PAlabC50
Three beads were washed with extraction buffer containing lysozyme (or lysostaphin for Staphylococcus sp.) and RNase A, incubated for 25 min at 37oC. Proteinase K and RNaseA were added and incubated for 5 min at 65oC. Genomic DNA was purified using an equal volume of SPRI beads and resuspended in EB buffer. DNA was quantified in triplicates with the Quantit dsDNA HS assay in an Ependorff AF2200 plate reader. Genomic DNA libraries were prepared using Nextera XT Library Prep Kit (Illumina, San Diego, USA) following the manufacturers protocol with the following modifications: two nanograms of DNA instead of one were used as input, and PCR elongation time was increased to 1 min from 30 seconds. DNA quantification and library preparation were carried out on a Hamilton Microlab STAR automated liquid handling system. Pooled libraries were quantified using the Kapa Biosystems Library Quantification Kit for Illumina on a Roche light cycler 96 qPCR machine. Libraries were sequenced on an Illumina instrument using a 250bp paired end protocol. Reads were adapter trimmed using Trimmomatic 0.30 with a sliding window quality cutoff of Q15. De novo assembly was performed on samples using SPAdes version 3.7, and contigs were annotated using Prokka 1.11.