title: GSM4875437: WT ovary mRNA; Drosophila melanogaster; OTHER
library_construction_protocol: The ovaries of wild-type and NSUN2 knockout flies were collected and lysed with lysis buffer (10 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2mM DTT, 100μg/ml CHX (CST), 50U/ml RNasin Plus RNase Inhibitor (Thermo Fisher) and cOmplete EDTA-free protease inhibitor (1 mini tablet per 10ml, Roche). Concentrations of total RNA in lysates were determined with Nanodrop, and 250 μg total RNA were digested with 3 U/μg micrococcal nuclease (Thermo, EN0181) in lysis buffer supplied with 100 mM NaCl and 5 mM CaCl2 for 40 min at 25°C in a Thermomixer (Eppendorf). Digestions were quenched by adding EGTA to a final concentration of 6.25 mM and placing the reactions on ice. 10–50% sucrose gradients were prepared in gradient buffer (20mM HEPES-KOH, pH7.4, 5mM MgCl2, 100mM KCl, 2mM DTT, 100μg/ml CHX, 50U/ml RNasin Plus RNase Inhibitor) using a Gradient Master (Biocomp). 200 μl lysates were loaded onto the gradients. Gradients were centrifuged at 36,000 rpm for 2 hours at 4℃ in an SW-40 Ti rotor, and then fractionated using the Gradient Master. The fraction that contains only the entire monosome peak was collected and then digested with 200 μg/ml Proteinase K supplied with 1 % SDS for 30 min at 42°C in a Thermomixer. The digested RNA was extracted by acid-phenol:chloroform. Ribosome-protected fragments (RPFs) between 28-30 nt were isolated using 15 % TBE-urea PAGE gel and depleted rRNA with biotinylated oligos49. rRNA-depleted RPFs were then treated with T4 PNK (NEB). Library was generated using VAHTS Small RNA Library Prep Kit for Illumina (Vazyme). For the RNA-seq control, poly(A)+ RNA was separated from total RNA and the library was constructed with NEBNext Ultra II Directional RNA Library Prep Kit (NEB).