New England Biolabs, E3330S, NEBNext UltraExpress® RNA Library Prep Kit

The NEBNext UltraExpress RNA Library Prep Kit is the latest generation of NEBNext RNA library prep, and it has a fast, streamlined workflow. With a 3-hour library prep protocol, the kit enables creation of high-quality RNA directional libraries in a single day, in conjunction with mRNA enrichment or rRNA depletion kits. Related Categories Library Preparation for Illumina,, RNA Library Prep for Illumina,, Next Generation Sequencing Library Preparation Specification Materials Required but not Supplied NEBNext Multiplex Oligos for Illumina® NEBNext Multiplex Oligos options can be found at www.neb.com/oligos. Alternatively, customer supplied adaptor and primers can be used; please see information in link: https://www.neb.com/faqs/2019/03/08/can-i-use-this-nebnext-kit-with-adaptors-and-primers-from-other-vendors-than-neb SPRIselect™ Reagent Kit (Beckman Coulter®, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881) Magnetic Rack (NEB S1515S, Alpaqua® cat. #A001322, or equivalent) 80% Ethanol (freshly prepared) Thermal cycler DNase-, RNase-free PCR strip tubes, for example TempAssure® PCR flex-free 8-tube strips (USA Scientific® #1402-4708) Bioanalyzer® or TapeStation® (Agilent® Technologies, Inc.) and associated reagents and consumables For use with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490): 1.5 ml Microcentrifuge tube and NEB #S1506 Magnet stand or equivalent (for washing beads only) For use with NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400) and other NEBNext RNA depletion kits that do not include beads (NEB #E7750, E7850, E7865): Agencourt® RNAClean® XP Beads (Beckman Coulter, Inc. #A63987) FAQ Q: Is the NEBNext UltraExpress® RNA library prep kit a directional (stranded) library kit? A: Yes, the NEBNext UltraExpress RNA library kit has a directional (stranded) RNA library workflow. Q: What is the difference between the NEBNext UltraExpress® RNA Library Prep Kit and the NEBNext® Ultra™ II Directional RNA Library Prep Kit? How do I choose the right one? A: The NEBNext UltraExpress RNA Library Prep Kit has been optimized for streamlined library prep processing with one adaptor concentration and one PCR cycling condition used for all samples within the starting amounts of 25-250 ng of total RNA. The NEBNext Ultra II Directional RNA Library Prep Kit has more customized conditions based on input amount, more steps and cleanups, a longer workflow time, and a broader input range recommendation (10 ng – 1 µg total RNA). Both kits produce high yields of high-quality libraries, but we recommend choosing NEBNext UltraExpress RNA for all of your standard samples within the stated input amount range, where speed is paramount. We recommend choosing NEBNext Ultra II Directional RNA for more complex and precious samples. Q: Is it possible to use total RNA input amounts outside the suggested range for the NEBNext UltraExpress® RNA library Kit? A: Yes. However, it is possible that doing so may result in higher adaptor dimers or lower yields. Optimization of adaptor dilutions and PCR cycles should be performed experimentally by the user and will be affected by sample quality. We suggest using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB #E7760/E7765) for total RNA inputs outside the ranges recommended for the NEBNext UltraExpress RNA Library Prep Kit. Q: Does the NEBNext UltraExpress® RNA Library Prep Kit include poly(A) mRNA isolation or rRNA Depletion reagents? A: No. For poly(A) mRNA isolation, we recommend the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). For rRNA Depletion, we recommend any of our RNA depletion kits (e.g., NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat)) (NEB #E7400/E7405). Q: Do the NEBNext UltraExpress® Library Prep kits come with beads? A: No, we recommend using the SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881). Q: Can I perform poly(A) mRNA isolation or rRNA depletion and then freeze my samples at -80°C before beginning library preparation with the NEBNext UltraExpress® RNA Library Prep Kit? A: Yes, however, some modifications will be required. If you plan to freeze your mRNA- or rRNA-depleted RNA before library prep, elution of your samples in 4 µl Tris [10 mM Tris HCl (pH 7.5), included with the NEBNext® Poly(A) mRNA Magnetic Isolation Module E7490] or nuclease free water is recommended. 2.5 µl of this eluate will then be transferred into a fresh tube and combined with an equivalent volume (2.5 µl) of NEBNext UltraExpress RNA Fragmentation Mix, for a total volume of 5 µl. Slightly greater sample loss is expected using this method compared to the standard protocol. However, adaptor input and cycling recommendations will remain the same. The 5 µl sample will then be fragmented in solution. Q: What should I do if I see adaptor dimer in my NEBNext UltraExpress® libraries? A: The phased cleanup step following PCR is specifically designed to reduce adaptor dimer; please follow the instructions for this step as written before attempting any additional recommendations. If you still see small amounts of adaptor dimer in samples, we recommend a 0.9X SPRI bead cleanup after pooling libraries or further optimization of the adaptor amount. Q: Can this kit be used with adaptors and primers from suppliers other 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®. Q: Are the USER enzyme included in the NEBNext UltraExpress® RNA Library Prep Kit (blue cap) and the USER enzyme included with NEBNext Index Primer sets (red cap) the same? A: The NEBNext UltraExpress RNA Library Prep Kit's USER formulation is a newer one. However, it can still be used interchangeably with the version included with the NEBNext adaptor. There is no difference in the activity of the enzyme. Q: What sample types and quality are compatible with the NEBNext UltraExpress® RNA Library Prep Kit? A: The NEBNext UltraExpress RNA Library Prep Kit has been extensively tested using Universal Human Reference RNA, bacterial RNA mix, and RNA extracted from human whole blood, but it is expected to be compatible with a range of sample types with good quality RNA. While successful libraries have been produced using low-quality samples, optimizing adaptor concentration and PCR cycles is necessary to achieve good quality libraries. We do not recommend using <100 ng of low-quality total RNA (e.g., from FFPE samples) with the NEBNext UltraExpress RNA Library Prep Kit. For highly fragmented RNA samples and low-input, low-quality samples, we recommend using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB #E7760/E7765). 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: 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.