Wu Hao, Wu Xiaoji, Zhang Yi
Howard Hughes Medical Institute, Boston, Massachusetts, USA.
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
Nat Protoc. 2016 Jun;11(6):1081-100. doi: 10.1038/nprot.2016.069. Epub 2016 May 12.
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.
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。