Next® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads |NEB酶试剂 New England Biolabs

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产品信息

Highly expressed transcripts with minimal biological interest, such as ribosomal RNA (rRNA) can dominate readouts, masking detection of more informative low-abundance transcripts. This second-generation NEBNext rRNA Depletion Kit is has been further optimized to incorporate reagent, probe and protocol improvements to the RNaseH-based workflow, resulting in superior depletion performance. 

  • The efficiency of this workflow, and close spacing of probes, enables effective depletion from both low- and high-quality RNA, with a broad range of input amounts from human, mouse and rat samples.
  • The rRNA-depleted RNA can be used in RNA-seq, random-primed cDNA synthesis, or other RNA analysis methods. 
  • The NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) depletes cytoplasmic (5S, 5.8S, 18S, 28S, human ITS, ETS) and mitochondrial (12S, 16S) rRNA.
  • The kit is also available without RNAClean® beads.     
Figure 1: The NEBNext rRNA Depletion Kit v2 enriches for RNAs of interest across a wide range of total RNA inputs in human, mouse and rat

Next® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads |

Universal human, mouse and rat reference total RNA (1 µg, 100 ng and 10 ng) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). RNA-seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75 bp). 10 Million reads were sampled (seqtk) from each library and reads were identified as ribosomal using mirabait (6 or more shared 25-mers) with rRNA sequences for human baits (5S, 5.8S, 12S, 16S, 18S, 28S, ETS/ITS) or mouse/rat baits (5S, 5.8S, 12S, 16S, 18S, 28S). Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. The data represents an average of 3 replicates. The NEBNext rRNA Depletion Kit v2 is efficient at depleting rRNA across species and input amounts.
Figure 2: Treatment with the NEBNext rRNA Depletion Kit v2 does not affect the abundances of non-targeted transcripts

Next® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads |

Universal human, mouse and rat reference total RNA (1 µg) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75bp). 10 million reads were sampled (seqtk) for depleted libraries and 100 million reads for undepleted libraries. GENCODE v27 transcript abundances were estimated using Salmon. Read Counts and R2 values for the linear fit are shown. Transcript expression is consistent after depletion across species.
Figure 3: Transcript expression correlation is preserved across a wide range of inputs

Next® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads |

Universal human, mouse and rat reference total RNA (1 µg) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75bp). GENCODE v27 transcript abundances were estimated using Salmon. Read Counts and R2 values for the linear fit are shown. Transcript expression correlation is consistent regardless of input amount.
Figure 4: The NEBNext rRNA Depletion Kit v2 efficiently depletes rRNA from degraded FFPE total RNA while preserving transcript abundances

Next® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads |

Human adult normal liver tissue FFPE Total RNA, RIN 2.3 (100 ng and 10 ng) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (A, B, C) or the TruSeq® Stranded Total RNA Gold kit (A). RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). 20 Million reads were sampled (seqtk) from depleted libraries and 200 million reads from undepleted libraries. (A) Reads were identified as ribosomal using mirabait (6 or more shared 25-mers) with rRNA sequences for 5S, 5.8S, 12S, 16S, 18S, 28S and ETS/ITS as baits. Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. The data represents an average of 3 replicates and error bars indicate standard error. (B) and (C) GENCODE v27 transcript abundances were estimated using Salmon. Read Counts and R2 values for the linear fit are shown. The NEBNext kit provides superior depletion of rRNA from FFPE (degraded) samples and offers the flexibility of lower total RNA input amounts. Transcript expression correlation is consistent after treatment and regardless of input amount.
Figure 5: The NEBNext rRNA Depletion Kit v2 depletes both mature and pre-rRNA from human total RNA

Next® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads |

Human universal reference total RNA (1 µg) was depleted of rRNA using the original NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310) and the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400). RNA-seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75 bp). 10 Million reads were sampled (seqtk) from each library. Reads were identified as ribosomal using mirabait (6 or more shared 25-mers) with rRNA sequences for 5S, 5.8S 12S, 16S, 18S, 28S and ETS/ITS as baits. Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. The data represents an average of 3 replicates. NEBNext rRNA Depletion Kit v2 provide superior depletion of rRNA.
Figure 6: Workflow

Next® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads |

产品类别:
RNA Depletion & mRNA Enrichment Products,
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation Products

