Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negates the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen tissue sections. We also demonstrate one tube UniNicE-seq for the FFPE tissue section for direct NGS library preparation without sonication and DNA purification steps. These refinements allowed reliable mapping of accessible chromatin for high-resolution genomic feature studies.