GSM4676790: WT_2; Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344; RNA-Seq
Total RNA was obtained from 10 ml cultures of S. Typhimurium expressing RcsB WT or mutant variants in exponential phase (OD600 ~ 0.25) using the TRIzol. The samples for the strains expressing each RcsB variant were prepared from three independent biological replicates. Total RNA was further purified by treatment with TURBO DNA-free kit (#AM1907, Invitrogen). Quality of RNA was checked by Bioanalyzer with final yields in the range of 25-40 µg per sample (concentration of 500-800 ng/µl) Purified RNA was then treated with Ribo-Zero™ rRNA Removal Reagent and magnetic core kit (Epicentre). rRNA-free RNA was resuspended in ethanol and subsequently used to prepare libraries using the ScriptSeq™ v2 RNA-Seq Library preparation kit (Epicentre and Illumina Company). The purifications were performed following manufacturer instructions and involving usage of MagSi-NGSPREP Plus (Magtivio). Size of the fragments composing the different libraries was monitored by capillary electrophoresis in a QIAxcel Advanced System (Qiagen) and quantification of the cDNA was made in a QuantiFluor dsDNA system (Promega)