The purpose of this tech-note is to describe the automation of the New England Biolabs NEBNext (Registered trademark) Ultra Directional RNA Library Prep Kit for Illumina on the Beckman Coulter Biomek FXP automated liquid handler. The Biomek NEB Ultra Directional RNA Method was designed to work with NEBNext Ultra Directional RNA Library Prep Kit for Illumina protocol (Catalog Number E7420S/L), and provides a solution for simultaneously constructing up to 96 individually bar-coded directional RNASeq libraries per method run.
The method provides users with the option to utilize either off-deck incubations using an external thermocycler, or to perform incubations on-deck with a Biometra TRobot thermocycler integrated to the Biomek liquid handler. The method employs individual Span-8 probes to deliver enzyme and reagent transfers while DNA cleanup, wash, and elution transfers are performed using the multi-channel 96 pipetting head. A static Peltier unit ensures that enzyme master mixes are kept cool during the course of the method.
The data presented below includes a comparison of library construction using the Biomek FXP method at 25ng and 100ng total RNA inputs. Automated sample preparation allows for the simultaneous construction of up to 96 directional RNASeq libraries in about 9.5 hours. For convenience, this method can be run with in a single nine and one-half hour run, or as a two day run, with day 1 consisting of sample preparation through cDNA synthesis and day 2 consisting of the sample preparation through the end of library construction. Figure 1 shows the workflow of the NEBNext Ultra Directional RNA Library Prep Kit for Illumina protocol.