Aspergillus fumigatus transcriptome during growth in SDB, SCFM2, and co-culture with Pseudomonas aeruginosa PA14
Aspergillus fumigatus transcriptome during growth in SDB, SCFM2, and co-culture with Pseudomonas aeruginosa PA14
A. fumigatus mono-cultures (50 mL) grown in SCFM2 and SDB, and SCFM2 co-cultures with P. aeruginosa were harvested after 32 h of growth (triplicates) by filtering culture contents over a 40 µm cell strainer into a 50 mL conical tube. Retained mycelial biomass was collected from the strainer using a sterile inoculation loop, transferred into 2 mL bead-beating tubes containing 0.1 mm and 1.0 mm Zirconia/Silica beads, and immediately snap-frozen in liquid nitrogen prior to RNA extraction. Snap-frozen A. fumigatus mycelia (triplicates per condition) were resuspended in 1 mL PureZOL™ RNA Isolation Reagent and mechanically lysed using a Mini-Beadbeater-16 (4 x 30 sec cycles), cooling on ice between cycles. Total RNA was extracted by chloroform-isopropanol precipitation and resuspended in RNase-free water. RNA (10 µg) was treated with TURBO DNase, ethanol precipitated, and resuspended in RNase-free water. Ribosomal RNA depletion (1 µg input RNA) was performed using the QIAseq FastSelect - rRNA Yeast Kit (Qiagen, #334215). Total RNA was DNase treated and ribosomal RNA depleted as described above. Sequencing libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer's instructions to generate stranded cDNA libraries suitable for paired-end sequencing.