发布: 2018年03月05日第8卷第5期 DOI: 10.21769/BioProtoc.2747 浏览次数: 7095
评审: Gal HaimovichOmar AkilAnonymous reviewer(s)
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Abstract
This protocol is designed to obtain base-resolution information on the level of 5-hydroxymethylcytosine (5hmC) in CpGs without the need for bisulfite modification. It relies on (i) the capture of hydroxymethylated sequences by a procedure known as ‘selective chemical labeling’ (see Szulwach et al., 2012) and (ii) the digestion of the captured DNA by exonucleases. After Illumina sequencing of the digested DNA fragments, an ad hoc bioinformatic pipeline extracts the information for further downstream analysis.
Keywords: 5-Hydroxymethylcytosine (5-羟甲基胞嘧啶)Background
The methylation of cytosine in genomic DNA can be read by proteins and is mainly translated into gene silencing. Most CpG dinucleotides in the genome are methylated, including those located in gene regulatory regions such as enhancers. However, when required, these CpGs can be demethylated through oxidation of the methyl group by Ten Eleven Translocation (TET) enzymes and replacement by unmethylated cytosines by the base excision repair system. 5-Hydroxymethylcytosine (5hmC) is the first oxidative derivative of 5-methylcytosine, and mapping this modified base in the genome provides information on the regions undergoing active demethylation. Although selective chemical labeling (SCL) allows very specific detection of 5hmC, the resolution of this technique is limited by the size of the DNA fragments, especially when several CpGs are present in the captured DNA. In order to improve resolution, we have introduced a digestion step using exonucleases which trim the DNA molecule up to close proximity of the hydroxymethylated cytosines (Sérandour et al., 2016). Appropriate bioinformatic treatment of the sequencing reads then assigns hydroxymethylation score to the captured CpGs.
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版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Sérandour, A. A., Avner, S. and Salbert, G. (2018). Coupling Exonuclease Digestion with Selective Chemical Labeling for Base-resolution Mapping of 5-Hydroxymethylcytosine in Genomic DNA. Bio-protocol 8(5): e2747. DOI: 10.21769/BioProtoc.2747.
分类
系统生物学 > 表观基因组学 > 5-羟甲基胞嘧啶
分子生物学 > DNA > DNA 修饰
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