Aim of this course is to prepare RNA libraries for Illumina sequencing by using the novel NEBNext Ultra II Directional RNA kit for low input applications. Throughout the course, we will be generating libraries from ultra- low as well as from normal amounts of RNA (1 ng to 50 ng) input to better understand the impact of input amounts and PCR cycles on the bioinformatics dealing with complexity, coverage, duplication rates and uniformity of libraries. The course participants will be guided with tips and tricks through the work flow. Finally, the bioinformatics analysis of the data will be discussed. Selected participants who – PRIOR to the course - will be able to prove their samples have required quality according to provided specifications will have the chance to bring maximum two of their own eukaryotic total RNA samples to process. At least 100ng of sample will be required for attendee samples.