Nanopore direct RNA sequencing on the MinION

Workflow notes for native RNA sequencing on an Oxford Nanopore MinION (SQK-RNA002 + R9.4.1 flow cell): input requirements, library prep at a glance, flow cell loading and QC. Follow the official ONT protocol for the full steps.

Version 1.0 · last updated 2026-10-05 · Used in 1 notebook entries ↓

ImportantUse the official protocol at the bench

This page is a planning and checklist summary. Run the library prep from Oxford Nanopore’s own protocol for your kit version (log in to the Nanopore Community). Nanopore kits change often, and newer direct RNA kits and flow cells may have replaced SQK-RNA002 / R9.4.1, so check what is current before ordering.

At a glance

Input	500 ng total RNA or 50 ng poly(A) RNA in 9 µL nuclease-free water
Output	Native-RNA reads (no RT or PCR bias; base modifications retained)
Kits	Direct RNA Sequencing Kit SQK-RNA002; Flow Cell Priming Kit EXP-FLP002; R9.4.1 flow cell FLO-MIN106; Flow Cell Wash Kit EXP-WSH004
Device	MinION Mk1B/Mk1C with MinKNOW
Time	~2 h library prep + ~15 min priming/loading, then sequencing

Why direct RNA

Use it to study native RNA (e.g. base modifications), to avoid reverse transcription and PCR bias, or for transcripts that are hard to reverse-transcribe. The RT step in the prep only makes a cDNA strand to stabilize the RNA; the RNA strand is what passes through the pore.

Third-party materials to have ready

• SuperScript III reverse transcriptase + 5× first-strand buffer + 0.1 M DTT
• 10 mM dNTPs
• NEBNext Quick Ligation Reaction Buffer; T4 DNA ligase (2 M U mL⁻¹)
• Agencourt RNAClean XP beads; freshly made 70% ethanol (nuclease-free water)
• Qubit RNA HS and dsDNA HS assays
• 1.5 mL DNA LoBind tubes, 0.2 mL PCR tubes, nuclease-free water
• Hula mixer, magnetic rack, thermal cycler, Qubit; Bioanalyzer/TapeStation optional

Before you start

• Check RNA quality: length, quantity and purity are critical (Qubit + TapeStation/Bioanalyzer).
• Check the flow cell: run a flow cell check in MinKNOW and confirm enough active pores.
• Install/update MinKNOW on the device or computer.

Workflow at a glance

Stage	What happens	Key amounts
1. RT adapter ligation	RNA + RT adapter (RTA) + RNA CS control, ligated 10 min at RT	9 µL RNA in a 15 µL reaction
2. Reverse transcription	Make the cDNA strand with SuperScript III: 50 °C 50 min → 70 °C 10 min → 4 °C	40 µL reaction
3. Bead cleanup	RNAClean XP beads, two 70% ethanol washes, elute	72 µL beads; elute in 20 µL water
4. RNA adapter ligation	Ligate the motor-protein adapter (RMX), 10 min at RT	40 µL reaction
5. Bead cleanup	RNAClean XP beads, 2× Wash Buffer (WSB) washes (no ethanol), elute	16 µL beads; elute in 21 µL Elution Buffer, 10 min
6. QC	Qubit dsDNA HS on 1 µL	Aim for ~20 ng recovered
7. Prime flow cell	Flush Tether into Flush Buffer; remove air bubble from the priming port; prime	800 µL, wait 5 min, then 200 µL
8. Load	Library in RNA Running Buffer, added dropwise to the SpotON port	37.5 µL RRB + 37.5 µL library (20 µL library + 17.5 µL water) = 75 µL
9. Sequence	Start the run in MinKNOW (basecalling in real time)

Critical tips

• Wash with WSB, not ethanol, after the RMX ligation (stage 5). Ethanol damages the motor protein on the adapter.
• Flick the beads during WSB washes rather than just adding and removing buffer; agitation removes free adapter much better.
• No air bubbles in the priming port or sensor array: draw back 20–30 µL before priming and pipette slowly.
• Mix RNA by flicking, not vortexing, to avoid shearing.
• Load the library dropwise, letting each drop enter the SpotON port before adding the next.

Notebook entries using this protocol

Changelog

Date	Version	Change
2026-10-05	1.0	Condensed from the 2023 notebook copy of the ONT protocol into a planning checklist that links to the official protocol
2023-10	–	Protocol notes for SQK-RNA002 on the MinION