2.7. Polysome fractionation

Polysome fractionation was carried out as described previously [25]. Three 15 cm plates with ATDC5 cells were used to generate a single sample at day 0, two plates at day 7 and one plate at day 14, and this was repeated four times to generate n = 4 biological replicates. At the day of sample collection, cells were pre-treated for 5 min with 100 μg/ml Cycloheximide (Sigma), washed twice in 0.9% NaCl with cycloheximide and collected by scraping with a rubber policeman in cold 0.9% NaCl. Pelleted cells were lysed for 10 min in 1.8 ml polysome extraction buffer (20 mM Tris-HCl (pH7.5), 100 mM KCl, 5 mM MgCl₂, 0.5% Nonidet P-40, 100 μg/ml Cycloheximide, complete protease inhibitor cocktail (Roche) and RNasin (Promega, 40U/ml)) on ice. Nuclei and cellular debris were removed by centrifugation at 12.000×g for 10 min at 4 °C and 9/10th of the total volume was transferred to fresh tubes and measured spectrophotometrically. Ten percent input was set aside and stored at −80 °C. Linear 10–50% sucrose gradients were made using the Gradient Master (BioComp) in SW41 ultracentrifuge tubes (Seton). A fixed amount of 160 μg cytoplasmic extract was loaded to each gradient, for each sample in the same volume. Gradients were run on a Beckman L60 ultra-centrifuge at 39.000 rpm for 1.5 h at 4 °C with max acceleration and deceleration 9. Samples were fractionated into 24 × 0.5 ml fractions using a Piston Gradient fractionator (BioComp) and fraction collector (Gilson FC203B) with continuous A260 monitoring (Triax FC-1).