GSM7018496: Ribo-seq_293AD, Vec, rep2; Homo sapiens; OTHER
293AD cells stably expressing empty vector or YAP 5SA subjected to serum deprivation were preincubated with cycloheximide (100 μg/ml, Sigma) for 10 min. The cells were then lysed in 600 μl of lysis buffer [Tris-Cl pH 7.4 (10 mM, Duchefa), MgCl2 (5 mM, Junsei), KCl (100 mM, Junsei), Triton-X-100 (1%, Sigma), cycloheximide (100 μg/ml, Sigma), dithiothreitol (1 mM, Sigma), RiboLock RNase inhibitor (0.2 U/μl, Thermofisher), and EDTA-free protease inhibitor cocktail (1x, Roche)]. Then 300 μl of the cell lysates were treated with RNase I (5 U/μl, Ambion) for 45 min at room temperature for Ribo-seq library construction. The remaining 300 μl cell lysates were aliquoted for mRNA-seq library construction. RNase I-treated cell lysates were then placed in the Illustra MicroSpin S-400 HR Columns (GE Healthcare) and centrifuged at 600 x g for 2 min. Next, RiboMinus TM Eukaryote System v2 (Thermofisher) was used for the removal of ribosomal RNAs. For processing 5- and 3-end of ribosome protected fragments (RFPs) for subsequent library construction, Antarctic phosphatase (NEB) and T4 polynucleotide kinase (NEB) were treated, followed by size selection of 26-32 nt RPFs using a 12% TBE-urea gel system (National Diagnostics). Library construction was performed using the TruSeq Small RNA Kit (Illumina)