GSM8919848: HepG2, siCtrl, FUBP1 RIP, rep1; Homo sapiens; RIP-Seq
For RNA-seq, HepG2 cell RNA was harvested by TRIzol™ reagent.  For RIP-seq, samples were  lysed by iCLIP lysis buffer (50 mM Tris pH 7.5, 100 mM NaCl, 1% NP-40 substitute, 0.1% SDS, 0.5% sodium deoxycholate, 100 × protein inhibitor cocktail and 100 × SUPERase•In™ RNase Inhibitor) at 4℃ for 15 mins with rotation, followed by centrifugation at 13,000 g for 15 mins. Protein G beads (Invitrogen, 10009D) were conjugated with 16 μg FUBP1 antibody (abcam, ab192867) by incubation at 4℃ for 1hr, then washed and mixed with lysate supernatant to rotate at 4℃ for 4 hrs. Beads were washed for three times with CLIP wash buffer (50mM Tris pH 7.5, 300 mM KCl, 0.05% NP-40 substitute, 1000 × protein inhibitor cocktail and 1000 × SUPERase•In™ RNase Inhibitor) and eluted by proteinase K digestion (Thermo Scientific, EO0491). Input samples harvested before immunoprecipitation was also digested with proteinase K for RNA recovery. The purified RNA was constructed for library with SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian.