Mediator Subunit 30 Directs Myc Oncogenesis [ChIP-seq]

Mediator Subunit 30 Directs Myc Oncogenesis [ChIP-seq] Overall design: To map the binding sites of Mediator component MED17, MED30 and MYC, these three factors ChIP-seq were performed in Mia PaCa-2 cells in normal culture condition. To test the effect of MED30 deletion in Myc's binding. MYC ChIP-seq was perform with or without three days 0.5mg/ml Dox treatment in teton shMED30 Mia PaCa-2 cells. 1-5 million cells were dual cross-linked with 2 mM disuccinimidyl glutarate (DSG, ProteoChem, #C1104-1GM) for 20min at room temperature and followed by 1% formaldehyde for 10 min for Mediator's ChIP. For transcription factors' ChIP, cells were only fixed in 1% formaldehyde for 10 min. In both situations, the cross-linking was quenched with 0.125M glycine for 5 min. Chromatin was fragmented using a Bioruptor to get 200-500bp fragments. Subsequently, the soluble chromatin was incubated with 1-3 mg antibodies at 4°C overnight. Immunoprecipitated complexes were collected using 20 ul Protein G Dynabeads (Life Technologies) per reaction. After washing, the protein-DNA complexes were eluted and de-crosslinked overnight at 65°C. and DNA was purified with QIAquick PCR Purification Kit (Qiagen, # 28104).