GSM2587292: 4_5_Input_PABP; Drosophila melanogaster; RIP-Seq
At collection, embryos were dechorionated with bleach, then selected through filtration. They were then washed with water and 0.1% Triton 100. Embryos were homogenized in Lysis Buffer B (150 mM KCl, 20 mM HEPES-KOH pH 7.4, 1 mM MgCl2, 1mM DTT, complete mini EDTA-free protease inhibitors), and were clarified at 15,000 rpm, at 4°C for 15 minutes. The supernatant was stored at –80°C. S2 cells were lysed using Buffer A (100 mM KCl, 0.1 mM EDTA, 20 mM HEPES-KOH pH 7.6, 0.4% NP-40, 10% glycerol, 1 mM DTT, complete mini EDTA-free protease inhibitors, SUPERase-In RNase inhibitors) 48 hours post transfection, clarified at 14000 rpm for 5 minutes at 4°C. After RNA extraction, the RNA underwent rRNA depletion using the Ribo-Zero Gold Kit (Illumina). Libraries for RNA sequencing were generated using Illumina's TruSeq stranded mRNA library preparation kit.