Mouse skin infection assay Mice were infected with S. aureus as previously described [14]. Briefly, six-week-old female BALB/c mice were infected by intradermal injection with 108 CFU of S. aureus. Mice were assessed and weighed daily for five days. Mice were culled on the 5th day and lesion size measured and CFU recovered from infected tissues by homogenization and colony enumeration on BHI. For each S. aureus strain, at least 10 mice were assessed. Genome sequencing Genome sequences for three ST93 strains Necrostatin-1 mw (TPS3104, TPS3105, TPS3106) were obtained from an Illumina GAIIx analyzer

using 100 bp paired-end chemistry with a mean fold coverage of 331×. Genome sequencing of the two laboratory-induced mutants JKD6159∆hla (TPS3265) and JKD6159_AraCr (TPS3268) was performed using Ion Torrent sequencing technology. Comparative genomics A read mapping approach was used to compare the sequences from all isolates used

in this selleck compound study, as previously described [14, 37]. Briefly, the reads from all genomes were aligned to the JKD6159 reference using SHRiMP 2.0 [38]. SNPs were identified using Nesoni v0.60 [ http://​www.​bioinformatics.​net.​au]. Using the whole genome sequence of JKD6159 as a reference, a global SNP analysis was performed, and allelic variability at any nucleotide position was tallied to generate a global SNP analysis for every genome compared to JKD6159. Quantitative RT-PCR for RNAIII expression To investigate activity of the agr locus (RNAIII) qRT-PCR was performed for RNAIII as previously described [37]. Briefly, RNA was prepared as previously described with two on-column DNase I digestion steps and cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen). Relative expression was determined as previously described and was normalised against gyrB. Results were obtained from P-type ATPase 3 biological replicates each performed in triplicate. RNA sequencing Staphylococcus aureus strains JKD6159 and JKD6159_AraCr were grown to early stationary culture as described above. For RNA protection, 0.5 volumes of RNAlater® RNA stabilization reagent (Qiagen) was added immediately to the liquid culture

and allowed to incubate with the bacterial suspension for 15 minutes at room temperature. Cells were pelleted at 5,000 × g for 5 minutes followed by RNA extraction using RNeasy mini kit (Qiagen) and two rounds of DNase I digestion (Qiagen) according to the manufacturer’s instruction. RNA concentration was quantified using Qubit® 2.0 Fluorometer and RNA quality assessed using Agilent 2100 Bioanalyzer. Ten μg of total RNA from the stationary growth phase with RNA intergrity number (RIN) greater than 7 was used in RNA-seq. Ribosomal depletion, cDNA Mdivi1 mw library preparation and pair ended sequencing using HiSeq2000 sequencing platform was performed by Beijing Genome Institute (Hong Kong, China). RNAseq was performed on two biological samples for each strain.