Crosslinking and Immunoprecipitation Protocols

CLIP, PAR-CLIP, and iCLIP were performed as previously described (Hafner et al. 2010 (link); Huppertz et al. 2014 (link)) with the variations described in the Supplemental Methods. We used 2 × 10⁸ cells for SUZ12, IgG, and GFP iCLIP and used 2.5 × 10⁷ cells for FUS and HNRNPC iCLIP. Five microliters of α-SUZ12 (Cell Signaling) or 5 μg of α-EZH2 (Cell Signaling), α-Flag (Sigma), α-MYC (Cell Signaling), α-FUS (Novus), nonspecific IgG (Santa Cruz), or α-GFP (Abcam) antibody were used per experiment. Crosslinked RNPs running 10–30 (SUZ12), 10–45 (FUS), 10–40 (HNRNPC), and 5–30 (GFP) kDa above the sizes of their respective proteins were isolated. For IgG, we isolated RNPs of the same molecular weight as for SUZ12. Libraries were sequenced using an Illumina HiSeq 2500 (50-bp single-end reads). Crosslinked RNA was quantified using a Typhoon phosphorimager (GE) and ImageQuantTL (GE). EZH2 PAR-CLIP data (GSE49435) were processed as previously described (Kaneko et al. 2013 (link)), retaining only those reads containing a T > C transition. A list of ezRNA genes was obtained from Kaneko et al. (2013) (link).