标签归档：rrna

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

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

The great majority of RNA in blood samples is comprised of cytoplasmic and mitochondrial ribosomal RNAs (rRNA) and globin mRNA. These highly abundant RNA species can conceal the biological significance of less abundant transcripts, and so their efficient and specific removal is desirable.

The NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) employs the NEBNext RNase H-based RNA depletion workflow to deplete adult, embryonic and fetal globin mRNA (HBA1/2, HBB, HBD, HBM, HBG1/2, HBE1, HBQ1 and HBZ), cytoplasmic rRNA (5S, 5.8S, 18S, 28S, ITS and ETS) and mitochondrial rRNA (12S and 16S).

The kit is effective with human, mouse and rat total RNA preparations, both intact and degraded. The resulting depleted RNA is suitable for RNA-seq, random-primed cDNA synthesis, or other downstream RNA analysis.

This kit can also be used following poly(A) mRNA enrichment (e.g., using the NEBNext poly(A) mRNA Magnetic Isolation Module NEB #E7490), so that the final depleted RNA contains only mRNA of interest and no non-coding RNA. 
 
The kit is also available without RNAClean® beads.
 
Features

  • Efficient, specific depletion of rRNA and globin mRNA (adult, fetal and embryonic)
  • Suitable for low-quality or high-quality RNA
  • Compatible with a broad range of input amounts: 10 ng – 1 μg
  • Optional integration with poly(A) mRNA isolation workflows for removal of globin RNAs, rRNAs, and noncoding RNAs
  • Fast workflow: 2 hours, with less than 10 minutes hands-on time

 

 

Figure 1. Depletion of globin mRNA and ribosomal RNA enriches for RNAs of interest across species

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

Human, mouse and rat whole blood total RNA (1 µg) was depleted of rRNA alone, or rRNA and globin mRNA transcripts, using the NEBNext Globin & rRNA Depletion Kit.  RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). Reads were identified as rRNA or globin mRNA using mirabait (6 or more, 25-mers), and levels of rRNA and globin mRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering.
Figure 2: Consistent Depletion Across Species and Across Inputs

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

Human, mouse and rat whole blood total RNA (1 µg, 100 ng and 10 ng) was depleted of rRNA and globin mRNA using the NEBNext Globin & rRNA Depletion Kit or Globin-Zero® Gold rRNA Depletion Kit (Illumina®). RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). Reads were identified as ribosomal or globin using mirabait (6 or more, 25-mers), and levels of rRNA and globin mRNA 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. The NEBNext Globin Depletion Kit is superior at depleting rRNA across species, and at depleting over 99% of globin mRNA.
Figure 3: Transcript expression correlation is maintained after depletion

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

Human whole blood total RNA (1 µg) was depleted of rRNA and globin mRNA using the NEBNext Globin & rRNA Depletion Kit or Globin-Zero® Gold rRNA Depletion Kit (Illumina®). RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). GENCODE v27 transcript abundances were estimated using Salmon. TPM (Transcript Per Million mapped reads) of protein coding transcripts, and R2 values for the linear fit are shown. Correlation analysis of the transcripts indicates better transcript expression correlation between depleted and undepleted samples for the NEBNext Globin & rRNA Depletion Kit. Treatment does not alter the abundances of non-targeted transcripts.
Figure 4: Transcript expression correlation is maintained across a range of input amounts

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

Human whole blood total RNA (1 µg, 100 ng and 10 ng) was depleted of rRNA and globin mRNA using the NEBNext Globin & rRNA Depletion Kit. RNA-seq libraries were prepared using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). GENCODE v27 transcript abundances were estimated using Salmon. TPM (Transcript Per Million mapped reads) and R2 values for the linear fit are shown. Correlation analysis of the transcripts indicates consistent transcript expression regardless of input amount.
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
    • E7755S     Multi-temperature    
        NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) E7750S -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® Globin and rRNA Depletion Solution E7751-2VIAL -20 1 x 0.018 ml 1 X
        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
    • E7755L     Multi-temperature    
        NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) E7750L -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® Globin and rRNA Depletion Solution E7751-3VIAL -20 1 x 0.072 ml 1 X
        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
    • E7755X     Multi-temperature    
        NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) E7750X -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® Globin and rRNA Depletion Solution E7751-4VIAL -20 1 x 0.288 ml 1 X
        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

    • Magnetic rack/stand such as the NEBNext Magnetic Separation Rack (NEB #S1515)
    • 80% Ethanol (freshly prepared)
    • Thermocycler
    • Thin wall 200 µl PCR tubes
    • Bioanalyzer®, TapeStation® (Agilent Technologies, Inc.) or similar fragment analyzer and consumables

操作说明、说明书 & 用法

  • 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。

    • manualE7750_E7755

工具 & 资源

  • Web 工具

    • NEBNext Selector

FAQs & 问题解决指南

  • FAQs

    1. What species can I use the NEBNext® Globin mRNA & rRNA Depletion Kit for? Will it work for other species?
    2. Which globin mRNAs and rRNAs are depleted with the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat)? 
    3. Can I use the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat) with degraded RNA or fragmented RNA?
    4. What is the total RNA input I should use?
    5. Why must the RNA be free of DNA?
    6. What is the expected RNA yield recovered after globin mRNA and rRNA depletion?
    7. What is the percentage of globin mRNA and rRNA left after depletion?
    8. How can I determine if the globin mRNA and rRNA depletion was efficient?
    9. I am only interested in coding RNA. Can I use the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat) to remove globin mRNA from previously enriched mRNA.
    10. Does the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat) work for ribosomal footprinting or profiling applications?
    11. Can the enriched RNA resulting from the NEBNext depletion kit be used in long read sequencing applications?

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

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

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?

Next® rRNA Depletion Kit (Bacteria) with RNA Sample Purification Beads |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

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 without RNAClean® XP 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)
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) with RNA Sample Purification Beads |

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) with RNA Sample Purification Beads |

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) with RNA Sample Purification Beads |

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) with RNA Sample Purification Beads |

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) with RNA Sample Purification Beads |

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) 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
    • E7860S     Multi-temperature    
        NEBNext® rRNA Depletion Kit (Bacteria) 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
        NEBNext® RNA Sample Purification Beads E6351S 4    
        NEBNext® RNA Sample Purification Beads E6351SVIAL 4 1 x 0.66 ml Not Applicable
    • E7860L     Multi-temperature    
        NEBNext® rRNA Depletion Kit (Bacteria) 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
        NEBNext® RNA Sample Purification Beads E6351L 4    
        NEBNext® RNA Sample Purification Beads E6351LVIAL 4 1 x 2.64 ml Not Applicable
    • E7860X     Multi-temperature    
        NEBNext® rRNA Depletion Kit (Bacteria) 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
        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

    • 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

操作说明、说明书 & 用法

  • 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

工具 & 资源

  • Web 工具

    • NEBNext Selector

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. Which species have been tested with the NEBNext rRNA Depletion Kit (Bacteria)?
    12. Can the enriched RNA resulting from the NEBNext depletion kit be used in long read sequencing applications?

  • 问题解决指南

    • Troubleshooting Guide for NEBNext® rRNA Depletion Kit (Bacteria) (NEB #E7860)

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?