RNA extraction and sequencing

Total RNA was extracted from 5–7 day-old cultures. This time period ensured that cultures did not reach the stationery phase, but gave enough time for sexual reproduction to take place. Nine MEA-ST plates were used per culture, allowing for three biological and three technical replicates. Mycelium was harvested from the vegetatively growing H. moniliformis isolate as well as the MAT1 and MAT2 H. omanensis isolates. A mixture of mycelium, ascomata and ascospores was harvested from the sexually reproducing H. moniliformis (unisexual) and H. omanensis (heterosexual) cultures.

The harvested tissue was flash frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. RNA extractions were performed using the RNeasy^® Mini Kit (Qiagen, Limburg, The Netherlands) following the manufacturer’s protocols with the following modifications: the RLC buffer was used as the extraction buffer and the optional on-column DNase 1 digestion was included. The integrity of the total RNA was assessed by 2% (w/v) agarose gel electrophoresis at 120V for 25 min and the concentration was estimated using an ND 1000 Spectrophotometer (ThermoScientific, Waltham, USA). The RNA was further subjected to quality testing using the Experion^™ automated electrophoresis system (BioRad Laboratories, California, USA) at the ACGT Microarray Facility (University of Pretoria, South Africa). Dynabead^®-based mRNA enrichment (ThermoFisher Scientific, Carlsbad, USA), cDNA synthesis and library preparation were performed at the Central Analytical Facilities (CAF) at the University of Stellenbosch (South Africa). The RNA sequencing was completed at the same facility using the Ion Proton Platform and PI^™ Chip system.