GSM8197305: A549 cells, RIP-seq, FTO_IP; Homo sapiens; RIP-Seq
RIP was performed using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (MilliporeSigma) with some modifications. A549 cells were harvested for each IP reaction and lysed in Complete RIP Lysis Buffer. 5% of the volume of cell lysate was saved as INPUT, mixed with 350 μl TRIzol. 50 μl of protein A/G magnetic beads were washed by RIP Wash Buffer, resuspended in 100 μl of RIP Wash Buffer containing 5 μg anti-METTL3 antibody  (Abcam),  anti-ALKBH5 antibody (Proteintech), anti-WTAP antibody (Abcam), anti-METTL14 antibody (Cell Signaling Technology) and anti-FTO antibody (Proteintech) at 4°C overnight, respectively. The antibody-bead mixture was washed and resuspended in 900 μl of RIP Immunoprecipitation Buffer and 100 μl of cell lysate and incubated for 4 h at 4°C. The beads were then washed 5 times and re-suspended with 350 μl TRIzol as IP. RNAs from INPUT and IP were extracted using RNeasy Mini Kit (QIAGEN). Library construction was conducted using SMARTer Stranded Total RNA-Seq Kit version 2-Pico Input Mammalian (Takara/Clontech).