PacBio WGS Sequencing of Knufia petricola
Knufia petricola strain A95 (CBS 123872) was grown at 25 C in constant darkness in liquid media at 80-100 rpm (in Kuhner ISF1-X shaker) for suspension cultures. Media were malt-extract medium (MEB/MEA) [2 % malt extract, 0.1 % peptone, 2 % glucose]. For inoculation, cells were taken from surface-grown colonies, dispersed using glass beads (3 to 5 mm) and a TissueLyser. Deep-frozen cells of K. petricola were ground with a mortar and pestle and homogenized with TNE buffer. Two extractions with phenol:chloroform:isoamyl alcohol (25:24:1) followed. Then, the aqueous phase was treated with RNase A. Following an extraction with chloroform:isoamyl alcohol (24:1), DNA was precipitated overnight from the aqueous phase with 0.7 volume of isopropanol at -20 C. The pellet was repeatedly washed with 70 % ethanol, dried and dissolved in nuclease-free water. PacBio single-molecule real-time (SMRT) sequencing was performed at DNA Link (Seoul, South Korea) on one SMRT cell with P6-C4 chemistry, and the MagBead OneCellPerWell protocol (Pacific Biosciences) of the PacBio RS sequencing platform.