Disrupted chromatin regulatory processes contribute to the development of cancer, in particular pancreatic ductal adenocarcinoma. The assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) is typically used to study chromatin organization. Here, we present a revised ATAC-seq protocol to study chromatin accessibility in adherent patient-derived cell lines. We provide details on how to calculate the library molarity using Agilent's Bioanalyzer and an analysis pipeline for peak calling and transcription factor mapping. For complete details on the use and execution of this protocol, please refer to Brunton et al. (2020).