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What is ChIP?

Chromatin immunoprecipitation (ChIP) is a technique used to study the association of specific proteins, or their modified isoforms, with defined genomic regions. It is a fast growing research technique and is commonly used for mapping the DNA-protein interactions in cells which are crucial for correct gene regulation. For example, they may be used to determine whether proteins such as transcription factors and modified histones bind to a particular region of DNA of living cells or tissues.

How does ChIP-seq work?

In a ChIP assay, fragments of the DNA-protein complex (chromatin) are cross-linked to retain the specific DNA-protein interactions. The chromatin is then extracted and sheared either by sonication or enzymatic digestion into smaller fragments. The DNA-protein fragments are selectively immunoprecipitated using specific antibodies directed against your protein of interest and the resulting fractions treated to separate the DNA and protein components.

What is ChIP-seq analysis used for?

For epigenetics scientists looking to reduce background noise and bias, while maintaining the high quality of the data they generate, a ChIP-Seq assay is the perfect choice. The ChIP-seq technique allows researchers to examine the way protein interacts with DNA and nucleic acids by looking at the physical bonds they form. After identifying the DNA bonds across a genome, they can study proteins and transcription factors in more detail.

As mentioned above, combining ChIP with next-generation sequencing has provided important insights into the role gene regulation events play in the development of different biological pathways and various diseases, such as cancer growth and advancement.

In addition to its genome-wide DNA capture of various histone modifications and transcription factors for any organism, the ChIP-Seq technique has a number of advantages. These include the ability to reveal gene regulatory networks when used alongside RNA sequencing and methylation analysis, the accurate definition of binding sites transcription factors and being compatible with a range of DNA samples.

Overview of ChIP-seq methods

In the field of epigenetics, ChIP is established as an effective method for conducting genome-wide investigation of cellular DNA-protein interactions. The popularity of ChIP is largely due to the fact that it helps researchers better understand epigenetic mechanisms and the dynamics of chromatin.

Prior to ChIP-seq becoming the method of choice, ChIP-on-chip was the preferred approach to studying DNA-protein. Merging chromatin immunoprecipitation with DNA microarrays, this method showcases the position of the bonds between protein and DNA in great detail. However, since ChIP-seq was first introduced in 2007, it has been widely adopted as the preferred method for analysing proteins in DNA largely due to its capacity for genome-wide analysis.

Technological advancements have made it possible to combine ChIP assay with next generation sequencing platforms and map out an entire genome to visualise the exact protein binding sites. ChIP-seq’s ability to pinpoint a specific gene sequence that a protein binds to aids epigenetic researchers in making their study of protein-DNA interactions even more focused and provides valuable insights into the development of diseases.

Developmental and differentiation ChIP-seq applications have led to more comprehensive understanding of the epigenetic processes behind gene transcription control, the factor function of transcription and the various cis regulatory elements.

What is a ChIP-seq library?

A ChIP-seq library is usually constructed from ChIP DNA using specific ChIP-seq protocols, including size selection, gel purification, single A-addition, end-repair and adapter-ligation, as well as a PCR using ChIP-seq specific primers.

To avoid overamplification in a ChIP-seq library, researchers can either reduce the amount of template DNA they use for PCR or the number of PCR cycles. If there’s uncertainty about whether overamplification has transpired, we recommend comparing the sizes of the PCR product and the adapter ligated product. If the PCR products are overamplified, there would be an increase in their size in comparison to the adapter ligated products.

There’s two ways to create a ChIP-seq library. As the name suggests, single end sequencing will generate short sequence readings from a single end of the DNA template. Paired-end sequencing, on the other hand, generates short sequence reads from both template ends. Both methods will successfully generate a ChIP-seq library, but paired-end sequencing offers several advantages over the single end alternative. 

In addition to better detection of fragment sizes and amplified sequencing coverage, paired-end sequencing enhances the efficiency of alignment to repetitive regions by obtaining more sequence information from each DNA template.

Sequencing both ends of the DNA template can also allow researchers to map the genome of interest more accurately if they encounter partially overlapping ChIP-enriched DNA fragments or repetitive sequences. Single-end sequencing, meanwhile, may cause the loss of repetitive sequences over the course of the analysis.

Factors such as the number of genome target sites and its antibody affinity determine the number of sequencing reads necessary for sound genomic coverage. Evaluating the saturation point (how many reads it takes before additional sequencing is no longer able to identify new binding or enrichment sites) is an alternative, more quantitative approach to determining the required depth of sequencing.

How many cells are needed for ChIP-seq?

In order to determine the starting number of cells needed for a ChIP-Seq analysis, scientists should consider several factors, including how abundant the protein or histone modification they’re investigating is, and the quality of the antibody they’re using. In general, a greater number of cells will produce a higher signal-to-noise ratio since the two are directly correlated.

This is why we would recommend determining the minimum number of cells empirically if possible. Typically, ChIP-Seq assays using one to ten million cells will produce 10–100 ng of ChIP DNA. For abundant proteins, one million cells is sufficient, but if the ChIP-seq assay targets histone modifications or less abundant proteins, up to ten million cells may be needed for conclusive results.

Researchers studying rare cell types may benefit from alternative protocols which have been developed to use 10–100 fold less cells than standard ChIP-seq protocols for genome-wide profiling of histone modifications distribution.

ChIP-seq kit advantages and cost

The Chromatrap ChIP-seq kits offer a new and improve method of performing ChIP which helps reduce hands on time and laborious manual handling steps while providing the highest signal-to-noise ratio compared to conventional kits. It is the only alternative kit on the market that enables researchers perform ChIP completely bead-free. What this means is that instead of beads in suspension driving the immunoprecipitation, we use a stationary, inert polymer filter to carry out the method. Our ChIP-seq kits are ideal for scientists new to ChIP or those struggling with current methods.

Some of the numerous advantages of a bead-free kit includes a user friendly experience due to a huge reduction of manual handling steps involved, significantly reduced sample loss between steps and no background, due to our bead-free inert filter technology. What’s more, our ChIP-seq kits can complete a ChIP assay in under 5 hours.

Starting from £450, Chromatrap offers a range of faster, easier and more sensitive kits for ChIP. Browse our full range of ChIP-seq kits above.