RNA extraction, selection and fragmentation for m6A-IP

Total RNA was isolated using TRIzol (Life Technologies), according to manufacturer’s instructions. RNA was resuspended in ultrapure water and treated with DNAse I (Ambion) for 30 min at 37°C and subjected to RNA clean up with RNeasy Midi Kit (Qiagen), according to manufacture’s protocol. RNA was eluted in ultrapure water. 800 µg of total RNA were subjected to two rounds of poly(A) RNA selection, using the MicroPoly(A) Purist kit, (Life Technologies) according to the manufacturer's protocol. For the second round of poly(A) RNA selection, the eluate of the first round of poly(A) RNA selection was used as starting material. 2.5 µg of poly(A)-selected RNA was incubated for 45 s with pre-warmed Zinc chloride buffer (10 mM ZnCl₂, 10 mM Tris-HCl, ph 7.0), to fragment the RNA to ~100 nucleotide long fragments. The reaction was stopped with 0.2M EDTA and immediately placed on ice. The fragmented RNA was recovered using the RNEasy Mini Kit (Qiagen).