Next® rRNA Depletion Kit (Bacteria) |NEB酶试剂 New England Biolabs

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产品信息

The great majority of RNA in bacteria is ribosomal RNA (rRNA). This highly abundant RNA can conceal the biological significance of less abundant transcripts, and so its efficient and specific removal is desirable. 

The NEBNext® rRNA Depletion Kit (Bacteria) employs the NEBNext RNase H-based RNA depletion workflow to deplete rRNA (5S, 16S, and 23S) from gram-positive and gram-negative organisms. View species tested to date.

The kit is effective with both intact and degraded RNA preparations, from monocultures or samples with mixed bacterial species (e.g., metatranscriptomic).

The kit is also available with RNAClean® beads.

Features

  • Efficient, specific depletion of bacterial rRNA (5S, 16S, 23S)
  • Compatible with both gram-positive and gram-negative organisms
  • Effective with monocultures and mix of bacterial species (e.g., metatranscriptome)
  • 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
  • Includes NEBNext RNA Sample Purification Beads (Agencourt® RNAClean® XP)

For use with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) (NEB #E7416), refer to the Protocols tab for UMI Adaptors-specific guidance.

Figure 1: Depletion of ribosomal RNA using the NEBNext rRNA Depletion Kit (Bacteria) enriches for RNAs of interest across a mock community of bacterial species and a range of input amounts

Next® rRNA Depletion Kit (Bacteria) |

Total RNA was extracted from a lyophilized pool of 20 different bacterial organisms (ATCC® #MSA-2002). Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit (Bacteria). 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 75 bp). Reads were aligned (Hisat2) to a composite reference genome containing the best matching strains in the NCBI genome database.  Alignments were duplicate marked (Picard) and assessed for transcript levels (ht-seq count). Effective depletion of sequences overlapping with annotated rRNA regions was observed at 100 ng and 10 ng of input RNA for most of the organisms.

Figure 2: Depletion of ribosomal RNA with NEBNext enriches for RNAs of interest across monoculture species

Next® rRNA Depletion Kit (Bacteria) |

Total RNA (100 ng) from Escherichia coli and Clostridum phytofermentans was depleted of rRNA using the NEBNext rRNA Depletion Kit (Bacteria). 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 75 bp). Reads were aligned to each reference genome (Hisat2), duplicate marked (Picard) and assessed for transcript levels (ht-seq count). Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. Effective depletion of sequences overlapping with annotated rRNA regions was observed for all species. 

Figure 3: NEBNext demonstrates consistent depletion of ribosomal RNA across a range of input amounts

Next® rRNA Depletion Kit (Bacteria) |

E.coli total RNA (1 µg, 100 ng, 10 ng) was depleted of rRNA using the NEBNext rRNA Depletion Kit (Bacteria).  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 75 bp). Reads were aligned to the E.coli MG1655 reference genome (Hisat2), duplicate marked (Picard) and assessed for transcript levels (ht-seq count). Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. Effective depletion of sequences overlapping with annotated rRNA regions was observed at 1 µg, 100 ng and 10 ng of input RNA. 

Figure 4. NEBNext maintains consistent transcript expression correlation after depletion and across a range of inputs: E. coli

Next® rRNA Depletion Kit (Bacteria) |

E.coli total RNA (1 µg, 100 ng and 10 ng) was depleted of rRNA using the NEBNext rRNA Depletion Kit (Bacteria).  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 75 bp). 4 Million read pairs were sampled (seqtk) from each library, mapped to the E. coli MG1655 reference genome (Bowtie 2.3.2) before counting reads on genes (htseq-count) and correlating their levels. Correlation analysis of the transcripts indicates consistent transcript expression regardless of treatment or input amount.

Figure 5. NEBNext maintains consistent transcript expression correlation after depletion and across a range of inputs: Mock bacterial community 

Next® rRNA Depletion Kit (Bacteria) |

Total RNA was extracted from a lyophilized pool of 20 different bacterial organisms (ATCC® #MSA-2002). Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit (Bacteria). 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 75 bp). 4 Million read pairs were sampled (seqtk) from each library, mapped to a composite genome (Bowtie 2.3.2) before counting reads on genes (htseq-count) and correlating their levels. Correlation analysis of the transcripts indicates consistent transcript expression regardless of treatment or input amount.

Figure 6: NEBNext rRNA Depletion Kit Workflow

Next® rRNA Depletion Kit (Bacteria) |

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
    • E7850S     -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® Bacterial rRNA Depletion Solution E7851-2VIAL -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
    • E7850L     -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® Bacterial rRNA Depletion Solution E7851-3VIAL -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
    • E7850X     -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® Bacterial rRNA Depletion Solution E7851-4VIAL -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

  • 特性和用法

    需要但不提供的材料

    • Magnetic rack (NEB #S1515) or magnetic plate (Alpaqua. cat. #A001322) or equivalent
    • 80% Ethanol (freshly prepared)
    • Thermocycler
    • Any thin wall 200 μl PCR tubes and 1.5 ml tubes
    • Bioanalyzer, Tapestation. (Agilent Technologies, Inc.) or similar instrument and consumables
    • Agencourt® RNAClean® XP Beads (Beckman Coulter, Inc. #A63987)

操作说明、说明书 & 用法

  • 操作说明

    1. Where can I find protocols for using NEBNext® RNA Library Prep reagents?

  • 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。

    • manualE7850_E7860

  • 使用指南

    • NEBNext rRNA Depletion Kit (Bacteria Species) compatibility

FAQs & 问题解决指南

  • FAQs

    1. Which bacterial rRNA subunits are depleted with the NEBNext® rRNA Depletion Kit (Bacteria)?
    2. Can the NEBNext® rRNA Depletion Kit (Bacteria) be used on metatranscriptomic samples?
    3. How can I remove rRNA from a mix of mammalian and bacterial samples?
    4. What is the percentage of bacterial rRNA remaining after depletion?
    5. How can I determine if the rRNA depletion was efficient?
    6. Can I use this product with degraded RNA or fragmented RNA?
    7. What is the total RNA input I should use?
    8. Why must the RNA be free of DNA?
    9. Which microbiome community was used in the depletion experiments described, and which references were used to assess the performance of the NEBNext® rRNA Depletion Kit (Bacteria) with this sample?
    10. Which species are compatible with the NEBNext® rRNA Depletion Kit (Bacteria)?
    11. Can I use Agencourt® AMPure® XP Magnetic Beads instead of NEBNext® RNA Sample Purification Beads (RNAClean® XP Magnetic Beads)?
    12. Can I use purification methods other than NEBNext® RNA Sample Purification Beads/Agencourt® RNAClean® XP Magnetic Beads?
    13. Which species have been tested with the NEBNext rRNA Depletion Kit (Bacteria)?
    14. Can the enriched RNA resulting from the NEBNext depletion kit be used in long read sequencing applications?