NEBNext® Ultra™ II Directional RNA Workflow

Learn more about the streamlined workflow for the NEBNext Ultra II Directional RNA Library Prep Kit.


The NEBNext Ultra II Directional RNA Library Prep kit is the next generation of strand specific library construction kit compatible with Illumina sequencing.  It enables production of high-quality libraries from low nanogram to microgram input amounts of total RNA using a streamlined workflow.

Before library construction, ribosomal RNA must be removed by either of two methods compatible with the kit; Poly(A) mRNA enrichment or ribosomal RNA depletion.  After removal of ribosomal RNA, the RNA of interest is fragmented by incubation at high temperature in the presence of divalent cations, followed by hybridization to the random hexamer primer for reverse transcription.  

Next, first strand cDNA synthesis occurs.  A reverse transcriptase extends from the hybridized primer along the length of the RNA molecule.  Actinomycin D is added to prevent second strand synthesis in this step.  

Second strand synthesis then takes place.  Strand specific library construction is different from standard library construction in this step.  As uracils are incorporated into the second strand and this gives the protocol its name.  the "dUTP method".  

After a clean up step, the double stranded DNA molecules are end repaired to create blunt phosphorylated ends and a single adenine overhang is added to the 3 prime end.  This enables efficient ligation to adapters with thymine overhangs.  These adaptors can be the hairpin loop-shaped NEBNext adaptor or other Illumina sequencing compatible adaptors.

The uracil containing strand is then selectively removed with the USER enzyme which is a mix of UDG and Endo VIII.  If applicable, USER also opens the hairpin loop of the NEBNext adaptor.  

What remains is a single strand in the final library, providing the library's strand-specificity.   Next, either a clean up for smaller fragments or a size selection for larger fragments is performed. 

The final step is PCR amplification of the library using a Q5 High fidelity DNA polymerase master mix with the recommended number of cycles based on input amount.  This step selects for molecules with an adaptor at each end, increases library yield, incorporates bar codes to enable multiplexing and incorporates P5 and P7 sequences required downstream  in the sequencing process.  

After a cleanup the library is ready to move to cluster generation and sequencing.

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