Unlike animals, plants do not have mobile defense cells and their immune systems are not naturally capable of developing and adapting to pathogens. The innate immune system of plants is very complex and organized in two important layers and extensively depends on signaling pathways. Each layer is composed of many components, which timely play their roles, chemical and physical barriers preventing pathogens or oxidative bursts, pathogen-related proteins, and programmed cell death that induced after pathogen recognition are all parts of plant complicated immune system. Transcriptome analysis of the plant pathogen interaction and listing differentially expressed genes (DEGs) shed light on the plant complicated responses to pathogens. The high throughput RNA-seq technology, developed after Next-generation sequencing arrival, is of the most used techniques in DEG analysis. Here, the basics and steps of the RNA-seq technique to study of the plant defense responses were summarized.