GSM3772893: SpenKO_RRM_plusDox_invivo_irCLIP_IgG; Mus musculus; RIP-Seq
nuclear RNA was isolated as previously described (Gagnon et al., 2014). 20 μg nuclear RNA was then fragmented (Ambion, AM8740), and incubated with 5 μg recombinant Spen RRMproteins (RRM2-4 WT or RRM2-4:RRM3 Mt) at 4C for 2 hours.  RNA-protein complexes were then transferred to a 3cm cell culture dish on ice and crosslinked (254nM UV-C) at 0.3J/cm2.  Following crosslinking, RNA-protein complexes were incubated with antibody-bound Protein A Dynabeads at 4C for 2 hours.  Following IP, beads were washed with IPP buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.05% NP-40, 5mM EDTA), low salt buffer (10 mM Tris-HCl pH7.4, 50 mM NaCl, 1mM EDTA, 0.1% NP-40), and high salt buffer (10 mM Tris-HCl pH7.4, 500 mM NaCl, 1 mM EDTA, 0.1% NP-40, 0.1%SDS).