title: GSM2692143: MEFs-control; Mus musculus; RIP-Seq
library_construction_protocol: PAR-CLIP was performed according to Spitzer et al., 2013, with the exception that cells were lysed in lysis buffer (10 mM Tris-HCl (pH 7.5), 260 mM sucrose, 100 mM KCl, 20 mM MgCl2, 1% digitonin) in the presence of RNase inhibitor (New England Biolabs) and protease inhibitors (Roche). Lysates were clarified by centrifugation and diluted in lysis buffer without digitonin until the final digitonin concentration was 0.2%. Diluted lysates were incubated with anti-FLAG M2 magnetic beads (Sigma, Cat. No. M8823) for 2.5 h at 4°C. Beads were washed in lysis buffer with 0.1% digitonin and then in lysis buffer without digitonin. RNA library preparation and sequencing was performed by Vertis Biotechnologie (Freising, Germany) using unique molecular identifiers to identify PCR duplicates according to Kivioja et al., 2011.