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

研究目的：探究CHD6在去势抵抗性前列腺癌（castration-resistant prostate cancer, CRPC）中关联核小体驱逐的作用。
研究方法：在C4-2细胞中，使用2条靶向CHD6或E2F1的短发夹RNA（short hairpin RNA, shRNA）分别沉默CHD6与E2F1的表达，设置3次生物学重复，以scramble RNA作为阴性对照。通过深度测序获取mRNA表达谱与全基因组染色质状态图谱；在C4-2细胞中开展CHD6与免疫球蛋白G（immunoglobulin G, IgG）的染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验；在CHD6沉默及未沉默的C4-2细胞中进行微球菌核酸酶（micrococcal nuclease, MNase）处理实验。
研究结果：CHD6敲低后，E2F1及其下游靶基因的表达显著下调；CHD6在染色质上的结合对于致癌通路转录激活过程中的核小体驱逐至关重要。
整体实验设计：通过RNA测序开展表达谱分析，获取C4-2细胞系中野生型及CHD6、E2F1敲低组的表达数据；使用酚氯仿抽提法从CHD6或IgG的染色质免疫沉淀产物中纯化DNA，以IgG作为ChIP实验的阴性对照；随后通过定量PCR对DNA进行扩增，并以input样本作为归一化参照。分别对对照组与CHD6敲低组的C4-2细胞进行微球菌核酸酶消化（细胞核分别孵育15 min与60 min），随后进行高通量测序（MNase-Seq）。