Production 2021 Tnseq

Citation: Sarsani, Aldikacti, et al.

Data: GSE244581

The repository for alignment/annotation and analysis of the Tnseq data in this app can be found here.

Tnseq Experiment Details

Transposon mutagenesis libraries were thawed and recovered in PYE or PYE + %0.2 Xylose overnight at saturating concentrations to minimize growth during recovery. Only for dnaK-NI library, overnights were recovered in saturating Xylose concentrations (%0.2), and treatment and T24 control conditions were grown in minimal Xylose concentrations. (%0.002)

Control condition: Libraries were back diluted to OD 0.008 into 7 ml of PYE or PYE+0.002% xylose and grown overnight until they reached saturation at OD ∼1.6.

Heat stress: Libraries diluted to OD of 1 and heat-stressed at low, medium, or high (37, 42, 43.8◦C, respectively) for 45 minutes in a Biorad Thermocycler. After 45 minutes, cells diluted back to a final OD of 0.008 in 7 ml media for 24-hour growth.

Oxidative stress: Libraries were directly diluted back to OD of 0.008 in 7 ml media that contains low, medium, or high (0.025mM, 0.05mM, 0.1mM) level hydrogen peroxide. Cells were grown for 24 hours in these chronic stress conditions.

L-canavanine stress: Libraries were directly diluted back to OD of 0.008 in 7 ml media that contains low, medium, or high (25ug/ml, 50ug/ml, 100ug/ml) levels of L-canavanine. Cells were grown for 24 hours in these chronic stress conditions.

Following overnight growth, 1 ml of saturated culture from each Tn library was pelleted at 8000xg for 2 minutes. Genomic DNA was extracted by Monarch Genomics DNA Preparation Kit (NEB) according to the manufacturer’s protocol.