title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of Escherichia coli MG1655 WT strain against strain deleted of the ettA gene and complemented strain
design: RNA-seq of Escherichia coli MG1655 WT strain against strain deleted of the ettA gene and complemented strain
library_name: WT MMAA NaCl_p
library_construction_protocol: 3 mL samples were collected for each strain for the transcriptomic analysis. Cultures were stopped by addition of two volumes of RNAprotect (Qiagen) then centrifuged at 5,000 xg for 5 min. Pellets were isolated and immediately stored at - 80 °C. Cultures of the three strains (WT, ∆ettA and CettA) in a volume of 125mL of MMAA 0.4 M NaCl medium were inoculated at 1/20 with overnight cultures prepared in LB-Difco medium. Cells were grown at 37 ºC with shaking. After 330 min of culture, 3 mL samples were collected for each strain for the transcriptomic analysis. Total RNA were extracted with the Direct-zol RNA MiniPrep kit (ZYMO RESEARCH). Ribosomal RNAs were specifically eliminated using the Ribo-Zero rRNA Removal kit (Illumina).  The library were constructed by the GATC company.Library preparation incorporated adaptor sequences and indexing compatible for Illumina sequencing technology, using proprietary methods of GATC Biotech. The cDNA library preparation was performed according to SOP S-11169. After first strand synthesis using random primers, the second strand synthesis is performed. The ends of the double stranded cDNA fragments are repaired and dATP ligated to the blunt ended fragments. Then, the sequencing adapters are ligated to the DNA fragments.