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NEXTFLEX Rapid Directional RNA-Seq Kit 2.0

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).

For research use only. Not for use in diagnostic procedures.
Part Number: NOVA-5198-43
Format: Automated
Unit Size: 48 rxn
Part Number: NOVA-5198-53
Format: Automated
Unit Size: 96 rxn
Part Number: NOVA-5198-01
Format: Manual
Unit Size: 8 rxns
Part Number: NOVA-5198-02
Format: Manual
Unit Size: 48 rxns
Part Number: NOVA-5198-03
Format: Manual
Unit Size: 96 rxns
    • High coverage uniformity with low duplication rate
    • Optimized for use with 5 ng – 5 µg total RNA with reverse transcriptase and cleanup/size selection beads included
    • Simple protocol validated with NEXTFLEX® poly(A) beads 2.0 and NEXTFLEX® RiboNaut rRNA depletion kit (human, mouse, rat)
    • Designed to work with NEXTFLEX® RNA-Seq 2.0 Unique Dual Index barcodes that allow a wide range of multiplexing (2 up to 1,526 samples in one run)
    • Automated on the Sciclone® G3 NGSx and Zephyr® G3 NGS workstations

    The NEXTFLEX® Rapid Directional RNA-seq kit 2.0 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 tested with the 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 (6.25 μM), which are color-balanced and have undergone proprietary purity QC metrics to generate reliable sequencing results for every sample.

  • rapid directional workflow

     

    Performance of Poly(A)-selected Libraries
    nextflex rapid directional RNA seq kit 2.0 fig1

    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.

    nextflex rapid directional RNA seq kit 2.0 fig2

    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.

    nextflex rapid directional RNA seq kit 2.0 fig3

    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.

    nextflex rapid directional RNA seq kit 2.0 fig4

    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.

    nextflex rapid directional RNA seq kit 2.0 fig5

    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).

    Performance of rRNA-depleted Libraries
    Nextflex rapid directional RNA-seq kit 2.0 fig-6

    Figure 6. 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.

    Nextflex rapid directional RNA-seq kit 2.0 fig-7

    Figure 7. 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.

    Nextflex rapid directional RNA-seq kit 2.0 fig-8

    Figure 8. 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.

    Nextflex rapid directional RNA-seq kit 2.0 fig-9

    Figure 9. 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).

  • Brand
    NEXTFLEX
    Format
    Manual
    Shipping Conditions
    Dual Temperature
    Type
    RNA-seq
    Unit Size
    48 rxns

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Flyer
New and improved library preparation kit for your RNA sequencing needs.

This flyer describes the benefits of the NEXTFLEX® Rapid Directional RNA-seq 2.0 Kit.