RNA preparation

Total RNA was purified from clarified cell lysates using the Direct-zol RNA MiniPrep kit (Zymo Research, USA). To generate cell lysates, 2 × 10⁸ cells were resuspended in 50 μl of Protoplasting buffer (15 mM Tris-HCl [pH 8.0], 0.45 M sucrose and 8 mM EDTA). Five μl of lysozyme (50 mg/ml) was added and the sample was incubated for 5 min at 25°C. A phenolic detergent (1 ml TRI Reagent; Molecular Research Center, USA) was added and vortexed for 10 sec before incubating for 5 min at 25°C. The RNA was isolated according to the manufacturer’s recommendations and dissolved in 30 μl of RNA storage buffer (Thermo Fisher Scientific, USA). RNA concentrations were determined by measuring the absorbance at 260 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).