Beckman polyallomer centrifuge tubes

For polysome profiling, cell extracts were prepared as previously described without the addition of RVC from exponentially growing or nutritionally stressed cells. 50–100 OD₂₆₀ of cell extract was layered on top of a 10–40% (w/v) sucrose gradient prepared in ribosome buffer A in SW 32Ti tubes (Beckman Polyallomer Centrifuge tubes 25 × 89 mm). The gradients were centrifuged in a Beckman SW 32Ti rotor (6 h at 25,000 rpm at 4 °C). Gradients were pumped out and fractions were collected every 16 s while continuously monitoring the absorbance at 260 nm. For downstream northern blot analyses the desired fractions were pooled and precipitated with EtOH before RNA extraction with 1 ml of TRI Reagent (Zymo Research). For 80S ribosome preparation, the fractions containing monosomes were pooled into a Beckman Optiseal Polyallomer tubes (volume 32.4 ml) and filled up with 1x ribosome buffer A. The ribosomes were pelleted by ultracentrifugation at 33,000 rpm (100,000 × g ) for 17 h at 4 °C (rotor type 60 Ti, Beckman). After centrifugation the pellet was resuspended in 200 μl ribosome buffer A. The concentration of ribosomes was determined by the absorption at 260 nm (1 A₂₆₀ = 18 pmol 80S).

Starch was suspended in 18.2 MΩ cm Milli-Q water at a concentration of 10 w/w%. Sodium Azide (Sigma Aldrich, USA) was added at a concentration of 0.02 w/w% to the starch suspension to eliminate mold growth during storage. Beckman Polyallomer centrifuge tubes (25 mm internal diameter × 64 mm height, Beckman Instruments, Inc., USA) were filled with ≈10 mL starch suspension and heat sealed prior to high pressure treatment.