The article describes a comprehensive protocol for analyzing RNA-seq data using the tools HISAT, StringTie, and Ballgown. These tools are designed to align reads to a genome, assemble transcripts, quantify gene expression levels, and identify differentially expressed genes and transcripts. The protocol covers the entire process from raw read data to the creation of lists of gene transcripts, expression levels, and differentially expressed genes and transcripts. It is suitable for experiments comparing two biological conditions, with each condition having six replicates. The protocol is demonstrated using human RNA-seq data from chromosome X, which is gene-rich and spans about 151 megabases. The tools are efficient and can handle large datasets, with the entire process taking under 45 minutes on a computer with appropriate resources. The article also provides detailed instructions for downloading and installing the necessary software, as well as troubleshooting tips. The results section includes examples of how to visualize the data using Ballgown's plotting tools.The article describes a comprehensive protocol for analyzing RNA-seq data using the tools HISAT, StringTie, and Ballgown. These tools are designed to align reads to a genome, assemble transcripts, quantify gene expression levels, and identify differentially expressed genes and transcripts. The protocol covers the entire process from raw read data to the creation of lists of gene transcripts, expression levels, and differentially expressed genes and transcripts. It is suitable for experiments comparing two biological conditions, with each condition having six replicates. The protocol is demonstrated using human RNA-seq data from chromosome X, which is gene-rich and spans about 151 megabases. The tools are efficient and can handle large datasets, with the entire process taking under 45 minutes on a computer with appropriate resources. The article also provides detailed instructions for downloading and installing the necessary software, as well as troubleshooting tips. The results section includes examples of how to visualize the data using Ballgown's plotting tools.