The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine.
The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine.
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5-羟基甲基胞嘧啶的抗CMS技术。
DOI：
10.1038/nprot.2012.103
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发表时间：
2012-10
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
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作者：
5-羟甲基胞嘧啶(5hmC)是最近在哺乳动物基因组中发现的一种碱基，由5-甲基胞嘧啶(5mC)在Tet蛋白的催化下氧化而成。5HmC及其进一步氧化产物的生物学功能正在深入研究中，因为它们可能是DNA去甲基化途径的中间产物。在这里，我们描述了一种在全基因组范围内分析5hmC的新协议。这种方法是基于亚硫酸氢钠介导的5HmC转化为胞嘧啶-5-亚甲基磺酸(CMS)；然后使用CMS特异性抗血清免疫沉淀含有CMS的DNA片段。与抗5hmC免疫沉淀(IP)相比，抗CMS技术具有高度的特异性和低本底，对5hmC浓度的依赖程度要低得多。此外，它不富含CA和CT重复序列，如使用5hmC抗体的5hmC DNA IP。抗CMS方案需要3天时间才能完成。
5-hydroxymethylcytosine (5hmC) is a recently discovered base in the mammalian genome, produced upon oxidation of 5-methylcytosine (5mC) in a process catalyzed by TET proteins. The biological functions of 5hmC and further oxidation products of 5mC are under intense investigation, as they are likely intermediates in DNA demethylation pathways. Here we describe a novel protocol to profile 5hmC at a genome-wide scale. This approach is based on sodium bisulfite–mediated conversion of 5hmC to cytosine-5-methylenesulfonate (CMS); CMS-containing DNA fragments are then immunoprecipitated using a CMS-specific antiserum. The anti-CMS technique is highly specific with a low background, and is much less dependent on 5hmC density than anti-5hmC immunoprecipitation (IP). Moreover, it does not enrich for CA and CT repeats, as noted for 5hmC DNA IP using antibodies to 5hmC. The anti-CMS protocol takes 3 d to complete.