New England Biolabs, E7765L, NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads

Do you need increased sensitivity and specificity from your RNA-seq experiments? Do you have ever-decreasing amounts of input RNA? To address these challenges, our next generation of strand-specific RNA library prep kits have been reformulated at each step, resulting in several fold higher yields of high quality libraries and enabling use of lower input amounts and fewer PCR cycles. The Ultra II Directional RNA kit uses the “dUTP method” (1, 2) for strand-specificity, and has a streamlined, automatable workflow. Related Categories Automation for NEBNext® NGS Library Prep,, RNA Library Prep for Illumina,, Next Generation Sequencing Library Preparation Specification Materials Required but not Supplied NEBNext Singleplex or Multiplex Oligos for Illumina (NEB.com/oligos) or customer supplied oligos Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua® 96S Super Magnet Plate (#A001322), or equivalent) SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881) 80% Ethanol (freshly prepared) Thermal Cycler NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) or NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400 or NEB #E7405) or NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7750 or NEB #E7755) or NEBNext® rRNA Depletion Kit (Bacteria) (NEB #E7850 or NEB #E7860) or NEBNext® RNA Depletion Core Reagent Set (NEB #E7865 or NEB #E7870) or NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310 or NEB #E6350) FAQ Q: I have USER enzyme from my library prep kit (blue cap) and USER enzyme from my NEBNext Index Primer kit (red cap). Are they the same? A: Yes, it is the same. We provide USER in the NEBNext Ultra RNA Directional kits since it is needed for the workflow (blue cap). USER enzyme with the red cap is provided in our oligo kits for use with the NEBNext adaptor. If you are using NEBNext Ultra I or Ultra II Directional RNA Library prep kit with NEBNext adaptor only one USER step is required and either vial of USER enzyme provided can be used. Q: What is the difference between the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (NEB #E7775) and the NEBNext Ultra II Directional RNA Library Prep with Sample Purification Beads (NEB #E7765)? A: The NEBNext Ultra II Directional RNA Library Prep workflow preserves information about RNA strand orientation while the NEBNext Ultra II RNA Library Prep workflow does not. The NEBNext Ultra II Directional RNA Library Prep contains dUTP in the second strand synthesis buffer that allows labeling of the second strand cDNA and subsequent excision with USER Enzyme. Q: What is the starting material I need to use when preparing libraries using the NEBNext Ultra II Directional RNA kit? A: The starting material is Total RNA (10 ng-1 μg); previously isolated mRNA (1-100 ng) or ribosomal-depleted RNA (1-100 ng). Q: Which kit can I use to isolate Poly (A) mRNA from Total RNA? A: To isolate poly (A) mRNA from Total RNA use the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Q: Which kit can I use to deplete ribosomal RNA (rRNA) from human, mouse or rat RNA? A: To deplete rRNA from human, mouse or rat, use the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #7400). Q: Can I use this NEBNext kit with adaptors and primers from other vendors than NEB? A: Yes, it should be possible to use adaptors and primers from vendors other than NEB with this NEBNext kit. The adaptor must be a T overhang adaptor and the final library yield may be slightly lower than when using the NEBNext adaptors for Illumina®. Instructions for use with non-NEBNext, full length barcoded adaptors and P5 and P7 universal PCR primers can be found in the manuals for the NEBNext indexed/ UMI adaptor manuals. These manuals can be found at neb.com/E7416 (RNA) or neb.com/E7395 (DNA). Choose the kit manual for the NEBNext kit you will be using. The manuals are written specifically for the NEBNext indexed/ UMI adaptors, the same protocol should work with other adaptors of similar design. We recommend performing a proof of principle experiment with the desired library prep kit and desired indexed adaptors and using expendable samples for optimization of experimental conditions. Q: What do I do if I see a precipitate in the Ultra II End Prep Reaction Buffer? A: It is not uncommon to see a white precipitation in the Ultra II End Repair Buffer. If this occurs, allow the mixture to come to room temperature and pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix. Q: Is it possible to reduce the volume of beads added during the second strand synthesis cleanup step? A: The total volume of the sample when beads are added during the second strand synthesis cleanup step is 224 µl, which may exceed the volume of some 0.2 ml PCR tubes and plates. In addition this volume may not allow for complete separation of the beads using some magnets. Many PCR plates and tubes with a nominal value of 0.2 ml have a larger actual capacity that will accommodate a 224 µl sample. However, if the plate or tube you prefer to use does not allow for a 224 µl sample volume it is possible to reduce the volume of beads to 1.5 x (120 µl) for a total liquid volume of only 200 µl. In our hands we did not see a reduction in final library yield. Q: What is the composition of the random primers? A: The random primers are random hexamers supplied at a 1.9 mM. Q: Does the kit include adaptor and primers? A: No. The kit can be used with the NEBNext Multiplex Oligos for Illumina. For a complete list, see NEBNext® Multiplex Oligos Selection Chart. For use with NEBNext library prep kits, consult the library prep kit manual. The multiplex oligo kit specific manuals also contain tips for setting up PCR reactions. For color balancing options, please see NEBNext® Index Oligo Selector for valid barcode combinations.