Imperial College : C. gattii Illumina resequencing project - WM276
Genomic DNA from all 23 Cryptococcus gattii strains was isolated with the EpiCentre MasterPureTM Yeast DNA Purification Kit according to a modified version of the instruction manual.   Briefly, the strains were grown in liquid YPD media for 24 h at 25°C rotating at 20 rpm. Cells from 3 ml of culture were harvested by centrifugation at 17,000 x g for 5 minutes.   Cells were lysed in 300ul of Yeast Cell Lysis solution by mechanical disruption with 0.1 mm silica spheres (FastPrepTM Lysing Matrix, MP Biomedicals) twice for 30 seconds at 6,800 rpm in a Precellys®24 and incubation at 65°C for 15 minutes.   Samples were cooled down on ice for 5 minutes and proteins removed by vortexing with 150ul of MPC Protein Precipitation Reagent and following centrifugation for 10 minutes at 17,000 x g.   DNA was recovered with 500ul isopropanol and centrifugation at 17,000 x g for 10 minutes. DNA was purified by RNase A treatment for 60 minutes at 37C followed by phenol:chloroform extraction and ethanol precipitation.   DNA yield and quality was determined by spectrophotometry. 2ug of genomic DNA were used for library preparation: DNA was fragmented to 150-500 bp using Covaris shearing and processed with the TruSeqTM DNA Sample Prep Kit (Illumina) according to the manufacturer’s  instructions.   Purification steps were performed with Agencourt AMPureXP magnetic particles (Beckham Coulter) on a magnetic stand (AmBio). Whole genomes were sequenced on an Illumina HiSeq2000 at the MRC Clinical Science Centre, Imperial College London (UK).