Performance of Poly(A)-selected Libraries

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.

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 Libraries

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.

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).

• Bond, D.M., Ortega-Recalde, O., Laird, M.K. et al. The admixed brushtail possum genome reveals invasion history in New Zealand and novel imprinted genes. Nat Commun 14, 6364 (2023). doi.org/10.1038/s41467-023-41784-8.
• Gaonkar, C.C., Campbell, L. De novo transcriptome assembly and gene annotation for the toxic dinoflagellate Dinophysis. Sci Data 10, 345 (2023). doi.org/10.1038/s41597-023-02250-8.
• Laudadio, I., Carissimi, C., Scafa, N. et al. Characterization of patient-derived intestinal organoids for modelling fibrosis in Inflammatory Bowel Disease. Inflamm. Res. 73, 1359–1370 (2024). https://doi.org/10.1007/s00011-024-01901-9.
• Manukjan, N., Chau, S., Caiment, F. et al. Wnt7a Decreases Brain Endothelial Barrier Function Via β-Catenin Activation. Mol Neurobiol 61, 4854–4867 (2024). doi.org/10.1007/s12035-023-03872-0
• Mulroney, T.E., Pöyry, T., Yam-Puc, J.C. et al. N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting. Nature 625, 189–194 (2024). doi.org/10.1038/s41586-023-06800-3
• Smith, C. H., Mejia-Trujillo, R., Breton, S., Pinto, B. J., Kirkpatrick, M., & Havird, J. C. (2023). Mitonuclear Sex Determination? Empirical Evidence from Bivalves. Molecular Biology and Evolution, 40(11), msad240.
• Tóvári, J.; Vári-Mező, D.; Surguta, S.E.; Ladányi, A.; Kigyós, A.; Cserepes, M. Evolving Acquired Vemurafenib Resistance in a BRAF V600E Mutant Melanoma PDTX Model to Reveal New Potential Targets. Cells 2023, 12, 1919. doi.org/10.3390/cells12141919.