New England Biolabs, E7400S, NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat)

The abundance of ribosomal RNAs (rRNAs), constituting 80–90% of total RNA, necessitates removal upstream of next generation sequencing and other applications. This second generation rRNA depletion kit incorporates reagent, probe and protocol improvements, resulting in superior depletion performance. The efficient RNAse-H-based workflow, and close spacing of probes, enables effective depletion from both low- and high-quality RNA, with a broad range of input amounts. Related Categories RNA Depletion & mRNA Enrichment,, Epitranscriptome Analysis,, RNA Library Prep for Illumina Specification Materials Required but not Supplied Pipettes Magnetic rack (NEB #S1515), magnetic plate (Alpaqua. cat. #A001322) or equivalent 80% Ethanol (freshly prepared) Thin wall 200 μl PCR tubes and 1.5 ml tubes Microcentrifuge Vortex mixer Thermocycler Bioanalyzer., TapeStation. (Agilent Technologies, Inc.) or similar instrument and consumables Agencourt. RNAClean. XP Beads (Beckman Coulter, Inc. #A63987) FAQ Q: What is the difference between the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) and the original NEBNext® rRNA Depletion Kit (Human/Mouse/Rat)? A: The NEBNext® rRNA Depletion Kit v2 features an improved probe design targeting all subunits of human pre-rRNA (5S, 5.8S, 18S, 28S, ITS, ETS), better coverage of mouse and rat mature rRNA, as well as improved enzyme formulations. Q: Which species are compatible with the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) for? Will it work for other species? A: The kit depletes rRNA from human, mouse and rat total RNA. It can work with samples from other species to the degree to which the rRNA sequences are similar to that of human, mouse and rat. Q: Which rRNA subunits are depleted with the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat)? A: The kit depletes both cytoplasmic (5S, 5.8S, 18S, and 28S, human ITS, ETS) and mitochondrial (12S and 16S) rRNA. 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 total RNA input should I use? A: The kit has been optimized for 5-1000 ng high quality total RNA (RIN = 8-9) and 10-1000ng degraded or fragmented RNA (RIN ≤ 2, e.g. FFPE RNA). 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: What is the expected RNA yield recovered after rRNA depletion? A: The percentage of RNA isolated after rRNA depletion will vary depending on the type of RNA (different sources or RNA species). Typically, we observe ~98% of rRNA will be removed. Q: What is the percentage of rRNA remaining after depletion? A: The percentage of rRNA remaining after depletion will vary by species. Typically, we observe between 1% (human) and 3% (mouse/rat) rRNA remaining. 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: Does the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) work for ribosomal footprinting or profiling applications? A: The protocol provided with this kit has not been validated for ribosomal footprinting or profiling. 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: To remove ribosomal RNA from total RNA, should I use the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) or the NEBNext rRNA Depletion Kit v2 (Human, Mouse, Rat)? A: The NEBNext rRNA Depletion Kits enrich for polyadenylated transcripts as well as non-polyadenylated RNA (such as non-coding RNAs and antisense transcripts). The NEBNext Poly(A) mRNA Magnetic Isolation Module only retains the polyadenylated transcripts. The NEBNext rRNA Depletion Kits can be used with low quality Total RNA samples. The NEBNext Poly(A) mRNA Magnetic Isolation Module requires high quality Total RNA. Q: Can I use more than one type of NEBNext Depletion Solution in the NEBNext RNA depletion workflow? A: Yes, it is possible to combine probes. Any combination of NEBNext Depletion Solutions (NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7400/E7405); NEBNext rRNA Depletion Kit (Bacteria) (NEB# E7850/E7860); NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7750/E7755) can be used at the probe hybridization step. The volume of the total sample during the probe hybridization step must remain constant (15 µl). The volume of the probe hybridization buffer must always remain 2 µl. The volume of the individual depletion solutions should be determined experimentally with an input titration experiment, since the optimal amount of probes will vary from sample type to sample type. In the absence of this experimental data, the probes can be added in a 1:1 ratio. Since the total volume of the reaction is 15 µl, the volume of total RNA that can be added is dependent on how much volume is left after adding the 2 µl Hybridization Buffer and the volume of the Depletion Solutions. If you are diluting your total RNA in nuclease free water prior to depletion, the volume of nuclease free water can be reduced to accommodate a higher volume of NEBNext Depletion Solution. RNA/PROBE HYBRIDIZATION REACTION VOLUME Total RNA in Nuclease-free Water (10 ng–1 μg) Variable, to 11 μl Combination of Depletion Solutions Variable (white) NEBNext Probe Hybridization Buffer 2 μl Total Volume 15 μl Please refer to the protocol in the NEB #E7400, NEB #E7750 or NEB #E7850 manual. 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.