GSM7035431
RIP assay was performed on PC3/ELAVL3-V5 and DU145/ELAVL3-V5 cells. About 1 × 107 cells in each dish were harvested and resuspended in 250 μL lysis buffer [20 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40, 10% glycerol, 2 mM EDTA, Protease inhibitor cocktail (11836145001, Roche), 0.5 mM PMSF (ST506, Beyotime, China), 2 mM Ribonucleoside Vanadyl Complexes (R0108, Beyotime, China), 2 μL RNase inhibitor (R0102, Beyotime, China)]. 10% volume of the lysates were separated into another two RNase-free tubes, labeled as input, and the rest lysates were used for RIP assay. All lysates were frozen rapidly at − 80°C overnight to gently lyse the cells. Then thawed the RIP lysates quickly and centrifuged at 15,000 rpm for 10 mins at 4°C. The supernatant was collected and incubated with V5 antibody (13202, Cell Signaling Technology) and protein A+G magnetic beads (P2108, Beyotime, China) at 4°C for 4 hours with rotation. The V5-Beads were washed with 500 μL lysis buffer for 5 times, rigorously. After the last washing, thawed the input lysates and added 1 mL Trizol reagent to the input lysates and the V5-Beads to extract total RNA. RNA was extracted using Trizol reagent according to the manufacturer's instructions. rRNAs were removed by using Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, CA, USA). RNA library was constructedusing rRNA-depleted RNAs with TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Sequencing was performed on the illumina NovaseqTM 6000 system (Illumina, San Diego, CA) at high out-put model according to the manufacturer's instruction. RIP-seq libraries were prepared following the SMARTseq protocol