Identification of Methylated Deoxyadenosines in Genomic DNA by dA6mDNA Immunoprecipitation.
dA6mDNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). 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 (Koziolet al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziolet al., 2015). This protocol describes the method to perform dA6mDNA immunoprecipitation (DIP), as was applied to characterize the first dA6mmethylome analysis in higher eukaryotes (Koziolet al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA6mis pulled down with an antibody that recognizes dA6min genomic DNA. After subsequent washes, DNA fragments that do not contain dA6mare eliminated, and the dA6mcontaining fragments are eluted from the antibody in order to be processed further for subsequent analyses.


BACKGROUND
This protocol was developed in order to identify regions in the genome that contain dA6m. It can be used to detect dA6min different genomes. As a guideline, this protocol was established from existing approaches used to detect adenosine methylation in RNA (Dominissiniet al.,2013). We developed this protocol and adapted it for the detection of dA6min 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 dA6min eukaryotic DNA.
Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK; Department of Zoology, University of Cambridge, Cambridge, UK.
Magdalena J
MJ
Koziol
Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK.
Charles R
CR
Bradshaw
Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK.
George E
GE
Allen
Medical Research Council Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge, UK.
Ana S H
AS
Costa
Medical Research Council Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge, UK.
Christian
C
Frezza