Battachariyya Sanchari, Tytarenko Ruslana, Heuck Christoph, Greally John, Verma Amit
Albert Einstein College of Medicine, Bronx, NY, USA.
University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Methods Mol Biol. 2018;1792:167-177. doi: 10.1007/978-1-4939-7865-6_12.
Here we describe a method for genome wide investigation of methylation and hydroxymethylation status of cytosines. This protocol is an improvement of the HELP-tagging protocol previously described by Suzuki et al. It involves the glucosylation of 5-hydroxymethylcytosines (5-hmC) with β-glucosyl transferase (β-GT), thus rendering them resistant to digestion by MspI. Parallel digestion of β-GT treated samples with MspI, untreated sample with MspI and another untreated sample with HpaII, followed by adapter ligation, parallel sequencing and bioinformatics processing results in a differential display of MspI digestion sites that allows the determination of the distribution of 5-methylcytosines (5-mC) and 5-hmC at these sites.
在此，我们描述了一种用于全基因组范围内胞嘧啶甲基化和羟甲基化状态研究的方法。该方案是对铃木等人先前描述的HELP标记方案的改进。它涉及用β-葡萄糖基转移酶（β-GT）对5-羟甲基胞嘧啶（5-hmC）进行糖基化，从而使其对MspI消化具有抗性。用MspI对经β-GT处理的样品、未经处理的样品用MspI以及另一个未经处理的样品用HpaII进行平行消化，随后进行接头连接、平行测序和生物信息学处理，从而实现MspI消化位点的差异显示，进而确定这些位点处5-甲基胞嘧啶（5-mC）和5-hmC的分布。
