With the evolution of high throughput sequencing technologies, the past decade has seen an exponential rise in the use of RNA sequencing (RNA-seq). RNA-seq has deepened our understanding of biological systems to unprecedented levels of resolution, identifying not only gene expression signatures but also regulatory RNA molecules that may play critical roles in disease pathogenesis. Pulmonary research has quickly incorporated this technology, from characterizing the IL-17 signature of steroid-unresponsive COPD patients [1] to discovering pathogen-host interactions of M. tuberculosis [2]. This mini-review provides an overview of core features and applications of RNA-seq to familiarize non-experts with the methodology and how it has impacted our understanding of lung pathophysiology.