title: NextSeq 500 sequencing; RNA-seq of pathogenic Escherichia coli strains CFT073 and EDL933  wild-type, rpoN mutant, and rpoN mutant + rpoN strains
design: RNA-seq of pathogenic Escherichia coli strains CFT073 and EDL933  wild-type, rpoN mutant, and rpoN mutant + rpoN strains
library_construction_protocol: Samples were grown in LB liquid media from OD600nm of 0.05 to OD 1 (mid-exponential). Cultures were mixed at a ratio of 1 : 19 phenol:ethanol solution at a 9 : 1 culture:solution ratio and then pelleted. Pellets were sent to Vertis Biotechnologie AG for further processing. Total RNA was isolated from the cell pellets using the RNAsnap protocol developed by Stead et al.(2012). The ribodepleted RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3 adapter as primer. The first strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/l using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics).