Biomek liquid handler

Total RNA from 4 biological replicates of each condition was poly(A)-enriched using Dynabeads Oligo(dT)₂₅ (Thermo Fisher) and fragmented to a mean size of 200-300 nucleotides by incubation in 50 mM MgCl₂ for 8 min at 95 °C. A portion of fragmented RNA was saved as input. Remaining RNA samples were incubated overnight at 4 °C rotating with protein G magnetic beads (Thermo Fisher) coated with EpiMark anti-m⁶A antibody (New England BioLabs). Washes and elution were performed on a Biomek liquid handler (Beckman Coulter). To remove unbound RNA, samples were washed five times with each of the following buffers: reaction buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O), low-salt reaction buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O), and high salt reaction buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40 in nuclease-free H2O). RNA was eluted with RLT buffer (Qiagen) and purified with MyOne Silane Dynabeads (Thermo Fisher). RNA libraries for the RNA input, collected supernatant, and IP were constructed using SMARTer PrepX Apollo NGS library prep system (Takara) following the manufacturer’s protocol. The size distributions of the resulting libraries were assessed using the Tapestation D1000 screen tape (Agilent Technologies), normalized, and sequenced on a NextSeq 550 (Illumina) using a single read 75 cycle kit.