Isolation of RNA for RNA Sequencing in Ustilago maydis

In the last 15 years, RNA sequencing (RNA-seq) has emerged as a popular tool for studying gene expression changes in an organism. In fungal plant pathology, this is a comprehensive method for identifying transcriptome changes to both the fungal pathogen and its host. RNA-seq generates a vast amount of data that can increase our understanding of the interaction between the fungus and its host, or how the fungal species adapts to various environmental conditions. This chapter emphasizes the importance of sample preparation for RNA-seq. It also describes a protocol for isolating high quality RNA from Ustilago maydis haploid, dikaryon, and teliospore samples, removing genomic DNA contamination and screening RNA to ensure its removal, and it provides a method for cDNA library preparation. Successful RNA-seq experiments require careful handling of RNA during isolation and its downstream steps. Careful handling enables RNA sequencing to generate vast amounts of information on transcriptome changes, which can provide meaningful insights into the growth and development of U. maydis.