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Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation.

Accepted version
Peer-reviewed

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Authors

Koziol, Magdalena J 
Bradshaw, Charles R 
Allen, George E 
Costa, Ana SH 

Abstract

dA6m DNA 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 (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 dA6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA6m is pulled down with an antibody that recognizes dA6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA6m are eliminated, and the dA6m containing 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 dA6m 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 dA6m 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 dA6m in eukaryotic DNA.

Description

Keywords

31 Biological Sciences, 3105 Genetics, Biotechnology, Genetics, Human Genome, 1.1 Normal biological development and functioning, 1 Underpinning research, Generic health relevance

Journal Title

Bio-Protocol

Conference Name

Journal ISSN

2331-8325
2331-8325

Volume Title

6

Publisher

Bio-Protocol
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/M022994/1)
Medical Research Council (G1001690)
MRC (unknown)
Medical Research Council (MR/K011022/1)
Wellcome Trust (092096/Z/10/Z)
Wellcome Trust (101050/Z/13/Z)
Cancer Research Uk (None)
Long-Term Human Frontiers Fellowship (LT000149/2010-L), Medical Research Council (Grant ID: G1001690), Isaac Newton Trust Fellowship (Grant ID: RG76588), Biotechnology and Biological Sciences Research Council (Grant ID: BB/M022994/1), Wellcome Trust (Grant ID: 101050/Z/13/Z), Wellcome Trust Core Grant (Grant ID: 092096/Z/10/Z), Cancer Research UK (Grant ID: C6946/A14492)