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    ChIP-Seq

    Overview

    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 industry standard MACS2 software and latest programs for motif prediction, peak annotation, functional analysis and data visualization.
  • Professional bioinformatics: A bioinformatics analysis team composed entirely of Ph.D. scientists 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.


    Figure 1 Distribution of MAPQ


    Figure 2 Plots of strand cross correlation


    Figure 3 Distance distribution of peaks to TSS


    Figure 4 Motif analysis


    Figure 5 Peak distribution in functional gene region


    Figure 6 GO enrichment


    Figure 7 KEGG enrichment scatter