Lin, Shih-ShunShih-ShunLinChen, YihuaYihuaChenLu, Mei-Yeh JadeMei-Yeh JadeLu2025-07-102025-07-10202597810716439769781071643983https://scholars.lib.ntu.edu.tw/handle/123456789/730688Degradome sequencing provides comprehensive insights into RNA degradation profiles. The method described here adapts the 5′-rapid amplification of cDNA ends (5′-RACE) technique, enabling the sequencing of degradome cDNA fragments using short-read next-generation sequencing (NGS). Degradome profiles can validate predicted miRNA-mediated cleavage of target genes and identify novel targets. Additionally, this approach offers valuable insights into RNA processing mechanisms, including the roles of RNA-binding proteins. This chapter outlines an optimized protocol for constructing a high-yield, high-quality degradome library, encompassing NGS and data analysis procedures. We anticipate that the degradome sequencing approach will be highly beneficial for studying RNA dynamics in non-model organisms.5′-RACEDegradomemicroRNANext-generation sequencingRNA degradationTarget RNAbook part10.1007/978-1-0716-4398-3_18