GSM2753222: 2_3_Input_PABP_PNG; 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. For P3D12 (PABP) pull downs, anti-FLAG beads were washed twice with 1xPBS and blocked overnight with anti-P3D12 antibody (purified according to Na et al, 2016) and ssDNA, at a ratio of 12.5 ul beads to 2.5 ug antibody. The next day, the beads were washed 3x with buffer (Buffer A for S2 cells, Buffer B for embryo lysate) and incubated with lysate (450 ul lysate to 12.5 ul beads). After incubation for 3 hours, the beads were washed 6x with the appropriate buffer, and RNA extraction was performed. For ME31B pull downs, protein G beads were used, blocked overnight in the same way as anti-FLAG beads were, but incubated with GFP antibody [Anti-GFP (clone 7.1 and 13.1), monoclonal mouse, from Roche, Cat#11814460001, RRID:AB_390913, Lot#14442000], pulling down GFP-tagged ME31B in eGFP-ME31B flies. The next day, the beads were washed 2x with buffer, lysate was added and incubated for 2 hours. After incubation, the beads were washed 6x with the approriate buffer, and RNA extraction was performed. During RNA extraction, 600 ng worm RNA was spiked in. 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.