GSM8191176: IgG, NPC, Rep2; Homo sapiens; RIP-Seq
Cellular RNAs bound by p-UPF1 were immunoprecipitated using anti-p-UPF1 [Millipore, anti-phospho-Upf1(Ser1127)] and Dynabeads protein A magnetic beads (Thermo Fisher Scientific). The RNA in bead-bound RNA–p-UPF1 complexes was digested by incubation for 20 min at 4°C with 1 U/μl RNase I (Ambion). To prepare RNA for cDNA library construction, after extensive washing, bound complexes were eluted using immunoprecipitation elution buffer (125 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 0.1% bromophenol blue, 10% 2-mercaptoethanol). RNAs were purified using TRIzol reagent followed by ethanol precipitation. In parallel, control immunoprecipitations using rIgG were performed. Additional control samples (input) were prepared without immunoprecipitation. Purified RNA fragments were used to make the library using the NEBNext Ultra II Directional Library Prep Kit (New England Biolabs), as per manufacturer's instructions. Libraries were sequenced (pair-end reads) with an Illumina NovaSeq 6000 platform for 100 cycles at the UCSD IGM core.