GSM7934064: MycWT_ChIP; Homo sapiens; ChIP-Seq
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). The ChIP-seq DNA library construction was performed using Illumina's ChIP-seq Sample prep kit or ABclonal's the Rapid DNA Lib Prep Kit (ABclonal, #RK20200). Library was usually amplified by 14 cycles of qPCR. Constructed libraries were further double size selected between 150-600 bp with AMPure XP (Beckman, #A63881) and sequenced on NovaSeq 6000 with 100 bp paired-end reads at the UCSD IGM Genomics Center.