Illumina HiSeq 4000 paired end sequencing; RNA-seq analysis of Clostridium beijerinckii DSM 6423 at early exponential, acetogenic, and solventogenic growth phase on rhamnose compared to glucose
RNA-seq analysis of Clostridium beijerinckii DSM 6423 at early exponential, acetogenic, and solventogenic growth phase on rhamnose compared to glucose
Total RNA was isolated from C. beijerinckii DSM 6423 for transcriptome studies. Samples were taken from duplicate 400-mL fermentations from cells in early exponential, acetogenic, and solventogenic phase. Cells were pelleted for 15 min at 3000 × g and 4 °C and stored at -80 °C until further use. Fermentations were performed at 37 °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 40.0 glucose or rhamnose. RNA was isolated using TRIzol reagent (Thermo Fisher Scientific) and the PureLink RNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. In short, the cell pellet was thawed on ice and resuspended in 5 mL TRIzol reagent for cell lysis. Next, 1 mL chloroform was added and after centrifugation for 15 min at 13,000 × g, the upper aqueous phase was mixed with an equal volume of 70 % ethanol. The solution was loaded on a spin cartridge, washed once and treated with 30 U DNase I. After two additional washing steps, the RNA was eluted in RNase-free water. Quality and quantity of the isolated RNA was checked by gel electrophoresis and NanoDrop, respectively. Afterwards, the samples were stored at -80 °C before being sent for sequencing.  Library construction and sequencing was performed by Novogene Co. Ltd. Messenger-RNA was depleted with the Ribo-Zero Magnetic Kit and a 250-300 bp insert cDNA library was constructed.
Experimental Factor: compound
rhamnose
Experimental Factor: dose
40
Experimental Factor: time
10