GSE131098
Hammer-seq to measure kinetics of DNA methylation maintenance in HeLa cells
Simultaneous measurement of the methylation status of parent and daughter strands of newly replicated DNA at the single molecule level has not been experimentally achieved, which is essential for measurements the kinetics of DNA methylation maintenance at base level genome wide. To achieve this, we developed a new method termed Hammer-seq (Hairpin assisted mapping of methylation of replicated DNA), which combines EdU labeling, biotin conjugation, immunopurification and hairpin bisulfite sequencing technologies. We were able to pulse label HeLa cells with EdU for as short as 4 min to capture newly synthesized DNA, and the paired-end hairpin bisulfite sequencing allowed us to simultaneously measure the methylation status of parent and daughter strands at the single molecular level with base resolution. Hammer-seq displayed strong enrichment of newly replicated regions and successfully captured maintenance intermediates, and also had robustness of measurements. Overall design: In this study, we arrested HeLa cells at the G1/S boundary with thymidine-aphidicolin double block, pulse labeled with EdU for 4 min immediately after cell cycle release, chased for various time points up to 24 h and performed Hammer-seq to measure the maintenance kinetics during and after S phase.