This Twist RNA Library Preparation Protocol details all the necessary steps to build sequencing libraries for target enrichment and sequencing on Illumina next-generation sequencing (NGS) systems. The steps include converting RNA to double-stranded cDNA and preparing cDNA libraries using the Twist Universal Adapter System. The Twist Universal Adapter system consists of Twist Universal Adapters and Twist Unique Dual Indexed (UDI) Primers. This kit is also compatible with the Twist HT Universal Adapter System, which consists of Twist Universal Adapters and Twist HT UDI Primers. This protocol is optimized for use with Twist Target Enrichment Kits and should only be performed with reagents specified or their equivalents.