m6A Immunoprecipitation and Sequencing

m⁶A-IP-Seq was performed as previously described [46 (link)]. Briefly, 5 µg of DNAse treated total RNA were subjected to fragmentation with ZnCl₂ incubation at 70 °C for 13 min. Following precipitation, fragmented RNA size distribution was assessed using RNA 6000 Nano Bioanalyzer kit (Agilent). Approximately 500 ng of sample were stored as input control. Fragmented RNA was subjected to two rounds of m⁶A immunoprecipitation for 2 h each using an anti-m⁶A antibody (ABE572, Merck) previously conjugated to protein-A magnetic beads (Thermo Fisher Scientific) and protein-G magnetic beads (Thermo Fisher Scientific) by incubation at 4 °C for at least 6 h. Following extensive washing, RNA was eluted from the beads using RLT buffer and RNeasy mini kit (Qiagen). RNA was quantified using RNA 6000 Pico Bioanalyzer kit (Agilent). To confirm m⁶A enrichment, cDNA was synthesised using the SensiFast cDNA synthesis kit (Bioline) and SETD7 and GAPDH levels measured by qPCR were used as positive and negative control respectively. Finally, library preparation was performed using the SMARTER Stranded Total RNA Seq kit v2-Pico Input Mammalian kit (Takara Bio) following the manufacturer’s instructions. Libraries were then sequenced using HiSeq2500 (Illumina) and a minimum of 20 million paired-end reads were obtained per sample. This experiment was performed in duplicates for each sample.