Illumina HiSeq 1500 paired end sequencing; Transcriptional analysis of regulatory effects in Corynebacterium glutamicum and a derived (p)ppGpp0 mutant strain
Transcriptional analysis of regulatory effects in Corynebacterium glutamicum and a derived (p)ppGpp0 mutant strain
After reaching an OD value of 2.5, cells were harvested by rapid centrifugation, supernatant removal, and immediate shock freezing in liquid nitrogen. Samples of stressed cells were treated identically. C. glutamicum CR099 and C. glutamicum CR099 relrelSrelH cells were grown in CGXII medium with 10 g/L glucose and 50 mg/L of all 20 proteinogenic amino acids with an initial optical density of 0.4. Cultures of 40 mL were incubated in 250 mL Erlenmeyer flasks at 30 °C and 300 rpm (2 inch deviation). RNA-isolation was performed using the RNeasy Mini Kit along with the DNase Kit (both Qiagen), using three DNA digestion steps. For initial cell disruption the cell pellet was resuspended in 800 µL RLT-buffer (containing 10 µL/mL ?-mercaptoethanol), transferred into lysing matrix B tubes (MP Biomedicals) and homogenized for 30 sec at 6500 rpm in a Precellys® 24 homogeniser (Peqlab). Illumina TruSeq® Stranded mRNA low sample protocol starting with rRNA-depleted total RNA
Experimental Factor: genotype
CR099 rel, relS, relH
Experimental Factor: growth condition
exponential phase
Experimental Factor: time
0