RNA Library Prep for Illumina

NEBNext has multiple library prep options to help you sequence all of your RNA samples.

Which way to RNA? 

  • The fastest, easiest way to generate high-quality RNA libraries is to use the NEBNext UltraExpress RNA Library Prep Kit (NEB #E3330). With a 3-hour workflow, when combined with poly(A) mRNA enrichment or rRNA depletion kits, you can go from total RNA to high-quality directional libraries in a day. A single protocol, with the same adaptor concentration and number of PCR cycles, can be used for all samples in the 25-250 ng total RNA input range, and with the use of master mixes and fewer cleanups, you’ll see savings in tips and tubes as well as time.
  • For input amounts that are outside of the 25-250 ng range, or are challenging (e.g., FFPE), the NEBNext Ultra II Directional RNA Kit is recommended. This tried and tested kit generates high-yield, high-quality strand-specific libraries from as little as 5 ng input.
  • If non-stranded libraries are preferred, the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (NEB #E7770) is available. 
  • The adaptors and primers in NEBNext Multiplex Oligos enable simple yet powerful NGS sample multiplexing

For microRNAs and other small RNAs, our novel Small RNA workflow has been optimized to minimize adaptor-dimers, while producing high-yield, high-diversity libraries.

For single cell and ultra-low input RNA, our simple and robust workflow generates full length cDNAs, followed by conversion to sequence-ready libraries using the Ultra™ II FS workflow, that incorporates enzymatic DNA fragmentation.



NEBNext® Ultra™ II Directional RNA Library Prep

NEBNext UltraExpress™ RNA Library Prep


Unprecedented performance, enabling a broader input range and lower bias; optimized for increased library complexity and improved transcript coverage; robust performance even with low quality RNA, including FFPE-treated samples

New! The latest generation of NEBNext RNA library prep offers a streamlined workflow and enables sample-to-library time under 1 day; high-quality libraries with fewer cleanups and pipetting steps; compatible with customized protocols, if desired

Workflow Time

5 hours for library prep; 1.5 days from input to library, including depletion/enrichment

3 hours for library prep; < 1 day from input to library, including depletion/enrichment

Input Amounts

10 ng – 1 µg of Total RNA

25 ng – 250 ng of Total RNA

Available with or without beads?

Modules available?

  • Ultra II Directional RNA Second Strand Synthesis Module (NEB #E7550)
  • Ultra II RNA First Strand Synthesis Module (NEB #E7771)

RNA Depletion & mRNA Enrichment

Both NEBNext Ultra II and NEBNext UltraExpress RNA Library Prep Kits are compatible with NEBNext RNA Depletion and mRNA Enrichment reagents, which can be found here.

Automation compatibility?



Stranded/directional workflow?

Yes. For a non-directional library, choose NEBNext Ultra II RNA Library Prep (NEB #E7770, #E7775)



NEBNext RNA library prep reagents are also available at scale for your high-throughput and/or development needs. Contact the Customized Solutions Team for more details on custom product options, large volumes, or OEM materials.

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Related Categories
NEBNext UltraExpress™ RNA Library Prep workflow
Workflow image
Single Cell/Low Input RNA Library Preparation Workflow for Illumina
Ultra II Non-directional RNA Library Preparation Workflow for Illumina
Ultra II Directional RNA Library Preparation Workflow for Illumina
Small RNA Library Preparation Workflow for Illumina
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