Abstract 3185: Rapid and efficient methods for preparing rRNA-depleted and directional RNA-Seq libraries from low-input and FFPE RNA samples

• Published in: Cancer research (Chicago, Ill.) Ill.), 2012-04, Vol.72 (8_Supplement), p.3185-3185
• Main Authors: Sooknanan, Roy, Hitchen, John
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2012-3185

Massively parallel sequencing of cDNA libraries (RNA-Seq) is rapidly becoming the preferred method for transcript profiling, and analysis of novel transcripts, novel isoforms, alternative splice sites, rare transcripts and cSNPs, compared to microarrays. Preparing NGS RNA-Seq libraries typically require first isolating rRNA-depleted RNA or enriching for poly(A)+ mRNA. However, total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) patient tissue samples is normally fragmented, making it not suitable for rRNA-depletion using some commercially available kits or poly(A)+ enrichment, which results in a 3′ sequence bias. Here, we present an efficient “single-pass” rRNA-depletion method (Ribo-Zero™ technology) for use with as little as 100 ng total RNA input from either intact or fragmented (e.g., FFPE) RNA samples. Additionally, an improved, more user-friendly version of the ScriptSeq™ RNA-Seq method (ScriptSeq™ v2) is used to rapidly prepare directional (∼99% strandedness) RNA-Seq libraries in about 2.5 hours, in a single-tube workflow, from either the intact or fragmented Ribo-Zero™ treated RNA samples. The ScriptSeq™ method does not require end-polishing, adaptor-ligation, cDNA fragmentation, or gel-size selection. The combined Ribo-Zero™ and ScriptSeq™ workflow is completed in about 5 hours, generating cluster-ready NGS libraries that contain