For any RNASeq experiment, the RNA used must be of high quality to achieve optimal sequencing results. Researchers from Beckman Coulter Life Sciences have recently described the automation of the New England Biolabs NEBNext Ultra Directional RNA Library Prep Kit for Illumina on the Beckman Coulter Biomek® FXP automated liquid handler (BFXP). The Biomek® NEB Ultra Directional RNA Method was developed to work with the library preparation kit protocol and enable the simultaneous construction of up to 96 directional RNASeq libraries for each method run.
This method allows users to choose whether they perform incubations off-deck using an external thermocycler or on-deck with an integrated Biometra TRobot thermocycler. The BFXP uses individual Span-8 probes to perform enzyme and reagent transfers and a multi-channel 96 pipetting head to carry out DNA clean-up, wash and elution transfers. The method also employs a static Peltier unit to ensure enzyme master mixes are kept cool.
Using the BFXP, Beckman Coulter scientists compared library construction at total RNA inputs of 25ng and 100ng Universal Human Reference (UHR) RNA for the library preparation kit. Once the UHR RNA was assayed to ensure the sample was of high quality, the purified adapter ligated library was amplified to 15 cycles in accordance with the library preparation kit protocol. The quality and size distribution of the libraries were then analyzed and their concentrations measured, before they were sequenced using a MiSeq® 2 x 100 cycle paired end sequencing run.
Reads were then assayed, transcript abundances measured and bioinformatic analysis performed, which showed a high degree of correlation between the 25ng and 100ng total RNA input libraries.
Automation of the New England Biolabs NEBNext Ultra Directional RNA Library Prep Kit for Illumina on the BFXP liquid handler enables the construction of high quality, reproducible directional RNASeq libraries from low sample inputs. Automation of sample preparation allows up to 96 directional RNASeq libraries to be constructed simultaneously in around 9.5 hours. For added convenience, the method can be run all in one go or over two days, with sample preparation through to cDNA synthesis being performed on day 1 and sample preparation through to end of library construction being performed on day 2.
For further information about this automation solution, please click here.
About Beckman Coulter
Beckman Coulter develops, manufactures and markets products that simplify, automate and innovate complex biomedical tests. More than a quarter of a million Beckman Coulter instruments operate in laboratories around the world, supplying critical information for improving patient health and reducing the cost of care.
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