RNA-Seq technology allows the rapid analysis of whole transcriptomes taking advantage of next-generation sequencing platforms. Moreover with the constant decrease of the cost of NGS analysis RNA-Seq is becoming very popular and widespread. Unfortunately data analysis is quite demanding in terms of bioinformatic skills and infrastructures required, thus limiting the potential users of this method. Here we describe the complete analysis of sample data from raw sequences to data mining of results by using NGS-Trex platform, a low user interaction, fully automatic analysis workflow. Used through a web interface, NGS-Trex processes data and profiles the transcriptome of the samples identifying expressed genes, transcripts, and new and known splice variants. It also detects differentially expressed genes and transcripts across different experiments.