Illumina HiSeq 4000 paired end sequencing; RNA-seq analysis of wildtype Aspergillus fumigatus A1160p+, ∆devR and ∆acdX in the presence and absence of Olorofim
RNA-seq analysis of wildtype Aspergillus fumigatus A1160p+, ∆devR and ∆acdX in the presence and absence of Olorofim
The null mutants were made as part of a 484-member transcription factor knockout library by the Bromley lab. Reference to how the null mutants were derived can be found in:   Furukawa, T., van Rhijn, N., Fraczek, M., Gsaller, F., Davies, E., Carr, P., Gago, S., Fortune-Grant, R., Rahman, S., Gilsenan, J.M. and Houlder, E., 2020. The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus. Nature communications, 11(1), pp.1-16. Aspergillus fumigatus A1160p+ (MFIG001) and transcription factor null mutants conidia were grown on Sabouraud Dextrose agar incubated at 37°C for 48 hours. Spores were harvested in PBS + 0.01% Tween-20 by filtration through Miracloth. Spores were counted using a haemocytometer. 1x10^6 spores/mL of A. fumigatus MFIG001, ΔdevR and ΔacdX were inoculated into 50 mL of Aspergillus complete media (ACM) and incubated for 18 hours at 37°C in a rotary shaker (180 rpm). Mycelia was harvested using filtration through Miracloth and washed in 1x RPMI-1640 medium. 1g of mycelia was added to shake flasks containing 50 mL RPMI-1640 medium, 165 mM MOPS buffer (pH 7.0), 2% glucose and incubated for 1 hour at 37°C in a rotary shaker (180rpm) in the presence or absence of 62.5 µg/L Olorofim. Mycelia was filtered through Miracloth and snapfrozen using liquid nitrogen and kept at -80°C until required. For RNA extractions, 1 mL of TRIzol reagent and 710-1180 μm acid washed glass beads were added to frozen mycelia and placed in a TissueLyser II® for 3 minutes at 30 Hz. The solution was centrifuged (12,000 rpm) for 1 minute at 4°C. The aqueous phase was added to 200 μL of chloroform and centrifuged (12,000 rpm) for 10 minutes at room temperature. The supernatant was added to 0.2 M sodium citrate, 0.3 M sodium chloride and 25% (v/v) isopropanol and left at room temperatures for 10 minutes. This solution was centrifuged (12,000 rpm) for 15 minutes at 4°C. The supernatant was removed; the pellet was washed in 70% (v/v) ethanol and resuspended in RNase free water. RNA samples were treated with RQ1 RNase-Free DNase and purified using a RNeasy Mini Kit. RNA quality and quantity were assessed using gel electrophoresis and using a NanoDropTM 2000/2000c Spectrophotometer. Sequencing libraries were prepared from mRNA using TruSeq® Stranded mRNA.