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使用MAB-seq和caMAB-seq对活性DNA去甲基化进行碱基分辨率分析。

Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq.

作者信息

Wu Hao, Wu Xiaoji, Zhang Yi

机构信息

Howard Hughes Medical Institute, Boston, Massachusetts, USA.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.

出版信息

Nat Protoc. 2016 Jun;11(6):1081-100. doi: 10.1038/nprot.2016.069. Epub 2016 May 12.

Abstract

A complete understanding of the function of the ten-eleven translocation (TET) family of dioxygenase-mediated DNA demethylation requires new methods to quantitatively map oxidized 5-methylcytosine (5mC) bases at high resolution. We have recently developed a methylase-assisted bisulfite sequencing (MAB-seq) method that allows base-resolution mapping of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), two oxidized 5mC bases indicative of active DNA demethylation events. In standard bisulfite sequencing (BS-seq), unmodified C, 5fC and 5caC are read as thymine; thus 5fC and 5caC cannot be distinguished from C. In MAB-seq, unmodified C is enzymatically converted to 5mC, allowing direct mapping of rare modifications such as 5fC and 5caC. By combining MAB-seq with chemical reduction of 5fC to 5hmC, we also developed caMAB-seq, a method for direct 5caC mapping. Compared with subtraction-based mapping methods, MAB-seq and caMAB-seq require less sequencing effort and enable robust statistical calling of 5fC and/or 5caC. MAB-seq and caMAB-seq can be adapted to map 5fC/5caC at the whole-genome scale (WG-MAB-seq), within specific genomic regions enriched for enhancer-marking histone modifications (chromatin immunoprecipitation (ChIP)-MAB-seq), or at CpG-rich sequences (reduced-representation (RR)-MAB-seq) such as gene promoters. The full protocol, including DNA preparation, enzymatic treatment, library preparation and sequencing, can be completed within 6-8 d.

摘要

要全面了解双加氧酶介导的DNA去甲基化的十一易位(TET)家族的功能,需要新的方法来高分辨率地定量绘制氧化的5-甲基胞嘧啶(5mC)碱基图谱。我们最近开发了一种甲基化酶辅助亚硫酸氢盐测序(MAB-seq)方法,该方法可以对5-甲酰基胞嘧啶(5fC)和5-羧基胞嘧啶(5caC)进行碱基分辨率图谱绘制,这两种氧化的5mC碱基是活跃DNA去甲基化事件的指示物。在标准的亚硫酸氢盐测序(BS-seq)中,未修饰的C、5fC和5caC都被读作胸腺嘧啶;因此,5fC和5caC无法与C区分开来。在MAB-seq中,未修饰的C被酶促转化为5mC,从而可以直接绘制罕见修饰如5fC和5caC的图谱。通过将MAB-seq与将5fC化学还原为5hmC相结合,我们还开发了caMAB-seq,一种直接绘制5caC图谱的方法。与基于减法的图谱绘制方法相比,MAB-seq和caMAB-seq所需的测序工作量更少,并且能够对5fC和/或5caC进行可靠的统计调用。MAB-seq和caMAB-seq可以适用于在全基因组规模(WG-MAB-seq)、富含增强子标记组蛋白修饰的特定基因组区域内(染色质免疫沉淀(ChIP)-MAB-seq)或富含CpG的序列(简化代表性(RR)-MAB-seq)如基因启动子处绘制5fC/5caC图谱。整个实验方案,包括DNA制备、酶处理、文库制备和测序,可以在6-8天内完成。

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