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RNA-seq (RNA sequencing) has become a powerful method for transcriptome analysis. Using next-generation sequencing (NGS) it is widely used for gene expression analysis, including detection of mutations, fusion transcripts, alternative splicing, and post-transcriptional modifications.  In fact, RNA-seq of the whole-transcriptome from patients provides a diagnostic approach to identify many of the mutations that affect RNA splicing or gene expression that led to clinical diseases. Beside the frequently studied mRNA molecules (messenger RNA), RNA-seq can also be applied to investigate different RNA populations, including small RNAs like miRNA and small non-coding RNAs, which are particularly useful in cancer prognosis and diagnosis. Overall, NGS-technologies have revolutionized sequence-based research and clinal studies through its advantages such as high throughput, high sensitivity, and high speed.

Getting started – Creating an RNA-Seq Library

For the commonly used Illumina® sequencing platforms, mRNA 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).

The NEBNext Ultra II workflow presents the heart of NEBs portfolio for next generation sequencing library preparation. NEBNext® reagents are available in multiple formats for each step in RNA library preparation workflows for the Illumina platform. The NEBNext RNA library prep kits are compatible with a wide range of inputs and sample qualities and produces RNA libraries of highest quality and yield.

Standard methods for RNA library preparation do not retain information on the DNA strand from which the RNA strand was transcribed. Therefore, NEB offers the ability to choose either the Ultra™ II Directional (#E7760/#E7765) or Non-directional RNA Library Prep kit (#E7770/#E7775). The ability to obtain information on the originating strand is useful for many reasons including the identification of antisense transcripts, determination of the transcribed strand of noncoding RNAs, and determination of expression levels of coding or non-coding overlapping transcripts. Overall, the ability to determine the originating strand can substantially enhance the value of an RNA-seq experiment.

An important factor to consider when building RNA libraries is the enrichment of RNA by either rRNA depletion or poly(A) mRNA isolation. The majority of RNA molecules present in a cell are ribosomal RNA (rRNA). These are generally not of interest and should be removed before making a library from the RNA of interest. Similarly, globin RNA is generally removed from blood samples and chloroplast RNA is often removed from plant leaf samples. 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 (NEBNext Poly(A) mRNA Magnetic Isolation Module #E7490.

All in all, the Ultra II workflows for RNA are streamlined for minimal hands-on time. The NEBNext products including the Ultra II RNA library preparation kits demonstrate robust performance even with low quality RNA and perform well and consistently resulting in higher quality libraries.


Streamline your RNA-seq workflow with NEBNext RNA Library preparation kits

Purchase a NEBNext Ultra II RNA kit from New England Biolabs and receive a 20% discount on an RNA enrichment kit of your choice.

NEBNext Ultra II RNA kits are available for directional (NEB#E7760)*1 and non-directional (NEB#7770)*2 library construction for the Illumina platform. With their streamlined and automatable workflow libraries of high yields and quality can be generated. In addition, NEBNext RNA Library preparation kits are compatible with a wide range of inputs, from single cell to microgram of total RNA, as well as high and low-quality samples. For optimal RNA sample preparation, RNA enrichment is embedded in the workflow through either ribosomal RNA depletion or poly(A) mRNA enrichment (NEBNext Poly(A) mRNA Magnetic Isolation Module #E7490).

*1 available with sample purification beads NEB#E7765

*2 available with sample purification beads NEB#E7775

Offer is valid until 31 March 2023. Use code RNAseq2023-Q1 when placing your order to get the discount.