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

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Chromatin immunoprecipitation followed by exonuclease digestion (ChIP-exo) offers high sensitivity and single-base-pair resolution for mapping transcription factor (TF) binding sites.
• ChIP-nexus is an advanced, simplified version of ChIP-exo, incorporating unique barcodes and single ligation for efficient adapter ligation via DNA circularization.

Purpose of the Study:

• To present an optimized protocol for generating next-generation sequencing (NGS)-ready, high-quality ChIP-nexus libraries.
• To specifically adapt the ChIP-nexus method for analyzing glucocorticoid receptor (GR) binding in bone marrow-derived macrophage (BMDM) cells.

Main Methods:

• The protocol involves DNA-protein cross-linking in intact BMDM cells.
• Key steps include chromatin shearing, ChIP, adapter ligation, lambda exonuclease digestion, single-stranded DNA purification, and library amplification for NGS.

Main Results:

• The described method yields high-quality ChIP-nexus libraries suitable for NGS analysis.
• The protocol is optimized for robust GR binding site identification in BMDM cells.

Conclusions:

• This optimized ChIP-nexus protocol provides a reliable and efficient approach for studying TF binding dynamics.
• The method facilitates detailed investigation of glucocorticoid receptor function in macrophages at single-nucleotide resolution.