As a revolutionary technology for life sciences, RNA-seq has many applications and the computation pipeline has also many variations. Here, we describe a protocol to perform RNA-seq data analysis where the aim is to identify differentially expressed genes in comparisons of two conditions. The protocol follows the recently published RNA-seq data analysis best practice and applies quality checkpoints throughout the analysis to ensure reliable data interpretation. It is written to help new RNA-seq users to understand the basic steps necessary to analyze an RNA-seq dataset properly. An extension of the protocol has been implemented as automated workflows in the R package ezRun, available also in the data analysis framework SUSHI, for reliable, repeatable, and easily interpretable analysis results.