DNAseq of Cryptococcus gattii CgZe121_iv
Cells were grown in YPD +2% glucose agar for 48h to obtain single colonies. One single colony was inoculated in YPD +2% glucose for 16h at 30C, with shaking at 200 rpm. DNA was isolated using a combination of phenol chloroform-isoamyl-alcohol method and the Qiagen DNA mini kit (QIAmp DNA mini kit, 51304). The samples were snap-frozen in liquid nitrogen, then heated to 75C for 10 min. Next, 200 l of chloroform:isoamylalcohol (24:1) was added and the lysate was centrifuged to separate the aqueous phase. A second chloroform:isoamylalcohol (24:1) wash was performed to remove residual phenol. The aqueous phase was precipitated with 2 volumes of absolute ethanol and incubated at -80C for at least 2 hours. The DNA was resuspended in 400 l of 10 mM Tris HCl pH 8.0 and treated with RNaseA (100 g/ml) at 37C for 30 min. DNA purification was completed using the Qiagen kit by adding 1.5 volumes of buffer AW1, followed by transfer to the DNeasy mini spin column. The column was centrifuged and washed with buffer AW2, followed by elution with 10 mM Tris HCl pH 8.0. Genomic libraries were prepared and sequenced on a NovaSeq 6000.