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Adv."],"published-print":{"date-parts":[[2026,10,23]]},"abstract":"<jats:p>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\u2013based studies into cellular regulation and disease.<\/jats:p>","DOI":"10.1126\/sciadv.aef2844","type":"journal-article","created":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T17:58:42Z","timestamp":1792778322000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":0,"title":["High-throughput droplet-based enzymatic DNA methylome profiling enables single-cell resolution of epigenetic heterogeneity"],"prefix":"10.1126","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-3869-1847","authenticated-orcid":true,"given":"Gaojie","family":"Li","sequence":"first","affiliation":[{"name":"China National Center for Bioinformation, Beijing 100101, China."},{"name":"Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China."},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-1207-3383","authenticated-orcid":true,"given":"Yang","family":"Yang","sequence":"additional","affiliation":[{"name":"China National Center for Bioinformation, Beijing 100101, China."},{"name":"Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China."},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China."},{"name":"Department of Pharmacy, Peking University First Hospital, Beijing 100034, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7367-7835","authenticated-orcid":true,"given":"Zhuoran","family":"Li","sequence":"additional","affiliation":[{"name":"School of Medicine, Nankai University, Tianjin 30071, China."},{"name":"Department of Urology, Chinese PLA General Hospital, Beijing 100039, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1973-8510","authenticated-orcid":true,"given":"Qi","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Urology, Chinese PLA General Hospital, Beijing 100039, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-1048-054X","authenticated-orcid":true,"given":"Ying","family":"Wang","sequence":"additional","affiliation":[{"name":"China National Center for Bioinformation, Beijing 100101, China."},{"name":"Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China."},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0748-7179","authenticated-orcid":true,"given":"Bin","family":"Guo","sequence":"additional","affiliation":[{"name":"China National Center for Bioinformation, Beijing 100101, China."},{"name":"Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China."},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-8582-5150","authenticated-orcid":true,"given":"Kunmeng","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Urology, Chinese PLA General Hospital, Beijing 100039, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-2424-2884","authenticated-orcid":true,"given":"Ming","family":"Tan","sequence":"additional","affiliation":[{"name":"SeekGene BioSciences Co. 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