RNA-Seq is the leading technology for analyzing gene expression on a global scale across a broad spectrum of sample types. However, due to chemical modifi cations by fi xation or degradation due to collection methods, samples often contain an abundance of RNA that is no longer intact, and the capability of current RNA-Seq protocols to accurately quantify such samples is often limited. We have developed an RNASeq protocol to address these key issues as well as quantify gene expression from the whole transcriptome. Furthermore, for compatibility with improved sequencing platforms, we use restructured adapter sequences to generate libraries for Illumina HiSeq, MiSeq, and NextSeq platforms. Our protocol utilizes duplexspecifi c nuclease (DSN) to remove abundant ribosomal RNA sequences while retaining other types of RNA for superior transcriptome profi ling from low quantity input. We employ the Illumina sequencing platform, but this method is described in suffi cient detail to adapt to other platforms.