NEW AND IMPROVED LIBRARY PREP KIT FOR YOUR RNA SEQUENCING NEEDS

NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0

For research use only. Not for use in diagnostic procedures.

  • NOVA-5198-01

    8 RXNS

  • NOVA-5198-02

    48 RXNS

  • NOVA-5198-03

    96 RXNS

New & Improved Library Preparation Kit for your RNA Sequencing Needs

The NEXTFLEX® rapid directional RNA-seq kit 2.0 produces libraries for Illumina® sequencing instruments with high coverage uniformity, low duplication rates, strand specificity and minimal rRNA contamination when used with the NEXTFLEX® Poly(A) Beads 2.0 (10 ng – 5 μg) or NEXTFLEX® RiboNaut rRNA depletion kit (human, mouse, rat) (5 ng – 1 μg). This kit includes reverse transcriptase, necessary library preparation reagents, and cleanup/size selection beads optimized to ensure reliable performance. The kit involves a simple library preparation protocol that has been validated with the updated NEXTFLEX® poly(A) beads 2.0 and NEXTFLEX® RiboNaut rRNA depletion kit (human, mouse, rat) to accommodate total RNA as input. The NEXTFLEX® rapid directional RNA-seq kit 2.0 is designed to be used with NEXTFLEX® RNA-Seq 2.0 Unique Dual Index Barcodes (6.25μM), which are color-balanced and have undergone proprietary purity QC metrics to generate reliable sequencing results for every sample.

PERFORMANCE OF POLY(A)-SELECTED LIBRARIES

rna fig 1

Figure 1. The NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 demonstrates even coverage along transcripts compared to the Competitor N kit. Poly(A) mRNA was isolated from Universal Human Reference RNA (Agilent #740000) using the NEXTFLEX® Poly(A) beads 2.0 and the Competitor N Poly(A) enrichment 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 paired-end mode (2×76 bp). Reads were trimmed using cutadapt and mapped to the Gencode v30 reference using bowtie2. The coverage along transcripts was calculated using the BBMap pileup tool.

fig 2 rapid rna

Figure 2. The NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 demonstrate low duplication rate compared to the Competitor N kit. Poly(A) mRNA was isolated from Universal Human Reference RNA (Agilent #740000) using the NEXTFLEX® Poly(A) beads 2.0 and the Competitor N Poly(A) enrichment 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 paired-end mode (2×76 bp). Reads were trimmed using cutadapt, mapped to the Gencode v30 reference using bowtie2, and randomly downsampled to 100k reads. Duplication rate was calculated using the fastp all-in-one FASTQ preprocessor.

fig 3 rapid rna

Figure 3. The NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 demonstrates comparable directionality to the Competitor N kit. Poly(A) mRNA was isolated from Universal Human Reference RNA (Agilent #740000) containing ERCC RNA Spike-In mix (Thermo Fisher Scientific #4456740) using the NEXTFLEX® Poly(A) Beads 2.0 and the Competitor N Poly(A) enrichment 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 paired-end mode (2×76 bp). Reads were trimmed using cutadapt and mapped to the ERCC92 reference using bowtie2. Strandedness was calculated using SAMtools.

figure 4 rna

Figure 4. The NEXTFLEX® Rapid Directional RNA-Seq kit 2.0 delivers libraries containing low levels of rRNA contamination than the Competitor N kit. Poly(A) mRNA was isolated from Universal Human Reference RNA (Agilent #740000) using the NEXTFLEX® Poly(A) Beads 2.0 and the Competitor N Poly(A) enrichment 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 paired-end mode (2×76 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.

figure 5 rna

Figure 5. Libraries prepared using the Zephyr® G3 NGS workstation and manually deliver comparable yields using the NEXTFLEX® Rapid Directional RNA-Seq kit 2.0. Poly(A) mRNA was isolated from Universal Human Reference RNA (Agilent® #740000) using the NEXTFLEX® Poly(A) Beads 2.0. 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).

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