Automating RNA library preparation at a core facility

This article is based on a poster originally authored by Huw Rees, Taylor C. Fennelly, Michael Benway, Siva Chavadi, Andrew Barry, and Robert Steen.

SPT Labtech’s firefly^® liquid handling platform has been used by the Biopolymer Facility (BPF) genomics core at Harvard Medical School to automate the NEBNext^® UltraExpress^® RNA Library Prep kits. This includes options for both rRNA depletion and polyadenylated mRNA enrichment workflows.

The NEBNext^® UltraExpress^® RNA Library Prep kits benefit from a single, uniform adapter dilution and a single PCR cycling condition across the entire input range. This system also requires fewer bead clean-up steps, enabling an addition-only library construction process and automation-friendly workflows.

This combination of features results in a more rapid process and reduced plastic waste.

This article highlights the ease of automating NEB’s UltraExpress^® kits with the intuitive firefly^® system, to produce reproducible, high-quality libraries and sequencing data.

In the examples presented here, libraries were prepared from 25 ng to 250 ng of total RNA, demonstrating consistently high data quality across this input range.

The rapid production of RNA libraries notably enhances the sequencing services provided by genomics core facilities like the BPF.

Image Credit: SPT Labtech

NEBNext^® UltraExpress^® RNA workflow

Combining the NEBNext^® UltraExpress^® NGS Library Prep kit with firefly^® yields a robust and streamlined means of generating high-quality, reproducible libraries for Illumina^® sequencing, using an RNA input from 25 to 250 ng.

This feature reduces incubation times and results in fewer clean-up steps, reducing total time and required consumables.

Figure 1. The NEBNext UltraExpress RNA workflow. Image Credit: SPT Labtech

Bead-based RNA purification

SPT Labtech’s firefly^® was utilized for all dispensing, aspiration, and mixing steps in a 96-well PCR plate (Biorad Hard Shell Plate).

RNA purification beads (80% ethanol in water) were dispensed via the non-contact dispense head (SPRIselect, Beckman Coulter). This dispense head was also used to dispense all master mixes (including the USER^® enzyme) and elution buffer (0.1X TE).

The pipetting head performed the initial RNA transfer, all mixing steps, and all transfers of supernatant to waste, while a 96-well plate magnet (MagnumFLX, Alpaqua) was employed in all bead separation steps.

Figure 2. TapeStation results of 24 undiluted sample libraries. Image Credit: SPT Labtech

Figure 3. Average size of library per sample. Image Credit: SPT Labtech

Conclusions

The robust combination of the NEBNext^® UltraExpress^® NGS Library Prep kit and firefly^® was found to yield a robust, accessible, efficient, and streamlined means of generating high-quality, reproducible libraries for Illumina^® sequencing. These libraries range from 25 – 250 ng of total input RNA.

High-throughput genomics core facilities working on a diverse range of projects are reliant on automation to deliver high-quality NGS data with rapid turnaround times. Automating the NEBNext^® UltraExpress^® kits on firefly^® at the BPF has delivered the facility’s most rapid method for RNA library production for Illumina^® sequencing, making NGS faster and more accessible to the wider research community.

Acknowledgments

Produced from materials originally authored by Huw Rees from SPT Labtech; Taylor C. Fennelly and Robert Steen from the Department of Genetics, Harvard Medical School; and Michael Benway, Siva Chavadi, and Andrew Barry from New England Biolabs.

About SPT Labtech

We design and manufacture robust, reliable, and easy-to-use solutions for life science.

We enable life scientists through collaboration, deep application knowledge, and leading engineering to accelerate research and make a difference together. We offer a portfolio of products within sample management, liquid handling, and multiplexed detection that minimize assay volumes, reduce material handling costs and put the discovery tools back in the hands of the scientist.

At the heart of what we do

Many of our innovations have been born out of the desire to create solutions to existing customer problems; and it’s this ethos that drives SPT Labtech’s R&D efforts. Our strengths come from the trust our customers have with us to develop truly unique, automated technologies to meet their needs. We combine cutting edge science with first-rate engineering to put customers at the heart of everything we do.

A problem-solving state of mind

The substantial breadth of expertise within our company enables us to be involved in the full life cycle of our products from the initial design concept, mechanical and software engineering and prototyping, to final manufacture and sale. These qualities allow us to offer the best possible technical and mechanical support to all the equipment that we supply, hence maintaining excellent client relationships.

Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.