Next® RNA Depletion Core Reagent Set |NEB酶试剂 New England Biolabs

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产品信息

RNA-seq samples can include a large dynamic range of transcript expression, and unwanted, abundant RNAs must be removed in order to achieve efficient sequencing of the RNAs of interest. While pre-designed kits are available for some sample types (e.g. human, mouse, rat, some bacteria) and abundant RNAs (e.g. rRNA, globin mRNA), a customized approach is required for rRNA removal from other sample types as well as for removal of specific non-rRNA RNAs. 

Customized depletion of unwanted RNAs is enabled by use of the NEBNext RNA Depletion Core Reagent together with custom probes, following this workflow:

STEP 1: Use the online NEBNext Custom RNA Depletion Design Tool to obtain custom probe sequences, by entering the sequence of your target RNA. 

STEP 2: Order ssDNA probe oligonucleotides from your trusted oligo provider. 

STEP 3: Use the probes with the NEBNext Custom RNA Depletion Core Reagent Set or in combination with other NEBNext RNA Depletion Kits.

The NEBNext RNaseH-based RNA depletion workflow together with the closely-tiled custom probes designed using the online tool, allow effective depletion from both low- and high-quality RNA, with a broad range of input amounts.

The kit is also available with RNAClean® beads.

Features

  • Customizable option to deplete unwanted RNA from any organism, using probe sequences designed with the NEBNext Custom RNA Depletion Design Tool.
  • 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

 

Figure 1: NEBNext customized RNA depletion enriches for RNAs of interest by efficiently removing targeted RNA from total RNA across species and a wide range of inputs

Next® RNA Depletion Core Reagent Set |

The NEBNext Custom RNA Depletion Design Tool was used to design probes against rRNA of the mosquito Aedes aegypti (28S, 18S, 5.8S, 5S, mt16S and mt12S), and the archaeal species Thermococcus kodakarensis and Pyrococcus furiosus (23S, 16S, 5SrRNA1, 5SrRNA2). Total RNA (1 µg, 100 ng or 10 ng) was used as input for rRNA depletion using the RNA Depletion Core Reagent Set with the designed probes. 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 each library. Read pairs were identified as ribosomal using mirabait (6 or more shared 25-mers), and 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 method efficiently depletes targeted rRNA across species and a wide range of total RNA input amount (1 µg–10 ng).

Figure 2: Removal of targeted RNA with NEBNext customized RNA Depletion does not affect the levels of non-targeted transcripts 

Next® RNA Depletion Core Reagent Set |

The NEBNext Custom RNA Depletion Design Tool was used to design probes against Aedes aegypti rRNA. Total RNA was extracted from adult Aedes aegypti mosquitos (Benzon Research) using the Monarch® Total RNA Miniprep Kit (NEB #T2010S). Total RNA (100 ng and 10 ng) was used as input for rRNA depletion using the NEBNext RNA Depletion Core Reagent Set with the designed probes. 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 from undepleted libraries. Transcript abundances were estimated using Salmon and transcripts from Vectorbase (AaegL5.2 assembly). Read counts and R2 values for the linear fit are shown. A) Treatment does not affect abundances of non-targeted transcripts. B) Transcript abundances are maintained between replicates and across input amounts. 

Figure 3: Combined probe pools efficiently deplete human rRNA and mitochondrial mRNA

Next® RNA Depletion Core Reagent Set |

The NEBNext Custom RNA Depletion Design Tool was used to design probes against human mitochondrial mRNA. The probes were used in combination with the probe pool from the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). 1 μg of total Universal Human Reference RNA (Agilent®) was depleted of mitochondrial RNA and rRNA using the combined probes. 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 each library. A. Read pairs were identified as ribosomal and mitochondrial using mirabait (6 or more, 25-mers), and levels of rRNA and mtRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. Both rRNA and mitochondrial RNA are efficiently depleted. B. Integrative Genome Viewer (IGV) visualization of read coverage across the human mitochondrial genes.

Figure 4: Workflow

Next® RNA Depletion Core Reagent Set |

 
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
    • E7865S     -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
        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
    • E7865L     -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
        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
    • E7865X     -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
        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

  • 相关产品

    相关产品

    • e7760-nebnext-ultra-ii-directional-rna-library-prep-kit-for-illumina
    • E7850 NEBNext rRNA Depletion Kit Bacteria
    • E7400 NEBNext rRNA Depletion Kit v2 Human Mouse Rat
    • E7405 NEBNext rRNA Depletion Kit v2 Human Mouse Rat with RNA 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
    • NEBNext® Ultra™ II RNA 非链特异性文库制备试剂盒

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。

    • manualE7865_E7870

  • 使用指南

    • Guidelines for use of Custom RNA Depletion Probe Pools with the NEBNext RNA Depletion Core Reagent Set

工具 & 资源

  • Web 工具

    • NEBNext Custom RNA Depletion Design Tool
    • NEBNext Selector

FAQs & 问题解决指南

  • FAQs

    1. How can I design DNA probes to use with the NEBNext RNA Depletion Core Reagent Set? 
    2. Are the probes designed by the NEBNext Custom RNA Depletion Design Tool sense or antisense?
    3. Can the input sequence into the NEBNext Custom RNA Depletion Design Tool contain IUPAC Ambiguity Code?
    4. Does the NEBNext Custom RNA Depletion Design Tool check for probes targeting transcripts other than the ones provided?
    5. I have used NEBNext Custom RNA Depletion Design Tool and obtained probe sequences, what’s the next step?
    6. How do I make a working probe pool to be used with the NEBNext RNA Depletion Core Reagent Set?
    7. What’s the maximum number of probes that can be combined in a pool?
    8. Can I use this product with degraded RNA or fragmented RNA?
    9. What total RNA input should I use with the NEBNext RNA Depletion Core Reagent Set? 
    10. Why must the RNA be free of DNA?
    11. How can I determine if the RNA depletion was efficient?
    12. Can I use Agencourt®AMPure XP Magnetic Beads instead of RNAClean® XP Magnetic Beads/NEBNext® RNA Sample Purification Beads?
    13. Can I use purification methods other than NEBNext® RNA Sample Purification Beads/Agencourt® RNAClean® XP Magnetic Beads?
    14. Does the NEBNext Custom RNA Depletion Tool check for probe redundancy or overlap between target RNA sequences entered?
    15. 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 use with the NEBNext® RNA Depletion Core Reagent Set and NEBNext Custom RNA Depletion Tool