title: GSM8410823: EBd8-WT-2.1-m6A; Mus musculus; OTHER
library_construction_protocol: Total RNA was extracted with RNeasy kit according to the manufacturer's instructions. Residual DNA removal was performed using the RNase-Free DNase Set. Polyadenylated RNA was enriched from 500 µg total RNA by one round of oligo-dT selection using the GenElute mRNA miniprep kit (Sigma-Aldrich). (m6A-immunoprecipitation) RNA was fragmented by mixing 90 µl RNA (at 0.9 µg/µl) and 10 µl fragmentation buffer 10X (100 mM Tris-HCl and 100 mM ZnCl2) in thin-walled 200-μl PCR tubes, then incubated at 94°C for 40 s in a pre-heated thermocycler block with the heated lid opened. 10 µl of 0.5 M EDTA was added and the tubes were placed on ice. RNA was then subjected to sodium acetate precipitation and resuspended in 300 μl RNase-free water. RNA fragmentation size was assessed by running 0.25 µg RNA on a 2100 Bioanalyzer (Agilent) with the RNA Nano kit (Agilent). Furthermore, 2-5 µg of fragmented RNA was kept at -80°C to later serve as input control. The RNA was denatured by heating (70°C for 5 min, then put on ice) and the immunoprecipitation mix was prepared as follows: polyA enriched fragmented RNA from cultured cells in 290 µl, 10µl proteinases inhibitors (Roche), 5 µl (200 units) RNasin® Ribonuclease Inhibitors (Promega), 5 µl of 200 mM Ribonucleoside Vanadyl Complex (RVC) (Sigma), 100 µl IP buffer 5X (50 mM Tris-HCl, 750 mM NaCl, 0.5% (vol/vol) Igepal), 3.5 µl anti-m6A antibody (Synaptic System, #202003) and 86.5 µl RNase-free water. The mix was incubated overnight at 4°C on a rotating wheel. The next day, 50 µl Dynabeads™ Protein G (Invitrogen, #10004D) were washed twice with IP buffer 1x supplemented with antiproteases (Roche) and blocked by incubating the beads in wash buffer supplemented with 0.5 mg/ml BSA for 1 h on a rotating wheel. The beads were washed again twice, then added to the immunoprecipitation mix and incubated for 2 h at 4°C on a rotating wheel. Then, the beads were washed three times with 1 ml washing buffer supplemented with 10 µl RNasin® and 10 µl RVC, and elution was performed by TriPure (Roche) extraction, so the RNA was resuspended in 9 µl RNase-free water. The sequence library was prepared with SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara) for both input and IP samples.