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.

在单分子水平上同时检测新复制DNA的亲本链与子链的甲基化状态，目前尚未在实验中实现，而该技术对于全基因组范围内碱基分辨率水平的DNA甲基化维持动力学研究至关重要。为达成这一目标，我们开发了一种名为Hammer-seq（Hairpin assisted mapping of methylation of replicated DNA）的全新方法，该方法整合了EdU标记、生物素偶联、免疫纯化以及发卡亚硫酸氢盐测序等多项技术。我们可通过最短4分钟的EdU脉冲标记HeLa细胞以捕获新合成的DNA，而双端发卡亚硫酸氢盐测序技术能够使我们在单分子水平上以碱基分辨率同时检测亲本链与子链的甲基化状态。Hammer-seq对新复制区域展现出极强的富集效果，可成功捕获甲基化维持中间体，同时具备优异的检测稳健性。

搜集汇总

背景与挑战

背景概述

该数据集使用Hammer-seq方法研究HeLa细胞中DNA甲基化维持的动力学，通过结合EdU标记和发夹亚硫酸氢盐测序技术，实现在单分子水平测量新复制DNA的母链和子链甲基化状态。数据集包含126个样本，主要来自人类宫颈上皮细胞，使用Illumina NovaSeq 6000平台进行ChIP-Seq和Bisulfite-Seq测序，旨在提供全基因组DNA甲基化维持的高分辨率数据。