title: GSM8491794: 32D cell eCLIP IP rep2; Mus musculus; RIP-Seq
library_construction_protocol: Cells were pelleted, flash frozen in liquid nitrogen and stored at -80°C. The pellet was lysed with lysis buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate and 1× protease inhibitor cocktail, Roche) followed by further RNase I, and Turbo DNase treatment as described. The lysate was incubated with specific 20 µg antibody overnight at 4°C for immunoprecipitation. eCLIP was performed as described previously (Nature methods, 2016, 13(6): 508-14.) Libraries were prepared according to NEB's instructions accompanying the NEBNext Ultra II DNA Library Prep Kit (E7103S) and NEBNext Multiplex Oligos for Illumina (E7335S).