Polysome Profile Analysis in Cells

Polysome profiles were obtained according to a previously published procedure (Göktaş et al., 2017 (link)). Briefly, cell lysis (3×10⁷ cells) was carried out in 5 mL lysis buffer [(100 mM NaCl, 10 mM MgCl₂, 30 mM Tris-HCl (pH 7), 1% Triton X-100, 1% NaDOC, 100 μg/mL cycloheximide (Applichem) and 30 U/mL SUPERase.IN RNase inhibitor (Ambion)], and the lysate was incubated on ice for 8 min. The cell debris and nuclei were removed by centrifuging the homogenates at 12,000 g at 4 °C for 8 min. Two-mL supernatant was loaded onto 5%–70% (w/v) sucrose gradients [100 mM NaCl, 10 mM MgCl₂, 30 mM Tris-HCl (pH 7), 200 U SUPERase.IN RNase inhibitor (Ambion)] and centrifuged at 27,000 rpm for 2 h 55 min at 4 °C in a Beckman SW28 rotor. Fractions were collected using Teledyne ISCO’s density gradient fractionation system (NE, USA) while recording the absorbance at A₂₅₄ to obtain the polysome profiles.