Transcriptomics refers to the comprehensive analysis of ribonucleic acid (RNA) molecules present in a cell or organism at a specific time and under defined conditions. Positioned, at a molecular level, between genomics and proteomics, transcriptomic studies offer a dynamic view of gene regulation and expression. RNA sequencing (RNA-seq) encompasses a range of technologies developed to identify and quantify diverse RNA species, playing a pivotal role in advancing plant research. These technologies have enabled deep insights into gene expression patterns, physiological states, developmental processes, and responses to environmental stimuli. The evolution of RNA-seq technologies is inseparable from the development of bioinformatic tools designed to manage and analyse the vast amounts of data these technologies produce. In this chapter, we provide an overview of the main RNA-seq technologies from a historical perspective, along with the key bioinformatics tools used throughout the typical RNA-seq analysis workflow. We also highlight specialized tools tailored to specific analyses, including differential gene expression, alternative splicing, small RNA characterization, and single-cell RNA-seq.