An improved RNA-Seq library prep workflow for FFPE RNA

Preparing high-quality, rRNA-depleted RNA-Seq libraries using RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue is notoriously difficult. FFPE RNA is typically degraded (resulting in small fragment sizes) and is not compatible with poly(A)⁺ enrichment methods to remove contaminating rRNA. We have recently evaluated Ribo-Zero™ Kits and the ScriptSeq™ v2 Kit for the preparation of RNA-Seq libraries from FFPE RNA.

In this study, two different FFPE RNA samples were treated with either the Ribo-Zero Kit (Human/Mouse/Rat), which removes cytoplasmic rRNA, or the Ribo-Zero Gold Kit (Human/Mouse/Rat), which removes both cytoplasmic and mitochondrial rRNA. The rRNA-depleted RNA was then used to prepare RNA-Seq libraries using the ScriptSeq v2 Kit. Libraries were sequenced on an Illumina^® GAIIx sequencer. 

Figure 1 (click to enlarge). Summary of sequencing metrics from FFPE RNA libraries.

As shown in Figure 1, the workflow yields high-quality RNA-Seq libraries, with excellent removal of rRNA and >98% directionality from FFPE RNA. Quality metrics surpass those obtained from FFPE RNA using standard ligation-based library preparation workflows.