Genome‐Wide Analysis of Protein Binding to DNA in Plastids by ptChIP‐seq

Abstract Plastids contain their own genomes encoding essential components of the photosynthetic apparatus and gene expression machinery. Transcription in plastids is mediated by two RNA polymerases, nuclear‐encoded (NEP) and plastid‐encoded RNA polymerase (PEP). Transcription, together with the binding of nuclear‐encoded plastid nucleoid‐associated proteins and dynamic tethering of nucleoids to plastid membranes, shapes nucleoid organization and function. Understanding these processes is critical for deciphering the regulation of gene expression in these organelles, which underpins chloroplast biogenesis, photosynthesis, and plant development. Here we present an optimized procedure for genome‐wide detection of protein‐binding sites in vivo using plastid chromatin immunoprecipitation coupled with high‐throughput sequencing (ptChIP‐seq). Compared with standard ChIP‐seq protocols, this method employs a high formaldehyde concentration for cross‐linking that maximizes DNA recovery without detectable nonspecific binding when proper controls are used. We use a custom Illumina‐compatible library preparation incorporating multiplexing strategies that enables pooling of many libraries per experiment, reducing per‐sample costs. Detailed procedures are provided for Arabidopsis thaliana at two developmental stages, but the method can be adapted to other species with appropriate modifications to chloroplast enrichment. Although we focus on detection of PEP, these protocols are applicable to detection of other endogenous and epitope‐tagged plastid proteins that directly interact with DNA. Unlike approaches relying on chloroplast ChIP‐qPCR or low‐resolution ChIP‐chip, ptChIP‐seq generates cost‐effective high‐resolution genome‐wide maps of binding sites of any DNA‐associated protein of interest in the plastid. This enables comprehensive investigation of active DNA metabolic processes within plastids, including transcription, replication, and DNA repair, and opens up avenues for exploring the dynamic interplay between nucleoid architecture, membrane association, and gene expression in response to developmental and environmental cues. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Plant material collection, chloroplast enrichment, and cross‐linking Alternate Protocol 1 : Working with young seedlings Basic Protocol 2 : Immunoprecipitation and reverse cross‐linking Basic Protocol 3 : DNA isolation, ptChIP validation, library preparation, and sequencing Basic Protocol 4 : Data analysis