Quantification of m6A RNA Modifications

Poly(A) RNA was purified from total RNA using a Dynabeads^TM mRNA purification kit (Thermo Fisher, USA) and then the purified mRNA was heated at 95 °C for 3 min. The mRNA was then used for Dot blot^{33 (link)}. The mRNA was spotted on an Amersham Hybond-N + membrane. After UV crosslinking, the membrane was blocked with 5% (w/v) non-fat milk in Tris-buffered saline with Tween-20 (TBST), and incubated with anti-m⁶A antibody (1:500; Synaptic Systems) overnight at 4 °C. After incubation with a secondary anti-rabbit antibody, the membrane was visualized using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).