GSM7018480: 293AD, F-YAP 5SA, rep2; Homo sapiens; RNA-Seq
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, and total RNA was extracted using TRIzol reagent (Invitrogen) Library construction was performed using the TruSeq Stranded mRNA Kit (Illumina)