title: GSM5048075: W-RIP-4; Drosophila melanogaster; RIP-Seq
library_construction_protocol: Following immunoprecipitation, RNA was isolated from IP and Input samples using a TRIzol-column hybrid approach adapted from (Rodriguez-Lanetty). To account for volume differences, samples were vigorously homogenized in TRIzol reagent (15596018, Thermo Fisher) at a ratio of either 1:10 (IP sample:TRIzol) or 1:3 (Input sample:TRIzol) and then incubated for 5 minutes at room temperature. All homogenized samples were clarified by centrifugation at 12,000×g at 4°C for 5 minutes, IP samples were magnetized to collect beads, and supernatant was isolated from all samples. After adding chloroform at a ratio of 0.2:1 (choloroform:TRIzol), samples were manually shaken and incubated at room temperature for 3 minutes. Samples were phase separated by centrifugation at 12,000×g at 4°C for 15 minutes, after which the aqueous layer was carefully isolated and mixed with an equal volume of 100% ethanol. RNA was further purified using an RNeasy Mini Kit (74106, QIAGEN) according to the manufacturer's instructions (RNeasy Mini Handbook, 4th Ed., June 2012) with the following deviations: for each sample, a final 30 µL elution was performed twice, isolating 60 µL of RNA in total into each collection tube. An on-column DNase digestion step was also performed under the same instructions using an RNase-Free DNase Set (79254, QIAGEN). Final RNA concentration and sample purity were determined via a NanoDrop 1000 spectrophotometer (Thermo Fisher). Once obtained, RNA samples were transferred on dry ice to the Georgia Genomics and Bioinformatics Core at UGA for library preparation and sequencing. There, IP samples were first concentrated using solid phase reversible immobilization (SPRI) beads. Then, the TruSeq Stranded Total RNA Library Prep Gold kit (20020598, Illumina) was used to deplete rRNA and prepare stranded cDNA libraries from all twenty-four samples. These uniquely barcoded cDNA libraries were then pooled by sample type, forming one IP library pool and one Input library pool. Each pool was sequenced on a separate NextSeq High Output Flow Cell (Illumina) for 150 cycles to generate paired-end, 75 base-pair (bp) reads.