GSM7597538: m6A_KD1_1; Homo sapiens; OTHER
total RNA harvest by Trizol method RNA was fragmented to around 200nt and purified using RNeasy Mini Kit (Qiagen). Dynabeads™ protein G beads (Invitrogen) were washed twice by IP buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O), resuspended in 500 μl of IP buffer with 5μg anti-m6A antibody at 4oC for 4 hours. Following 2 washes in IP buffer, the beads was resuspended in 500μl of the IP reaction mixture containing 10μg fragmented total RNA, 100μl of 5×IP buffer, and 5μl of SUPERase·In™ RNase Inhibitor (Invitrogen) and incubated for 2 hours at 4 oC. A low/high salt-washing method was used: the RNA reaction mixture was washed twice in 1ml IP buffer, twice in 1ml low-salt IP buffer (50 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O), and twice in 1ml high-salt IP buffer (500 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O) for 5 minutes each at 4 oC. After extensive washing, the m6A-enriched fragmented RNA was eluted from the beads in 350 μl of RLT buffer supplied in RNeasy Mini Kit (Qiagen) for 10 minutes at room temperature. A magnetic separation rack was used to pull beads to the side of the tube. Supernatant was collected to a new tube and 350μl 70% ethanol was added to it. RNA was purified following the manufacturer's protocol. Equal volume of m6A-enriched fragmented RNA from control and knockdown samples was used for library construction using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) and sequenced using Hiseq X platform.