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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 Demo Reports

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 & Demo Reports and contact us for customized requests.

Project Workflow

Sample Quality Control

Library Quality Control

Data Quality Control

Sample Preparation

Sample Preparation

Sample Preparation

Sample Preparation

E6 Protein Expressed by High-Risk HPV Activates Super-Enhancers of the EGFR and c-MET Oncogenes by Destabilizing the Histone Demethylase KDM5C

Background:

Cervical cancer is one of the most common gynecological tumors, which seriously threatens women’s health. Infection by the high-risk (HR) types of HPV, HPV-16 and HPV-18, is the major cause of anogenital carcinomas in women and men, as well as a fraction of head and neck cancer. As a noncanonical function, HR HPV E6 plays an important role in regulating certain oncogene expression, such as EGFR and c-MET. However, the molecular mechanisms underlying the upregulation of these two proto-oncogenes are unknown. An emerging role in proto-oncogene activation is the abnormal epigenetic modifications. Previous studies have shown that HR HPV E6 interacts with both histone methyltransferases and acetyltransferases. Moreover, the generation and activation of super-enhancers can be a persistent regulatory element that drives the uncontrolled proliferation in cancer cells. A number of super-enhancers have been identified in various types of cancer, but it has not been reported in cervical carcinomas. It was demonstrated that the presence of HPV16 E6 is sufficient to upregulate the EGFR and c-MET super-enhancers, further elevating the expression of these two protooncogenes.

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

Figure 1. Epigenetic adaptations in the anterior cingulate cortex following child abuse.

Figure 2. DNA methylation was measured in sorted populations of NeuN+ and Sox10+ nuclei, focusing on differentially methylated loci identified by RRBS.
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 (fig. 1).

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 (Fig. 2).

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.

ChIP-Seq and RIP-Seq FAQs

1.1 Sample requirements

1.1.1 Which species can you provide ChIP-seq or RIP-seq service?
We require the sample should be diploid species that have a chromosome-level assembled reference genome (NCBI) and completed gene annotation (GTF file).

1.1.2 What are the requirements for different types of samples to proceed Novogene ChIP-Seq and RIP-seq service?
ChIP-seq:
Sample Type Required Amount Volume Concentration Fragment size Purity
Enriched DNA Sample ≥50 ng ≥10 μL ≥2ng/μL 100 bp~500bp OD260/280=1.8-2.0
No degradation or contamination of RNA or protein
Through we require 50ng, the success rate to prepare the lib with 30ng is more than 90%. We also could do the lib prep with 5ng. The PCR cycle number will be increased for low amount library preparation, which may affect the data quality and analysis.
RIP-seq:
Sample Type Required Amount RIN
(before IP) Peaks Distribution Volume Concentration Purity
Enriched RNA Sample ≥100 ng ≥8 (Animal)
≥7 (Plant, Fungi) For unfragmented sample, the main peak should be higher that 1000bp. ≥20 μL ≥3 ng/μL OD260/280>2.0
No degradation or contamination of DNA or protein”

1.1.3 What is the relationship between Input and IPed sample?
Input and IPed sample are two different samples. A portion of the sample is taken as Input before immunoprecipitation. They are QC tested, library constructed and sequenced in parallel in the early stage of the project. However, in the analysis, the sequencing data of the two samples need to be integrated and analyzed to get the final peak results and carry out subsequent analysis.

1.1.4 What is role of the Input control?
Input sample can verify the effects of IP assay throughout the experiment. It is very important and necessary for ChIP-seq and RIP-seq experiments.
Input control and Mock (IgG) control are both negative controls, both of which could exclude the false positive result.

1.1.5 What methods can be used to send IPed DNA or RNA samples?
You have the following options for shipping samples:
a) The DNA can be lyophilized for shipping at room temperature.
b) Samples can be packed with ice packs/blue ice.
c) Best method: pack samples in dry ice, using sufficient dry ice to allow for the consumption (sublimation) of 5 kg per day, through FedEx or other carrier of choice.

1.1.6 Since gDNA samples are relative stable, can I ship the samples under room temperature?
Even though DNA is more stable compare to RNA, the quality still can be affected by surrounding environment. We strongly recommend our client to include sufficient dry ice in the package to allow for the consumption (sublimation) of 5 kg per day and ship the samples via FedEx second day delivery service.

