RNA-seq, ChIP-seq and Mnase-Seq analysis of CHD6-silenced prostate cancer cells C4-2

Purpose: Study of the role of CHD6 linking nucleosome ejection in castration-resistant prostate cancer(CRPC) Method: The expression of CHD6 or E2F1 was silenced by 2 shRNAs (3 replicates) targeted at CHD6 or E2F1 in C4-2 cells, scramble RNA as control. mRNA profiles and genome-wide chromatin-state maps were generated by deep sequencing. CHD6 and IgG ChIP was conducted in C4-2 cells. MNase before and after CHD6-silenced was conducted in C4-2 cells. Results: E2F1 and E2F1 downstream genes were significantly down regulated by CHD6 knockdown. The binding of CHD6 on chromatin is required for nucleosome eviction in transcriptional activation of oncogenic pathways. Overall design: Expression profiling by RNA-seq, profiling of CHD6 and E2F1 (wildtype and knockdown) from C4-2 cell lines. DNA was purified from chromatin immunoprecipitates for CHD6 or IgG using the phenol/chloroform extraction. IgG was used as a negative control for ChIP. Then, DNA was amplified by quantitative PCR and normalized to input. Micrococcal nuclease digestion was performed (Nuclei were incubated for 15 min and 60 min) followed by high-throughput sequencing (MNase-Seq) in both C4-2 control cells and CHD6 knockdown cells.