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ChIP-Seq: A Versatile Tool for Epigenomics

What Is ChIP-Seq?

ChIP-seq is one of the most important techniques for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes. It offers higher resolution, less noise and greater coverage than ChIP-chip. ChIP-seq combines next-generation sequencing (NGS) with Chromatin immunoprecipitation. ChIP-seq has been used as a versatile tool in epigenomics. On one hand, it can detect histone modifications; on the other, it can reveal the DNA-protein interactions in vivo. It is essential for deciphering gene regulatory networks, allowing a full understanding of transcriptional regulation.

The Contribution of ChIP-Seq to Epigenome mapping

1. Transcription factors binding maps
Decipher the gene regulatory networks that underlie various biological processes

2. Histone modification maps
Such as methylation, trimethylation, acetylation, etc.

3. Nucleosome maps
Reveal the role of nucleosomes in transcriptional regulation.

How Does ChIP-Seq Work?

In a ChIP experiment, the DNA-binding protein is cross-linked to DNA in vivo through formaldehyde treatment, and the chromatin is sheared into 200-600 bp fragments. Micrococcal nuclease (MNase) digestion without crosslinking is often used to fragment the chromatin since MNase treatment removes linker DNA more efficiently than sonication. However, MNase digestion is found to have a more pronounced sequence bias. An antibody specific to the protein of interest is used for immunoprecipitation. The quality of the antibody is very important to generate high-value ChIP or ChIP-seq data. Subsequently, the crosslinks are reversed and the DNA is released for NGS library preparation. The immunoprecipitated DNA is first subjected to size selection (commonly in the 150-300 bp range). Finally, ChIP-seq data are generated on the NGS platforms such as Illumina platforms. An increased sequencing depth enables the detection of more rare sites.

Non-Histone ChIP-seq and Histone ChIP-Seq

ChIP-seq can be divided into non-histone ChIP-seq and histone ChIP-seq depending on the antibody used in the ChIP step.

  Non-histone ChIP-seq Hiostone ChIP-seq
Antibody Specific to transcription factors such as p300 and Pol II, or other chromatin-associated proteins Specific to modified histones such as H3K4me3, H3K27me3, H3K4me1, H3K27ac, and H3K36me3
Applications
  • Screening of the specific DNA sites that interact with chromatin binding proteins
  • Tumor suppressor gene profiling
  • Screening of epigenetic writers, readers, erasers
  • Transcriptional repressor profiling
  • Histone modification mapping
  • Active promoter profiling
  • Inactive promoter profiling
  • Enhancer profiling
  • Active gene body profiling

Bioinformatics Pipeline for ChIP-Seq

Raw sequences need to be processed to interpret the ChIP-seq experiments.

(i) Filtering poor-quality reads

First, it is necessary to remove poor-quality reads and contaminants, and trim adapter sequences.

(ii) Align reads to the genome

Second, align reads to gene genome using BWA, Bowtie, GSNAP, or Wikipedia list of aligners.

(iii) Filter artifacts and reads aligned to multiple sites

Third, filter artifacts created in the PCR amplification step and reads aligned to multiple locations.

(iv) Peak calling

Tools including MACS, PeakSeq, and ZINBA can identify genomic regions of signal enrichment such as a bound protein, histone modification or open chromatin. Differential peak calling can be implemented using edgeR, DESeq, baySeq, or SAMSeq.

* For Research Use Only. Not for use in diagnostic procedures.
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