Introduction to ChIP-Seq
ChIP-sequencing (also known as ChIP-seq), which combines chromatin immunoprecipitation (ChIP) assays with DNA sequencing, is a powerful technique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes. ChIP is a type of immunoprecipitation (IP) experimental method used to isolate specific DNA sites in direct physical interaction with transcription factors and other proteins. In ChIP, specific antibodies are used to enrich DNA fragments bound by particular proteins or nucleosomes.
ChIP-seq was one of the early applications of NGS (next-generation sequencing), and the first study of large-scale profiling of the genome-wide histone methylations using ChIP-seq was published in 2007 (Barski et al., 2007). The sequencing of this study was performed on the platform of Solexa 1G genome analyzer. In the same year, Johnson et al. (2007) used ChIP-seq to generate the genome-wide mapping of transcription factor binding sites. Robertson et al. (2007) developed ChIP-seq to identify mammalian DNA sequences bound by transcription factors in vivo. These two papers also demonstrated the increased sensitivity and specificity of ChIP-seq. Owing to the rapid progress of NGS technology and the decreasing cost of sequencing, ChIP-seq has become an indispensable tool for characterization of epigenomes and gene regulation study (Park, 2009).
Comparison of ChIP-chip and ChIP-seq
ChIP-chip, ChIP coupled with microarrays, and ChIP-seq are two standard techniques for identification of the genome wide DNA-proteins binding interactions. Taking advantage of sequencing technologies, ChIP-seq offers many advantages over ChIP-chip, as summarized in Table 1 (Park, 2009; Schones and Zhao, 2008).
Table 1. Comparison of ChIP-chip and ChIP-seq.
|Array-specific, generally 30-100 bp
|Limited by sequences on the array; repetitive regions are usually masked out
|Limited only by alignability of reads to the genome; increases with read length; many repetitive regions can be covered
|Dependent on available products; multiple arrays may be needed for large genomes
|Genome-wide assay of any sequenced organism
|Source of platform noise
|Cross-hybridization between probes and nonspecific targets
|Some GC bias can be present
|Single- or double-channel, depending on the platform
|Profiling of selected regions; when a large fraction of the genome is enriched for the modification or protein of interest (broad binding)
|Large genomes; when a small fraction of the genome is enriched for the modification or protein of interest (sharp binding)
|Required amount of ChIP DNA
|High (a few micrograms)
|Low (10-50 ng)
|Lower detection limit; saturation at high signal
|Less required; single-molecule sequencing without amplification is available
Workflow of ChIP-seq
The workflow of ChIP-seq used to profile the specific DNA binding sites for transcription factors, DNA-binding enzymes or other DNA-associated proteins (non-histone ChIP) and DNA sites correspond to modified nucleosomes (histone ChIP) is illustrated in Figure 1 (Park, 2009). Following ChIP protocols, the chromatin is fragmented and crosslinked proteins or modified nucleosomes immunoprecipitated using an antibody specific to the protein or the histone modification. After DNA purification and library construction, DNA fragments can be sequenced simultaneously on any of the sequencing platforms, such as Illumina Solexa Genome Analyzer, Roche 454 and Applied Biosystems (ABI) SOLiD platforms, and HeliScope by Helicos, as illustrated in Figure 1. With the tremendous progress of NGS technology, the Illumina platform, such as Hiseq, has been the most widely used platform for sequencing.
Figure 1. Overview of a ChIP-seq experiment (Park, 2009).
Experimental Design of ChIP-seq
The Encyclopedia of DNA Elements (ENCODE) and model organism ENCODE (modENCODE) consortia have developed a set of working standards and guidelines for ChIP-seq experiments based on experience of hundreds of ChIP-seq experiments (Landt et al., 2012). To obtain high-quality ChIP-seq data, there are several technical aspects should be considered in the ChIP-seq experimental design, including antibodies, cell number, controls, replicates, chromatin fragmentation, library construction and sequencing (Kidder et al., 2011).
Considering the above technical aspects, a ChIP-seq experimental design that would obtain high-quality data is illustrated in Figure 2 (Kidder et al., 2011). At first, the appropriate controls for antibody specificity should be determined before ChIP. Chromatin is sheared into an ideal size range by sonication or enzymatic means after isolation of the ideal number of cells. Next, high-quality antibodies are used for ChIP. After purification of ChIP-enriched DNA, a library is constructed to allow sequencing on NGS platforms.
Figure 2. ChIP-seq experimental design (Kidder et al., 2011).
At CD Genomics, we provide you with high-quality sequencing and integrated bioinformatics analysis for your ChIP-Seq project, enabling accurately screen and determine the protein binding sites in the whole genome. If you have additional requirements or questions, please feel free to contact us.