RNA-binding protein immunoprecipitation sequencing and quantitative PCR (RIP-Seq and RIP-qPCR)

EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) was used according to the manufacturer's protocol, in order to detect the RNAs that bind IGF2BP1 proteins. AN3CA cells (1×10⁷) were washed in PBS twice and then lysed in 500 μL IP lysis buffer (containing 1× Protease Inhibitor Cocktail and 5 μL RNAse inhibitors). The supernatant was collected centrifugally and 10% was reserved as the input. To the 90% supernatant remaining, flag antibody (5 μg) and Magnetic Beads (40 μL) complex were added and samples were incubated at 4 °C overnight. Beads were washed in a buffer and eluted with an RNA purification kit (Qiagen), according to the manufacturer's protocol. The Ultra II RNA kit (NEB) was used to prepare the library according to the manufacturer's instructions. The samples were sequenced using the HiSeq PE150 platform. qPCR was performed with a 7900HT real-time instrument. All primers are listed in Table S1.