High-throughput droplet-based enzymatic DNA methylome profiling enables single-cell resolution of epigenetic heterogeneity.

DNA methylation is a fundamental epigenetic modification that regulates gene expression, cellular identity, and disease-associated regulatory programs. However, high-throughput single-cell DNA methylation technologies have been limited by DNA degradation, low CpG recovery, and the poor scalability of bisulfite-based methods. Here, we present scEM-seq, a droplet-based single-cell enzymatic methylome sequencing method that integrates barcode ligation within a single emulsification step, followed by postemulsion cytosine-to-uracil conversion. This design substantially reduces DNA loss and enables robust profiling of tens of thousands of single cells per experiment. scEM-seq generates high-quality single-cell methylomes with moderately improved CpG coverage and balanced read utilization compared with other droplet-based microfluidic methods. Applied to peripheral blood mononuclear cells, scEM-seq accurately resolves major immune lineages, and in prostate cancer tissues, it reveals diverse malignant cell states and patient-specific epigenetic and copy number architectures. scEM-seq enables scalable single-cell dissection of epigenetic heterogeneity across diverse biological and disease contexts, broadening the scope of DNA methylation-based studies into cellular regulation and disease.