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 availablewith RNAClean®beads.
Features
Efficient, specific depletion of bacterial rRNA (5S, 16S, 23S)
Compatible withboth 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 amountsTotal 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 speciesTotal 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 amountsE.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. coliE.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 communityTotal 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
This product is related to the following categories:
RNA Depletion & mRNA Enrichment,
RNA Library Prep for Illumina,
Next Generation Sequencing Library Preparation
This product can be used in the following applications:
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