An Optimized High-Resolution Mapping Method for Glucocorticoid Receptor-DNA Binding in Mouse Primary Macrophages

Methods Mol Biol. 2024:2846:91-107. doi: 10.1007/978-1-0716-4071-5_6.

Abstract

ChIP-exo is a powerful tool for achieving enhanced sensitivity and single-base-pair resolution of transcription factor (TF) binding, which utilizes a combination of chromatin immunoprecipitation (ChIP) and lambda exonuclease digestion (exo) followed by high-throughput sequencing. ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode, and single ligation) is an updated and simplified version of the original ChIP-exo method, which has reported an efficient adapter ligation through the DNA circularization step. Building upon an established method, we present a protocol for generating NGS (next-generation sequencing) ready and high-quality ChIP-nexus library for glucocorticoid receptor (GR). This method is specifically optimized for bone marrow-derived macrophage (BMDM) cells. The protocol is initiated by the formation of DNA-protein cross-links in intact cells. This is followed by chromatin shearing, chromatin immunoprecipitation, ligation of sequencing adapters, digestion of adapter-ligated DNA using lambda exonuclease, and purification of single-stranded DNA for circularization and library amplification.

Keywords: Bone marrow–derived macrophages; ChIP-exo; ChIP-nexus; Exonuclease digestion; Glucocorticoid receptor.

MeSH terms

  • Animals
  • Binding Sites
  • Chromatin Immunoprecipitation* / methods
  • DNA* / genetics
  • DNA* / metabolism
  • High-Throughput Nucleotide Sequencing* / methods
  • Macrophages* / metabolism
  • Mice
  • Protein Binding
  • Receptors, Glucocorticoid* / genetics
  • Receptors, Glucocorticoid* / metabolism

Substances

  • Receptors, Glucocorticoid
  • DNA