GSM6395335: NCI-H1299 cells, DMSO 1; Homo sapiens; OTHER
Cells were lysed in ice cold lysis buffer (20 mM Tris-HCl, pH 7.8, 100 mM KCl, 10 mM MgCl2, 1% Triton X-100, 2 mM DTT, 100 μg ml−1 cycloheximide, 1× EDTA-free Complete protease inhibitors) and the lysate was clarified by centrifugation at 5000 rpm. The clarified lysate was treated with 2U/μl RNase I for 45 min at room temperature and loaded on a linear sucrose gradient (7% to 47%) and centrifuged using the SW-41Ti rotor at 36,000 rpm for 2 h. Fractions containing monosomes were pooled, treated with proteinase K. RNA fragements thus obtained were purifed using Trizol reagent as per manufacturer's recommendations. RNA thus obtained was loaded on a 10% polyacrylamide urea (7M) gel and the 30-33 nt long RNA fragments were excised and purified by ethanol precipitation. The Ribosome protected RNA fragements were 3'-dephosphorylated by treating with T4 PNK for 6 h at 37C, and 3' adaptor ligated using T4 RNA ligase 1 for 2.5 h at 37C. 3' adaptor ligated fragements were 5'phosphorylated by treating with T4PNK for 30 min at 37C, 5' adaptor ligated using T4 RNA ligase 1 for 18 h at 22C.