Nanopore DNA sequencing in a model of oxidative stress

Cytosine DNA methylation in the CpG context (5mCpG) is associated with the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mCpG has a regulatory role in this context. The main aim of this work was to develop and validate a novel tool for determining methylation of mtDNA and to corroborate its existence across different biological contexts. Here, we profiled the human cell lineHEK 293T, before and after oxidative stress, using long-read nanopore sequencing. Oxidative stress was induced in vitro with hydrogen peroxide (H2O2) alone or in combination with N-acetyl-cysteine (NAC) as a rescue treatment. mtDNA was enriched by sequential gradient centrifugation and, after linearization with BamH1, samples were barcoded and sequenced with a MinION device (ONT).