N6-adenine DNA-immunoprecipitation-based genomic profiling in eukaryotes

The detection of low-abundance DNA N6-methyladenine (DNA-m6A) remains challenging, limiting our understanding of this novel base in eukaryotes. To address this, we introduce an approach for systematically validating the selectivity and sensitivity of antibody-based DNA-m6A methods, revealing most commercial antibodies as poorly selective towards DNA-m6A. Finally, we validate selective anti-DNA-m6A antibodies with sensitivity <2 ppm, and expose distinct pathways mediating endogenous DNA-m6A in C. reinhardtii, A. thaliana, and D. melanogaster. Overall design: The first part of this series is the evaluation of antibody-based DNA-m6A methods for identifying endogenous DNA-m6A-modified genomic loci using exogenously methylated control samples. Specifically, we applied DNA-m6A immunoprecipitation followed by massively parallel sequencing (m6A-IP-seq) to gDNA isolated from Drosophila S2 cell nuclei treated with 0.6 units, 0.075 units or no non-specific N6-adenine methyltransferase. For the second part of this series, we applied m6A-IP-seq to gDNA derived from three organisms with previously reported endogenous DNA-m6A (C. reinhardtii A. thaliana cauline leaves, and dechorionated D. melanogaster 45-minute embryos).