title: Sample11_Orc33f2
organism: Homo sapiens
description: Protocols: Huh-7 cells were treated with DMSO for 90 min. Cells were washed twice with PBS, UV-crosslinked at 254nm and lysed in lysis buffer (100mM NaCl, 50 mM TRIS-HCl pH7.5, 1mM MgCl2, 0.1 mM CaCl2, 1% NP40, 0.1% SDS, 0.5% Na-deoxycholate, protease inhibitors cocktail). iCLIP protocol was then followed (Koenig et al, 2010). Shortly, lysates were sonicated, cleared and treated with RNase I and DNAse I. Immunoprecipitation was performed with antibodies coupled to Protein G Dynabeads for 1 hour at 4 C. Beads were washed twice with lysis buffer, twice with high salt buffer (1M NaCl, 1mM EDTA, 1% NP40, 0.1% SDS, 0.5% Na-deoxycholate, protease inhibitors cocktail) and twice with low salt buffer (50mM NaCl, 50mM TRIS-HCl pH7.5, 10 mM MgCl2, 0.5% NP40). For linker ligation, beads were incubated at 16 C overnight, washed twice with low salt buffer, radioactively labeled with T4 polynucleotide kinase and eluted by boiling. Complexes were resolved on PAGE, RNAs isolated and used for cDNA production. Libraries were generated as described in Koenig et al, 2010 [PMID: 20601959].