GSM8266980: 𝚫rrpA/B_24_1; Aspergillus fumigatus; ncRNA-Seq
Total RNA was isolated from three biological replicates of CEA17ΔakuBKU80, ΔppdA/B, ΔdclA/B, and ΔrrpA/B knockout strains 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. These samples were collected simultaneously as matched samples with those reported in GEO Series GSE223618. Libraries were constructed by Novogene. According to the manufacturer, 3' and 5' adaptors were ligated to 3' and 5' ends of small RNA, repsectively. Then the first strand cDNA was synthesized after hybridization with reverse transcription primer. The double-stranded cDNA library was generated through PCR enrichment. After purification and size selection, libraries with insertions between 18-40 bp were ready for sequencing with SE50.