Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution.
Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution.
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最近发现胞嘧啶修饰5-羟甲基胞嘧啶（5 hmC）和5-甲酰胞嘧啶（5 fC）存在于多种哺乳动物细胞类型的基因组DNA中。现在重要的是要了解它们在正常生物功能和疾病中的作用。在这里，我们介绍了还原亚硫酸氢盐测序（redBS-Seq），一种以单碱基分辨率定量解码DNA中5 fC的方法，该方法基于5 fC选择性化学还原为5 hmC，然后进行亚硫酸氢盐处理。在对合成和基因组DNA进行广泛验证后，我们将redBS-Seq和氧化亚硫酸氢盐测序（oxBS-Seq）相结合，以生成小鼠胚胎干细胞中5-甲基胞嘧啶，5 hmC和5 fC的第一个组合基因组图谱。我们的实验表明，与5 hmC和5 mC相比，5 fC在某些基因组位置以相对较高的水平存在。这些化学方法的结合可以量化和精确地绘制基因组中的这三种胞嘧啶衍生物，并将有助于深入了解它们的功能。
The cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were recently found to exist in the genomic DNA of a wide range of mammalian cell types. It is important to now understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a method to quantitatively decode 5fC in DNA at singlebase resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. Following extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that 5fC is present at relatively high levels in certain genomic locations, in comparison to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.
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DOI：
10.1126/science.1170116
发表时间：
2009-05-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Tahiliani M;Koh KP;Shen Y;Pastor WA;Bandukwala H;Brudno Y;Agarwal S;Iyer LM;Liu DR;Aravind L;Rao A
通讯作者：
Rao A
影响因子：
14.9
作者：
Hashimoto H;Hong S;Bhagwat AS;Zhang X;Cheng X
通讯作者：
Cheng X
影响因子：
5.6
作者：
BIRD, AP
通讯作者：
BIRD, AP
影响因子：
4.8
作者：
Maiti, Atanu;Drohat, Alexander C.
通讯作者：
Drohat, Alexander C.
影响因子：
64.5
作者：
Mellen, Marian;Ayata, Pinar;Heintz, Nathaniel
通讯作者：
Heintz, Nathaniel