Next Generation Sequencing (NGS) has revolutionized the way that we address complex biological questions. As sequencing output rapidly increases and experimental scales get bigger, library preparation becomes one of the major bottlenecks for NGS. This course provides a comprehensive hands-on training on how to prepare high quality libraries for Illumina HiSeq2500 and MiSeq sequencing platforms. Participants are encouraged to bring their own total RNA template and, by the end of the course, will have a library ready for NGS sequencing. Lectures will cover the entire workflow including sample QC/QA, as well as the basic principles of NGS technology and consideration for experimental design meeting current publication standards. The course will highlight applications such as mRNA-seq, ChIP-seq, de novo sequencing, re-sequencing, mutation discovery, single cell genomics, and PacBio RSII. A case example of combinatorial usage of NGS technology to meet specific biological goals of sequencing will be explored.