The goal of this course is to teach the fundamentals of NGS library preparation and validation using the Illumina TruSeq stranded total RNA sample preparation kit. We will start with an introduction into Illumina NGS technology and the library preparation workflow. The main part will be the preparation of libraries from total RNA, with a focus on the critical steps and potential pitfalls of the protocol. Finally we will quantify and quality control the prepared libraries, before discussing solutions for total RNA sequencing data analysis.