Abstract Background: Epigenetic changes, such as nucleosome positioning and DNA methylation, control gene expression and are altered in cancer. Therefore, accurately mapping these changes between normal and tumor tissue will provide novel information for identification of epigenetic driver genes, biomarker discovery, and anti-tumor therapy development for clear cell renal cell carcinoma (ccRCC), the most common subtype of renal carcinoma. Current methods are expensive and require a considerable amount of biological tissue. Methods: We have developed a novel assay (AcceSssIble) to determine DNA methylation and nucleosome occupancy in a single experiment. It is rapid and cost-effective, requiring the Infinium HumanMethylation450 BeadChip platform and the CpG methyltransferase M.SssI. Preliminary studies using fresh and frozen kidney tumors show that the epigenetic profile is maintained in frozen tissues, making this method suitable to interrogate archived clinical samples. Results: We have used AcceSssIble to assay the epigenomes of 5 fresh ccRCC tumors and their adjacent normal tissue, and have uncovered 257 genes that are associated with changes in chromatin accessibility in at least 4 ccRCC samples; 155 of these genes also show a change in endogenous DNA methylation levels in at least 4 tumors. These chromatin accessibility and DNA methylation changes occurred in the promoter regions, open reading frames, and the 3’ untranslated regions (UTRs) of the genes. Cross-referencing our results to the expression data of 72 matched ccRCC samples from The Cancer Genome Atlas (TCGA), we discovered that 29% (74/257) of these genes exhibited a significant change in gene expression, over half of which had been previously implicated in various cancers, including ccRCC. Furthermore, we observed chromatin accessibility changes in intergenic regions, including several which contain known transcription factor binding sites and ncRNA promoter regions. These sites might serve as potential enhancers for neighboring genes important to the development of ccRCC. Conclusions: In these studies, we were able to identify epigenetic changes in ccRCC samples associated with known oncogenes and tumor suppressor genes, as well as uncover novel targets. These targets, associated both within genes as well as in intergenic regions, undergo changes in chromatin structure and may be important in the development of ccRCC. Linking these findings to gene expression data revealed functionally relevant targets for ccRCC treatment. Expansion of these studies into a larger number of clinical samples across tumor grades will allow us to uncover biomarkers that can be used for personalized medicine. Citation Format: Elinne Becket, Christopher Duymich, Yin-Wei Chang, Kurinji Pandiyan, Peter A. Jones, Gangning Liang. Simultaneous evaluation of chromatin accessibility and DNA methylation in clear cell renal cell carcinoma by a newly developed assay, AcceSssIble. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-130. doi:10.1158/1538-7445.AM2014-LB-130