1.1.7 Can I send the samples in 96-well plates or PCR stripe tubes?
96-well plates and PCR stripe tubes are NOT acceptable. Samples should be shipped in 1.5 ml or 2.0 ml Eppendorf tubes. Otherwise additional $5/sample will be charged as container transferring fee

1.1.8 What documents do I need to submit to Novogene before shipping out the samples?
Email the following documents/information to Novogene sales AND technical support PRIOR TO shipping:
i. Sample information form (both hard-print and electronic version, completed excel file).
ii. Purchas order (PDF copy, with billing information).
iii. Tracking number for the package.
iv. A copy of Gel Electrophoresis picture or Bioanalyzer trace (QC file), if available.
v. Any instructions on the samples, if any.

1.1.9 What other types of samples do you accept? Can I send tissues or body fluids for you to perform the DNA extractions?
We DON NOT provide IP assay service for ChIP-seq and RIP-seq project. If you have a need for nucleic acid extractions and immunoprecipitation, please contact your Novogene representative to discuss your specific needs.

1.1.10 How will Novogene perform sample QC on IPed DNA or RNA samples?
Novogene utilizes 2 major QC methods for DNA sample qualification:
i. Qubit® 2.0 flurometer quantitation for accurate measurement of DNA concentration;
ii. If the total of the sample is more than 20ng (the standard amount for a ChIP-Seq library preparation), agarose gel electrophoresis will be done to analyze the DNA purity, integrity and the size of the main band;
Novogene utilizes 3 major QC methods for RNA sample qualification:
i. Nanodrop for preliminary quantitation and purity (OD260/280) detection of RNA.
ii. Agilent 2100 for detection of the RNA integrity.
iii. If the total of the sample is more than 100ng, agarose gel electrophoresis will be done to analyze the RNA purity, integrity and the size of the main band;

1.2 Library Preparation

1.2.1 What is the library preparation kit that Novogene uses for ChIP-seq and RIP-seq?
DNA library prep kit: NEB Next® Ultra™ II DNA Library Prep Kit
RNA library prep kit: NEB Next® Ultra™ II RNA Library Prep Kit

1.2.2 What is the fragment size that gDNA samples will be sheared to?
If the mian band is higher than 1000bp, the IPed DNA of each sample will be randomly sheared into short fragments, around 250bp.
If the mian band is higher than 1000nt, the IPed RNA of each sample will be randomly sheared into short fragments, around 250nt.”

1.2.3 Can Novogene sequence with longer insert fragment libraries?
Yes, we have successfully construct libraries with up to 800 bp long.

1.3 Sequencing

1.3.1 What sequencing strategy do Novogene use for ChIP-Seq or RIP-seq services?
HiSeq OR NovaSeq platform, paired-end 150 bp.

1.3.2 Can Novogene sequence all my samples within the same lane on the flow cell?
For full-service projects, because we guarantee the data output and quality for each individual sample, we do not guarantee to pool the samples within the same lane.

1.4 Data Analysis

1.4.1 What kinds of analysis can Novogene provide for ChIP-Seq or RIP-seq?
For ChIP-seq service, our standard analysis package includes:
Standard Analysis
Data quality control (get rid of reads containing adapter or with low quality; Q20, Q30, error rate distribution, GC distribution, total bases)
Mapping onto reference genome (mapping rate, reads distribution)
Peak calling
Motif prediction
Peak annotation (downstream or overlapping gene, TSS)
Functional analysis of peak-associated genes (Gene Ontology, pathway)
Visualization of ChIP-seq data

For RIP-seq service, our standard analysis package includes:
Standard analysis
Data quality control (get rid of reads containing adapter or with low quality; Q20, Q30, error rate distribution, GC distribution, total bases)
Mapping onto reference genome (mapping rate, reads distribution, rRNA content)
Peak calling
Motif prediction
Peak annotation (downstream or overlapping gene, peak distribution in functional region of gene and transcript)
Functional analysis of peak-associated genes (Gene Ontology, pathway)
Visualization of RIP-seq data”

1.4.2 What is the data output per sample do you recommend for each analysis?
We recommend 12Gb/sample and 6G/sample is required for ChIP-seq.
We recommend 6Gb/sample and 3G/sample is required for RIP-seq.

1.4.3 Can Novogene provide any customized analysis based on client’s requirement?
The customized analysis needs to be estimated the feasibility by the BI team before the project initiate.

1.4.4 What files do Novogene also provide along with the analysis report?
We also provide FASQ, BAM, Annotation and VCF files with the report.

1.5 Others

1.5.1 What is the turnaround time of my project?
Please refer to Novogene TAT table to estimate the turnaround time based on number of samples and​​ data amount.


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