Glia are integral components of the central nervous system. Astrocytes, microglia, oligodendrocytes, and oligodendrocyte progenitor cells are numerous, ubiquitous cells that perform vital and wide-ranging functions from ion homeostasis to immune defense. However, neuroscience research historically adopted a neuron-centric framework, so tools and techniques developed to understand neuronal biology were not as quickly applied to glia. This dissertation utilizes next generation sequencing of RNA from various glial cell types to expand our understanding of glial biology in a several different contexts. In the first chapter, I examined the transcriptomic signatures of acutely purified human astrocytes at baseline and in pathological contexts because little is known about the biology of astrocytes in humans in health and disease. Peritumor astrocytes had decreased expression of synapse-related genes as well as a number of cell surface receptors, suggesting that astrocytes contribute to circuit dysfunction in the tumor microenvironment. I also mapped how gene expression of human astrocytes changes during maturation and aging, and identified sex-specific gene signatures of human astrocytes. In the second chapter, I tested whether altering the peripheral immune system would alter glia in the brain. In recent years, there has been a boom in research uncovering interactions between the immune system and the brain, which was previously considered an immune-privileged organ. I performed RNA sequencing on four classes of glia in immunodeficient Rag2-/- mice that lack mature T and B cells required for adaptive immune responses. These mice had altered RNA expression in oligodendrocytes without notable changes in other cell types, including microglia, the brain-resident immune cells. These results revealed novel interactions between lymphocytes and oligodendrocytes. In the third and final chapter, I tested whether the gene Serpine2 was important for glial transcription in astrocytes and microglia. Serpine2 is substantially expressed in multiple brain cell types, particularly astrocytes, and it codes for a secreted serine protease inhibitor that regulates several enzymes, including thrombin. Microglia upregulated antimicrobial genes in the absence of Serpine2, revealing a novel mechanism by which microglial antimicrobial genes are regulated. In contrast astrocyte transcriptome did not change in the absence of Serpine2. Intriguingly, I did not find any gene expression associated with Serpine2 in microglia or astrocytes after presenting an inflammatory stimulus. In total, this dissertation represents multiple lines of inquiry that improves our understanding of glial transcriptome in health and disease.