Rnasin plus rnase inhibitor 90 u

TREx BCBL1-Rta cells expressing Scr shRNA, BUD23 specific shRNAs or ORF11-GFP were treated with cycloheximide (Sigma) at 100 μg/ml for 3 min at 37 °C. A total of ∼50 × 10⁶ 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, 10 mM 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 min. Lysates were clarified by centrifugation (12,000 × g, 10 min, 4 °C) and the resulting supernatants 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 3.5 h, 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).

RA was added to SH-SY5Y cells 3 d prior to harvesting. Cells were treated with cycloheximide (Sigma) at 100 µg/mL for 3 min at 37°C, washed (1× PBS, 100 µg/mL cycloheximide) and trypsinized for 5 min at 37°C. Subsequently, cells were pelleted, washed (1× PBS, 100 µg/mL cycloheximide), and resuspended in ice cold lysis buffer (Supplemental Methods); 50 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 1% IGEPAL, 100 µg/mL cycloheximide, Turbo DNase 24 U/µL (Invitrogen), RNasin Plus RNase Inhibitor 90U (Promega), cOmplete Protease Inhibitor (Roche), for 45 min. Cells were then subjected to centrifugation at 17,000g for 5 min, to pellet nuclei. Cytoplasmic lysates were loaded onto 18%–60% sucrose gradients (∼70 × 10⁶ cells per gradient) at 4°C and subjected to ultracentrifugation (121,355 × g_avg 3.5 h, 4°C) in SW-40 rotor. Gradients were fractionated using Gradient Station (Biocomp) and absorbance at 254 nm was monitored using a Bio-Rad detector.