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Summary: Open or accessible regions of the genome are the primary positions of binding sites for transcription factors and chromatin regulators. Transposase-accessible chromatin sequencing (ATAC-seq) can probe chromatin accessibility in the intact nucleus. Here, we describe a protocol to generate ATAC-seq libraries from fresh Arabidopsis thaliana tissues and establish an easy-to-use bioinformatic analysis pipeline. Our method could be applied to other plants and other tissues and allows for the reliable detection of changes in chromatin accessibility throughout plant growth and development.For complete details on the use and execution of this protocol, please refer to Wang et al. (2020).