Information

This protocol is explicitly for the new SQK-RNA004 Nanopore kit. If you are using the discontinued SQK-RNA002 kit and either an R9.4.1 flow cell or flongle, please see the old Nanopore RNA Sequencing Protocol (SQK-RNA002).

This protocol is a modified version of the Nanopore SQK-RNA004 protocol available here, and the first part series of my m6A Detection Protocol. This protocol will cover the library preparation portion and beginning of the sequencing run, while the m6A detection will be handled in a separate article.

Introduction

This protocol is designed to guide you through the preparation of an RNA library for sequencing through nanopore, adapted from the Oxford Nanopore Technologies Direct RNA Sequencing SQK-RNA004 protocol. Following the sequencing run, you can move onto the analysis protocol for the alignment and detection of RNA modifications using m6ANet or Dorado. At the end of this protocol, I've also included a Troubleshooting section to cover some common issues I've had while sequencing to hopefully streamline your runs. Where possible, I have highlighted deviations from the original protocol in bold red terms to make it easy to identify my changes.

Requirements

Below is a list of required reagents and equipment you will need to carry out the protocol. Note that some of these are not necessary for the sequencing run but are highly recommended, such as the Qubit fluorometer, which checks your RNA library's sample quality prior to burning a flow cell. I do not have access to a Qubit, so I will use a total RNA gel and nanodrop results to eyeball RNA quality, but I recommend you use one if you have access to it.

Materials and Equipment
Type	Items
Materials	330 ng of poly(A)-tailed RNA or 1 μg of total RNA in 8 μL. RT Adapter (RTA, Blue Cap). RNA CS (RCS, Yellow Cap). RNA Adapter (RLA, Green Cap). Wash Buffer (WSB, Orange Cap). RNA Elution Buffer (REB, Black Cap).
Consumables	NEBNext® Quick Ligation Reaction Buffer (NEB, B6058). T4 DNA Ligase 2M U/mL (NEB, M0202T/M). 0.2 mL thin-walled PCR tubes. Nuclease-free water (e.g. ThermoFisher, AM9937). Agencourt RNAClean XP beads (Beckman Coulter™, A63987). RNaseOUT (Invitrogen, 10777019). Freshly prepared 70% ethanol in nuclease-free water. 1.5 mL Eppendorf DNA LoBind tubes. SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, 18080044). 10 mM dNTP solution (e.g. NEB N0447). Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851).
Equipment	Magnetic separator, suitable for 1.5 mL Eppendorf tubes. Hula mixer (gentle rotator mixer). Thermal cycler.
Optional Equipment	Qubit fluorometer (or equivalent for QC check).

RNA Library Preparation

WARNING

You need to test your mRNA purity before proceeding. This can be done by saving some of the total RNA and running it on a gel (more information on interpreting those results here) as well as checking the concentration of total and mRNA on a nanodrop. You should have a decent concentration (with mRNA 3-6% of the measured total RNA) with 260/230 and 260/280 ratios of 2.0 or better (mor information on interpreting those results here). A low 260/230 ratio indicates organics in your sample, which can damage the flow cell pores, whereas a low 260/280 ratio indicates proteins, which can clog them. You may want to dilute your input sample so that you're loading your ideal amount in 8 μL of sample, which will also help dilute and contaminants. Higher mRNA amounts than expected (greater than 3-6% of the total RNA) suggests contamination by total RNA or DNA.

Regarding samples that have been frozen: I highly recommend that you pipette your sample 40+ times using an appropriate pipette to draw the full volume of the solution. You should also remeasure the concentration of your sample on the nanodrop, rather than relying on the concentration you obtained prior to freezing. I have had sample library preparations that were sub-par because the concentration dropped following a freeze/thaw.

Prepare the RNA in nuclease-free water.
- Transfer 330 ng† of poly(A)-tailed RNA or 1 μg of total RNA into a 1.5 mL Eppendorf DNA LoBind tube.
- Adjust† the volume to 8 μL with nuclease-free water.
- Mix thoroughly by flicking the tube to avoid unwanted shearing.
- Spin down briefly in a microfuge.

†Note: For my best run, I diluted the sample and loaded 9 μL of pure mRNA directly. This was around 333 ng total in 9 μL (~37 ng/μL).

WARNING

Only poly(A)-tailed RNA will eventually be read by the nanopore system. Therefore, if you are interested in non-poly(A)-tailed RNA (such as long non-coding RNA), you will need to poly(A)-tail it separately and purify it prior to the run. Additionally, the old protocol called for 500 ng of poly(A)-tailed RNA instead of 50 ng (it is unclear at the present on why this was changed). My best run varied from both of these amounts. Finally, according to ONT, there is no difference in using total or mRNA for human samples, however, they acknowledge that this is not the case for other species (such as yeast) and that some users have reported better results with purified mRNA across species.

In a 0.2 mL thin-walled PCR tube, mix the reagents in the following order:

Prepping Sample for Adapter Ligation
Reagent	Volume
NEBNext Quick Ligation Reaction Buffer (see below warning)	3.0 μL
RNA Sample	9.0 μL
RNA CS (RCS, Yellow Cap), 110 nM	0.5 μL
RT Adapter (RTA, Blue Cap)	1.0 μL
T4 DNA Ligase	1.5 μL
Total	15 μL

WARNING

The NEBNext Quick Ligation Reaction Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for several seconds to ensure the reagent is thoroughly mixed.

