GSM2152771: iCLIP_T7_Cont1; 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 agarose beads. A T7 Tag antibody Agarose from Novagen was used for the precipitation. 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. Using different barcodes in the RT primers, the three libraries obtained for T7-Acinus S* iCLIP were sequenced on a single lane while the equivalent control was sequenced on a different lane
GEO Accession
GSM2152771