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Summary: The isolation of sufficient amounts of intact nuclei is essential to obtain high-resolution maps of chromatin accessibility via assay for transposase-accessible chromatin using sequencing (ATAC-seq). Here, we present a protocol for tag-free isolation of nuclei from both cell walled and cell wall-deficient strains of the green model alga Chlamydomonas reinhardtii at a suitable quality for ATAC-seq. We describe steps for nuclei isolation, quantification, and downstream ATAC-seq. This protocol is optimized to shorten the time of isolation and quantification of nuclei. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.