PromethION reads from Schizosaccharomyces pombe strain EBC725 (inversion II:2.157Mb-his5): single cell colony culture
High-weight genomic DNA was isolated from single colony cultures and protoplast were obtained by digesting cell walls in 1M Sorbitol with 100 mM EDTA pH 8.0 and 100mg/ml Lallzyme MMX (Flor-Parra et al. 2014) by incubation for 30 min at 32C with gentle agitation. Protoplasts were spun down and resuspended in 1ml of Qiagen buffer G2 with 50l protinase K (>600 mAU/ml, Qiagen) and incubated at 37C for 30min. From this suspension DNA was extracted using a phenol-chloroform extraction method (Sambrook et al. 1989). DNA was precipitated by addition of ice cold isopropanol, spooled on a pipette tip, washed in 70% ethanol and dissolved in water. Multiplexing libraries were generated and sequenced on PromethION chip. Raw reads were base called using Dorado 0.3.0 duplex and demultiplexed.