Digital-scRRBS: A cost-effective, highly sensitive and automated single-cell methylome analysis platform via digital microfluidics

Single-cell DNA methylation sequencing is a powerful method for elucidating important physiological and pathological processes, identifying cell subpopulations, and constructing epigenetic regulatory networks. Existing methylome analyses typically require substantial starting materials, complex operations, and high cost and are susceptible to contamination. These problems have impeded the development of single-cell methylome technology for rare cell profiling. In this work, we report Digital Microfluidics-based single-cell Reduced Representation Bisulfite Sequencing (Digital-scRRBS), the first microfluidics-based single-cell methylome library construction platform, which is an automatic, efficient, reproducible, and reagent-economy approach to dissect the single-cell methylome. Taking advantage of our uniquely designed digital microfluidic chip, we realized efficient single-cell isolation in less than 15 seconds. Furthermore, with the advantages of a confined environment, superhydrophobic surface, and nano-scale reaction volume of our digital microfluidic chip, more amplifiable DNA is retained for library construction compared to other approaches. We have successfully constructed single-cell methylation sequencing libraries with a unique genome mapping rate of up to 53.6%, covering up to 2.26 million CpG sites. The application of Digital-scRRBS allows us to discriminate cell identity and dynamically monitor DNA methylation levels during drug administration. Digital-scRRBS provides the technology for widespread application of single-cell methylation methods as a versatile tool for epigenetic analysis in rare cells and highly heterogeneous populations. In total, 25 GM12878 cell line samples and 20 K562 cell line samples were analyzed. We examined genome-wide DNA methylation profiles of GM12878 cells, including 8 low-input samples with starting DNA sample amounts in the range of 0.3-10ng, 8 singe-cell samples, pooled-2, 3, 4, 10, 15, 20 cells samples using Digital-scRRBS, and 3 single-cell samples in the tube. K562 cells were treated with decitabine in a final concentration of 300 nM to induce demethylation, and 8 treated samples and 12 untreated sample were analyzed to reveal the mechanism of demethylation.