Cross-Linking Immunoprecipitation (CLIP) Protocol

CLIP was performed as previously reported^{18 (link)} with some modifications. Briefly, the whole cell lysate from cross-linked (twice by 150 mJ per cm² of 365 nm UV light) PANC-1 cells were isolated and sonicated, followed by treatment with DNase I (0.5 U/μl, 37 °C for 5 min) and RNase TI (0.2 U/μl, 22 °C for 15 min). Pre-washed Dynabeads protein A/G (Millipore) conjugated with 10 μg antibodies against CSTF2, METTL3, or IGF2BP2 were then incubated with the extraction at 4 °C overnight with rotating. After substantial washing of beads, end repair was performed by using T4 PNK (NEB). RNA was then treated with proteinase K (37 °C for 30 min), acidic phenol/chloroform extraction, and ethanol precipitation, and was subsequently used for library construction by using NEBNext small RNA library prep kit (E7330S) and sequenced on Illumina Hiseq4000. For CLIP-qPCR, the input and immunoprecipitated RNA samples were recovered as described above. cDNA was synthesized with SuperScript III RT (Invitrogen) and random hexamer primers (Invitrogen) and subject to qRT-PCR using specific primers shown in Supplementary Table 3.