GSM7018593: NEO (exposed to an inaccessible nonedible object), rep 6; Mus musculus; RIP-Seq
Nucleus arcuate (ARC)-enriched microdissections were ice-cold homogenized in 0.25 ml homogenization buffer (50 mM Tris, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 200 U/mL Promega RNasin, 1 mg/mL heparin, 100 mg/mL cycloheximide, Sigma protease inhibitor mixture at pH 7.5). To pull down, 2 ml of mouse monoclonal anti-HA antibody (HA.11, ascites fluid; Covance) was added to the homogenate and allowed to rotate for 2 h at 4°C. Then, 200 ml of Dynabead protein G magnetic beads (Invitrogen) were added and further incubated for 2 h at 4°C with rotation. Immunoprecipitates (IPs) were washed 3 times for 10 min with gentle rotation at 4°C in high-salt buffer (50 mM Tris, 300 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 100 mg/mL cycloheximide at pH 7.5). After the final wash, RNA was stripped from the beads with Quiagen RLT buffer and total RNA was prepared according to manufacturer's instructions using the RNeasy-plus Mini kit (Qiagen). Yield and concentration were measured with the Quant-iT™ RiboGreen™ RNA assay kit (Invitrogen). RNA integrity was assessed on a 2100 Bioanalyzer device (Agilent Technologies) using the RNA 6000 Pico kit (Agilent Technologies). Only samples with RIN > 8 were used for RNA-Seq analysis. cDNA libraries were synthetized from 0.5 ng RNA with the SMARTer Ultra Low Input RNA kit v4 (Clontech). Sequencing libraries were prepared with the NexteraXT kit (Illumina)