The stable maintenance of DNA methylation patterns during mitotic cell division is crucial for cell identity. Precisely determining the maintenance kinetics and dissecting the exact contributions of relevant regulators requires a method to accurately measure parent and daughter strand DNA methylation at the same time, ideally at the single-molecule level. Recently, we developed a method referred to as Hammer-seq (hairpin-assisted mapping of methylation of replicated DNA) that fulfils the above criteria. This method integrates 5-ethynyl-2′-deoxyuridine (EdU) labeling of replicating DNA, biotin conjugation and streptavidin-based affinity purification, and whole-genome hairpin bisulfite sequencing technologies. Hammer-seq offers the unique advantage of simultaneously measuring the methylation status of parent and daughter strands within a single DNA molecule, which makes it possible to determine maintenance kinetics across various genomic regions without averaging effects from bulk measurements and to assess de novo methylation events that accompany methylation maintenance. Importantly, when combined with mutant cell lines in which mechanisms of interest are disrupted, Hammer-seq can be applied to determine the functional contributions of potential regulators to methylation maintenance, with accurate kinetics information that cannot be acquired with other currently available methods. Hammer-seq library preparation requires ~100 ug EdU-labeled genomic DNA as input (~15 million mammalian cells). The whole protocol, from pulse labeling to library construction, can be completed within 2–3 d, depending on the chasing time.

在有丝分裂细胞分裂过程中稳定维持 DNA 甲基化模式对于细胞身份至关重要。精确确定维持动力学并剖析相关调节剂的确切贡献需要一种同时准确测量父链和子链 DNA 甲基化的方法，最好是在单分子水平上。最近，我们开发了一种称为 Hammer-seq（复制 DNA 甲基化的发夹辅助作图）的方法，该方法满足上述标准。该方法集成了复制 DNA 的 5-乙炔基-2'-脱氧尿苷 (EdU) 标记、生物素偶联和基于链霉亲和素的亲和纯化，以及全基因组发夹亚硫酸氢盐测序技术。Hammer-seq 提供了同时测量单个 DNA 分子内母链和子链的甲基化状态的独特优势，这使得确定跨基因组区域的维持动力学而不需要批量测量的平均效应和评估从头甲基化事件成为可能伴随甲基化维持。重要的是，当与感兴趣的机制被破坏的突变细胞系结合使用时，Hammer-seq 可用于确定潜在调节剂对甲基化维持的功能贡献，并提供其他当前可用方法无法获得的准确动力学信息。Hammer-seq 文库制备需要约 100 ug EdU 标记的基因组 DNA 作为输入（约 1500 万哺乳动物细胞）。整个协议，