2025-03-14
ENA first public
ERC000011
ENA-CHECKLIST
Quadram Institute Bioscience
INSDC center name
public
INSDC status
ERS23403225
SRA accession
E-MTAB-14856:Sample 9
Submitter Id
ArrayExpress
broker name
not collected
collection date
Protocols: 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).
description
not collected
geographic location (country and/or sea)
not applicable
isolate
Salmonella enterica subsp. enterica serovar Typhimurium
organism
Salmonella enterica subsp. enterica serovar Typhimurium
organism
whole organism
organism part
1
replicate
Salmonella enterica subsp. enterica serovar Typhimurium
scientific_name
0.125% Sodium nitrite
stimulus
Sample 9
title