Name: GSM8266956
Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Total RNA was isolated from three biological replicates of CEA17ΔakuBKU80 from conidia, 24-h-, and 48-h-old mycelium. For A. fumigatus conidia, the spores were first homogenized in a bead beater (0.5 mm beads; FastPrep-24, MP Biomedicals) in Trizol. 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. 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. To all samples, chloroform was added to the fungal samples in TRIzol, 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 1 volume of phenol/chloroform/isoamyl alcohol (25:24:1, v/v). Brief vortexing preceded centrifugation for 5 min at 4°C. RNA was precipitated using 400 µl isopropanol for 20 min, followed by pelleting by centrifugation for 20 min at 4°C. The pellet was washed with 700 µl 70% ethanol and air dried at 37°C for 5 min prior to resuspension in RNase free water. The RNA isolation was followed by a DNase treatment using 2 units of TURBO DNase (Thermo Fisher) per 10 µg RNA for 30 min at 37°C in 100 µl total volume. Total RNA was then collected using the RNA Clean and Concentrator-25 kit (Zymo Research) according to the manufacturer's instructions. tRF&tiRNA-seq (CD Genomics) The following treatments were performed before library preparation for total RNA samples: 3'-aminoacyl (charged) deacylation to 3'-OH for 3' adaptor ligation, 3'-cP (2',3'-cyclic phosphate) removal to 3'-OH for 3' adaptor ligation, 5'-OH (hydroxyl group) phosphorylation to 5'-P for 5'-adaptor ligation, m1A and m3C demethylation for efficient reverse transcription. Sequencing libraries were size-selectedfor the RNA biotypes to be sequenced using an automated gel cutter.The libraries were qualified and absolutely quantified using Agilent BioAnalyzer2100.