ChIP-Seq Library Prep

Properly generating robust, high-quality libraries for ChIP-seq will prepare your samples for Illumina sequencing and an overall successful ChIP-seq experiment. It is often challenging to prepare DNA for a next-gen ChIP-seq library because it’s difficult to obtain a large amount of DNA after ChIP; this is especially true when a researcher is using antibodies against transcription factors that are not highly abundant. After the chromatin is immunoprecipitated with a specific antibody, a typical ChIP-seq library preparation calls for fragmentation, repairing DNA fragment ends, dA-tailing, ligating adaptors onto DNA fragments, and enriching/amplifying with PCR.

chromatin immunoprecipitation sequencing workflow for ChIP-seq
ChIP-seq library preparation workflow.
For investigating DNA binding proteins, the ideal method for uniform and precise fragmentation of DNA is shearing via sonication, but it can also be done enzymatically or chemically. Alternatively, when investigating histone modifications, researchers can use micrococcal nuclease for fragmentation. For dA-tailing, the 3’ ends are typically A-tailed using Taq polymerase. If heating is not desired, which may be the case when preparing certain libraries, Klenow Fragment (exo-) may be used, although this is not as efficient as Taq for A-tailing. During the adaptor ligation step of preparing the library, keep in mind that too much adaptor may create adaptor dimers which can present some challenges in the following PCR amplification step. After size-selection, the sequences are then analyzed against a reference genome which maps the specific binding locations of the protein of interest or histone modifications.

Numerous commercial kits exist for preparing an adaptor ligated NGS library for ChIP-seq and other downstream applications. Some of these kits can make a complex library from as little as 0.2 ng, but because less starting material means more amplification, library complexity is typically better with larger amounts of starting material. For magnetic bead separation of DNA for DNA library prep, size selection, and cleanup, there are several magnetic bead technologies commercially available, including the EpiNext DNA Purification HT System. This enables high throughput DNA or PCR amplicon purification and DNA size selection.

Keeping these tips in mind will help you to prepare a high-quality ChIP-seq library for NGS. Although sequencing machines and various technical steps of the ChIP-seq procedure may lead to biases, researchers can take appropriate measures during ChIP, library preparation, and bioinformatics to combat these biases. For more info, take a look at these helpful ChIP-seq resources or read the following articles on NGS library construction and sequencing.

Helpful Articles

Library construction for next-generation sequencing: Overviews and challenges

  • This paper from Head et al. discusses the importance of library quality in light of advances in next-generation or high-throughput sequencing capabilities. This is a useful guideline that lists challenges and tips researchers should consider when preparing NGS libraries from DNA and other sources.

Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

  • These researchers evaluate sources of bias during Illumina library preparation, especially PCR amplification bias, and find ways to ameliorate the effects.