design: Using TraDIS-Xpress to find the genes that affect susceptibility to common food preservatives
library_construction_protocol: Approximately 10^7 CFU/mL of the Salmonella enterica serovar Typhimurium library described by Holden et al (2022) was added to 5 mL lysogeny broth (LB) made without salt and treated with subinhibitory concentrations of each preservative: this equated to 5% sodium chloride (NaCl), 10% potassium chloride (KCl), 5% sodium lactate (SL) and 0.125% sodium nitrite (SN), alongside an untreated control. Transcription from the transposon-located promoter was induced in each condition with 1 mM IPTG and all conditions used two independent replicates. Cultures were incubated at 37 ºC shaking at 200 rpm for 24 hours before being centrifuged at 3000 x g for 10 minutes to pellet the cells for DNA extraction.  DNA was extracted following the protocol described by Trampari et al. (2021) and quantified by Qubit HS dsDNA kit.  Total DNA was normalised to 11.1 ng/µL, tagmented using a MuSeek DNA fragment library preparation kit (ThermoFisher) and purified using AMPure XP beads (Beckman Coulter). DNA containing the transposon was amplified using primers customised for the tagmented ends and biotinylated primers specific to transposon. Following another purification step, biotinylated PCR products were incubated for 4 hours with streptavidin beads (Dynabeads® kilobaseBINDER™, Invitrogen) to capture only fragments containing the transposon. A second PCR step using this captured DNA used customised barcoded Illumina sequencing primers specific to the tagmented ends and to the transposon. Beads were removed from the reaction with a magnet and DNA was purified and size-selected using AMPure beads. Fragment length was quantified using a Tapestation (Aligent).