mechanisms. It develops a highly multiplexed, efficient, integrated, and automated sample preparation platform for single-cell miRNA sequencing based on digital microfluidics, which is applied to explore miRNAs involved in the ossification of mouse skeletal stem cells after bone fracture.image

Automated summary

Single-cell microRNA (miRNA) sequencing provides comprehensive insights into the abundance and complex networks of miRNAs and their dynamic regulation of cellular events. However, current benchtop-based technologies for single-cell miRNA sequencing have limitations in terms of throughput, efficiency, manual operations, and reagent costs. In this study, a highly multiplexed and automated sample preparation platform called Hiper-seq is introduced, which is based on digital microfluidics (DMF). The platform allows for the construction of miRNA sequencing libraries through digital control of addressable droplets on the DMF chip, enabling selective isolation and parallel processing of multiple single cells. This platform offers higher throughput and efficiency for single-cell miRNA measurement, with a smaller reaction volume and lower reagent cost compared to benchtop methods. Furthermore, it has been successfully applied to explore miRNAs involved in the ossification of mouse skeletal stem cells after bone fracture, leading to the discovery of previously unreported miRNAs that regulate bone repairing.