• 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.

Performance of Poly(A)-selected LibrariesFigure 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.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.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. 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. 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 LibrariesFigure 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.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.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.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).