Ribo-Seq of homo sapiens: SH-SY5Y neuroblastoma cell line
Poly-Ribo-Seq. Cells were treated with cycloheximide (Sigma) at 100g/ml for 3 min at 37oC, washed (1X PBS, 100g/ml cycloheximide) and trypsinised for 5 min at 37oC. Subsequently, cells were pelleted, washed (1X PBS, 100g/ml cycloheximide), and resuspended in ice cold lysis buffer (50mM Tris-HCl pH8, 150mM NaCl, 10mM MgCl2, 1mM DTT, 1% IGEPAL, 100g/ml cycloheximide, Turbo DNase 24U/L (Invitrogen), RNasin Plus RNase Inhibitor 90U (Promega), cOmplete Protease Inhibitor (Roche)), for 45 min. Cells were then subjected to centrifugation at 17,000 xg for 5 min, to pellet nuclei. Cytoplasmic lysate was loaded onto 18%- 60% sucrose gradients (~70x106 cells per gradient) at 4oC and subjected to ultracentrifugation (121,355 x gavg 3.5h, 4oC) in SW-40 rotor. Polysome fractions were pooled and diluted in either Buffer A (50mM Tris-HCl pH8, 150mM NaCl, 10mM MgCl2) or Buffer B (100mM Tris-HCl pH8, 30mM NaCl, 10mM MgCl2). RNaseI (either AM2295 at 10-20U/million cells EN601, 10U/l 0.7-1U/million cells) was subsequently added incubated either for 1 hour at RT or overnight at 4oC. RNaseI was deactivated using SUPERase inhibitor (200U/gradient) for 5min at 4oC. Samples were concentrated using 30 kDa molecular weight cut-off columns (Merck) and loaded on sucrose cushion (1M sucrose, 50mM Tris-HCl pH8, 150mM NaCl, 10mM MgCl2, 40U RNase Inhibitor) and subjected to ultracentrifugation at 204,428 x gavg at 4oC for 4h (TLA110). Pellets were resuspended in TRIzol (Ambion, Life Technologies) and processed for RNA purification followed by TURBO DNase treatment (Thermofisher) (according to manufacturers instructions), acidic phenol/chloroform RNA purification and ethanol precipitation at -80oC overnight. RNA concentration was determined by Nano-drop 2000 software. 2834 nt ribosome footprints were gel purified in 10% (w/v) polyacrylamide-TBE-Urea gel at 300V for 3.5h in 1X TBE. Ribosome footprints were subjected to rRNA depletion (Illumina RiboZero rRNA removal kit).