Short read illuminated sequencing of Acinetobacter spp
Pure cultures of each strain are grown in plates or broth. Cells are harvested and resuspended in a tube with cryoperservative (MicrobankTM, Pro-Lab Diagnostics UK, United Kingdom) or with DNA/RNA Shield (Zymo Research, USA) following MicrobesNG strain submission procedures. Strain containing tubes are sent to MicrobesNG for sequencing.Five to forty microlitres of the suspension are lysed with 120 L of TE buffer containing lysozyme (final concentration 0.1 mg/mL) and RNase A (ITW Reagents, Barcelona, Spain) (final concentration 0.1 mg/mL), incubated for 25 min at 37C. Proteinase K (VWR Chemicals, Ohio, USA) (final concentration 0.1mg/mL) and SDS (Sigma-Aldrich, Missouri, USA) (final concentration 0.5% v/v) are added and incubated for 5 min at 65C. Genomic DNA is purified using an equal volume of SPRI beads and resuspended in EB buffer (Qiagen, Germany).DNA is quantified with the Quant-iT dsDNA HS kit (ThermoFisher Scientific) assay in an Eppendorf AF2200 plate reader (Eppendorf UK Ltd, United Kingdom).