Improved Small RNA Library Preparation Workflow for Next-Generation Sequencing

Next-generation sequencing (NGS) is used to analyze microRNA (miRNA), a class of small non-coding RNAs that are important therapeutic targets and diagnostic markers. Commercially available small RNA sequencing library preparation kits require large inputs (>100 ng) and a laborious gel purification step. Additionally, commercial kits are hindered by adapter dimer formation, where 5' and 3' adapters ligate without an intervening RNA insert. Adapter dimers preferentially amplify during PCR. This is exacerbated at low RNA inputs where adapter dimer formation can greatly diminish usable sequencing reads.

We describe an optimized workflow which suppresses adapter dimers, works with low RNA input and eliminates the need for a gel purification step. Our workflow introduces chemically modified adapters that efficiently form libraries while reducing adapter dimer formation. TriLink’s modified adapter workflow allows RNA inputs as low as 1 ng with less than 1% adapter dimer reads when gel purified. Furthermore, when our workflow is combined with bead-based size selection purification, automation becomes possible.