Illumina HiSeq 4000 paired end sequencing; RNA-seq of S. enterica serovar Typhimurium SL1344 and ∆acrB after 1, 3 and 5 hours of growth
RNA-seq of S. enterica serovar Typhimurium SL1344 and ∆acrB after 1, 3 and 5 hours of growth
4 biological replicates of each strain were grown for 5 hours. At 1 hour of growth, 6 mL of culture (for each replicate) was centrifuged and cells were harvested and snap frozen. At 3 hours and 5 hours of growth, 1 mL of culture for each replicate was centrifuged and harvested, with snap freezing of the pellets. All centrifugation steps were at 3500 x g and samples were stored at -80°C. Strains were grown in MOPs minimal media (supplemented with histidine) for 5 hours at 37°C with shaking at 150 RPM. Strains were grown from a 4% inoculum of an overnight culture. GENEWIZ Inc. carried out the RNA extraction. Total RNA was extracted from cell pellets using RNeasy Plus Universal kit (Qiagen).To remove rRNA, the ribozero Removal Kit was used (Illumina).RNA quality control was carried out using Qubit 2.0 Fluorometer to measure total RNA concentration and Agilent TapeStation. Library construction was done by GENEWIZ Inc. The NEBNext Ultra II RNA Library Prep Kit (Illumina) was used for library preparation, following the manufacturer's protocol. For library preparation, cDNA was synthesised, end repaired and adenylated at the 3' ends. Universal adapters were ligated to cDNA and library enrichment was carried out using limited cycle PCR.