Transcriptome-wide identification of IGF2BP binding RNA by RIP-seq: sequencing data (human). Homo sapiens

We applied RIP-seq to the insulin-like growth factor-2 (IGF2) mRNA-binding proteins 1, 2, and 3 (IGF2BP1/2/3) for exploring their targets. RNA immunoprecipitation was performed as previous described with some modifications. Briefly, cells seeded in 10 cm dish at 70-80% confluency were cross-linked by UV and harvested by trypsinization. Nuclear extraction was isolated and sonicated. One microgram of Flag (Sigma-Adrich), IGF2BP1 (CST), or IGF2BP2 (CST) antibody or a corresponding control IgG (mouse IgG from Millipore for Flag, rabbit IgG from Thermo Fisher Scientific for IGF2BP1 and IGF2BP2) was conjugated to Protein A/G Magnetic Beads (Thermo Fisher Scientific) by incubation for 4 hours at 4 °C, followed by 3x wash and incubation with pre-cleared nuclear extraction in RIP buffer [150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 1x protease inhibitor] at 4 °C overnight. After washing with RIP buffer for three times, beads were resuspended in 80 μL PBS, followed by DNA digestion at 37 °C for 15 min and incubation with 50 μg of Proteinase K (Thermo Fisher) at 37 °C for 15 min. Input and co-immunoprecipitated RNAs were recovered by TRIzol (Invitrogen) extraction and and seqeunced by Illumina. Overall design: IGF2BP1/2/3 RIP-seq in human 293T cells.