Introduction

This method is based on selectively isolating poly(A) molecules from a sample of total RNA. The method is very quick to perform (~15 mins) 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.

Materials

• ≥75 µg total RNA extract (can be scaled)
• DYNAL™ Dynabeads™ Kit (Invitrogen™: 61006)
• Magnetic rack
• Nuclease-free or DEPC-treated water, or 10 mM Tris-HCl, pH 7.5
• Heat block
• HulaMixer Sample Mixer
• Small centrifuge for microcentrifuge tubes
• Nuclease-free 1.5 ml microcentrifuge tubes
• RNA HS Qubit kit (Invitrogen™)
• Qubit meter (Invitrogen™)

Before starting: recommendations to reduce RNase digestion or RNA degradation

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.

Method

1. Using 75 µg of total RNA, continue with the DYNAL™ Dynabeads™ protocol as directed (page 1) to step 4 under the heading "Isolate mRNA".
2. When proceeding with the final elution of poly(A)+ selected RNA (step 4 of DYNAL protocol), we recommend a final elution step at ≤70°C for 2 minutes to ensure longer transcripts and reduce possible degradation.
3. (Optional step) A second elution step (repeat of Step 2.) can be performed to ensure the maximum amount of poly(A)+ RNA has been isolated. This can provide an additional 0.1% of final product.
4. Measure the concentration of the eluate using HS RNA Qubit kit. The final amount after poly(A) isolation ought to be ~2% of starting input.
5. Store final product at -80°C.

Results

• Yield: 1.5 µg (2%) of enriched poly(A)+ RNA
• Sequencing performance:

Sequencing libraries were prepared from 1000 ng total RNA or 500 ng poly(A)+ selected 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 an increase in the median poly(A) tail length was detected after poly(A)+ selection (Figure 1B). This suggests that some bias may be involved during poly(A)+ selection, wherein molecules with longer tails are more readily targeted than molecules with shorter tails.

Figure 1. Aligned read count and median poly(A) tail length for total RNA and poly(A)+ 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 enriched human sample was a little lower than for total RNA. An increase in the median poly(A) tail length was detected after poly(A)+ selection (Figure 1B). This suggests that some bias may be involved during poly(A)+ selection, wherein molecules with longer tails are more readily targeted than molecules with shorter tails.

Change log

Version	Change
v1, June 2019	Initial protocol publication
v2, August 2023	Updated URL link