GSM8689652: 293T_IFIT1:FLAGoe_FLAG_IP_VSV_rep2; Homo sapiens; OTHER
eCLIP was performed as previously described (Blue SB, et al. Nature Protocols 2022). Briefly, 10 million 293T or MEF cells were UV crosslinked (254 nM, 400 mJ/cm2), snap frozen, and lysed as previously described. RNA fragmentation was performed, 2% of the sample was removed for size-matched input, and the remainder was subjected to immunoprecipitation of IFIT-bound RNA with antibodies against FLAG (M2 / F1804, Sigma) and HA (HA.11 / 901502, Biolegend) for 293T experiments, and IFIT2 (PA3-845, ThermoFisher) and IFIT3 (ABF1048, Millipore) for MEF experiments. Dephosphorylation, RNA adapter ligation, SDS-PAGE electrophoresis and nitrocellulose membrane transfer, RNA isolation, reverse transcription, and PCR amplification were performed as previously described (Blue, SM. et al, Nat Protocols 2022). Libraries are as previously described for eCLIP (Blue, SM. et al, Nat Protocols 2022), based on the Illumina RNA TruSeq HT adapter strategy.