Single-CpG resolution mapping of 5-hydroxymethylcytosine by chemical labeling and exonuclease digestion (SCL-exo)

We report the setup of a new method to map 5-hydroxymethylcytosine (5hmC) genome-wide at CpG resolution. The method combines selective chemical labeling by 5hmC b-glucosyltransferase and exonuclease digestion of the DNA molecules bound to streptavidin beads after biotinylation of the 5-glucosylmethylcytosines. Associated with a straightforward bioinformatic analysis, this new procedure provides a cost-effective and fast method for mapping 5hmC at high resolution. Overall design: Genomic DNA from P19 mouse embryonal carcinoma (EC) cells treated with retinoic acid (RA) for 48hrs, or from undifferentiated E14 mouse embryonal stem cells, was extracted and submitted to the SCL-exo protocol. In parallel, a SCL reaction was run without exonuclease digestion for P19 cells. Reads were mapped to the mouse mm8 genome for P19 cells or mm9 genome for E14 cells and treated to extract a single-CpG signal file indicating the relative levels of hydroxymethylation.