Lysate Preparation and Centrifugation in Sucrose Gradient

The oocytes were homogenized with 20 strokes using Eppendorf pestle in the lysis buffer (20 mM Tris HCl, pH 7.5, 50 mM KCl, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 1% Triton X-100), supplemented with 0.1 mg/ml cycloheximide and complete EDTA-free protease inhibitors (Roche). For the oocytes of ∼2 and ∼5 mm, the volume of the lysis buffer was 400 µl, for the oocytes of ∼10 mm, the volume was 1 ml. In parallel, the T. scripta liver lysate was used as a centrifugation quality control. The liver was mechanically disrupted in 1 ml of lysis buffer with the Dounce homogenizer (20 strokes with pestle B). To remove cellular debris, the homogenates were centrifuged at 12,000 × g for 10 min at +4 °C. After that, the lysates were loaded on a 10–50% linear sucrose gradient in SW41 tubes (Beckman Coulter, USA). Gradients were made in buffer (20 mM Tris HCl, pH 7.5, 50 mM KCl, 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT). After centrifugation at 35,000 rpm for 3 h at +4 °C, the gradients were analyzed by measuring absorbance at 260 nm and the selected fractions were collected manually. The gradient from liver lysate served as a reference to determine the position of monosome (80S) and polysome fractions. Further analysis using RT-qPCR was performed on six pooled fractions. The scheme of gradient fractionation is shown in supplementary figure S7, Supplementary Material online.