GSM9487705: Ribo-seq data of the mutant detaPoxTMA7,biol rep1; Penicillium oxalicum; OTHER
Mycelia from detaPoxTMA7 and WT strains collected after culturing in medium containing 1% Avicel, then powdered using liquid nitrogen with mortar and pestle. The same mass of powdered mycelia (1 g) was mixed with cold polysome extraction buffer, and lysed on ice for 25 min. Cell lysates were separated by centrifugation at 14,000 rpm for 20 min, and OD260 was measured using a NanoDrop instrument Cell lysates were then treated with 0.5 U/OD260 RNase I at 28°C for 1 h. The digests were separated by Amersham MicroSpin S-400 HR columns  and eluates were collected. The collected samples were mixed with 3-fold volume of anhydrous ethanol and placed at -80°C for 4-5 h. Next, RNA was extracted by the TRIzol method as described above. Resuspended RNA was separated by 16% TBE-urea PAGE, and the 26-34 nucleotide region was excised. RNA extraction buffer containing 0.025% SDS (w/v) was added to the excised gel slice, incubated at -80°C for 1 h, and rotated at room temperature for 3-4 h. Samples were separated from gel slices using Costar Spin-X Centrifuge Tube Filters (. RNAs (26-34 nucleotides) were extracted using the TRIzol method, and extracted RNAs were considered ribosome protected frames (RPFs). Construction of Ribo-Seq library was measured using a QEZ-seq 1.0 kit (Hangzhou NeoRibo Biotechnology, Hangzhou, China), according to the manufacturer's protocol. RPFs (20-500 ng) were adenylated by adding 2 μL adenylation buffer and 4 μL adenylation enzyme at 37°C for 30 min, followed by incubating at 70°C for 10 min, and put on ice immediately. Adenylated RPFs were ligated with linker by adding 8 μL ligation buffer and 1 μL ligation enzyme, incubated at 25°C for 90 min. Ligation products (RPFs-linker) were purified via streptavidin beads at room temperature. cDNA was synthesized by adding 6 μL RT buffer, 1 μL RT enzyme and 1 μL RT primer, incubated at 50°C for 20 min, and put on ice immediately. 10 μL 2ⅹ PCR mix, 0.5 μL forward primer and 0.5 μL reverse primer were added into mixture to amplify library. Library was purified by 8% TBE-PAGE and sequenced.