title: GSM9075126: adult brain, ZT12, tim>RpL3-3xFLAG co-IP, replicate 1; Drosophila melanogaster; RIP-Seq
library_construction_protocol: Each sample utilized 75 adult female and 75 adult male flies. Fly heads were isolated from bodies using flash freezing followed by vortexing. Immediately after collection, the head tissue was frozen on dry ice and stored at -80C until all samples are ready for processing in one batch. The frozen tissue was homogenized in 300 µl of lysis solution (10 mM HEPES pH 7.4, 150 mM KCl, 5 mM MgCl2, 100 µg/ml Cycloheximide), supplemented with 0.5% Triton-X100, 1U/µl ANTI-RNase (ThermoFisher Scientific, AM2690), and EDTA-free protease inhibitor (Sigma, COEDTAF-RO). A 20 µl aliquot of the resulting lysate was saved as the INPUT sample. The remaining lysate was used for co-immunoprecipitation with anti-FLAG magnetic beads following protocol described in Vaziri et al. Sci Adv 2020, yielding the FLAG sample. Each sample was subjected to total RNA extraction employing TRIzol LS Reagent (ThermoFisher Scientific, 10296010) followed by column-based purification (Zymo, R2050) with on-column DNase treatment. Purified RNA was quantified by Nanodrop and Qubit RNA and saved at -80C until library construction. For each sample, 10ng (INPUT) or 100ng (FLAG) of purified total RNA was used for library construction using the NEBNext Low-Input cDNA Synthesis Module (New England Biolabs). cDNA library was subject to quality control by Tapestation (HSD5000) and Qubit (High Sensitivity DNA) for mean size of ~1500bp. Next, cDNA library was tagmented to prepare Illumina sequencing library (Illumina Nextera XT, UDI indexing). Quality control was performed with Qubit (High Sensitivity DNA) and Tapestation (HSD1000) for mean size of ~300bp. Samples were loaded in equal molar with 3% spike-in PhiX DNA and sequenced on an Illumina NextSeq 1000 platform with paired-end 100bp cycle.