ChIP-Seq: technical considerations for obtaining high-quality data

Abstract

Chromatin immunoprecipitation followed by next-generation sequencing analysis (ChIP-Seq) is a powerful method with which to investigate the genome-wide distribution of chromatin-binding proteins and histone modifications in any genome with a known sequence. The application of this technique to a variety of developmental and differentiation systems has provided global views of the cis-regulatory elements, transcription factor function and epigenetic processes involved in the control of gene transcription. Here we describe several technical aspects of the ChIP-Seq assay that diminish bias and background noise and allow the consistent generation of high-quality data.

Figure 1. ChIP-seq experimental design

Chromatin immunoprecipitation followed by next-generation sequencing (NGS) is a powerful tool to investigate protein-DNA interactions on a global scale. Before performing ChIP-seq it is important to determine the appropriate controls for antibody specificity. After isolation of an ideal number of cells, chromatin is sheared into an ideal size range by sonication or enzymatic means (MNase). Next, ChIP is performed using high-quality antibodies to enrich for factor-occupied DNA sequences. A reference genome should also be included in this step to control for the ChIP experiment. Following purification of ChIP-enriched DNA, library construction is performed to allow for sequencing on next-generation sequencing platforms. Library construction typically includes end-repair, single A addition, adapter ligation, and PCR using primers compatible with the sequencing platform. Following cluster generation, single or paired-end sequencing is performed on NGS platforms.

Figure 2. Common procedures for ChIP-seq data analysis

After base-calling, short read sequences are aligned to a reference genome. Data quality is inspected by a combination of various strategies such as visual inspection using a genome browser, motif identification and Q-PCR validation. The initial inspection or prior knowledge provides information about whether the peaks are broad, sharp or both. Different algorithms have been developed to identify peaks from these three groups, of which several representatives are shown in the figure.