NEW rRNA DEPLETION FOR TOTAL RNA-SEQ APPLICATIONS
NEXTFLEX® RiboNaut™ rRNA Depletion Kit (Human / Mouse / Rat)
- Hybridization and bead-based protocol for rRNA depletion
- Optimized for use with 5 ng – 1 µg of total RNA as starting material
- Validated using the NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0 and automated
on the Sciclone® G3 NGSx and Zephyr® G3 NGS workstations
NOVA-512961
8 rxns
NOVA-512962
48 rxns
NOVA-512963
96 rxns
The NEXTFLEX® RiboNaut™ Kit Ensures Effective rRNA Depletion using Hybridization Technology
The NEXTFLEX® RiboNaut™ rRNA depletion kit (human / mouse / rat) is an effective method to remove rRNA contamination while enabling labs to interrogate additional RNA species in a sample, not only limited to intact mRNAs. The kit utilizes subtractive hybridization technology consisting of a mixture of biotinylated oligos complementary to rRNAs to pull them out of the sample using streptavidin-coated magnetic beads. The kit has been optimized for use with 5 ng – 1 µg of total RNA as starting material to deplete cytoplasmic and mitochondrial rRNAs. The kit has been verified to be compatible with human, mouse and rat, and it may be compatible with other mammalian species.
Figure 1: Example of 1 µg of Universal Human Reference RNA (Agilent® #740000) after rRNA depletion. 1 µL of rRNA-depleted RNA was run on the LabChip® GXII Touch™ HT instrument using the RNA Pico Assay Reagent Kit (# CLS960012) and a DNA/RNA/Charge Variant Assay (# 760435).
Optimized to Produce Robust Performance Data from Total RNA Samples
The NEXTFLEX® RiboNaut™ rRNA Depletion kit (human / mouse / rat) shows robust performance data using total RNA samples for gene coverage along a transcript, duplication rate, directionality, and rRNA contamination. Prior to rRNA depletion and subsequent RNA-seq, qualification of total RNA integrity using the LabChip® GXII Touch™ HT instrument.
See data tab below for performance of rRNA-depleted libraries using the NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 kit and NEXTFLEX® RiboNaut™ rRNA depletion kit.
As an alternative mRNA-Seq solution for intact poly(A)-tailed mRNA species, PerkinElmer offers the NEXTFLEX® Poly(A) Beads 2.0 for use NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 or other applications.
KIT CONTENTS
- NEXTFLEX® Bait Hybridization Buffer
- NEXTFLEX® Cleanup Buffer
- NEXTFLEX® RiboNaut™ Bait Mix
- NEXTFLEX® Cleanup Beads XP
- NEXTFLEX® RiboNaut™ Beads
- Nuclease-free water
- NEXTFLEX® RiboNaut™ Beads Wash Buffer
REQUIRED MATERIALS NOT PROVIDED
- 5 ng – 1 μg total RNA
- 96-well PCR plate magnetic stand
- 1.5 mL – 2 mL magnetic rack (optional)
- 15 mL tube magnetic rack (optional)
- 50 mL tube magnetic rack (optional)
- Thermal cycler
- 80% Ethanol
- 10, 20, 200 μL pipettes
- RNase-free pipette tips
- 96-well PCR plate
- 96-well PCR plate seals
- Microcentrifuge
- Vortex
Figure 1. The NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 demonstrates even coverage along transcripts compared to the Competitor N kit. rRNA-depleted total RNA was isolated from Universal Human Reference RNA (Agilent® #740000) using the NEXTFLEX® RiboNaut™ rRNA depletion kit (human, mouse, rat) and the Competitor N rRNA-depletion kit. Libraries were generated using the NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 and the Competitor N’s library preparation kit. The resulting libraries were sequenced on the Illumina® MiSeq® sequencer using single-end mode (1×151 bp). Reads were trimmed using cutadapt, mapped to the Gencode v30 reference transcriptome using bowtie2, and randomly downsampled to 720k reads. The coverage along transcripts was calculated using the BBMap pileup tool.
