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Anal Chem. 2018 Nov 20;90(22):13200-13206. doi: 10.1021/acs.analchem.8b02832. Epub 2018 Nov 2.

Mirror Bisulfite Sequencing: A Method for Single-Base Resolution of Hydroxymethylcytosine.

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Zymo Research Corporation , 17062 Murphy Avenue , Irvine , California 92614 , United States.
School of Life Sciences , Guangzhou University , Guangzhou 510006 , China.
Division of Human genetics , Cincinnati Children's Hospital Medical Center , 3333 Burnet Avenue , Cincinnati , Ohio 45229 , United States.


Although the role of 5-methylcytosine has been well studied, the biological role of 5-hydroxymethylcytosine still remains unclear because of the limited methods available for single-base detection of 5-hydroxymethylcytosine (5hmC). Here, we present mirror bisulfite sequencing for 5hmC detection at a single CpG site by synthesizing a DNA strand to mirror the parental strand. This semiconservative duplex is sequentially treated with β-glucosyltransferase and M.SssI methylase. The glucosyl-5hmCpG in the parental strand inhibits methylation of the mirroring CpG site, and after bisulfite conversion, a thymine in the mirroring strand indicates a 5hmCpG site in the parental strand, whereas a cytosine indicates a non-5hmC site. Using this method, the 5hmC levels of various human tissues and paired liver tissues were mapped genomewide.

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