BioSample

Protocols: Total RNA was isolated from C. beijerinckii NCIMB 8052 for transcriptome studies. Samples were taken from biological triplicate of 400-mL fermentations from cells in early exponential, acetogenic, and solventogenic phase. Cells were pelleted for 15 min at 3000 × g and 4 °C, washed 3 times with sterile H2O and stored at -80 °C until further use. Fermentations were performed at 35 °C in 400 mL working volume of CM2 medium containing (in g L-1): yeast extract, 1.00; KH2PO4, 1.00; K2HPO4, 0.61; MgSO4 × 7 H2O, 1.00; FeSO4 × 7 H2O, 0.0066; para-aminobenzoic acid, 0.10; and ammonium acetate, 2.90 and with either 50.0 glucose. Frozen samples were thawed on ice and RNA was isolated using high pure RNA isolation kit (Roche Diagnostics, Basel, Switzerland). Quality and concentration of RNA samples were checked using a nanodrop machine (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The absence of DNA in the RNA samples was evaluated by qPCR analysis performed with BioRad CFX 96 Touch™. The RNA quality and integrity were determined using the Qsep 100 bioanalyzer (Bioptic Inc.). Messenger-RNA was depleted with the Ribo-Zero Magnetic Kit and a 250-300 bp insert cDNA library was constructed using the Illumina True seq stranded kit.