Whole genome sequencing of CRISPR-Cas9 edited Candida parapsilosis
One isolate from each targeted gene disruption was sequenced. Genomic DNA for Whole-Genome Sequencing (WGS) was extracted from an overnight culture incubated at 30C in YPD medium with shaking, using phenol:chloroform:isoamyl alcohol (25:24:1). Briefly, cells were resuspended in 200 L of Extraction Buffer (Triton X 100 2% m/v; NaCl 100 mM; Tris 10 mM pH 7.4; EDTA 1 mM; SDS 1 % m/v) and 200 L of phenol:chloroform:isoamyl alcohol (25:24:1), and lysed by mechanical beating with 0.3 g acid-washed glass beads (0.45-0.52 mm, Sigma). The mixture was microcentrifuged at full speed for 10 minutes, and the aqueous phase transferred in a new tube. Two hundred L of TE Buffer (pH 8.0) and 200 L of phenol:chloroform:isoamyl alcohol (25:24:1) were added, and the suspension was mixed in a bead-beater for 30 seconds. After centrifugation, the aqueous phase was transferred to a new tube and subjected once again to bead-beating in the presence of 200 L phenol:chloroform:isoamyl alcohol (25:24:1). The aqueous phase was then transferred to a new tube, and nucleic acids precipitated by addition of 1 mL of 100% Isopropanol + 80  L of 7.5 M Ammonium Acetate. The sample was centrifuged at max speed, and the pellet washed with 1 mL of 70% Ethanol. The air-dried pellet was resuspended in 400 L of TE buffer and 1 uL of RNAse A (100 mg/mL) and incubated at 37C for 1 hour. DNA was precipitated with 1 mL of 100% Isopropanol + 80  L of 7.5 M Ammonium Acetate, washed with 70% Ethanol, dried and resuspended in 100 L of deionized water. The DNA was then cleaned with the Genomic DNA Clean and Concentrattor-10 kit (Zymo Research), according to the manufacturers instructions.