Optimized Genome-Wide Profiling of Centromeric Chromatin Complexes by ChIP-seq

Centromeres are specialized chromosomal regions essential for faithful chromosome segregation and genome stability. Their highly repetitive DNA composition and epigenetically defined chromatin state present significant challenges for genome-wide profiling approaches such as chromatin immunoprecipitation followed by sequencing (ChIP-seq). Standard ChIP-seq workflows often perform poorly at centromeric regions, resulting in low signal-to-noise ratios and limited reproducibility. This article describes a ChIP-seq protocol optimized to enrich for and analyze centromeric chromatin from cell lines. The procedure builds on established ChIP-seq methodologies while incorporating centromere-focused modifications in nuclei isolation, chromatin fragmentation, immunoprecipitation, library preparation, and data analysis. Detailed step-by-step instructions, critical parameters, recommended controls, and troubleshooting guidance are provided to support reproducible implementation. When applied carefully, this protocol enables reliable profiling of centromere-associated chromatin and can be integrated into broader studies of chromosome organization, centromere function, and genome stability. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sucrose gradient–based isolation of nuclei. Support Protocol 1: Titration of micrococcal nuclease (MNase). Basic Protocol 2: MNase digestion and immunoprecipitation of CENP-A chromatin. Alternate Protocol 1: Chromatin sonication and immunoprecipitation of CENP-C chromatin complexes. Support Protocol 2: NET blocking and washing of Protein G Sepharose beads. Basic Protocol 3: DNA extraction. Alternate Protocol 2: Reversal of cross-links and DNA purification. Support Protocol 3: Bead clean-up. Basic Protocol 4: Library preparation and size selection. Support Protocol 4: qPCR to assess α-satellite DNA enrichment. Basic Protocol 5: ChIP-seq data processing and analysis.