GSM9107340: IP rep2; Mus musculus; RIP-Seq
Livers were pulverized on dry ice and the resulting liver powder was thinly spread on a petri dish and UV-crosslinked at 254 nm with 400 mJ/cm² using a Stratagene Stratalinker UV 1800, keeping samples frozen throughout. Crosslinked samples were stored at -80 °C until processing. The eCLIP protocol was performed as described in Blue et al., 2022 (PMID: 35322209) and Nostrand et al., 2016 (PMID: 27018577). Briefly, cells were lysed in lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.5% NP-40, 0.1% SDS, 0.5 mM DTT, 1 mM PMSF, and protease inhibitors). RNA was partially digested using RNase I (Thermo Fisher Scientific cat: AM2295). Lysates were centrifuged at maximum speed and the supernatant was subjected to immunoprecipitation with FLAG antibody (Sigma-Aldrich cat: F3165) conjugated to protein A/G magnetic beads. Input samples were saved prior to washing. Next, the RNA-protein complexes were washed with high salt wash buffer (50 mM HEPES pH 7.4, 1 M NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40), low salt wash buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40), and PNK buffer (50 mM Tris pH 7.4, 10 mM MgCl2, 0.5% NP-40). RNA-protein complexes were ligated to 3' adapters, reverse transcribed using SuperScript III (Thermo Fisher Scientific cat: 12574026), and cDNA was purified with Agencourt AMPure XP beads (Beckman Coulter). Libraries were PCR amplified using Q5 High-Fidelity DNA Polymerase (NEB cat: M0541) with barcoded primers, purified again with AMPure XP beads, and sequenced.