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ChIP 2.0: Guide to advanced chromatin immunoprecipitation techniques

This guide outlines the latest advanced ChIP-based techniques, and describes how their application has enabled new understanding of epigenetic mechanisms.

Chromatin immunoprecipitation (ChIP) is the basis for much of modern chromatin analysis technologies. The central principle of ChIP is the use of an antibody targeting a protein or histone modification of interest to pull down DNA regions with which it associates. The adaptability and versatility of ChIP allow it to be applied to diverse research questions. This versatility is twofold: diversity of antibody choice and diversity of technology (qPCR, microarray, sequencing etc.) (Collas, 2010).

Coupling of ChIP to microarray (ChIP-chip) has been pivotal to our understanding of the human genome (Kim et al., 2005). ChIP-seq has been instrumental for the ENCODE project which has mapped much more of the human genome (Landt et al., 2012). Furthermore, innovation of data analysis techniques can allow reanalysis or integration of data sets (Klein et al., 2014). This allows for new and often more reliable information to be uncovered.

Traditional ChIP has acted as a springboard to develop novel adaptations, which often combine ChIP with other techniques to address diverse biological problems. Restriction digestion of cross-linked chromatin allows for ligation of interacting fragments. This can be used to determine the three-dimensional organization of the genome. ChIP can also be combined with epigenetic technologies such as bisulfite conversion to interrogate the interplay between histone modification and DNA methylation.

Find out more about the latest ChIP-based techniques using the links below.


ChIP-loop detects DNA-DNA interactions mediated by a specific protein of interest. This is achieved by adding an immunoprecipitation (IP) step to a relatively standard Chromatin Conformation Capture (3C) protocol:

  • Cells are formaldehyde cross-linked followed by chromatin isolation and restriction digestion.
  • Cross-linked chromatin is immunoprecipitated using an antibody against a protein of interest.
  • The sticky DNA fragment ends are ligated while the chromatin fragments are still bound to the antibody,
  • The cross-links are reversed and the DNA is purified. A specific DNA-DNA interaction of interest can then be detected using PCR,

The result is that predicted DNA-DNA interactions that are mediated by a protein of interest are confirmed.

The primary advantage of ChIP-loop over ChIP or 3C alone is specificity. For one, a reduction in background noise over a simple 3C assay is achieved by removing a large amount of genomic DNA by IP. Further, by targeting analysis to a specific protein of interest only specific, biologically relevant interactions are detected. 

As a drawback, some argue that ChIP-loop may not be fully informative on its own, and that ChIP-loop should be cross-referenced against ChIP data. For example, if ChIP-loop interacting DNAs are not both enriched in a ChIP experiment, then the interaction should not be considered valid (Simonis et al., 2007).

ChIP and 3C were first combined in 2005 to study a mouse model of Rhett Syndrome (Horike et al., 2005). These researchers found that formation of a silent-chromatin loop is important for the function of the Dlx50Dlx6 locus. Further, they found that this loop was mediated by MECP2 and that this interaction is lost in individuals with Rhett Syndrome.

ChIP-loop is ideally suited to examine DNA loops mediated by transcription factor is a master regulator of chromatin-loop structure at the Kcnq5 locus.

ChIP-loop tips and tricks

Minimize ligation bias by having sufficient starting material. By performing the ligation when the DNA is concentrated in a small volume, undesirable cross-ligation can occur between chromatin fragments.

Validate with ChIP alone if possible. As stated above, cross-referencing ChIP-loop with ChIP data greatly reduces false-positives in the ChIP-loop assay.

Use appropriate controls.

  • Make a control template that contains ligation products in equal amounts. 
  • Determine interaction frequency between known loci of increasing distance to estimate random interaction.
  • When comparing two conditions, determine interaction frequency for a region expected to be the same in the two states (similar to how Gapdh or ActB is used for gene expression analysis) (Dekker, 2006).


Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) is a hybrid of ChIP and 3C techniques. ChIA-PEET is a high-throughput version of ChIP-loop that is capable of detecting long-range chromatin interactions via a protein of interest across the genome. The first steps in the protocol are very similar to a standard ChIP experiment.

