New England Biolabs, E7850L, NEBNext® rRNA Depletion Kit (Bacteria)

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. Related Categories RNA Depletion & mRNA Enrichment,, Epitranscriptome Analysis,, RNA Library Prep for Illumina, Specification 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) FAQ Q: Which bacterial rRNA subunits are depleted with the NEBNext rRNA Depletion Kit Bacteria A: The kit depletes 5S, 16S and 23S rRNA subunits. Q: Can the NEBNext® rRNA Depletion Kit (Bacteria) be used on metatranscriptomic samples? A: Yes, since the probes are designed against rRNA from several species, the depletion will be effective on both monocultures and mixed microbial communities (gram-positive and gram-negative). Q: How can I remove rRNA from a mix of mammalian and bacterial samples A: Probes from NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400/E7405) or NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7750/E7755) can be mixed in equal amounts with probes from the NEBNext® rRNA Depletion Kit (Bacteria) kit for depletion in a mixed sample. Depending on the amount of mammalian rRNA relative to the bacterial rRNA, the probe amounts can be further titrated. Please refer to the information here. Q: What is the percentage of bacterial rRNA remaining after depletion? A: The percentage of bacterial rRNA remaining after depletion will vary by sample. For several different species we expect to see ~1-5% rRNA remaining. However, depending on the diversity of the species and its rRNA sequence, higher %rRNA can also be seen. Q: How can I determine if the rRNA depletion was efficient? A: To assess depletion efficiency, design RT qPCR primers for the sample species’ rRNA, and primers for a housekeeping gene. Compare rRNA content before and after depletion. Q: Can I use this product with degraded RNA or fragmented RNA? A: Yes, this kit can be used with degraded or fragmented RNA. Q: What is the total RNA input I should use? A: The kit has been optimized for 10-1,000 ng total RNA input (100 ng minimum for fragmented or degraded RNA). For RNA-seq, we recommend using inputs higher than 100 ng to increase library complexity and reduce duplication rate. Q: Why must the RNA be free of DNA? A: Contaminating DNA can cause inaccurate RNA quantification and impede proper globin mRNA and rRNA removal. Q: 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? A: The ATCC 20 Strain Even Mix Whole Cell Material (ATCC® MSA-2002™) was used in the depletion experiment. A composite reference genome was constructed from NCBI genomes to assess the performance of the kit. The composite reference is available at https://doi.org/10.5281/zenodo.3386404 Q: Can I use Agencourt® AMPure® XP Magnetic Beads instead of NEBNext® RNA Sample Purification Beads (RNAClean® XP Magnetic Beads)? A: Yes, it is possible to use AMPure XP Beads. However, we recommend using the RNAClean XP Beads/ NEBNext® RNA Sample Purification Beads (supplied in NEB #E7860) as these beads have been manufactured and tested to eliminate RNase contamination. A version of the kit includes these beads: NEBNext® rRNA Depletion Kit (Bacteria) with RNA Sample Purification Beads (NEB #E7860). Q: Can I use purification methods other than NEBNext® RNA Sample Purification Beads/Agencourt® RNAClean® XP Magnetic Beads? A: Although we recommend using RNAClean® XP beads NEBNext® RNA Sample Purification Beads (supplied in NEB #E7755), rRNA depleted samples can alternatively be purified by ethanol precipitation or using RNA column cleanups such as the Monarch Spin RNA Cleanup Kit (NEB #T2030), For use with other columns, check the column specification for RNA size range to ensure any fragmented RNA will be retained during the cleanup. Q: Which species have been tested with the NEBNext rRNA Depletion Kit (Bacteria)? A: Information on species tested to date can be found here. Q: Can the enriched RNA resulting from the NEBNext depletion kit be used in long read sequencing applications? A: Yes, but note that RNA is being somewhat fragmented during the depletion process. This can reduce read length.