Quantifying m6A Levels in Mycn Transcript

We identified a putative m⁶A site within the Mycn MeRIP peak using the m⁶A-Atlas database (http://www.xjtlu.edu.cn/biologicalsciences/atlas)(36 (link)). We then measured m⁶A by RT-qPCR, following a method previously described(37 (link), 38 (link)), which takes advantage of the diminished capacity of Bst enzymes to retrotranscribe m⁶A residues compared to MRT control enzyme, and RT primers targeting just before or after the site (primer + or −). Each cDNA was generated with ~100ng of total RNA, 100nM primer (+ or −), 50μM dNTPs and 0.1U of Bst3.0 (NEB) or 0.8U of MRT (ThermoScientific). The cycling conditions were 50°C for 15min, 85°C for 3min, then 4°C. RT-qPCR data were then acquired on a QuantStudio 5 (Thermo Fisher Scientific) and normalized as [2^−(Ct_Bst− − Ct_MRT−) − 2^-(Ct_Bst+ − Ct_MRT+)] / 2^−(Ct_Bst− − Ct_MRT−). Negative values were considered below the detection threshold and rounded to 0.