  • Cells are cross-linked using formaldehyde, linking integrating DNA regions and proteins. 
  • The chromatin is digested with a restriction enzyme (or sonicated, see tips and tricks) and pulled down with an antibody against a protein of interest.
  • During ligation, linker sequences are added that introduce type I restriction enzyme recognition sites into the DNA (Zhang et al., 2012).
  • These sites allow the subsequent isolation of ligation junctions by paired-end sequencing. This creats a library of only the hybrid, interacting DNA fragments.
  • The library is sequenced on a next-generation platform to both identify interacting regions, but also to quantify their frequency of interaction.

ChIA-PET provides researchers with genome-wide interactions involving a protein of interest.

ChIA-PET has many advantages over similar techniques. The use of ChIP reduces the amount of DNA to be sequenced, reducing complexity and increasing the specificity of the sequencing reaction. ChIA-PET works with all the tag-based next generation platforms (Roche 454 pyrosequencing, SOLiD, Helicos etc.). ChIA-PET can be used to interrogate the interactions of any protein, given a suitable antibody.

Like any ChIP technique, ChIA-PET is limited by the specificity of the antibody. It is also limited to known genomes, since the calls must be mapped back to a reference genome. Finally, while ChIA-PET can identify if a protein is present in an interacting complex, it cannot determine if that protein actually mediates the interaction. For example, the protein may bind two other proteins that themselves bind the DNA.

ChIA-PET workflow. Adapted from: Zhang et al., Nature (Nov 2013).

ChIA-PET was introduced in a 2009 paper by (Fullwood et al., 2009). These researchers developed ChIA-PET to map the binding sites of oestrogen receptor alpha (ER-alpha) in human cells. They found that ER-alpha mediates the formation of long-range looping at gene promoters, bringing genes together for coordinated transcription. 

ChIA-PET has also been used to characterize the binding dynamics of other transcription factors such as CTCF (Li et al., 2013). Li et al. found that CTCF may have a role in organizing recently identified topological domains of chromatin. Chen et al. (2013) used ChIA-PET to examine miRNA regulation, and found that miRNA expression is coordinated in large chromatin domains.

​​​ChIA-PET tips and tricks

Use sufficient starting material. The ChIP step is critical for generation of the ChIA-PET library. At least 100 ng of ChIP DNA is needed to generate a sufficiently complex ChIA-PET library. If using cells from culture, use at least 108 cells to obtain 100-300 ng of ChIP DNA (IP will need to be optimized for cell type and protein of interest). 

Sonication or restriction enzyme digest: choose carefully. Sonication gets around the sequence bias and limitations of using a restriction enzyme to shorten protein-cross-linked chromatin. It also improves resolution; however, it is rather harsh, and can cause the loss of long-range interactions.

If long-range interactions are vital to your experiment, consider restriction digest. If sequence bias and resolution are issues, consider sonication.

Analysis is tricky, adapt another ChIP program, or use a previously developed software. The same group that developed ChIA-PET provide the software they used for analysis (Li et al., 2010).


​Very slight modifications to traditional ChIP allow ChIP-exo to detect protein binding at single-nucleotide resolution. ChIP-exo uses restriction to digest and next-generation sequencing to determine genome-wide protein binding with high resolution.

  • Cells are first formaldehyde cross-linked and the chromatin is sheared and isolated.
  • After IP with an antibody against the DNA binding protein of interest, adapters are ligated to the DNA fragments.
  • The DNA is then digested with lambda 5'→3' exonuclease. The exonuclease removes all the 5' DNA tails protruding from protein-DNA complexes, leaving only the DNA bound to the protein and the 3' tails (Rhee and Pugh, 2012a).
  • The cross-links are reversed and the DNA is amplified, then another adaptor is ligated to the 5' ends.
  • Following next-generation sequencing, sequences adjacent to 2nd adaptors can be identified. These sequences are the binding sites of the protein of interest.

