GSM5008978: Control_rep3; Aspergillus luchuensis; RNA-Seq
Total RNA was extracted as follows. The conidia of A. kawachii control and Tet-S-nrdA strains were inoculated into YES medium with Dox and pre-cultured and then transferred to YES medium with or without Dox and further cultivated. After incubation, mycelia were collected and ground to a powder in the presence of liquid nitrogen. Total RNA was extracted using RNAiso Plus (Takara Bio) according to the manufacturer's protocol. Next, immunoprecipitated RNA was prepared as follows. The conidia of A. kawachii Tet-S-nrdA strain were inoculated into YES medium with Dox and pre-cultured and then transferred to medium with Dox and further cultivated. After incubation, mycelia were collected and ground to a powder in the presence of liquid nitrogen. The powdered mycelia was dissolved in the ice-cold nuclear extraction buffer (25 mM HEPES [pH 6.8], 1 M sorbitol, 250 μg/ml phenylmethylsulfonyl fluoride [PMSF], cOmplete [EDTA-free protease inhibitor cocktail, Roche, Basel, Switzerland]) and vigorously mixed using a vortexer. Cell debris was removed by filtration using miracloth, and then centrifuged. The supernatant was removed and the pellet was dissolved in the ice-cold nuclear solubilization buffer (25 mM HEPES [pH 6.8], 1 M sorbitol, 0.5% NP-40, 250 μg/ml phenylmethylsulfonyl fluoride [PMSF], cOmplete). After incubation, the debris was removed by centrifugation. The resultant supernatant was added 25 µl of protein A sepharose beads (50% slurry contained PBS buffer) (GE healthcare) and the resulting mixture was gently mixed for 1 h at 4°C using a rotator. The mixture was separated by centrifugation, and supernatant was mixed with anti-S-tag antibody (MBL) or normal rabbit IgG (MBL) cross-linked protein A sepharose. Then, these mixtures were gently mixed for 3 h at 4°C using a rotator. Immunoprecipitated RNA was purified using RiboCluster Profiler (MBL) according to the manufacturer's protocol. RNA libraries were prepared for sequencing using standard Illumina protocols