This RNA depletion and RNA Library Preparation Protocol details all the necessary steps to build sequencing libraries sequencing on Illumina next-generation sequencing (NGS) systems. The steps include ribosomal and hemoglobin transcript depletion, 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 details steps to remove ribosomal RNA and hemoglobin from total RNA to generate amplified indexed cDNA libraries that can undergo whole transcriptome sequencing. If desired, whole transcriptome libraries are compatible with Twist target enrichment workflows. This protocol has been optimized for use with the reagents specified and should only be performed with them or their equivalents.