Hypoxia-induced DNA Methylation Analysis

Neuroblastoma cells were grown for 72 h at atmospheric or hypoxic conditions, and the extracted gDNA was sonicated on M220 Focused-ultrasonicator (Covaris, Woburn, MA, United States) in 10 mM Tris-HCl (pH 8.5) buffer to yield fragments with a peak size of ∼200 bp. 5hmC glycosylation was carried out in a 100-μl reaction mixture with 800–1,000 ng fragmented gDNA supplemented with 50 μM UDP-6-azide-glucose (Jena Bioscience) and 10 U T4 β-glucosyltransferase (TS) for 2 h at 37°C followed by enzyme inactivation at 65°C for 20 min and column purification [GeneJet PCR Purification kit (TS)]. Azide-tagged DNA was processed as described previously (Gibas et al., 2020 (link)). DNA libraries were amplified for 12 cycles, size selected for ∼300 bp fragments (MagJET NGS Cleanup and Size Selection kit, TS), and subjected to Ion Proton (TS) sequencing.