m6A RNA Immunoprecipitation and Quantification

m⁶A IPs were carried out as described previously [32 (link)]. Briefly, TREx-BCBL1-Rta cell total RNA was fragmented into 100–200 nucleotide segments using RNA fragmentation reagents (Ambion, Austin, TX, USA) and sodium-acetate-precipitated overnight at −80 °C. A 5% input was collected prior to immunoprecipitation. The remaining RNA was combined with 25 μL of Magna ChIP Protein A+G magnetic beads (Merck Millipore, Burlington, MA, USA) coated in 5 μL of anti-m6A antibody (Merck Millipore, Burlington, MA, USA) and incubated at 4 °C overnight with rotation. RNA from inputs and m⁶A immunoprecipitations was eluted using proteinase K (ThermoFisher, Waltham, MA, USA) followed by Trizol LS: chloroform extraction. RNA immunoprecipitations and inputs were converted to cDNA using the LunaScript RT SuperMix kit (NEB, Hitchin, UK). m⁶A-immunoprecipitated samples were normalised to their respective input samples, and m⁶A content at a particular region was calculated relative to an unmodified control region within the same transcript.