Ion Torrent Proton sequencing
RNA isolated from aerobic batch cultivation of Brettanomyces naardenensis CBS 7540 was used to guide genome annotation.
Cells were pelleted by centrifugation at 4°C for 2 min  and frozen in liquid nitrogen and stored at −80°C before extraction of RNA. RNA was isolated using an RNAeasy kit (Qiagen, Germany) according to the manufacturer's instructions. The RNA was treated with the RiboMinus™ Eukaryote Kit (Thermo Fisher Scientific) to remove ribosomal RNA and purified using Agencourt RNAClean XP Kit (Beckman Coulter). The size and quantity of RNA fragments were assessed on the Agilent 2100 Bioanalyzer system (RNA 6000 Pico kit, Agilent). Libraries were prepared using the Ion Total RNA-Seq kit for the AB Library Builder System (Life Technologies). Samples were then quantified using the Agilent 2100 Bioanalyzer system (High Sensitivity DNA kit, Agilent) and pooled followed by emulsion PCR on the Ion OneTouch™ 2 system using the Ion Proton™ Template OT2 200 v2 Kit (Life Technologies) chemistry. Templated Ion Sphere particles were enriched using the Ion OneTouch™ ES (Life Technologies). Samples were loaded on a two Ion PI v2 Chips and sequenced on the Ion Proton™ System using Ion Proton™ Sequencing 200 v2 Kit (200 bp read length, Life Technologies) chemistry.