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NEXTFLEX Rapid Directional RNA-Seq Kit 2.0

The NEXTFLEX™ Rapid Directional RNA-seq library prep kit 2.0 produces libraries for Illumina® and Element Biosciences® 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.

View product information ビュー Related Reagents

Feature	Specification
Automation Compatible	Yes
Product Group	RNA-seq

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

View product information ビュー Related Reagents

Product Variants

Unit Size: 8 rxns

フォーマット

Manual

Part #:

NOVA-5198-01

Unit Size: 48 rxns

フォーマット

Manual

Part #:

NOVA-5198-02

Unit Size: 96 rxns

フォーマット

Manual

Part #:

NOVA-5198-03

Unit Size: 96 rxns

フォーマット

Automation Friendly Volumes

Part #:

NOVA-5198-53

Request More Information

Product information

Overview

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)
Up to 96 UDI-UMI Barcodes now available to improve gene expression analysis by removing PCR duplicates.
Automated on the Sciclone® G3 NGSx and Zephyr® G3 NGS workstations
Compatible with Illumina® and Element Biosciences® sequencing platforms

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.

Additional product information

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).

Specifications

Other Specifications

Automation Compatible	Yes
Format	Automation Friendly Volumes
Product Group	RNA-seq
Shipping Conditions	Dual Temperature
Unit Size	96 rxns

Citations

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.