GSM2152778: iCLIP_AciS1; Homo sapiens; RIP-Seq
RNAs were extracted following the published protocol (Konig et al. 2011). iCLIP experiments were carried out following a published protocol (Konig et al. 2011), with minor modifications. After cell lysis, extracts were sonicated for 5 min (30 secoff and 30 secon) then centrifuged at 10,000g for 3min. Supernatants were treated with various RNAse dilutions. Before the immunoprecipitation, each extract was pre-cleared for 1h with 10uL of Dynabeads protein A. The immunoprecipitation (IP) was carried out using Acinus (Ab-2) PC552 Calbiochem antibody coupled to Dynabeads Protein A, whereas rabbit IgG was used for the control. Dephosphorylation of the RNA 3’ ends was performed on beads using Alkaline Phosphatase, Calf Intestinal (CIP) following the supplier’s protocol. The RNA linker (5’Phosphate-UGAGAUCGGAAGAGCGGTTCAG-3’Puromycin) ligated to the 3’ end of the RNAs was described in (König et al. 2010). The obtained libraries were purified with Agencourt Ampure XP system (Beckman Coulter) and quantified using High sensitivity DNA ChIP on Agilent 2100 Bioanalyzer. The four libraries for the endogenous Acinus iCLIP (2 replicates for Acinus L and 2 replicates for Acinus S/S’) were sequenced together with the control libraries on a separate lane.
GEO Accession
GSM2152778