title: GSM8824357: strain KX155R biol rep 1; Caenorhabditis elegans; RIP-Seq
library_construction_protocol: Worm pellets were subsequently crushed in a mortar and pestle under liquid N2 with an equal volume of mRNP immunoprecipitation buffer (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol (DTT), 800 U of RNAsin/ml (Promega), 4 mM Vanadyl-ribonucleoside complex, 2X HALT complete protease inhibitor (ThermoFisher), 14 mM E64 protease inhibitor (SigmaAldrich), then centrifuged at 14,000 × g at 4°C for 15 min. The resultant supernatant was subjected to 20-50 μL of packed equilibrated GFP or RFP-targeted Nano-Trap agarose beads (Chromotek) and bound for 1.5 hrs. at 4°C while rotating. All immunoprecipitations were done on 6-10 mg of total C. elegans protein. After binding, beads were washed 4X in medium salt high detergent buffer (50 mM Tris-HCl [pH 7.8], 300 mM NaCl, 0.5% NP-40, 0.5% deoxycholic acid). The Trap beads (or an aliquot of the initial homogenate for unenriched control) were extracted with Trizol. Total RNA was also isolated from the same frozen worm samples by standard methods using Trizol. Resuspended precipitate was further extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and twice with chloroform:isoamyl alcohol (24:1), then ethanol precipitated. GlycoBlue (Invitrogen) was used as a co-precipitant. RNA quality and quantity was determined using NanoDrop® ND-1000 spectrophotometer. RNA sequencing services were outsourced to Admera Health, LLC. RNA (>1 ug) from three replicates were sent. Library type: RNA (rRNA depleted), NEBNext Ultra II non-directional with Qiaseq Fast Select HMR.