Next Generation Sequencing

Sabrina Shore, Jordana M. Henderson, Anton P. McCaffrey

Next-generation small RNA sequencing is a valuable tool which is increasing our knowledge regarding small noncoding RNAs and their function in regulating genetic information. Library preparation protocols for small RNA have thus far been restricted due to higher RNA input requirements (>10 ng), long workflows, and tedious manual gel purifications. Small RNA library preparation methods focus largely on the prevention or depletion of a side product known as adapter dimer that tends to dominate the reaction. Adapter dimer is the ligation of two adapters to one another without an intervening library RNA insert or any useful sequencing information. The amplification of this side reaction is favored over the amplification of tagged library since it is shorter. The small size discrepancy between these two species makes separation and purification of the tagged library very difficult. Adapter dimer hinders the use of low input samples and the ability to automate the workflow so we introduce an improved library preparation protocol which uses chemically modified adapters (CleanTag) to significantly reduce the adapter dimer. CleanTag small RNA library preparation workflow decreases adapter dimer to allow for ultra-low input samples (down to approx. 10 pg total RNA), elimination of the gel purification step, and automation. We demonstrate how to carry out this streamlined protocol to improve NGS data quality and allow for the use of sample types with limited RNA material.