Mix by pipetting and spin down.
Incubate the reaction for 10 minutes at room temperature.
Mix the following reagents together to make the reverse transcription master mix:

Reverse Transcription Master Mix
Reagent	Volume
Nuclease-free water	9.0 μL
10 mM dNTPs	2.0 μL
5x first-strand buffer	8.0 μL
0.1 M DTT	4.0 μL
Total	23.0 μL

Add the master mix to the 0.2 mL PCR tube containing the RT adapter-ligated RNA from the "RT Adapter ligation" step. Mix by pipetting.
Add 2 μL of SuperScript III Reverse Transcriptase to the reaction and mix by pipetting.
Place the tube in a thermal cycler and incubate at 50°C for 50 minutes, then 70°C for 10 minutes, and bring the sample to 4°C before proceeding to the next step.

Note

This can be found on the thermal cycler near Tie's bench as the program RT Ligation Nanopore. This is also the point where you can take a break, since this step will take approximately 1 hour and 6 minutes to complete.

Transfer the sample to a clean 1.5 mL Eppendorf DNA LoBind tube.
Resuspend the stock of Agencourt RNAClean XP beads by vortexing.
Add 72 μL of resuspended Agencourt RNAClean XP beads to the reverse transcription reaction and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
Prepare 200 μL of fresh 70% ethanol in nuclease-free water (i.e., mix 140 μL pure ethanol with 60 μL PCR water in a clean Eppendorf tube).
Spin down and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant (use a p100 if possible to avoid pulling in RNA beads).

Note

For the spin down, I used 2,500g for 1 minute.

Keep the tube on magnet, and wash the beads with 150 μL of freshly prepared 70% ethanol without disturbing the pellet as described below.

Tip

Rotate the tube very fast to avoid the pellet from smearing. You want it to migrate directly across the tube through the ethanol, but otherwise remain intact.

Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and form a pellet. Wait 2.5 minutes.
Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet. Wait 2.5 minutes.

Remove the 70% ethanol using a pipette and discard.
Spin down and place the tube back on the magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off any residual ethanol.

Note

For the spin down, I used 2,500g for 1 minute.

Remove the tube from the magnetic rack and resuspend pellet (gentle pipetting) in 20 μL nuclease-free water. Incubate for 5 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colourless.
Remove and retain 20 μL of eluate into a clean 1.5 mL Eppendorf DNA LoBind tube.
In the same 1.5 mL Eppendorf DNA LoBind tube, mix the reagents in the following order:

RNA Adapter Mix
Reagent	Volume
NEBNext Quick Ligation Reaction Buffer (see below warning)	8.0 μL
RNA Adapter (RMX, Green Cap)	6.0 μL
Nuclease-free water	3.0 μL
T4 DNA Ligase	3.0 μL
Total (including all reagents)	40 μL

WARNING

The NEBNext Quick Ligation Reaction Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for several seconds to ensure the reagent is thoroughly mixed.

Mix by pipetting.
Incubate the reaction for 10 minutes at room temperature.
Resuspend the stock of Agencourt RNAClean XP beads by vortexing.
Add 16 μL of resuspended Agencourt RNAClean XP beads to the reaction and mix by pipetting.

Tip

Check to ensure that the Elution Buffer is thawing at this point. In my experience, even on ice it tends to remain frozen.

Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

Note

For the spin down, I used 3,000g for 1 minute (note the change!).

Tip

Start thawing the Flush Buffer, Flush Tether, and RNA Running Buffer at room temperature in preparation for flow cell priming and loading. If you are loading using the Flongle reagents, also set the Sequencing Buffer, Flongle Flush Buffer, and Loading Beads II out to get to room temperature.

Add 150 μL of the Wash Buffer (WSB) to the beads. Close the tube lid and resuspend the beads by flicking the tube. Return the tube to the magnetic rack, allow the beads to pellet and pipette off the supernatant.
Repeat the previous step.

WARNING

Agitating the beads results in a more efficient removal of free adapter, compared to adding the wash buffer and immediately aspirating.

Tip

Make sure the Elution Buffer is completely thawed at this point.

Remove the tube from the magnetic rack and resuspend pellet in 21 μL Elution Buffer by the gently flicking the tube and pipetting. Incubate for 10 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colourless.
Repeat the above two steps to increase your yield using the same 21 μL of Elution Buffer. Mix by pipetting and incubate for 10 minutes prior to pelleting on magnet.
Remove and retain 21 μL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1 μL of reverse-transcribed and adapted RNA using the Qubit fluorometer DNA HS assay - recovery aim ~20 ng. If you have omitted the reverse transcription step, please use the Qubit RNA HS Assay Kit instead. However, please note that the kit will measure all RNA present, including any non-adapted RNA that has been carried through in the RNAClean XP bead clean-up. The reported quantity of RNA may therefore not fully represent the amount of sequenceable RNA.

Note that you can view the concentration of your library on the Nanodrop and may or may not get a reading. For a good run, I recorded a slight "hill" peak around 260 nm and around 2.6 ng/μL final concentration. Since your library is double stranded, use the DNA setting on the nanodrop. If you use RNA, you will get an inaccurate reading.

Note that for the loading step, you will want to load the following amounts of RNA for a good sequencing run:

RNA Loading Amounts
Flow Cell	Amount of mRNA (in fmol)	Amount in ng (if N50 = 1,400 bases)
Flongle	3-20 fmol; 10-15 fmol ideal	~ 6.751 ng (my best run loaded 2.6 ng/μL in 7 μL, for 18.2 ng total)
Standard MinION	44.44 fmol	20 ng

You can also calculate your own loading amounts using the NEBio RNA mass calculator if your RNA is of a different length.