Direct-mRNA-Seq of Cryptotrichosporon argae strain CM19 to aid genome annotation.
Yeast cells were grown in liquid medium (per L: 3g yeast extract, 3g malt extract, 5g soy-peptone, 10g glucose) in baffled Erlenmeyer flasks (23C, 125rpm shaking with 50mm throw) to an OD600nm of about 1.3. A ca. 200L cell-pellet was ground to a powder while frozen with liquid nitrogen. RNA extraction was done using a 'magnetic mRNA isolation kit' (NEB#S1550) and the single-strand cDNA-library was produced using the ONT 'direct RNA sequencing kit' (ONT#SQK-RNA002) with its recommended 3rd party consumables. Raw data generation was done on a R9 flowcell (FLO-MIN106D) using a MinION Mk1C platform. Basecalling was done on the MinION device using the on-board MinKNOW v.23.04.5 application with a high accuracy model and minimum qscore filter of 7. The primary purpose of this sequencing was to fine-tune gene-models for genome-annotation.