VRC 2
Protocols: Fungal mycelia were quickly scraped off the surface of the cellophane membranes using sterille pipette tip. The harvested biomass was immediately transferred to pre-chilled tubes, flash-frozen in liquid nitrogen to stop transcriptomic updates, and immediatelly processed for total RNA extraction. Aspergillus cultures were inoculated onto cellophane membranes placed over RPMI medium (solidified with agar 2% m/v). Cultures were incubated under controlled conditions at 37 °C for a period of 40 hours to allow for standard vegetative mycelial development. Following the initial growth period, the cellophane-bound Aspergillus colonies were transferred to fresh media supplemented with antifungal compounds => 4 mg/L itraconazole, 2 mg/L voriconazole, 0.5 mg/L posaconazole. Stock solution were prepared to DMSO. Control samples received DMSO 1% v/v. All cultures were incubated for an additional 4 hours before immediate biomass harvesting. Total RNA was extracted from Aspergillus samples using a hybrid isolation approach blending TRI Reagent (Merck, Germany) and a column-based purification system. Briefly, 1 mL of TRI Reagent was added to the sample and incubated at room temperature for 15 minutes with continuous vortexing. Following centrifugation at 12,000 × g for 10 minutes at 4 °C, the supernatant was transferred to a clean microcentrifuge tube and mixed with 200 μL of chloroform (Mikrochem, Slovakia). The mixture was vortexed, incubated on ice for 5 minutes, and centrifuged at 12,000 × g for 15 minutes at 4 °C to achieve phase separation. The upper aqueous phase containing the RNA was carefully collected and mixed with 400 μL of ethanol (Mikrochem, Slovakia). Final RNA purification was completed using the GenUp™ Plant RNA Kit (biotechrabbit, Germany) following the manufacturer's column-binding and washing protocol starting from the step of ethanol addition. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, first-strand cDNA was synthesized using random hexamer primers, followed by second-strand cDNA synthesis using dTTP to create a non-strand-specific library. The non-directional library was completed following standard steps of end repair, A-tailing, adapter ligation, size selection, PCR amplification, and final magnetic bead purification. Completed libraries were quantified using Qubit and real-time PCR, and size distribution was checked using an Agilent Bioanalyzer.
ENA first public
2026-08-06
INSDC center name
Institute of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava
INSDC status
public
Submitter Id
E-MTAB-17342:VRC 2
broker name
ArrayExpress
collection date
not collected
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
mycelium
replicate
C2
sample name
E-MTAB-17342:VRC 2
scientific_name
Aspergillus fumigatus Af293
strain
Af293