RNA-seq

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.