GSM8973101: CEA10_37°C_2; Aspergillus fumigatus; RNA-Seq
Fungal mycelium was collected from liquid culture using Miracloth (Millipore) and disrupted in liquid nitrogen using a precooled mortar and pestle. Roughly 0.5 g of homogenized mycelium was transferred into a 2-mL Eppendorf tube. An amount of 800 µL TRIzol was added to the ground mycelia and vortexed vigorously. Tubes were frozen briefly for 5 sec in liquid nitrogen and allowed to thaw on ice. An amount of 160 µL of chloroform was added to the thawed mixture, vortexed, and centrifugated for 5 min at 4°C at full speed. The aqueous upper phase was transferred to a fresh 2-mL tube without disturbing the interphase. RNA extraction from aqueous phase was done with one volume of phenol/chloroform/isoamyl alcohol (25:24:1, v/v). Brief vortexing preceded centrifugation for 5 min at 4°C. Extraction was repeated until no more interphase was observable, and this was followed by another extraction with 400 µL of chloroform. RNA was precipitated using 400 µL of isopropanol for 20 min, followed by pelleting by centrifugation for 20 min at 4°C. The pellet was washed with 700 µL of 70% ethanol and air dried for 5 min at 37°C prior to resuspension in RNase-free water. Total RNA was DNase-treated according to the manufacturer's recommendation (ThermoFisher, RNase-free DNase) and DNase treated RNA was purified using the RNA Clean and Concentrator™ kit (Zymo Research, USA) after the manufacturer's recommendations. For poly(A)-RNA-Seq, library preparation was performed by NovoGene, UK according to standard protocols. Briefly, mRNA was purified from total RNA using poly-(T) oligo-attached magnetic beads. Following fragmentation, first strand cDNA was synthesized using random hexamer primers. The second strand cDNA was synthesized using dUTP. The directional library was considered complete after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and finally purification. The library was then checked by Qubit and real-time PCR for quantification and by bioanalyzer for size distribution, prior to pooling for Illumina sequencing using an Illumina Novaseq 6000.
GEO
GSM8973101_r1