GSM1960354: NORAD-KO; Homo sapiens; RIP-Seq
4-thiouridine-labeled cells were washed with cold PBS and crosslinked using 365 nm UV with 150 mJ/cm2 total energy in a Spectrolinker XL-1500 (Spectroline).  A total of ~720 million cells (36 150 mm dishes) per CLIP condition were collected and resuspended in NP-40 lysis buffer (50 mM HEPES-KOH, pH 7.5, 150 mM KCl, 2mM EDTA-NaOH, pH 8.0, 1 mM NaF, 0.5% NP-40 substitute, 0.5 mM DTT, and Complete EDTA-free protease inhibitor cocktail).  After centrifugation, the soluble fraction was filtered through a 5 mm syringe filter and incubated with 1 U/m RNase T1 at 22ºC for 15 min.  100 mg PUM2 antibody (K-14, sc-31535, Santa Cruz) was conjugated to Protein G magnetic beads and incubated with RNase-treated lysate at 4ºC for 4 hours.  Bead-bound PUM2 RNP complexes were washed with IP wash buffer (50 mM HEPES-KOH, pH 7.5, 300 mM KCl, 0.05% NP-40 substitute, 0.5 mM DTT and Complete EDTA-free protease inhibitor cocktail) followed by an additional RNase T1 treatment (1 U/mL at 22ºC for 15 min).  Beads were further washed with high-salt wash buffer (50 mM HEPES-KOH, pH 7.5, 500 mM KCl, 0.05% NP-40 substitute, 0.5 mM DTT and Complete EDTA-free protease inhibitor cocktail) and the 5'  ends of PUM2 bound RNAs were labeled with 32P using calf intestinal alkaline phosphatase followed by T4 PNK and [gamma-32P]-ATP.  100 mM unlabeled ATP was added after radiolabeling to ensure all RNA species were 5'  phosphorylated.  Labeled RNP complexes were eluted from beads by boiling in 1X SDS-PAGE loading buffer (62.5 mM Tris HCl pH 6.8, 1.5% SDS, 8.3% Glycerol, 0.005% Bromophenol blue) and resolved on an SDS-PAGE gel.  After autoradiography, bands corresponding to the PUM2 RNP size (~120 kDa) were excised and electro-eluted using D-tube dialyzer tubes (Milipore) in MOPS-SDS running buffer.  Eluted samples were then digested with 1.2 mg/mL Proteinase K (Sigma) at 55ºC for 30 min.  RNA was isolated using phenol/chloroform extraction followed by ethanol precipitation. Sequencing libraries were constructed using the TruSeq Small RNA Library Preparation Kit (Illumina).  Sequencing was performed on a NextSeq 500 (Illumina).
GEO Accession
GSM1960354