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Ansari, S.A. ; Uhlenhaut, N.H.

An optimized high-resolution mapping method for glucocorticoid receptor-DNA binding in mouse primary macrophages.

In: Chromatin Immunoprecipitation. Berlin [u.a.]: Springer, 2024. 91-107 (Methods Mol. Biol. ; 2846)
DOI PMC
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.
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Publication type Article: Edited volume or book chapter
Corresponding Author
Keywords Bone Marrow–derived Macrophages ; Chip-exo ; Chip-nexus ; Exonuclease Digestion ; Glucocorticoid Receptor
ISSN (print) / ISBN 1064-3745
e-ISSN 1940-6029
Book Volume Title Chromatin Immunoprecipitation
Journal Methods in Molecular Biology
Quellenangaben Volume: 2846, Issue: , Pages: 91-107 Article Number: , Supplement: ,
Publisher Springer
Publishing Place Berlin [u.a.]
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Diabetes and Endocrinology (IDE)