DIRECT: Digital Microfluidics for Isolation‐Free Shared Library Construction of Single‐Cell DNA Methylome and Transcriptome

Abstract Simultaneous profiling of DNA methylation and gene expression within single cells is a powerful technology to dissect complex gene regulatory network of cells. However, existing methods are based on picking a single‐cell in a tube and split single‐cell lysate into two parts for transcriptome and methylome library construction, respectively, which is costly and cumbersome. Here, DIRECT is proposed, a digital microfluidics‐based method for high‐efficiency single‐cell isolation and simultaneous analysis of the methylome and transcriptome in a single library construction. The accuracy of DIRECT is demonstrated in comparison with bulk and single‐omics data, and the high CpG site coverage of DIRECT allows for precise analysis of copy number variation information, enabling expansion of single cell analysis from two‐ to three‐omics. By applying DIRECT to monitor the dynamics of mouse embryonic stem cell differentiation, the relationship between DNA methylation and changes in gene expression during differentiation is revealed. DIRECT enables accurate, robust, and reproducible single‐cell DNA methylation and gene expression co‐analysis in a more cost‐effective, simpler library preparation and automated manner, broadening the application scenarios of single‐cell multi‐omics analysis and revealing a more comprehensive and fine‐grained map of cellular regulatory landscapes.