GSM6965727: 48_h_3; Aspergillus fumigatus; ncRNA-Seq
Total RNA was isolated from three separate biological experiments using A. fumigatus conidia and mycelium after 24 and 48 hours of growth using the Universal RNA Kit (Roboklon) with slight modifications. Namely, conidia and mycelia at different time points were harvested and resuspended in RL buffer followed by bead beating 3x for 30 s each using 0.5 mm glass beads (Carl Roth). The total RNA was then isolated according to the yeast protocol provided by the manufacturer (Roboklon) using an on-column DNase treatment (Lucigen Baseline-ZERO DNase). The RNA isolation was followed by an additional DNase treatment using 4 units of TURBO DNase (Thermo Fisher) for 30 min at 37°C in 100 µl total volume. The small RNAs were then collected using the RNA Clean and Concentrator-25 kit (Zymo Research) according to the manufacturer's instructions for small RNA enrichment. Since the 5' end of A. fumigatus small RNAs are not well-defined, the isolated RNA was treated with 1 µl (5 units) RppH enzyme (New England Biolabs) in NEB Buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM DTT at pH 7.9) for 1 h at 37°C to facilitate removal of a pyrophosphate from the 5' end of triphosphorylated RNA. The reaction was ended with EDTA for 5 min at 65°C followed by cleanup using the RNA Clean and Concentrator-25 kit (Zymo Research) as described above. RNA was quantified using the Qubit 4 Fluorometer (Thermo Fisher). Small RNA libraries were then prepared from 10 ng of small RNA using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs #E7300S) according to the manufacturer's recommendation using NEBNext Multiplex Oligos for Illumina (Index Primer Set 1, #E7335S; Table S5). Size selection was performed according to the manufacturer using AMPure XP beads (Beckman Coulter). Libraries were validated for the correct size by polyacrylamide gel electrophoresis. The barcoded libraries were pooled without further adjustment, followed by 50-cycle-single-end sequencing using the Illumina HiSeq 4000 platform (Eurofins Genomics).