GSM8721381: 𝚫rrpA/B_IRT_1; Aspergillus fumigatus; ncRNA-Seq
Mycelium was collected with Miracloth (EMD Millipore Corp., USA) and rinsed with sterile MilliQ water. After being pressed tightly in between paper towels to remove moisture, the dried mycelium was transferred to a 2-mL screw-cap tube filled 1/3 full with 0.5 mm soda lime glass beads (BoSpec). 1 mL of TRIzol (Thermo Fischer Scientific, Dreieich, Germany) was added to efficiently lyse the cells using the FastPrep-24™ Homogenizer by subjecting the cells to three rounds of 30 s, 4.0 m/s homogenization (1 min break between repetitions) and 5 min of incubation at room temperature. 200 μL of chloroform was added to each sample, vigorously mixed and incubated for 3 min at room temperature before centrifuging the samples at 12,000 x g for 15 min at 4°C. The upper aqueous phase containing the RNA was carefully transferred to a clean RNase-free tube and an equal volume of pure ice-cold ethanol was added and mixing thoroughly. Purification of RNA was carried out by using the RNA Clean and Concentrator™ kit (Zymo Research GmbH, Freiburg, Germany) following the manufacturer's instructions. To eliminate DNase contamination, total RNA was treated with DNase I, RNase-free (Thermo Fisher Scientific, Darmstadt, Germany). For every 1 μg of RNA, 1 U of the enzyme was added along with 2 μL of 10x Reaction Buffer with MgCl2 and adjusted with DEPC-treated water to make up to 20 μL final volume. Following a 1-hour incubation at 37°C, the reaction was terminated by adding 2 μL of 50 mM EDTA and further incubated 65°C for 10 min. DNase treated RNA was purified using the RNA Clean and Concentrator™ kit (Zymo Research, USA) after the manufacturer's recommendations. 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. 50-bp single read sequencing sRNA-seq