NGS small RNA library preparation has historically suffered from two major drawbacks: substantial bias introduced during ligation steps and necessity of size-selecting libraries by PAGE, which greatly limits throughput and ease of automation. The NEXTflex Small RNA Sequencing Kit v3 uses adapters with randomized ends to substantially reduce ligation bias, and employs a two-pronged adapter-dimer reduction strategy to greatly reduce formation of adapter-dimer, allowing gel-free library preparation. Following 3' adapter ligation, excess 3' adapter is depleted with a size-based selection, followed by inactivation of any leftover 3' adapter with an enzymatic method. This two-pronged strategy results in negligible adapter-dimer product in the post-PCR sample when a limited number (≤18) of amplification cycles are used, allowing final selection by SPRI beads rather than with PAGE. This gel-free protocol significantly improves the throughput and potential for automation for small RNA library preparation.
Substantial reduction of adapter-dimer also allows a library preparation protocol from low inputs (≥1 ng total RNA), as substantially more PCR cycles can be performed without adapter-dimer dominating the final library. At least two independent studies have shown that PCR introduces negligible bias into small RNA library preparation, so reliable data may still be generated with high PCR cycle numbers, although size selection by PAGE will typically be necessary. This low input protocol allows reduced-bias small RNA libraries to be created from samples with limited amounts of available RNA that previously would have been impossible, greatly increasing the accessibility and applicability of small RNA-Seq.
In conclusion, the NEXTflex Small RNA Sequencing Kit v3 combines reduced bias with either gel-free or low-input library preparation, allowing greater throughput, automation potential, and creation of libraries from a greater variety of sample types where limited starting material previously made small RNA-Seq impossible.
