GSE192792
Enhanced CLIP-seq analysis of PTBP1 and IGF2BP2 binding targets
To investigate the binding targets of PTBP1 (PTB) and IGF2BP2 (IMP2) in glioma cells, we carried out an unbiased analysis of transcripts bound by PTBP1 and IGF2BP2 using enhanced crosslinking immunoprecipitation followed by high-throughput sequencing (eCLIP) in A172 glioma cells. We repeated the eCLIP method twice in each experiment, and found that the ratio of PTBP1 and IGF2BP2 binding mRNAs overlap was very high. Overall design: eCLIP studies were performed according to the published protocol (Eric L Van Nostrand et al., 2016). 2 × 107 A172 cells incubated for 3 hours with 500 µM 4-thiouridine (4SU, T4509, Sigma) were crosslinked with 2× 400 mJ/cm2 365 nm UV light, and snap frozen. Cells were then lysed by 1ml CLIP lysis buffer (50 mM Tris-HCl pH7.4, 100 mM Nacl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) with temporary addition of 1 mM DTT (P2325, Gibco), 5.5 µl 200 × protease inhibitor cocktail ? (539134-1SET, Calbiochem/MercK) and 11 µl RNase inhibitor (N2515, Promega)  and treated with RNase I (AM2295, Invitrogen by Thermo Fisher Scientific) to fragment RNA. PTBP1 or IGF2BP2 antibody (RN011P/RN008P, MBL Beijing Biotech co., LTD) was then pre-coupled to Protein A Dynabeads(10002D, Invitrogen by Thermo Fisher Scientific), added to lysate, and incubated overnight at 4?. Prior to immunoprecipitation, 2% of the sample was taken as the paired input sample. The remaining samples were ligated to the RNA adapter, washed and separated magnetically.