Abstract Solid tumors consist of heterogeneous cell types including a subpopulation of stem like cells. Starting from bulk tumor mass, the analysis of these cancer stem cells (CSCs) is hampered by their rare frequency and their characteristics remain hidden. To address this issue, we have developed a method for the semi-automated dissociation of human tumor tissue yielding a viable single cell suspension. Subsequently, a magnetic cell sorting (MACS) strategy was established allowing for the isolation of CD24-/CD44+ breast CSCs. Using these methods, we isolated breast CSCs from an invasive ductal carcinoma at a purity of 94%. Due to the limited starting material, a few hundred CSCs as well as bulk tumor cells were used for global PCR based RNA amplification. The amplified cDNA was sheared, size selected, and processed for high throughput gene expression analysis on the Illumina Genome Analyzer system. Samples were prepared in triplicate and over 10 million individual purity-filtered reads were analyzed per sample. In addition, Agilent whole genome microarrays were used for validation of expression differences. Analysis of the sequence tags allowed us to identify novel putative markers overexpressed in breast CSCs. GeneOntology analysis was used to determine functional groups of genes which are differentially regulated between CSCs and bulk tumor cells. We found a strong overrepresentation of genes connected to TGF-beta as well as Wnt/GSK3 signaling supporting the correlation of the CSC phenotype with epithelial-mesenchymal-transition (EMT). Other differentially expressed genes are known to be involved in pluripotency, cell migration, and apoptosis further indicating a stem cell-like fate.Our proof of principle experiment demonstrates the advantage of isolating rare cell populations for subsequent molecular analysis by high throughput sequencing. The analysis of purified CSCs revealed new insights on the regulation, maintenance, and biology that would not have been observed when assessing only the bulk tumor mass. Analyzing the genetic set up of defined tumor subpopulations by Next Generation Sequencing can improve our understanding of cancer and could pioneer targeted diagnostic and therapeutic approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2011-3329