GSM5964226: Repeat 1: Scrambled shRNA - ribosome footprint RNA; Homo sapiens; Human gammaherpesvirus 8; OTHER
Cells were treated with cycloheximide (Sigma) at 100 μg/ml for 3 min at 37°C. A total of ∼50 × 10^6 cells were pelleted and washed (1 x PBS, 100 μg/ml cycloheximide) then lysed in ice cold buffer [50 mM Tris-HCl pH8, 150 mM NaCl, 10mM MgCl2, 1 mM DTT, 1% IGEPAL, 100 μg/ml cycloheximide, Turbo DNase 24 U/mL (Invitrogen), RNasin Plus RNase Inhibitor 90 U (Promega), 1 × protease inhibitor cocktail (Roche)] for 45 minutes. Lysates were clarified by centrifugation (12,000 × g, 10 min, 4°C) and 20% saved as a total mRNA input for downstream processing and analysis. The resulting supernatants were applied to continuous sucrose gradients of either 5% − 45% or GFP-ORF11 lysates applied to 5% − 30% (10 mM MgCl2, 50 mM Tris/HCl (pH 7.6), 150 mM NaCl, 1 mM DTT, 100 μg/ml cycloheximide and 1× protease inhibitor cocktail). Gradients were then subjected to ultracentrifugation (121,355 × g 3.5h, 4°C) in SW-40 rotor. Fractions from each gradient were collected using a Gradient Fractionator (BioComp) and the RNA profile was measured by absorbance (254 nm) across the gradient in real time using an EM-1 Econo UV Monitor (Bio-Rad). Total nucleic acid was extracted from input samples using TRIzol™ LS (Invitrogen™), as described by the manufacturer. Sucrose fractions containing 80S and polysome ribosomes were used for poly-ribo-seq. Fractions for each sample were pooled and diluted (100 mM Tris/HCl (pH 7.6), 30 mM NaCl and 10 mM MgCl2) to a 10% sucrose concentration and ribosomes footprinted with 1 U/1×10^6 cells RNase I (Thermo Fischer), rotating overnight at 4°C. The reaction was stopped with SuperRNaseIN (200U/gradient, Thermo Fischer), rotating for 5 minutes at room temperature. Sucrose samples were concentrated with 30 kDa cut off filter units (Millipore) and loaded onto 5% − 45% sucrose gradients. Gradients were processed as described above and just the 80S fraction collected containing footprinted ribosomes and the RNA precipitated 1:1 isopropanol, 300 mM NaCl and glycoblue, overnight at -80°C. Fractions for each were pooled through resuspension in 100 μl Nuclease-Free Water, and the same done for the mRNA input samples. Samples were treated with TURBO DNase (Thermo Fischer) according to manufacturer's instructions and the RNA purified with TRIzol™ LS (Invitrogen™), as described by the manufacture. PolyA RNA was selected from input mRNA samples using a Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen™), according to manufacturer's instructions and the RNA fragmented in 2 mM EDTA, 10mM Na2CO3 and 90mM NaHCO3 for 20 minutes at 95°C. 28–34 nt ribosome footprints and 50-80 nt polyA RNA samples were gel purified in 10% (w/v) polyacrylamide-TBE-urea gel at 300 V for 3.5 h in 1X TBE. Fragment ends were repaired using T4 PNK treatment (New England Biolabs) as described by the manufacturer. Ribosome footprints were subjected to rRNA depletion using a NEBNext® rRNA depletion kit (New England Biolabs) according to the manufacturers protocol. The NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs) was used to generate the libraries for next generation sequencing. Resulting cDNA was PCR amplified and gel purified prior to sequencing.