ChIP-exo has many benefits over traditional ChIP. Most obviously, the resolution of ChIP-exo is greater than almost all other ChIP-based techniques. The single-nucleotide resolution achieved is robust across many diverse DNA-binding proteins (Rhee and Pugh, 2011; Rhee and Pugh, 2012a). Most importantly, the high degree of both experimental resolution and scale does not come with the drawbacks of comparable techniques, specifically noise associated with genome-wide approaches.

ChIP-exo workflow. Adapted from: Rhee and Pugh, Current Protocols in Molecular Biology (Oct 2012).

​​Noise is greatly reduced by the DNA digestion step: all non-protein bound DNA is digested by the lambda exonuclease. An additional single-strand specific exonuclease (such as RecJ) can also be used to digest background DNA more efficiently. The removal of this DNA greatly reduces the noise, and therefore the sequencing depth required, reducing cost. 

Another major advantage is sensitivity. ChIP-exo can detect weaker DNA-protein interactions that other techniques, allowing more biologically relevant interactions to be discovered (Rhee and Pugh, 2011).

The only substantial disadvantage of ChIP-exo is related to multiple binding events by a single protein. If a protein binds several DNA loci simultaneously creating a ring or other 3D structure, such structures cannot be detected; any and all multiple-binding events are lost. ChIP-exo will simply read these events are single-bindings. This may be a problem depending on the protein, since the significance of complex ring structures in transcription is becoming apparent (Gibcus and Dekker, 2013).

ChIP-exo was presented in a 2011 Cell paper (Rhee and Pugh, 2011). These researchers examined the binding patterns of the yeast transcription factors Reb1, Gal4, Phd1, Rap1 and human CTCF. They found that each yeast transcription factor had multiple binding patterns and mechanisms. For example, they found that many low-affinity sites had high occupancy. This suggests that sequence alone cannot predict factor binding. This is explained in part by the finding that clustered sites bind more factors than lone sites, suggesting that a combination of effects including high concentrations and direct and indirect cooperation affect transcription factor binding. In a follow-up paper, these researchers also use ChIP-exo to identify TATA-box-like features in promoters that were thought to be TATA-less (Rhee and Pugh, 2012b).

ChIP-exo tips and tricks

Assess if resolution is paramount before starting. ChIP-exo is superior to ChIP-seq in almost all metrics, but it is labor and cost intensive. If the extra resolution and better signal-to-noise ratios are not vital, consider ChIP-seq for your experiment.

Consider use of magnetic beads. Serandour et al. (see additional reading) adapted the Rhee protocol to include the use of magnetic beads. Magnetic beads are believed to better pull down weak interactions and large protein complexes than agarose.

ChIP-BS-seq and BisChIP-seq

ChIP-BS-seq and BisChIP-seq are two nearly identical techniques that combine ChIP with bisulfite sequencing. 

Bisulfite conversion is perhaps the most informative DNA methylation technology available. Sodium bisulfite chemically converts unmodified cytosine to uracil while leaving methylated cytosine intact (Frommer et al., 1992). Bisulfite-seq combines bisulfite treatment and next-generation sequencing platforms, providing single-nucleotide methylation resolution on a genome-wide scale. 

In combination with ChIP, 5mC information can be targeted to regions bearing a specific histone modification or chromatin-binding protein of interest: 

  • The first step in ChIP-BS-seq and BisChIP-seq is ChIP against a protein or histone modification of interest. 
  • The sample is then size-selected and adapter-ligated. Small differences in the order and type of ligations and size selection differentiation one technique from the other.
  • The library is bisulfite treated and sequenced on a next-generation platform.
  • After comparison to a reference sequence using bioinformatic software, single-nucleotide methylation information of regions that contain the histone modification of interest are obtained (Brinkman et al., 2012; Statham et al., 2012). 

It is becoming increasingly clear that co-occurence of epigenetic marks is vital to their function. Various marks act in concert to modulate gene expression, and often co-regulate one another (Ernst et al., 2011). As such, examining two or more epigenetic marks in the same biological samples can give enhanced insight into the research problem. This is the key advantage of ChIP-BS-seq and BisChIP-seq.

