NEBNext® mRNA Library Prep Reagent Set for Illumina®


The NEBNext® mRNA Library Prep Reagent Set for Illumina® contains enzymes and buffers that are ideally suited for sample preparation for next-generation sequencing, and for preparation of expression libraries. Each of these components must pass rigorous quality control standards and are lot controlled, both individually and as a set of reagents.

Functional Validation
Each set of reagents is functionally validated together through construction and sequencing of an RNA library on the Illumina sequencing platform.

Lot Control

The lots provided are managed separately and qualified by additional functional validation. Individual reagents undergo standard enzyme activity and quality control assays, and also meet stringent criteria in the additional quality controls listed on each individual component page

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
NEBNext RNA Fragmentation Buffer10X
NEBNext RNA Fragmentation Stop Solution10X
Random Primers
NEBNext First Strand Synthesis Reaction Buffer5X
NEBNext Second Strand Synthesis Enzyme Mix
NEBNext Second Strand Synthesis Reaction Buffer10X
Klenow Fragment (3´→5´ exo-)
Quick T4 DNA Ligase-20
Nuclease-free Water-20
Phosphorylation Reaction Buffer-2010X
T4 Polynucleotide Kinase-20
Deoxyadenosine 5´- Triphosphate (dATP)-201 mM
NEBuffer 2 for Klenow Fragment (3´→ 5´ exo)10X
Deoxynucleotide Solution Mix-2010 mM
T4 DNA Polymerase-20
DNA Polymerase I, Large (Klenow) Fragment-20
ProtoScript® II Reverse Transcriptase200,000 units/ml
Quick Ligation Reaction Buffer-202X
NEBNext® Q5® Hot Start HiFi PCR Master Mix-20
NEBNext High-Fidelity 2X PCR Master Mix (SAMPLE)2X

Properties and Usage

Materials Required but not Supplied

  • 80% Ethanol (freshly prepared)
  • 0.1X TE, pH 8.0
  • 10 mM Tris-HCl, pH 7.5–8.0 (optional)
  • NEBNext Singleplex or Multiplex Oligos for Illumina (NEB #E7350, #E7335, #E7500, #E7710, #E7730, #E6609 or #E7600)
  • Agencourt® AMPure® XP Beads (Beckman Coulter, Inc., #A63881)
  • Agencourt RNAClean XP Beads (Beckman Coulter, Inc., #A63987)
  • Agilent Bioanalyzer

Storage Temperature



  1. What type of sample prep/library construction can I use these Sets for?
  2. How much starting material do I need to use when preparing libraries using the NEBNext® RNA sets?
  3. Do I need any reagents that are not supplied in the kit?
  4. How do I know the concentration of my index primers? 


  1. Please see manual (NEB #E6100) for protocols


The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

Usage Guidelines & Tips

Interactive Tools

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.