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Current benchtop\u2010based technologies for single\u2010cell 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\u2010cell miRNA sequencing based on digital microfluidics (DMF), named Hiper\u2010seq. 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\u2010structures 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\u2010cell 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\u2010seq to explore miRNAs involved in the ossification of mouse skeletal stem cells after bone fracture and discovered unreported miRNAs that regulate bone repairing.<\/jats:p>","DOI":"10.1002\/smtd.202301250","type":"journal-article","created":{"date-parts":[[2023,11,28]],"date-time":"2023-11-28T20:33:58Z","timestamp":1701203638000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Highly Multiplexed, Efficient, and Automated Single\u2010Cell MicroRNA Sequencing with Digital Microfluidics."],"prefix":"10.1002","volume":"8","author":[{"given":"Yingwen","family":"Chen","sequence":"first","affiliation":[{"name":"State Key Laboratory for Physical Chemistry of Solid Surfaces Key Laboratory for Chemical Biology of Fujian Province Key Laboratory of Analytical Chemistry and Department of Chemical Biology College of Chemistry and Chemical Engineering Xiamen University  Xiamen 361005 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