Global gene expression analysis is a powerful method for identifying biological networks and regulatory mechanisms that govern cellular or tissue-level responses to physiologic stress. In the context of tumor biology, differential gene expression studies have provided information about the growth, aggressiveness, prognosis, and therapeutic response of tumors in patients. Scientists are using these valuable data to investigate pathways that can be targeted therapeutically with the goal of improving patient outcome. RNA sequencing enables nucleotide resolution of expression of whole transcriptomes, but arrives with a new set of challenges surrounding the management and analysis of large datasets. This chapter aims to review technical advancements to current methods for isolating high-quality RNA for sequencing studies directly from hypoxic tissues and introduces select widely used applications for gene expression analyses of next-generation sequencing data.