Next generation sequencing (NGS) can be used to determine the presence and quantity of RNA species in a sample enabling sensitive and accurate gene expression analysis. For the commonly used Illumina® sequencing platform, mRNA libraries are prepared by construction of a cDNA library, followed by DNA library preparation steps: repair of 3´ and 5´ ends followed by the addition of a non-templated dA-tail before ligation with an adaptor. Libraries are size selected after adaptor ligation, and amplified via polymerase chain reaction (PCR).

Small RNA libraries are constructed using a different workflow, in which adaptors are ligated directly to the small RNA molecules, followed by reverse transcription, PCR amplification and size selection.

Libraries are then clonally amplified and sequenced using sequencing by synthesis (sbs) methods, such as the Illumina sequencing platform.

NEBNext® reagents are available for each step in mRNA and small RNA library preparation workflows for the Illumina platform. Reagents from NEB are available in a set, master mix or module format. Each of these reagents has been functionally validated by preparation of an RNA library from an accepted RNA reference sample, followed by Illumina sequencing.

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FAQs for RNA-seq

Protocols for RNA-seq

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  • Latest NEB Expressions

    This edition of NEB expressions discusses breakthroughs in Golden Gate Assembly research, that now enables 20+ fragment assembly with high efficiency and accuracy. New products include the NEBNext® Single Cell/Low Input RNA Library Prep Kit and the NEBNext Direct® Custom Ready Panels for NGS Target Enrichment. Lastly, learn about the importance of basic research at NEB in an interview with NEB’s Research Director.

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    Learn more about NEB's products for RNA synthesis, which range from template generation to poly(A) tailing.

  • RNA Technical Guide

    Find in depth information on tools designed to streamline your RNA workflows, including RNA extraction & purification, qPCR and RT-qPCR, RNA-seq, RNA synthesis and gRNA synthesis.

  • Usage Guidelines

  • Getting Started with RNA-Sequencing (RNA-Seq)

Ultra II Non-directional RNA Library Preparation Workflow for Illumina

Ultra II Directional RNA Library Preparation Workflow for Illumina

RNA Product Details for Illumina

Small RNA Library Preparation Workflow for Illumina

Small RNA Product Details for Illumina