ChIP-Seq

概要
デモ結果

概要

Chromatin Immunoprecipitation Sequencing (ChIP-Seq) provides genome-wide profiling of DNA targets for histone modification, transcription factors, and other DNA-associated proteins. It combines the selectivity of chromatin immuno-precipitation (ChIP) to recover specific protein-DNA complexes, with the power of next-generation sequencing (NGS) for high-throughput sequencing of the recovered DNA. Additionally, because the protein-DNA complexes are recovered from living cells, binding sites can be compared in different cell types and tissues, or under different conditions. At Novogene, we provide high-quality sequencing and comprehensive bioinformatics solutions for your ChIP-Seq projects.

Service Specifications

Applications

Applications range from transcriptional regulation to developmental pathways to disease mechanisms and beyond.

Advantages

Cost-effective: Rapid and efficient genome-wide profiling of multiple samples, using only 1/100 of the amount of DNA required for ChIP-chip.
Unsurpassed data quality: We guarantee that ≥ 80% of bases have a sequencing quality score ≥ Q30, exceeding Illumina’s official guarantee of ≥ 75%.
Comprehensive analysis: Expert bioinformatics analyses utilizing widely accepted MACS2 software and latest programs for motif prediction, peak annotation, functional analysisand data visualization.
Professional bioinformatics:A bioinformatics analysis team composed of Ph.D. scientists entirely for Chip-Seq data analysis.

Sample Requirements

Sample Type	Required Amount	Fragment size	Purity
Enriched DNA Sample	≥50 ng (Concentration ≥2ng/μL)	100 bp~500bp	OD260/280=1.8-2.0

Sequencing Parameter and Analysis

Platform	Illumina Novaseq 6000
Read length	Pair-end 150
Recommended Sequencing Depth	≥ 20 million read pairs per sample for the species with reference genome
Standard Data Analysis	Data quality control Mapping onto reference genome Peak calling Motif prediction Peak annotation and functional analysis of peak-associated genes Summary of differential peaks and functional analysis of differential peak related genes Visualization of ChIP-seq data

Note: For detailed information, please refer to the Service Specifications and contact us for customized requests.

Project Workflow

Sampling & Sequencing Strategy:

2.1. Samples：
CaSki-pHAGE cells, CaSki-KDM5C cells

2.2. Library preparation:
ChIP-seq library and RNA-seq library

2.3. Sequencing:
Illumina HiSeq

2.4. Bioinformatics analysis:
ChIP-seq standard analysis and gene expression analysis

Results:

1) Whole genome ChIP-seq revealed existence of the EGFR and c-MET super-enhancers in the human cervical cancer cell line
The existence of possible super-enhancers was screened by ChIP-seq analysis of H3K27Ac in the CaSki cells, as well as the CaSki-vector control and CaSki-KDM5C cells. It was found that there the two protooncogenes, EGFR and c-MET, each contained a super-enhancer. The EGFR super-enhancer is located in the first intron while the c-MET super-enhancer resides within the c-MET gene 50 region through intron 2 (Figure 1).

Figure 1. KDM5C regulates super-enhancers activity and gene expression of the EGFR and c-MET.

2) KDM5C regulates cervical cancer cell EGFR and c-MET expression by modulating their super-enhancer H3K4 methylation dynamics
ChIP-qPCR of KDM5C was performed and displayed a direct correlation between the restoration of KDM5C and its enrichment. KDM5C restoration led to specific increased H3K4me1 in global super-enhancers rather than in adjacent regions, confirming KDM5C as a specific enhancer regulator (Figure 2).

Figure 2. KDM5C were enriched at super-enhancer or other regions of EGFR and c-MET.

Conclusion:

This study generated a carcinogenic model of HPV infection: HPV16 E6 binds to KDM5C and form an E6‒E6AP‒KDM5C complex, thereby degrading KDM5C in a polyubiquitin-dependent manner. As a result, the super-enhancers of key protooncogenes, EGFR and c-MET, become highly upregulated, increasing their expressions and promoting tumor cell growth. This finding has provided novel insights into virus-induced cancer.

Reference: Chen X, Loo J X, Shi X, et al. E6 Protein Expressed by High-Risk HPV Activates Super-Enhancers of the EGFR and c-MET Oncogenes by Destabilizing the Histone Demethylase KDM5C[J]. Cancer Research, 2018, 78(6): 1418-1430.