Reducing Ribosomal rRNA Reads in RNA-seq Libraries 

Several approaches have been used to remove ribosomal RNA (rRNA) from total RNA samples for RNA-Seq library preparation. Removal of rRNA avoids the waste of reagents and bioinformatics resources to sequence and align ~85% of total RNA comprising rRNA (which is generally not a target of interest in NGS sequencing experiments). Revvity’s NEXTFLEX Poly(A) beads 2.0 kit offers an easy and cost-effective method for removing rRNA in RNA-Seq libraries. This product takes advantage of the tract of adenosine residues present at the 3’ ends of a vast majority of protein-coding mRNAs. Hybridization of these poly(A) tails to tracts of complementary thymidines (“oligo dT”) immobilized on solid supports allows the retrieval of poly(A)+ RNAs by recovering the solid support along with the hybridized RNA complexes.

Figure 1. Example of 1 µg of Universal Human Reference RNA (Agilent^® # 740000) after mRNA enrichment using the NEXTFLEX Poly(A) Beads 2.0.  3 µL Poly(A) enriched RNA was run on the LabChip^® GX Touch^™ HT Nucleic Acid Analyzer using the RNA Pico Assay Reagent Kit (# CLS960012) and a DNA/RNA/Charge Variant Assay LabChip (# 760435)

(A) mouse brain

(B) MCF7 cells

Figure 2. NEXTFLEX Poly(A) Beads 2.0 yield clean mRNA with minimal rRNA carry-over from 5 µg of total RNA input from (A) mouse brain and (B) MCF7 cells. ~1.5% rRNA carry-over was observed based on sequencing data.

For total RNA-Seq solution not limited to only mRNA-Seq, Revvity offers the NEXTFLEX RiboNaut^™ rRNA depletion kit (Human / Mouse / Rat) for use NEXTFLEX Rapid Directional RNA-Seq Kit 2.0 or other applications.