The Wellcome Trust Sanger Institute has recently re-evaluated its RNA-seq library preparation methods and has chosen to implement the NEBNext Ultra II Directional RNA Library Prep Kit. Our previous RNA-seq library preparation required a minimum input of 500 ng total RNA, but the high efficiency of the NEBNext Ultra II kit has allowed us to reduce this to either 100 ng for a standard pipeline, or down to just 5 ng for a low input option, enabling RNA-seq to be carried out from just 5,000-10,000 cells.

At The Earlham Institute we process many sample types; these include plant, microbial and animal. We trialed NEB’s Ultra II Directional RNA Library Prep using three plant species that have previously been problematic with other RNA-seq kits. We were thrilled with how well and how consistently this kit performed. The NEBNext^® Poly(A) mRNA Magnetic Isolation Module was also effective and SortMeRNA showed that we had less than 2% contamination for each plant species. The strand specificity was also good at 98%. We were so pleased with the results that we are automating this protocol ready for production with in the EI Genomics Lab.

The Ultra II RNA kit has allowed us to reduce the input for directional polyA+ RNAseq libraries by a factor of 10 or more. We can now make a library with only 10 ng high quality total RNA and get the same gene expression profile as for 1 µg input. We've even pushed the input as low as 1 ng for very high quality total RNA. The new Ultra II RNA kit makes RNAseq achievable for low yield samples, we actually need more RNA for quality control than for library prep! Furthermore, the library prep protocol is streamlined compared to the previous Ultra RNA kits, including a reduction in AMpure bead cleanups and PCR cycles, resulting in better libraries for less time and resources.

I used the NEBNext Ultra II Directional RNA Library Prep Kit to process very low input (7–8 ng total RNA) samples from difficult to obtain tissue for one of our customers, and I am very pleased with the results.

The Ultra II Directional RNA kit is very impressive... I’ve pushed the lower limit of input down to 1 and 2 ng and still achieved libraries with a good depth in complexity. Importantly, I've also noticed a reduction in adaptor-dimers and duplication in sequencing. The Ultra II is my “go-to” kit for all RNA-seq library preparations.