This second edition details new and updated methods used for studying prokaryotic non-coding RNAs and their protein accomplices. Chapters detail discovery of ncRNAs, characterization of their structure, functions, and their interactomes. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.