RNA-seq of MG1655 dnaA46 ackA::kan strain, LB, 30C, sample B
The Escherichia coli MG1655 strain and its derivatives were grown at 30ĄC in LB medium. The overnight culture was diluted 1:1000 in a fresh LB medium and cells were grown with aeration until the OD600 of the culture reached 0.3. To each sample (109 cells), ice-cold Ethanol/Phenol (5% Phenol) solution was immediately added (1:8 ratio of the stop solution: culture) to prevent mRNA degradation; the cells were then harvested by centrifugation and the pellets were frozen in liquid nitrogen. RNA was extracted with the RNeasy Mini kit (Qiagen) and treated with Turbo DNase (Life Technologies) according to the manufacturersŐ instructions. Subsequent rRNA depletion was carried out with a Ribo-Zero kit (Illumina) and 0.5 g of the enriched mRNA of each sample was sent for RNA-seq analysis (Illumina HiSeq 2000).