ChIPseq

Chromatin immunoprecipitation sequencing (ChIP-seq) is a method used to analyze protein-DNA interactions on a genome-wide scale. It combines chromatin immunoprecipitation with high-throughput sequencing to identify binding sites of DNA-associated proteins, such as transcription factors and histone modifications, across the genome. The technique has become a central tool in epigenetics and gene regulation research because it can map binding landscapes in diverse cell types and conditions.

The typical workflow includes crosslinking of proteins to DNA in cells (often with formaldehyde), fragmentation of chromatin by sonication or MNase digestion, immunoprecipitation using an antibody specific to the protein of interest, washing and recovery of the bound DNA, reversing crosslinks, and sequencing of the recovered DNA. Sequenced reads are aligned to a reference genome and enriched regions, or peaks, are identified by computational software (for example MACS2), usually using an input or IgG control to distinguish signal from background. The resulting peaks indicate candidate binding sites or modified histone domains.

Applications include mapping transcription factor binding sites, profiling histone modifications to define chromatin states, enhancers and promoters, and studying changes in regulatory landscapes across conditions, development, or disease. The data are the basis for integrating with RNA-seq to interpret regulatory consequences and for comparative epigenomics.

Limitations and considerations: antibody specificity, crosslinking efficiency, resolution limited by fragment size, potential biases from chromatin accessibility and sequencing depth, and the need for sufficient starting material. Newer variants such as ChIP-exo and ChIP-nexus provide higher resolution by using exonuclease treatment to narrow binding footprints; alternative methods like CUT&RUN and CUT&Tag offer different approaches with several advantages in sensitivity and background.