Introducing a new solution for customized depletion of abundant RNAs.
In RNA-seq, highly expressed transcripts with minimal biological interest, such as ribosomal RNA (rRNA) can dominate readouts and mask detection of more informative low-abundance transcripts. This challenge is amplified when working with sample types for which pre-designed RNA depletion kits are not available. The NEBNext RNA Depletion Core Reagent set provides a customized approach to deplete unwanted RNA from any organism, using probe sequences designed with the user-friendly NEBNext Custom RNA Depletion Design Tool.
The efficient RNase-H-based workflow, and close tiling of probes designed using the online tool, enables effective depletion from both low- and high-quality RNA, with a broad range of input amounts.
Compatible with a broad range of input amounts: 10 ng - 1µg
Suitable for low-quality or high-quality RNA
Fast workflow: 2 hours, with less than 10 minutes hands-on time
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 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.
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
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
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
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
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