GSM8271125: CPT_2; Staphylococcus aureus; OTHER
The frozen bacteria pellets from the transposon library screen were thawed on ice and genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). Genomic DNA was sheared to 350 bp by sonication using a Covaris LE220 and prepared by sequencing using the homopolymer tail-mediated ligation PCR (HTML-PCR) as outlined in van Opijnen et. al. with the modification of the use of the KAPA HiFi HotStart DNA Polymerase (Roche) for the PCR cycles and the use of the transposon-specific primers olj510 and olj511 as previously outlined. Replicates were all individually barcoded and then multiplexed for sequencing. A transposon library was constructed in the JE2 strain using the plasmids pBursa and pMG020 as described in Grosser et. al. The library construction process was repeated multiple times until enough transposon mutants could be pooled together to make a single library. This pooled library was grown for an additional 4 hours in TSB (without antibiotics) before being frozen in individual aliquots at a concentration of roughly 1x1011 CFUs/ml at -80°C. Spot plating of the inoculum on TSA plates containing antibiotics (either 10 µg/ml tetracycline or 10 µg/ml chloramphenicol 10 µg/ml) confirmed curing of plasmids pBursa and pMG020 in greater than 99.98% of bacterial cells. The library was confirmed to have roughly 150,000 independent transposon mutants as verified by Illumina sequencing analysis. Pooled aliquots of this high-density library were frozen at -80°C until used.