GSM5050741: Argonaute rep5; Mus musculus; RIP-Seq
Crosslinked explants were lysed in low salt buffer. Lysates were sequentially treated with RNase-free DNase and micrococcal nuclease. EGTA was added to stop digestion. Lysates were cleared by centrifugation. Cleared lysates were immunoprecipitated with anti-Argonaute mAb or IgG, RNA was dephosphorylated, then phosphorylated with T4 Polynucleotide Kinase. Protein-RNA complexesb (100-150kd) were isolated by SDS-PAGE, digested with Proteinase K, and RNA recovered by acidic phenol/chloroform extraction and ethanol precipitation. Libraries were prepared (input from 6 pooled retinas for each) using an IlluminaTruSeq Small RNA Library Preparation Kit according the manufacturer's directions, with the following modification: after reverse transcription 80 to 150nt cDNA was gel purified and precipitated by with 2-propanol, then used for subsequent steps.