Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: For each treatment, 1 ml of culture was transferred to lysing matrix S tubes (MP Biomedicals, LLC, Santa Ana, CA, USA), centrifuged at 10,000 x g for 5 min at 4°C, and the supernatant discarded. Lysis buffer (PureLink® RNA Mini Kit, Thermo Fisher Scientific) was immediately added to the fungal pellets, and the samples were homogenized using the FastPrep-24™ 5G Instrument (MP Biomedicals) at a speed of 6 m/sec with two segments of homogenization for 30 seconds with a 1 min rest in between. The total RNA was extracted following the manufacturer's instructions (PureLink® RNA Mini Kit, Thermo Fisher Scientific). Isolated RNA samples were DNase treated (TURBO DNA-free™ Kit, Thermo Fisher Scientific), tested by conventional PCR for the complete removal of DNA, and checked for quality (RNA integrity number > 6.0) using an Agilent 2100 Bioanalyzer (Agilent Technology, Waldbronn, Germany). Twelve libraries (wild type and mutant ΔFVEG_10494-6.4 treated with and without 1.5 mM DETA NONOate for 30 min, with three biological replicates each) were prepared and sequenced on an Illumina NextSeq (75 Cycles) High Output Flow Cell (paired end, 75 bp read length) by the Georgia Genomics Facility at the University of Georgia.