Illumina HiSeq 2000 sequencing
Ribo-Seq
Ribosome footprints libraries were prepared starting from five million C2C12 cells, seeded in 10cm dishes. After 24 hours, cells were treated with 100 microgram/ml cycloheximide (C7698-1G, Sigma) for 10 minutes or with 2 microgram/ml harringtonin (sc-204771A, Santa Cruz Biotechnology) for 5 minutes followed by 10 minutes of cycloheximide treatment. After drug treatment at 37C, dishes were transferred on ice and cells were washed with ice-cold PBS supplemented with 100 microgram/ml cycloheximide. Cells lysis was performed using 1 ml ice-cold lysis buffer (1X salt buffer [10 mM Tris, 120 mM MgCl2, 1.4 M NaCl, pH 7.4], 0.5% IGEPAL) supplemented with RnaseOUT (500 U/ml, Invitrogen), DDT (1.5 mM), cOmplete Protease Inhibitor Cocktail (40 microliter of 25X stock, Roche) and cycloheximide (100 microgram/ml). Nuclei were removed by centrifugation at 13,000 g for 10 minutes. Supernatant was digested with RnaseI (1500U/ml, Ambion) for 30 minutes at room temperature. Digestion was blocked with SuperaseIN (600U/ml, Ambion) and lysate was layered on frozen sucrose gradients (7-46% sucrose) and separated by ultracentrifugation at 35,000 g for 3 hours at 4C. Twelve fractions (750 microliter each) were collected from the top, and digested with proteinase K (0.15 mg/750 microliter) for 30 minutes at 42C in the presence of 1% SDS. RNA was extracted by acid phenol (Ambion) purification followed by ethanol precipitation. For each sucrose gradient separation, an undigested lysate was used to monitor the polysome profile, determined on the Bioanalyzer (Agilent) with the RNA 6000 Nano kit. Fractions containing monosomes (corresponding to fractions nine and ten) were combined. Cytoplasmic rRNAs and mitochondrial rRNAs were removed using Ribo-Zero Magnetic Gold Kit (Epicentre) according to manufacturer's instructions, with the following modifications: Removal Solution was incubated for 4 minutes at 68C prior RNA addition, then mixed with RNA and kept at 37C for 15 minutes; RNA hybridized to Removal Solution was incubated with magnetic beads at room temperature for 5 minutes, followed by 1 minute at 50C and 4 minutes at 37C. Size selection of footprints with length 28-32 nt was performed on 15% TBE-urea gel (Invitrogen). Footprints were dephosphorylated with T4 polynucleotide kinase (10U, NEB) and ligated to double stranded RNA adapters at both ends (SOLID Total RNA-Seq Kit, Ambion). RNA was reverse transcribed and amplified using indexed custom primers adapted for Illumina Hiseq 2000 (5'-AATGATACGGCGACCACCGATGGGCAGTCGGTGAT-3', 5'-GCGGAACCGGCATGTCGTC|index|AGCATACGGCAGAAGACGAA-3'). Sequencing libraries were size selected for amplicons of 120 bps on 4-12% PAGE gel (Novex TBE, Life Technologies).