RNA expression profiles produced by next-generation sequencing (NGS) technology 
(RNA-seq) allow comprehensive investigation of transcribed sequences within a 
cell or tissue. RNA-seq is rapidly becoming more cost-effective for 
transcriptome profiling. However, its usage will expand dramatically if one 
starts with low amount of RNA and obtains transcript directionality during the 
analysis. Here, we describe a detailed protocol for the creation of a 
directional RNA-seq library from 100 ng of starting total RNA.