MiSeq 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. 3 g air-dried cells of K. petricola in 30 ml TRI Reagent solution were passed twice through a French press, extracted with phenol-chloroform and precipitated with 100 % ethanol. Air-dried pellets were dissolved in 8 mM NaOH, centrifuged, neutralized with 1 M HEPES and again precipitated with 1/10 volume of 3 M sodium acetate and 0.7 volume of isopropanol. Ethidium bromide and solid CsCl were added, the mix was centrifuged for 4 h at 525,000 g and 4 C. Genomic DNA was precipitated, washed several times with 70 % ethanol, dried and finally dissolved in nuclease-free water. The sequencing library was prepared with the Nextera XT DNA Library and sequenced on a MiSeq sequencer using MiSeq Reagent Kit V2.