For use with the Illumina® sequencing platform, RNA libraries are prepared by depletion of abundant RNAs from total RNA, and synthesis of cDNA 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). For more information, choose either the Ultra™ II Directional or Non-directional RNA Library Preparation Workflow for Illumina tab below.
Abundant RNAs can be removed from total RNA directly using an RNA depletion product specific for the sample species being used, or by enrichment of mRNA by a poly(A)-based method.
For single cell and ultra-low input RNA samples, libraries are constructed with a template-switching method to generate full-length cDNAs, followed by Ultra II library preparation. For more information, choose the Single Cell/Low Input RNA Library Preparation Workflow for Illumina tab below.
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. In the novel NEBNext® workflow, the introduction of the RT primer ahead of ligation of the 5’ adaptor prevents formation of contaminating adaptor-dimers. For more information, choose the Small RNA Library Preparation Workflow for Illumina tab below.
NEBNext reagents are available for each step in RNA and small RNA library preparation workflows for the Illumina platform. Reagents from NEB are available in multiple formats, and a wide range of index primers for multiplexing is available. Each of these reagents has been functionally validated by library preparation followed by Illumina sequencing.
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Learn more about the streamlined workflow for the NEBNext Ultra II Directional RNA Library Prep Kit.
Watch as Deyra explains how to optimize your RNA inputs for successful NGS library preparation.
This video will help you optimize fragmentation for RNA workflows in the NEBNext product line.
The common problem of adaptor dimer formation during small RNA library construction can be avoided by using NEBNext® protocols. Learn about this technique, and how it improves both performance and sensitivity in library construction.
Learn how Biomek automation of NEBNext RNA library preparation enables high-throughput, reproducible production of libraries with high yield and complexity. In this webinar Zach Smith from Beckman Coulter describes strand-specific RNA library preparation using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina and discusses Biomek automation of this method.