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  • NEBNext® rRNA Depletion Kit (Human/Mouse/Rat)

    Description






    The NEBNext rRNA Depletion Kit (Human/Mouse/Rat) employs an RNaseH-based method (1), (2) to deplete both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human total RNA preparations. This product is suitable for both intact and degraded RNA (e.g. FFPE RNA). The resulting rRNA-depleted RNA is suitable for RNA-Seq, random-primed cDNA synthesis, or other downstream RNA analysis applications.
    Figure 1. Efficient Removal of rRNA from Intact and Degraded RNA (FFPE), While Retaining Coding and Non-coding Transcripts
    Figure 1. Efficient Removal of rRNA from Intact and Degraded RNA (FFPE), While Retaining Coding and Non-coding Transcripts
    RNA-seq libraries were generated from Universal Human Reference Total RNA (UHR, Agilent) or Breast Cancer FFPE RNA (with an archive age of 1 year and 10 years). RNA
was either untreated or treated with the NEBNext poly(A) mRNA Magnetic Isolation Module (NEB #E7490) or the NEBNext rRNA Depletion Kit. RNA-seq libraries were made using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420). Reads were mapped to the hg19 genome and read distributions were determined using Picard RNA-seq Metrics. Libraries generated from rRNA-depleted RNA result in comparable low rRNA reads to poly(A) mRNA-enriched RNA, while also retaining more noncoding reads. rRNA depletion efficiency is achieved even with FFPE RNA.


    Figure 2. NEBNext rRNA Depletion Kit WorkflowFigure 2. NEBNext rRNA Depletion Kit Workflow
    Figure 3. NEBNext rRNA Depletion Kit Workflow TimesFigure 3. NEBNext rRNA Depletion Kit Workflow Times
    Figure 4. Depletion Efficiency with Intact or Degraded RNA
    Figure 4. Depletion Efficiency with Intact or Degraded RNA
    Ribosomal RNA was depleted from intact Universal Human Reference Total RNA (UHR, Agilent) (RIN>9) and degraded UHR Total RNA (RIN<3) using either the NEBNext rRNA Depletion Kit, Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre) or Ribo-Zero Gold provided within the TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (Illumina). rRNA- depleted RNA libraries were made using either the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420. Green and blue bars) or the TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (Illumina, red bars). Total rRNA-aligned reads were determined using Bowtie 2.0 (local, sensitive). NEBNext rRNA-depleted libraries contain a minimal percentage of rRNA reads regardless of the quality of the RNA.
    Figure 5. Depletion Efficiency with FFPE RNAFigure 5. Depletion Efficiency with FFPE RNA

    0.1 μg, 0.5 μg or 1 μg of breast cancer FFPE RNA samples (with archive ages of one year and 10 years) were depleted of rRNA using either the NEBNext rRNA Depletion Kit or the TruSeq® Stranded Total RNA Kit with Ribo- Zero Gold (Illumina #RS-122-2301). rRNA-depleted RNA libraries were made using either the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420)(green bars) or the TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (blue bars). Total rRNA-aligned reads were determined using Bowtie 2.0 (local, sensitive). NEBNext rRNA-depleted FFPE libraries result in a minimal percentage of rRNA reads, regardless of the archive age of the FFPE RNA.
    Figure 6. rRNA Depletion Efficiency on Mouse and Rat RNAFigure 6. rRNA Depletion Efficiency on Mouse and Rat RNA

    Ribosomal RNA was depleted from mouse and rat kidney total RNA (two technical replicates) using the NEBNext rRNA Depletion Kit. RNA-seq libraries were made from non-depleted Total RNA or rRNA-depleted RNA, using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420). Total rRNA-aligned reads were determined using Bowtie 2.0 (local, sensitive). NEBNext rRNA-depleted mouse and rat libraries contain a minimal percentage of reads mapping to rRNA.
    Figure 7. Transcript Expression Correlation with Non-depleted LibrariesFigure 7. Transcript Expression Correlation with Non-depleted Libraries

    Libraries were made from UHR RNA (Agilent) and Breast Cancer FFPE RNA (with archive age of one year and 10 years), both non-depleted and depleted rRNA using the NEBNext rRNA Depletion Kit. All libraries were made using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420). TopHat2 and Cufflinks were used for read mapping and transcript assembly and quantification. FPKM (Fragments Per Kilobase of transcript per Million mapped reads) correlation analysis indicates very good transcript expression correlation (R >0.93) between Depleted and Non-Depleted libraries. NEBNext rRNA depletion does not affect transcript expression levels.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    RNase H-20
    RNase H Reaction Buffer-2010X
    NEBNext rRNA Depletion Solution-20
    NEBNext Probe Hybridization Buffer-2010X
    DNase I (RNase-free)-20
    DNase I Reaction Buffer-2010X
    Nuclease-free Water-20

    Properties and Usage

    Materials Required but not Supplied

    • Magnetic Rack (Alpaqua, Cat. #A001322 or equivalent)
    • Thermal Cycler
    • Agencourt® RNAClean® XP (Beckman Coulter, Inc., Cat. #A63987)

    Storage Temperature

    -20°C

    References

    1. Adiconis, X. et al (2013). Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nature Methods 10. 623-629.
    2. Morlan,J.D. et al. (2012). Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue. PLoS One 77. e42882.

    FAQs

    1. What is the expected RNA yield recovered after rRNA depletion?
    2. What is the total RNA input I should use?
    3. What is the percentage of rRNA left after depletion?
    4. Can I use purification methods other than Agencourt RNAClean XP Magnetic Beads?
    5. Can I use Agencourt DNA AMPure XP Magnetic Beads instead of RNAClean XP Magnetic Beads?
    6. Can I use this product with degraded RNA or fragmented RNA?
    7. To remove ribosomal RNA from total RNA, should I use the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) or the NEBNext rRNA Depletion Kit (NEB #E6310)?

    Protocols

    1. NEBNext rRNA Depletion Kit (Human/Mouse/Rat) Protocol (E6310)

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Application Notes

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.