GSM6701471: Sample 22_HeLa cells, mRNA, eIF3e KD, rep2; Homo sapiens; RNA-Seq
Sample1 -Sample12: For footprint libraries cycloheximide was added to a final concentration of 100 µg/ml 1 min prior to harvesting. Cells were washed with ice cold 1x PBS and lysed in buffer A (10 mM HEPES [pH 7.5], 62.5 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 1mM PMSF, 1 µg/ml Aprotinin, 1 µg/ml Leupeptin, 1 µg/ml Pepstatin, Complete Mini EDTA-free [Roche] – 1 tablet/5 ml, 1% Triton X-100, 100 µg/ml Cycloheximide) on the dish. The resulting lysate was collected and cleared by centrifugation. Aliquots of twelve absorbance units (AU) OD260 of WCE were flash frozen in liquid nitrogen and used for footprint isolation. One aliquot of 12 AU OD260 was thawed on ice, digested with 5 µl RNAseI (Ambion) for 30 min at 24 °C with mild shaking (300 rpm) and inactivated with SuperaseIN (Ambion). 80S ribosomes were separated by high-velocity sedimentation through a 5% to 45% sucrose gradient at 39,000 rpm for 2.5 h using the SW41Ti rotor. The gradients were scanned at 254 nm to visualize the ribosomal species and fractions containing 80S ribosomes were collected and mixed with RNA Blue (TopBio). RNA was isolated according to vendor's instructions, FP RNA was size selected on 15% TBE-Urea PAGE (Biorad) in range 20-40 nt. Sample13-Sample28: For mRNA libraries the growth media was aspirated and cells were harvested into 5 ml of RNA Blue per Ø 15 cm dish. RNA was isolated according to vendor's instructions. mRNA was isolated from the total RNA using Poly(A)purist MAG kit (Ambion), fragmented using RNA Fragmentation Reagents (Ambion) and size selected on 15% TBE-Urea PAGE in range 50-100 nt. Sample1 -Sample12: According to Ingolia et al., 2010, Methods Enzymology Sample13-Sample28: library was prepared using SMARTer smRNA-Seq Kit (TAKARA) according to vendor's instructions