GSM7746909: L570-5hmU_chemical_mapping_mild_oxid; Breviolum minutum; OTHER
Chemical mapping of 5-hmU as carried out following the previously described by Kawasaki et al. chemical conversion method. Briefly, sheared genomic DNA was used as input and end prep and adapter ligation were carried out using the NEBNext Ultra II DNA Library Prep Kit. After the ligation step, DNA was purified using AMPure XP beads and eluted in 50 µL of H2O. DNA denaturation was performed by adding NaOH to a final concentration of 0.05M and incubating at 37◦C for 30 minutes. Oxidation was carried out by adding 2 µL of KRuO4 solution (15 mM in 0.05 M NaOH) and incubating for 30 minutes at room temperature. Oxidized DNA was purified using AMPure XP beads and extension was carried out by mixing 13.5 µL DNA, 1.6 µL 100 mM MgSO4, 2 µL NEB Index Primer, 2 µL 10× ThermoPol Reaction Buffer (NEB), 0.5 µL 10 mM dNTP mix, and 0.4 µL Bst DNA Polymerase, Large Fragment (NEB), then incubating for 1 hour at 37◦C PCR amplification was carried out using the NEB Ultra DNA Library Prep Kit, with 12 cycles of PCR. Final libraries were purified using AMPure XP beads.