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 modifications by fixation 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.
Researchers at the Indiana University School of Medicine have developed an RNA-Seq protocol to address these key issues as well as quantify gene expression from the whole transcriptome. Furthermore, for compatibility with improved sequencing platforms, they use restructured adapter sequences to generate libraries for Illumina HiSeq, MiSeq, and NextSeq platforms. Their protocol utilizes duplex-specific nuclease (DSN) to remove abundant ribosomal RNA sequences while retaining other types of RNA for superior transcriptome profiling from low quantity input. The researchers employ the Illumina sequencing platform, but this method is described in sufficient detail to adapt to other platforms.
Detailed description of library construction steps
This figure shows a detailed step-by-step library sequence modifications with adapter and primer locations and sizes including potential adapter dimers (modified from Miller et al., 2013)