title: Ion Torrent Proton sequencing; RNA-seq of the prototrophic non-hyphal smooth-33 mutant (FKP514) and its prototrophic control strain (FKP391)
design: RNA-seq of the prototrophic non-hyphal smooth-33 mutant (FKP514) and its prototrophic control strain (FKP391)
library_construction_protocol: Samples for RNA analysis were rapidly taken when the chemostats reached steady state, defined as stable CO2 and O2 outflow and optical density, which was achieved after circa 120 h. Samples were frozen in liquid nitrogen and stored at −80°C. Chemostat cultivation was performed with a dilution rate of 0.05 per hour at 30 °C in a 1.2 L bioreactor (DASGIP, Jülich, Germany) with a working volume of 750 ml at pH 3.5, controlled with 2 M KOH. Dissolved oxygen was kept above 30% with a stirrer rate of 600 rpm and an air flow rate of 1 v.v.m. Growth medium contained 25 g/L glucose, 0.5 g/L (NH4)2SO4, 5.96 g/L K2SO4, 3 g/L KH2PO4, 0.5 g/L MgSO4·7 H2O, vitamins and trace metal solutions and 125 μl antifoam 204 (Sigma-Aldrich, St Louis, MO, USA). Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions with additional mechanical disruption of the cells using a FastPrep homogenizer (MP Biomedicals, Santa Ana, CA, USA) and 1 mm silica beads. Samples were depleted of rRNA using RiboZero