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Abstract Background The “Assay for Transposase Accessible Chromatin sequencing” (ATAC-seq) is an efficient and easy to implement protocol to measure chromatin accessibility that has been widely used in multiple applications studying gene regulation. While several modifications or variants of the protocol have been published since it was first described, there has not yet been an extensive evaluation of the effects of specific protocol choices head-to-head in a consistent experimental setting. In this study, we tested multiple protocol options for major ATAC-seq components (including three reaction buffers, two reaction temperatures, two enzyme sources, and the use of either native or fixed nuclei) in a well-characterized cell line. With all possible combinations of components, we created 24 experimental conditions with four replicates for each (a total of 96 samples). In addition, we tested the 12 native conditions in a primary sample type (mouse lung tissue) with two different input amounts. Through these extensive comparisons, we were able to observe the effect of different ATAC-seq conditions on data quality and to examine the utility and potential redundancy of various quality metrics. Results In general, native samples yielded more peaks (particularly at loci not overlapping transcription start sites) than fixed samples, and the temperature at which the enzymatic reaction was carried out had a major impact on data quality metrics for both fixed and native nuclei. However, the effect of various conditions tested was not always consistent between the native and fixed samples. For example, the Nextera and Omni buffers were largely interchangeable across all other conditions, while the THS buffer resulted in markedly different profiles in native samples. In-house and commercial enzymes performed similarly. Conclusions We found that the relationship between commonly used measures of library quality differed across temperature and fixation, and so evaluating multiple metrics in assessing the quality of a sample is recommended. Notably, we also found that these choices can bias the functional class of elements profiled and so we recommend evaluating several formulations in any new experiments. Finally, we hope the ATAC-seq workflow formulated in this study on crosslinked samples will help to profile archival clinical specimens.