DMF-DM-seq: Digital-microfluidics enabled dual-modality sequenc-ing of single-cell mRNA and microRNA with high integration, sensi-tivity, and automation.

Multi-modality measurement of single cells provides deep insights into the intricate relationships between individual molecular levels and regulatory mechanisms underlying intercellular variations. Here, we developed a highly integrated, sensitive, and automated sequencing library preparation platform for single-cell mRNA and microRNA (miRNA) co-sequencing based on digital microfluidics, named DMF-DM-seq. This platform highly integrated the process of single-cell isolation, lysis, component separation, and simultaneous sequencing library preparation of mRNA and miRNA all within a single DMF device. Compared with the current half-cell measuring strategy, DMF-DM-seq enabled a complete separation of single-cell mRNA and miRNA via magnetic field application, which endows DMF-DM-seq with a higher miRNA detection ability. DMF-DM-seq revealed differ-ential expression patterns between single cells of non-cancerous breast cells, non-invasive and aggressive breast cancer cells at both mRNA and miRNA levels, and demonstrated the anti-correlated relationship between miRNA and their targeted mRNA. Further, we unraveled the tumor growth and metastasis-associated biological processes enriched by miRNA-targeted genes, alongwith important miRNA-interaction networks involved in significant signaling pathways. We also deconstructed miRNA regulatory mechanisms underlying different signaling pathways across different breast cell types. In summary, DMF-DM-seq offers a powerful tool to comprehensively study the expression heterogeneity of single-cell mRNA and miRNA, which is antic-ipated to be widely applied in basic and clinical research.