title: Illumina HiSeq 4000 paired end sequencing; RNA-seq analysis of Aspergillus fumigatus response to low and supra-MIC concentrations of voriconazole
design: RNA-seq analysis of Aspergillus fumigatus response to low and supra-MIC concentrations of voriconazole
library_construction_protocol: Mycelium were harvested (avoiding conidia as much as possible) from plates without voriconazole disc (No Drug), from the area equidistant from the border of the plate and the inhibition halo (Low Drug) and the colonies in the halo (Supra-MIC = Persistence). Persister colonies were combined from 20 plates per replicate in order to obtain sufficient material for RNA extraction. Additionally, mycelia were harvested from A1160 with No and Low Drug in the same conditions. Mycelia were scratched using a spatula and immediately frozen and grounded in liquid nitrogen. Two independent replicates were assayed per isolate and condition. Spores were inoculated on top of a 0.45 µm pore nylon membrane placed on an RPMI solid plate. The disc with voriconazole (0.8 mg/mL) was placed on the membrane and incubated for 5 days at 37°C. Standard halos of inhibition formed on the membrane, and persister colonies appeared for the PD-47-104 strain, but not for the A1160 strain.  RNA was extracted and purified with the Plant RNeasy Mini Kit (Qiagen) following the manufacturer's instructions for filamentous fungi, and using on-column DNase treatment. Library preparation of samples was performed using the Illumina Stranded mRNA Prep. Ligation kit (Illumina, Inc.)