Another advantage of these two techniques over traditional bisulfite-seq is the reduction in DNA used for bisulfite sequencing. This means that fewer reads are necessary in sequencing, dramatically reducing costs. However, this does mean that a good deal of input sample is needed, so ChIP-BS-seq/BisChIP-seq is not well suited to low-cell count applications.

An additional drawback of these techniques is that like all antibody-based techniques, the specificity of the experiment is determined by the quality of the antibody.

ChIP-BS-seq and BisChIP-seq were independently developed by two groups and published in the same issue of Genome Research (Brinkman et al., 2012; Statham et al., 2012). Brinkman et al. used ChIP-BS-seq to look at histone H3 lysine 27 trimethylation (H3K27me3) and DNA methylation. They found that H3K27me3 and DNA methylation are mutually exclusive at CpG islands. Statham et al. used BisChIP-seq to examine DNA methylation and H3K27me3 in normal and cancerous prostate cells. Surprisingly, they found that unmethylated and methylated DNA can be associated with H3K27me3 regions. This imples that DNA methylation status is not depended on the presence of the repressive histone mark, a widely held assumption.

​​ChIP-BS-seq and BisChIP-seq tips and tricks

Data analysis can be tricky. Since these techniques are relatively new no software has been specifically designed for ChIP-BS-seq/BisChIP-seq data. Statham et al. released the custom pipeline they created available here

Adapt reference sequence to allow read mapping. Replace all the cytosines in the reference sequence with thymidines to allow the converted bisulfite sequence to be mapped.

Additional reading

  • Collas P (2010). The current state of chromatin immunoprecipitation. Mol Biotechnol, 45, 87-100.

    This review describes the basic principles of ChIP as well as many of its derivative technologies, focusing on how each varies from the basic protocol.
  • Furey TS (2012). ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions. Nat Rev Genet, 13, 840-852.

    This review is focused on the principles of ChIP-seq, but is also a good resource for other related techniques. The author describes how analysis and data output differ for each technique.
  • Splinter E, de Wit E, van de Werken HJ, Klous P and de Laat W (2012). Determining long-range chromatin interactions for selected genomic sites using 4C-seq technology: from fixation to computation. Methods, 58, 221-230.

    ​This text book chapter focuses on many 3C technologies and includes a section on the basis and application of ChIP-loop.
  • de Wit E and de Laat W (2012). A decade of 3C technologies: insights into nuclear organization. Genes Dev, 26, 11-24.

    Sajan SA and Hawkins RD (2012). Methods for identifying higher-order chromatin structure. Annu Rev Genomics Hum Genet, 13, 59-82.

    These reviews cover various ChIP and 3C technologies including ChIP-loop and ChIA-PET. They also examine the strengths and weaknesses of each, and how the techniques fits into the larger picture of chromatin analysis.
  • Serandour AA, Brown GD, Cohen JD and Carroll JS (2013). Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties. Genome Biol​, 14, R147.

    This paper describes the adaptation of ChIP-exo to be used on the common Illumina sequencing platform. This adaptation outperformed ChIP-seq on Illumina in all metrics, and is specifically designed for human experiments.
  • Gao F, Ji G, Gao Z, Han X, Ye M, Yuan Z, Luo H, Huang X, Natarajan K, Wang J, Yang H and Zhang X (2014). Direct ChIP-bisulfite sequencing reveals the role of H3K27me3 mediating aberrant hypermethylation of promoter CpG islands in cancer cells. Genomics​, 103, 204-210.

    This primary paper uses ChIP-BS-seq to explore the roles of H3K27me3 and H3K4me3 in regulating DNA methylation at CpG islands in TCGA primary cancer cells.


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  • Chen D, Fu LY, Zhang Z, Li G, Zhang H, Jiang L, Harrison AP, Shananan HP, Klukas C, Zhang HY, Ruan Y, Chen LL and Chen M (2014). Dissecting the chromatin interactome of microRNA genes. Nucleic Acids Res, 42, 3028-3043.
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  • Collas P (2010). The current state of chromatin immunoprecipitation. Mol Biotechnol​, 45, 87-100.
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