This method is based on selectively depleting ribosomal RNA from a sample of total eukaryotic RNA (originally optimised for the removal of human, mouse and rat rRNA) and it yields a sample enriched for mRNA that is suitable for downstream library preparation and nanopore sequencing. We tested this protocol on RNA extracted from Saccharomyces cerevisiae cells (S288c) and human GM12878 cells using TRIzol-based extraction methods.

• ≥ 10 µg total RNA extract (can be scaled)
• RiboMinus™ Eukaryote Kit v2 Kit (Invitrogen™: A15020)
• RiboMinus™ Concentration Module (Invitrogen™: K155005)
• Magnetic rack
• Vortex mixer
• Nuclease-free or DEPC-treated water, or 10 mM Tris-HCl, pH7.5
• Heat block
• HulaMixer Sample Mixer
• Centrifuge fitted for microcentrifuge tubes
• Nuclease-free 1.5 ml microcentrifuge tubes
• RNA HS Qubit kit (Invitrogen™)
• Qubit meter (Invitrogen™)

1. Wear clean gloves at all times to avoid degradation of RNA. Change to fresh gloves if they are suspected to be contaminated.
2. Clean bench and equipment (pipettes, racks, heat-block, etc) with a RNase neutralising chemical such as RNaseZAP™.
3. Keep RNA sample on ice unless otherwise indicated.
4. Use clean, RNase-free solutions.

1. Aliquot ≥10 µg total RNA into 1.5 ml microcentrifuge tubes to achieve a final mass of 5 µg/tube (e.g. for 10 µg total RNA, this will be 2x tubes with 5 µg each). Place on ice.
2. Continue with the RiboMinus™ Eukaryote Kit v2 protocol as directed (page 3), following the specifications for the larger, 5 µg starting material protocol for each tube. NOTE: The final volume at the end of this protocol will be approximately 300 µl of ribo-depleted RNA. This will need to be concentrated to reduce overall volume. We recommend continuing with the RiboMinus™ Concentration Module.
3. Set up one column per initial tube (containing 5 µg) to concentrate ribo depleted RNA using the RiboMinus™ Concentration Module. (e.g. for 10 µg starting material, 2x Concentration Module columns will be needed). Follow the protocol instructions as specified by the kit. Elute ribo-depleted RNA from each column using 15 µl of nuclease-free water.
4. Pool final eluate in a fresh 1.5 ml microcentrifuge tube.
5. Measure the concentration of the eluate using HS RNA Qubit kit. The final amount after ribo-depletion ought to be ~5-10% of starting input.
6. Store final product at -80°C.

• Yield: 0.5-1.0 µg (5-10%) of ribo-depleted RNA
• Sequencing performance:

Sequencing libraries were prepared from 1000 ng total RNA or 500 ng ribo-depleted RNA using the Direct RNA Sequencing Kit and run on MinION. Alignment was performed by minimap2-x map-ont to the Ensembl transcriptomes keeping primary alignments with minimum mapping quality of 5. We observed a higher yield of aligned reads for the enriched yeast samples compared to the yeast total RNA, although this was not the case for the human samples (Figure 1A). The length of the poly(A) tail for each read was also measured and little variation in the median poly(A) tail length was detected after ribo-depletion (Figure 1B).

Figure 1. Aligned read count and median poly(A) tail length for total RNA and ribo-depleted RNA samples from yeast and human extractions. We observed a higher yield of aligned reads for the enriched yeast sample compared to the yeast total RNA, although the aligned read count for the ribo-depleted human sample was a little lower than for total RNA. Little variation was detected in the median poly(A) tail length between total RNA and ribo-depleted runs.