Identification of Methylated Deoxyadenosines in Genomic DNA by dA^6m DNA Immunoprecipitation

dA^6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N⁶-methyldeoxyadenosine (dA^6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol et al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol et al., 2015). This protocol describes the method to perform dA^6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA^6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA^6m is pulled down with an antibody that recognizes dA^6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA^6m are eliminated, and the dA^6m containing fragments are eluted from the antibody in order to be processed further for subsequent analyses.

This protocol was developed in order to identify regions in the genome that contain dA^6m. It can be used to detect dA^6m in different genomes. As a guideline, this protocol was established from existing approaches used to detect adenosine methylation in RNA (Dominissini et al., 2013). We developed this protocol and adapted it for the detection of dA^6m in DNA, rather than detecting adenosine methylation RNA. This was required, as no protocol was available at that time to allow the genome-wide identification of dA^6m in eukaryotic DNA.