Genome Wide Mapping of Methylated and Hydroxyl-Methylated Cytosines Using a Modified HpaII Tiny Fragment Enrichment by Ligation Mediated PCR Tagged Sequencing Protocol
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.
Key words5-methylcytosine 5-hydroxymethylcytosine Sequencing β-glucosyl transferase HpaII MspI DNA methylation