title: GSM7984660: mod5 knockout mycelium_FC 3 [D3]; Aspergillus fumigatus; RNA-Seq
library_construction_protocol: Total RNA was isolated from three biological replicates of CEA17ΔakuBKU80 and Δmod5 knockout from 24-h-old mycelium either unstressed, or stressed for the final 8 hours with 5-fluorocytosine. Afterwards, mycelia were harvested using Miracloth (EMD Millipore Corp., USA), rinsed with sterile water and same amounts of mycelium were transferred to 2-mL screw cap tubes for RNA isolation. All centrifugation steps were performed at 4°C and full speed (20,817 x g). 800 µL of TRIzol (Thermo Fischer Scientific, Dreieich) were added to each tube, along with glass beads (1/3 of the tube) and the mycelium was mechanically disrupted by 3 rounds of 30 sec, 4.0 m/sec homogenization using the FastPrep-24™ Homogenizer. Subsequently, the samples were incubated for 5 min on ice, followed by additional 5 min at room temperature. After addition of 160 µL of chloroform, the samples were vigorously vortexed for 10 sec and centrifuged for 5 min. The resulting aqueous phase containing the RNA was carefully transferred to a new RNase-free tube without disturbing the interphase. The RNA phase was then purified using the same amount of phenol/chloroform/isoamyl alcohol and centrifuged for 5 min. The purification step was repeated 2-3 times until the interphase remained clear. In the next step, RNA was further purified with 400 µL of chloroform and centrifugation for 5 min. The aqueous phase was again transferred to a new RNase-free 2-mL tube, precipitated by addition of 500 µL isopropanol for 20 min, and RNA was pelleted by 20 min centrifugation. The RNA pellet was washed with 70% ethanol (v/v) and air-dried at 37°C. Finally, RNA was dissolved in 30-50 µL DEPC water at 65°C. The RNA was purified using the RNA Clean and Concentrator™ kit (Zymo Research, USA) following the manufacturer's instructions. Up to 10 µg of total RNA was afterwards treated with 2 U of DNase I endonuclease (Thermo Fisher) for 30-60 min at 37°C in a volume of 20 µL. RNA concentration was then quantified with the QubitTM RNA BR Assay kit (Thermo Fisher Scientific) on the Qubit Flex Fluorometer (Thermo Fisher Scientific). Quality was assessed either by agarose gel electrophoresis or QUBITTM Integrity and Quality (IQ) kit (Thermo Fisher). Poly-(A) selection and directional sequencing of mRNA was performed by Novogene using paired-end 150-bp read sequencing on a NovaSeq 6000.