Simultaneously measuring the methylation of parent and daughter strands of replicated DNA at the single-molecule level by Hammer-seq, Nat Protoc, 8 Mar 2021
Simultaneously measuring the methylation of parent and daughter strands of replicated DNA at the single-molecule level by Hammer-seq
Xuan Ming, Bing Zhu & Zhuqiang Zhang
Abstract
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.
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Simultaneously measuring the methylation of parent and daughter strands of replicated DNA at the single-molecule level by Hammer-seq, Nat Protoc, 8 Mar 2021
Nature Protocols, 8 March, 2021, DOI：https://doi.org/10.1038/s41596-020-00488-9
Simultaneously measuring the methylation of parent and daughter strands of replicated DNA at the single-molecule level by Hammer-seq
Xuan Ming, Bing Zhu & Zhuqiang Zhang
Abstract
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.
文章链接：https://www.nature.com/articles/s41596-020-00488-9
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