2024-06-01
ENA first public
ERC000011
ENA-CHECKLIST
Quadram Institute Biosciences
INSDC center name
public
INSDC status
ERS19897109
SRA accession
E-MTAB-13967:Sample 20
Submitter Id
ArrayExpress
broker name
not collected
collection date
Protocols: Each preservative stress condition was made up of eight lineages: two preservative-exposed planktonic lineages (P1 and P2), two unexposed biofilm lineages (C1 and C2), and four exposed biofilm lineages (A, B, C, D) (Trampari et al., 2021). Biofilm lineages were comprised of three 5 mm stainless steel beads (from Simply Bearings) in 5 mL LB broth without salt. Planktonic lineages were set up the same but did not contain beads. Approximately 10^5 CFU/mL of Salmonella enterica subsp. enterica serovar Typhimurium strain 14028S was inoculated into each condition and cultures were incubated at 30°C for 72 hrs horizontally with mild agitation at 40 rpm. Cultures were exposed to preservatives at subinhibitory concentrations, corresponding to 5% Sodium chloride, 7 % Potassium chloride, 5 % Sodium lactate or 0.06 % Sodium nitrite. Subinhibitory concentrations were selected as those that inhibited the growth of S. Typhimurium by 25-35 %.   Cultures were passaged into new media every 72 hours. For biofilm lineages, one steel bead was washed in sterile PBS and transferred to fresh media to seed the next passage. For planktonic conditions, 5 µL of culture was added to fresh media. A sample of each condition was stored at each passage: this consisted of 1 mL of planktonic culture or one bead from the biofilm lineages. Beads were washed with sterile PBS to remove planktonic cells and vortexed in fresh sterile PBS to resuspend the biofilm. Both planktonic cultures and resuspended biofilms were mixed with 7.5 % DMSO and stored at -20 °C. To prepare cultures for DNA extraction, frozen stocks were inoculated in 5 mL LB broth and grown for 18 hours at 37 °C with shaking. To extract genomic DNA for sequencing, selected strains were grown O/N in a 96-deep-well plate in LB, at a final volume of 1.5 mL. Cells were recovered by centrifugation at 3500 × g and were resuspended in 100 μL of lysis buffer (5 μg/mL lysozyme, 0.1 mg/mL RNAse in Tris-EDTA, pH 8) per well. The resuspended cells were then transferred in a new semi-skirted, low-bind polymerase chain reaction (PCR) plate, secured with an adhesive seal and incubated at 37 °C, 550 × g for 25 min. Ten microliters of a lysis additive buffer (5% sodium dodecyl sulfate, 1 mg/mL proteinase K, 1 mg/mL RNAse in Tris-EDTA, pH 8) was added to each well and the plate was sealed with PCR strip lids before being incubated at 65 °C, 550 × g for 25 min. The plate was briefly centrifuged and 100 μL were moved to a new PCR plate. For the DNA isolation, 50 μL of DNA-binding magnetic beads (KAPA Pure Beads, Roche Diagnostics) were added to each well and were incubated at room temperature for 5 min. The plate was then placed on a magnetic base and the supernatant was removed by pipetting. The beads were washed three times with 80% freshly prepared ethanol. After removing the last wash, the beads were left to dry for 2 min before eluting the DNA. For the DNA elution, the plate was removed from the magnetic apparatus and 50 μL of 10 mM Tris-Cl, pH 8.5, were added to each well. The beads were pulled using the magnetic apparatus and the isolated DNA was transferred to a new PCR plate. DNA concentration was determined using the Qubit ds DNA HS Assay Kit (Q32851) following the manufacturer’s instructions. Genomic DNA was normalised to 0.5 ng/µL with 10 mM Tris-HCl. A measure of 0.9 µL of TD Tagment DNA Buffer (Illumina, Catalogue No. 15027866) was mixed with 0.09 µL TDE1, Tagment DNA Enzyme (Illumina, Catalogue No. 15027865) and 2.01 µL PCR grade water in a master mix and 3 µl added to a chilled 96-well plate. Two microlitres of normalised DNA (1 ng total) was mixed with the 3 µL of the Tagmentation mix and heated to 55 °C for 10 min in a PCR block. A PCR master mix was made up using 4 µl KAPA2G buffer, 0.4 µL dNTPs, 0.08 µL polymerase and 4.52 µL PCR grade water, contained in the KAPA2G Robust PCR Kit (Sigma, Catalogue No. KK5005) per sample and 11 µL added to each well need to be used in a 96-well plate. Two microlitres of each P7 and P5 of Nextera XT Index Kit v2 index primers (Illumina, Catalogue No. FC-131-2001 to 2004) were added to each well. Finally, the 5 µL Tagmentation mix was added and mixed. The PCR was run with 72 °C for 3 min, 95 °C for 1 min, 14 cycles of 95 °C for 10 s, 55 °C for 20 s and 72 °C for 3 min. Following the PCR reaction, the libraries were quantified using the Quant-iT dsDNA Assay Kit, High-Sensitivity Kit (Catalogue No. 10164582) and run on a FLUOstar Optima plate reader. Libraries were pooled following quantification in equal quantities. The final pool was double-SPRI size selected between 0.5 and 0.7× bead volumes using KAPA Pure Beads (Roche, Catalogue No. 07983298001). The final pool was quantified on a Qubit 3.0 instrument and run on a High Sensitivity D1000 ScreenTape (Agilent, Catalogue No. 5067-5579) using the Agilent Tapestation 4200 to calculate the final library pool molarity.
description
not collected
geographic location (country and/or sea)
Potassium chloride
growth condition
not applicable
isolate
Salmonella enterica
organism
Salmonella enterica
organism
whole organism
organism part
Biofilm
phenotype
B
replicate
Salmonella enterica
scientific_name
Salmonella enterica subsp. enterica serovar Typhimurium
strain
Sample 20
title