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E7405S     Multi-temperature    
        NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) E7400S -20    
        NEBNext® DNase I E7753-2VIAL -20 1 x 0.015 ml 20,000 units/ml
        NEBNext® Thermostable RNase H E7752-2VIAL -20 1 x 0.012 ml 5,000 units/ml
        NEBNext v2 rRNA Depletion Solution E7401-2VIAL -20 1 x 0.012 ml Not Applicable
        RNase H Reaction Buffer E6312-2VIAL -20 1 x 0.012 ml Not Applicable
        DNase I Reaction Buffer E6315-2VIAL -20 1 x 0.03 ml Not Applicable
        Nuclease-free Water E6317-2VIAL -20 1 x 0.4 ml Not Applicable
        NEBNext Probe Hybridization Buffer E6314-2VIAL -20 1 x 0.012 ml Not Applicable
        NEBNext® RNA Sample Purification Beads E6351S 4    
        NEBNext® RNA Sample Purification Beads E6351SVIAL 4 1 x 0.66 ml Not Applicable
    • E7405L     Multi-temperature    
        NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) E7400L -20    
        NEBNext® DNase I E7753-3VIAL -20 1 x 0.06 ml 20,000 units/ml
        NEBNext® Thermostable RNase H E7752-3VIAL -20 1 x 0.048 ml 5,000 units/ml
        NEBNext v2 rRNA Depletion Solution E7401-3VIAL -20 1 x 0.048 ml Not Applicable
        RNase H Reaction Buffer E6312-3VIAL -20 1 x 0.048 ml Not Applicable
        DNase I Reaction Buffer E6315-3VIAL -20 1 x 0.12 ml Not Applicable
        Nuclease-free Water E6317-3VIAL -20 1 x 1.5 ml Not Applicable
        NEBNext Probe Hybridization Buffer E6314-3VIAL -20 1 x 0.048 ml Not Applicable
        NEBNext® RNA Sample Purification Beads E6351L 4    
        NEBNext® RNA Sample Purification Beads E6351LVIAL 4 1 x 2.64 ml Not Applicable
    • E7405X     Multi-temperature    
        NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) E7400X -20    
        NEBNext® DNase I E7753-4VIAL -20 1 x 0.24 ml 20,000 units/ml
        NEBNext® Thermostable RNase H E7752-4VIAL -20 1 x 0.192 ml 5,000 units/ml
        NEBNext v2 rRNA Depletion Solution E7401-4VIAL -20 1 x 0.192 ml Not Applicable
        RNase H Reaction Buffer E6312-4VIAL -20 1 x 0.192 ml Not Applicable
        DNase I Reaction Buffer E6315-4VIAL -20 1 x 0.48 ml Not Applicable
        Nuclease-free Water E6317-4VIAL -20 1 x 6 ml Not Applicable
        NEBNext Probe Hybridization Buffer E6314-4VIAL -20 1 x 0.192 ml Not Applicable
        NEBNext® RNA Sample Purification Beads E6351X 4    
        NEBNext® RNA Sample Purification Beads E6351XVIAL 4 1 x 10.6 ml Not Applicable

  • 特性和用法

    需要但不提供的材料

    • Pipettes
    • Magnetic rack (NEB #S1515), magnetic plate (Alpaqua. cat. #A001322) or equivalent
    • 80% Ethanol (freshly prepared)
    • Thin wall 200 μl PCR tubes and 1.5 ml tubes
    • Microcentrifuge
    • Vortex mixer
    • Thermocycler
    • Bioanalyzer., TapeStation. (Agilent Technologies, Inc.) or similar instrument and consumables

  • 优势和特性

    Features

    • Superior depletion of rRNA from human, mouse and rat RNA
    • Depletion of cytoplasmic (5S, 5.8S, 18S, 28S, human ITS, ETS) and mitochondrial (12S and 16S) rRNA
    • Compatible with a broad range of input amounts: 10 ng – 1 µg
    • Suitable for low-quality or high-quality RNA
    • Fast workflow: 2 hours, with less than 10 minutes hands-on time

  • 相关产品

    相关产品

    • e7760-nebnext-ultra-ii-directional-rna-library-prep-kit-for-illumina
    • e7765-nebnext-ultra-ii-directional-rna-library-prep-with-sample-purification-beads
    • E7750 NEBNext Globin and rRNA Depletion Kit Human Mouse Rat
    • E7755 NEBNext Globin and rRNA Depletion Kit Human Mouse Rat with RNA Sample Purification Beads
    • E7850 NEBNext rRNA Depletion Kit Bacteria
    • E7860 NEBNext rRNA Depletion Kit Bacteria with RNA Sample Purification Beads
    • e7490-nebnext-polya-mrna-magnetic-isolation-module

操作说明、说明书 & 用法

  • 操作说明

    1. Where can I find protocols for using NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) with NEBNext Ultra II RNA Library Prep (NEB #E7400 & #E7405)?

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE7400_E7405

工具 & 资源

  • Web 工具

    • NEBNext Selector

FAQs & 问题解决指南

  • FAQs

    1. What is the difference between the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) and the original NEBNext® rRNA Depletion Kit (Human/Mouse/Rat)?
    2. Which species are compatible with the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) for? Will it work for other species?
    3. Which rRNA subunits are depleted with the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat)?
    4. Can I use this product with degraded RNA or fragmented RNA?
    5. What total RNA input should I use?
    6. Why must the RNA be free of DNA?
    7. What is the percentage of rRNA remaining after depletion?
    8. How can I determine if the rRNA depletion was efficient?
    9. Does the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) work for ribosomal footprinting or profiling applications?
    10. 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 v2 (Human, Mouse, Rat)?
    11. Can I use more than one type of NEBNext Depletion Solution in the NEBNext RNA depletion workflow?
    12. Can the enriched RNA resulting from the NEBNext depletion kit be used in long read sequencing applications?