title: GSM8774648: siDENR HEK293T #2 Ribo-seq replicate 1; Homo sapiens; OTHER
library_construction_protocol: RNA-seq extract protocol Total RNA was extracted from cell lysates with the use of a PureLink RNA Mini Kit (Thermo Fisher Scientific), and the quality of the purified RNA was assessed with a 2100 Bioanalyzer (Agilent). Ribo-seq extract protocol cells were seeded at a density of 3.0 × 106 per 10-cm dish and cultured overnight to ensure subconfluence. The culture medium was removed, and the cells were immediately placed on ice, washed once with ice-cold PBS, and lysed with a lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 1% Triton X-100, and EDTA-free protease inhibitor cocktail] supplemented with cycloheximide (100 μg/ml). The lysates were incubated on ice for 10 min with TURBO DNase (Thermo Fisher Scientific) and then centrifuged at 20,380 × g for 10 min at 4°C, and the resulting supernatants were collected, assayed for RNA concentration with a Qubit RNA BR Assay Kit (Thermo Fisher Scientific), incubated with RNase I (20 U per 10 μ