Ua 6 gradient fractionator

10⁷ cells were treated with 100 μg/ml cycloheximide for 5 min at RT to stabilize existing polysomes before washing with ice-cold PBS and harvesting by scraping in 400 μl polysome lysis buffer (20 mM Tris–HCl, pH 7.4, 5 mM MgCl₂, 150 mM NaCl, 1 mM DTT, 1% Triton X-100, 100 μg/ml cycloheximide, 1 × Complete Protease Inhibitors (Roche)). Lysates were rotated end-over-end for 10 min at 4°C and cleared by at 10 000 rpm for 10 min at 4°C. 40 μl of supernatant lysate was saved as input before loading the lysates to linear 17.5 to 50% sucrose gradients in 20 mM Tris–HCl (pH 7.4), 5 mM MgCl₂, 150 mM NaCl. Centrifugation was carried out at 35 000 rpm for 2.5 h at 4°C in a Beckmann SW60 rotor. Gradients were eluted with an ISCO UA-6 gradient fractionator, and polysome profiles were recorded by continuously monitoring the absorbance at 254 nm using PeakTrak software. During gradient elution, fractions of ∼300 μl were collected every 14 s. For RNA isolation, 300 μl urea buffer (10 mM Tris, pH 7.5, 350 mM NaCl, 10 mM EDTA, 1% SDS, and 7 M urea) and 300 μl phenol:chloroform:isoamylalcohol (25:24:1) were added to each fraction. After phase separation, RNA was isolated from the aqueous phase and precipitated using isopropanol and GlycoBlue (Thermo Fisher Scientific).