CLIP Assay for Protein-RNA Interactions

Cross-linking immunoprecipitation (CLIP) assays were as described in Ule et al. [29 (link)]. Briefly, cells or isolated mitoplasts were UV-irradiated on ice, harvested and lysed in Sigma FLAG lysis buffer adjusted with 0.1% SDS (v/v), Roche EDTA-free Protease Inhibitor Cocktail and Promega RNaseIn. Specific RNP complexes were immunoprecipitated. RNA species bound to the protein of interest were dephosphorylated, ligated to the 3′-RNA linker and end-labelled with γ³²P. Protein–RNA complexes were resolved on SDS–PAGE, transferred to nitrocellulose (BA-85 Whatman) and subjected to autoradiography. Appropriately sized RNP complexes were excised and proteinase K-treated. Following ligation of a 5′ RNA linker RNA, CLIP tags were amplified by RT-PCR, and then cloned, sequenced and analyzed as described in ref. [14 (link)], or IonTorrent-sequenced as described in ref. [30 (link)].