New England Biolabs, E7770S, NEBNext® Ultra™ II RNA Library Prep Kit for Illumina®

Do you prefer to use non-strand-specific library prep methods, but need increased sensitivity and specificity from your RNA-seq experiments, from ever-decreasing amounts of input RNA? To address these challenges, our next generation of non-directional RNA library prep kit has 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. Related Categories RNA Library Prep for Illumina 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) 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: 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. Q: Is the Monarch® Total RNA Miniprep Kit compatible with NEBNext® reagents for RNA Library Prep? A: Yes. RNA purified with this kit can be used for RNA library preparation using NEB's NEBNext RNA Library Prep reagents. Monarch-purified RNA can be used to prepare high quality RNA-seq libraries for gene expression analysis. Transcript levels in Universal Human Reference RNA (UHRR, Agilent) are compared before and after re-purification using either Qiagen RNeasy® or the Monarch Total RNA Miniprep Kit. Strong correlation with untreated UHRR is observed for both methods (Pearson R > 0.99 for both samples). All samples display consistent end-to-end coverage of transcripts indicating an absence of detectable degradation during purification. Poly-A selected RNA was selected from 100 ng of untreated, Qiagen and Monarch samples using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). RNA-seq libraries were then prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina® (NEB #E7760) before sequencing on a Miseq® instrument (2 x150). 1.6M reads were randomly sampled from each library and adaptor trimmed (Seqprep v1.1). Levels of all Gencode v26 transcripts were assessed using Salmon (0.4) and plotted above (panel A). Average 5'-3' Coverage of Gencode v26 transcripts (assessed by Picard's CollectRnaSeqMetrics 1.56 after mapping to the GRCh38 reference genome with Hisat v2.0.3 and marking duplicates with Picard's MarkDuplicates 1.56 ) is shown below (panel B). 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: 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: Is it possible to reduce the volume of beads added during the second strand synthesis cleanup step of the protocol for E7760, E7765, E7770, and E7775? 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 magnet types. 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. 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 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: What is the difference between the NEBNext Ultra II RNA Library Prep Kit (NEB #E7770) and the original NEBNext Ultra RNA Library Prep Kit (NEB #E7530)? A: Each step in the workflow for the NEBNext Ultra II RNA Library Prep Kit has been optimized, including the incorporation of several new reagents. This enables the use of 10 to 20-fold lower input amounts and fewer PCR cycles compared to the original kit, and results in library yields that are substantially higher, without compromising library quality. 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 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.