title: GSM5047184: Ec55989 Exponential 3 (Exp3); Escherichia coli 55989; RNA-Seq
library_construction_protocol: The RNA extraction process of invitro cells started with the centrifugation of cells and the subsequent addition of TRIzol (sigma T-9424) for lysis. For invivo samples TRIzol (sigma T-9424) was subsequently added to the frozen samples which were homogenized (Oligo-Macs, Mylteny). The total RNA of both in vitro and in vivo samples was purified through a standard organic extraction (phenol/chloroform) followed by ethanol precipitation. Afterwards RNA was submitted to RNeasy mini kit (Qiagen) for better purification and remaining genomic DNA was removed using on-column RNAse-free DNAse set protocol (Qiagen). RNA integrity was assessed with bioanalyser system (Agilent) and RNA Integrity Number (RIN) and the ribosomal ratio (23S/16S) was considered. The rRNA-depleted RNA were obtained by using the RiboZerorRNA depletion kit, for eucaryote and procaryote called 'epidemiology' (Illumina). Libraries preparation was performed with TruSeq Stranded RNA LT prep kit (Illumina) with final validation on a DNA Agilent Chip (bioanalyser system) and final DNA quantifications were performed with sensitive fluorescent-based quantitation assays ("Quant-It" assays kit and QuBitfluorometer, Invitrogen). Samples were then normalized to 2nM concentrations and multiplexed. Afterwards samples were denatured at a concentration of 1nM using 0.1N NaOH, at room temperature and was finally diluted at 9,5 pM Single-read sequencing was performed using HiSeq 2500 Illumina sequencing machine (65 cycles, with 7 index base read).