NEBNext® rRNA Depletion Kit (Bacteria)#E7850

Catalog # Size Price (£)
E7850L 24 REACTIONS £994.00
E7850S 6 REACTIONS £273.00
E7850X 96 REACTIONS £3574.00

Ribosomal RNA (rRNA) is highly abundant in bacterial samples, and its removal is desirable in order to reveal the biological significance of less abundant transcripts. Specific enrichment of bacterial mRNAs is challenging due to their lack of poly(A) tails, and so the converse - efficient and specific removal of bacterial rRNA - is necessary.

The NEBNext® rRNA Depletion Kit (Bacteria) employs the NEBNext RNase H-based RNA depletion workflow to target removal of rRNA from gram-positive and gram-negative organisms. The method is effective with intact and degraded RNA, from monocultures or samples with mixed bacterial species (e.g., metatranscriptomic).

  • 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

The kit is also available with RNAClean® beads.

Description

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. 

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)



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



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



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



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



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 



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



Kit Components

The following reagents are supplied with this product:

Store at (°C)

Concentration

NEBNext® DNase I

-20

NEBNext® Thermostable RNase H

-20

NEBNext® Bacterial rRNA Depletion Solution

-20

RNase H Reaction Buffer

-20

10 X

DNase I Reaction Buffer

-20

10 X

Nuclease-free Water

-20

NEBNext Probe Hybridization Buffer

-20


Properties and Usage

Materials Required but not Supplied

  • 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)

Storage Temperature

-20°C

Quality Control

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.


Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]


Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]


Safety Data Sheet

The following link is to a PDF Safety Data Sheet (SDS) that applies to this product to help you use it safely.



This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.



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