NEX Poly(A) beads 2.0

• 10 ng – 5 µg total starting RNA
• Magnetic bead-based protocol
• No organic solvents or precipitation step required
• Tested using the NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0
• Optimized for use with 10 ng – 5 µg of total RNA as starting material
• Automated on the Sciclone^® G3 NGSx and Zephyr^® G3 NGS workstations

Poly(A)-tailed mRNA is isolated via magnetic beads conjugated to oligo(dT), and separation using a magnetic stand allows for high mRNA recovery. The intact mRNA is eluted in small volumes, thereby eliminating the need for precipitation. These beads have been validated with the new and improved NEXTFLEX^® Rapid Directional RNA-Seq Kit 2.0.

• 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 Poly(A) 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.