Acidic phenol chloroform

Triplicate cultures of the wild-type strain h^−S (972) were grown at 30°C in either EMM2 (Edinburgh minimal medium) or YES (yeast extract with supplements) to a density of 5 × 10⁶ cells/ml (32 ). For nitrogen starvation, cells were transferred by vacuum filtration from EMM2 to EMM2 without ammonium chloride and incubated for 16 h at 30°C. Heat shock was imposed by transferring YES cultures to 39°C for 15 min. Oxidative stress was inflicted by treating YES cultures with 0.5 mM H₂O₂ for 15 min at 30°C. Total RNA was extracted from 10⁸ cells. In brief, cells were harvested by 5 min of centrifugation at 3000 rpm. Pellets were resuspended in TES (10 mM TrisHCl pH7.5, 10 mM ethylenediaminetetraacetic acid and 0.5% sodium dodecyl sulphate) and transferred to 65°C preheated acidic phenol-chloroform (Sigma P-1944). After 1 h of incubation at 65°C with mixing every 10 min, RNA was extracted with chloroform-isoamyl alcohol (Sigma C-0549), ethanol precipitated and re-suspended in water. All RIN-values were above 8.8. As CAGE requires a certain amount of input RNA concentration, RNA from nitrogen-starved cultures was additionally concentrated by vacuum centrifuge (reported RIN-scores are after concentration). See Supplementary Table S1 for an overview of libraries.