title: GSM5097942: Con MO m6A-IP; Xenopus laevis; OTHER
library_construction_protocol: PolyA+ RNAs were fragmented using fragmentation buffer (Ambion #AM8740) to yield ~200 nt lengths RNA fragments. Polyclonal anti-m6A antibody (Synaptic Systems) was incubated with fragmented RNAs in RIP buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP40) supplemented with RNase inhibitors, RNasin (promega) and Superase-in (Ambion), for 4 hours at 4 °C. Along with this, Dynabeads protein A (Invitrogen #10002D) was washed and pre-blocked with 0.5 mg/ml BSA for 2 hours at 4 °C. Beads were then added to antibody-RNA complex and incubated for an additional 1 hour at 4 °C. Bound RNA was eluted after extensive washing with RIP buffer by competitive elution with buffer containing 7 mM m6A (Abcam #ab145715) twice each for 1 hour at 4 °C. Eluted RNAs were then precipitated with ethanol and used for m6A-sequencing library preparation. Sequencing libraries were prepared using SMARTer Stranded RNA-seq kit (Clontech) according to the manufacturer's protocol. Purified m6A-seq libraries were sequenced on an Illumina MiSeq platform with 10-20% PhiX control library (Illumina).