Low Duplication Rate
Figure 2. The NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 demonstrate low duplication rates compared to the Competitor N kit. rRNA-depleted total RNA was isolated from Universal Human Reference RNA (Agilent® #740000) using the NEXTFLEX® RiboNaut™ rRNA depletion kit (human, mouse, rat) and the Competitor N rRNA-depletion kit. Libraries were generated using the NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 and the Competitor N’s library preparation kit. The resulting libraries were sequenced on the Illumina® MiSeq® sequencer using single-end mode (1×151 bp). Reads were trimmed using cutadapt, mapped to the Gencode v30 reference transcriptome using bowtie2, and randomly downsampled to 28k reads. Duplication rate was calculated using the fastp all-in-one FASTQ preprocessor.
Reliable Directionality
Figure 3. The NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 demonstrates comparable directionality relative to the Competitor N kit. rRNA-depleted total RNA was isolated from Universal Human Reference RNA (Agilent® #740000) using the NEXTFLEX® RiboNaut™ rRNA depletion kit (human, mouse, rat) and the Competitor N rRNA-depletion kit. Libraries were generated using the NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 and the Competitor N’s library preparation kit. The resulting libraries were sequenced on the Illumina® MiSeq® sequencer using single-end mode (1×151 bp). Reads were trimmed using cutadapt and mapped to the Gencode v30 reference transcriptome using bowtie2. Reads from respective samples were combined and downsampled for a total of 800k reads each. Strandedness was calculated using the fastp all-in-one FASTQ preprocessor.
Low Levels of rRNA Contamination
Figure 4. The NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 delivers libraries containing low levels of rRNA contamination compared to the Competitor N kit. rRNA-depleted total RNA was isolated from Universal Human Reference RNA (Agilent® #740000) using the NEXTFLEX® RiboNaut™ rRNA depletion kit (human, mouse, rat) and the Competitor N rRNA-depletion kit. Libraries were generated using the NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 and the Competitor N’s library preparation kit. The resulting libraries were sequenced on the Illumina® MiSeq® sequencer using single-end mode (1×151 bp). The reads were trimmed using cutadapt and the percent of rRNA was determined by using bowtie2 to map reads to human rRNA. The NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 demonstrated superior removal of 5S, 5.8S, 12S, 16S, 18S, and 28S rRNA species compared to the Competitor N kit.
Automated vs. Manual Prep
Figure 5. Libraries prepared using the Sciclone® G3 NGSx workstation and manually deliver comparable yields using the NEXTFLEX® Rapid Directional RNA-Seq kit 2.0. rRNA-depleted total RNA was isolated from Universal Human Reference RNA (Agilent® #740000) using the NEXTFLEX® RiboNaut™ rRNA depletion kit. Libraries were generated using the NEXTFLEX® Rapid Directional RNA-Seq kit 2.0. Final library concentrations were quantified using the Qubit® 2.0 fluorometer (Thermo Fisher® Scientific #Q32866).
The NEXTFLEX® RiboNaut™ rRNA Depletion kit (human, mouse, rat) contains enough material to deplete rRNA from 8, 48 or 96 total RNA samples for subsequent total RNA-seq library prep or other applications. The kit ships with a minimum viable shelf life of at least six months. All components can be safely stored at 4°C, except the NEXTFLEX® Bait Hybridization Buffer, NEXTFLEX® RiboNaut™ Beads Wash Buffer, and Nuclease-free water, which should be stored at room temperature.
RELATED PRODUCTS
- NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0
- NEXTFLEX® Poly(A) Beads 2.0
- NEXTFLEX® RNA-Seq 2.0 Unique Dual Index Barcodes (1-384)
- NEXTFLEX® Rapid RNA-Seq Kit
- NEXTFLEX® Rapid Directional RNA-Seq Kit