Optiseal polyallomer 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).

For SIP incubation, AOA subcultures were additionally supplemented with 2.5 mM NaHCO₃ (¹²C or ¹³C), and pH was adjust to 6.5 as normal cultivation using 1 M HCl. The NaH¹³CO₃ (99 atoms%) was purchased from Sigma-Aldrich (USA) Co. After one cycle of incubation, 10% of the medium was used as inoculation for a new cycle, and the rest were all used for DNA extraction. Two cycles of incubation with two replicates were performed totally. Extracted DNA (~2 μg) was added into CsCl gradients with an initial density of 1.696 g ml⁻¹. Density gradient centrifugation was performed in 4.9 ml OptiSeal polyallomer tubes (Beckman Coulter, USA) in a VTi 90 vertical rotor, subject to centrifugation at 56200 rpm for 24 h at 20 °C58 (link). Centrifuged gradients were fractionated into 24–25 equal volumes (~200 μl) as described in detail by Zhang et al.59 (link). Nucleic acids were precipitated by using PEG 6000, and then dissolved in 30 ml of TE buffer.