Illumina Sequencing of Metarhizium acridum ARSEF 324 L2
Liquid culture filtrate was ground with liquid nitrogen in a mortar and ca. 500 mg finely ground powder transferred to 50 mL Falcon tube. DNA was extracted using a CTAB method by adding 17.5 mL CTAB buffer with 0.1% 2-Mercaptoethanol and 125 l Proteinase K and incubated for one hour at 60 C. After centrifugation for 20 min. at 5.000g at 4 C, the supernatant was removed and washed with 1 volume of Phenol/chloroform/isoamyl alcohol (25:24:1) followed by two rounds of 1 volume chloroform/isoamyl alcohol (24:1) and centrifuged for 10 min at 11.000 rpm at each step. To remove RNA, 150 l RNAse was added to the aqueous phase and incubated for 120 min. at 37 C. To precipitate DNA, 0.6 vol. 2-propanol was added and incubated at -20 C overnight, before centrifugation at 11.000 rpm at 4 C for 30 min. The resulting pellet was washed twice with 2 mL 70% Ethanol and centrifuged for 10 min at 11.000 rpm at 4 C before being suspended in 500 l TE-buffer.   A single SMART Cell library was sequenced on a PacBio Sequel platform using the Sequel Sequencing Kit 2.1 v2 (Sequencing Kit par number 101-309-500). Each continuous long read (CLR) had one passage of sequencing; subsequently, subreads were generated by the removal of adapters and bases were called with the basecaller v5.0.0.6236.