Optimized Methodology for the Generation of RNA-Sequencing Libraries from Low-Input Starting Material: Enabling Analysis of Specialized Cell Types and Clinical Samples.

RNA sequencing (RNA-seq) has become an important tool for examining the role of the transcriptome to biological processes. While RNA-seq has been widely adopted as a popular approach in many experimental designs, from gene discovery to mechanistic validation of targets, technical issues have largely limited the use of this technique to abundantly available sample sources. However, RNA-seq is becoming increasingly utilized for more specialized applications, such as flow cytometry-sorted cells and clinical specimens, due to protocol advances enabling the use of very low input material ranging from 10 pg to 10 ng of total RNA or 1-1000 intact cells. In this chapter, we present an optimized and detailed approach to RNA-seq for use with low abundance samples.