Nov 01, 2024
Direct RNA sequencing (SQK-RNA004) for ONT PromethION
This protocol is a draft, published without a DOI.
- Oxford Nanopore Technologies1
- 1Oxford Nanopore Technologies
Protocol Citation: Oxford Nanopore Technologies 2024. Direct RNA sequencing (SQK-RNA004) for ONT PromethION. protocols.io https://protocols.io/view/direct-rna-sequencing-sqk-rna004-for-ont-promethio-dqwz5xf6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 30, 2024
Last Modified: November 01, 2024
Protocol Integer ID: 111289
Keywords: ONT, Nanopore, Direct RNA, dRNA, RNA modification profiling, RNA modification, Long read RNA-seq, direct rna sequencing kit, total rna, native rna, total rna sample, direct rna, rna, mrna purification, ribodepleted rna, rna004, rna flow cell, sequencing adapter, dynabeads mrna purification, complementary cdna strand, reverse transcription, stability via reverse transcription, cdna hybrid
Abstract
The following protocol outlines how to sequence native RNA using the Direct RNA Sequencing Kit (SQK-RNA004) allowing exploration of attributes such as modified bases. It accommodates both total RNA and enriched samples, such as poly(A)+ or ribodepleted RNA, without requiring fragmentation. For information on how to perform poly(A)+ selection on your total RNA sample, please refer to "Dynabeads mRNA Purification" found here.
Starting with either poly(A) tailed RNA or total RNA, a complementary cDNA strand is synthesized for stability via reverse transcription. Sequencing adapters are then attached to the RNA-cDNA hybrid for sequencing on MinION or PromethION RNA Flow Cells (FLO-MIN004RA / FLO-PRO004RA).
Attachments
Materials
Materials
- 300 ng of poly(A) tailed RNA or 1 μg of total RNA in 8 μl
- Direct RNA Sequencing Kit (SQK-RNA004)
Consumables
- Induro Reverse Transcriptase (NEB, M0681)
- 10 mM dNTP solution (e.g. NEB, cat #N0447)
- NEBNext Quick Ligation Reaction Buffer (NEB, B6058)
- T4 DNA Ligase 2M U/ml (NEB, cat #M0202M)
- Murine RNase Inhibitor (NEB, M0314)
- Agencourt RNAClean XP beads (Beckman Coulter, cat # A63987)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit RNA HS Assay Kit (ThermoFisher, cat #Q32852)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat# Q32851)
- Qubit Assay Tubes (Invitrogen, Q32856)
Equipment
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Microfuge
- Vortex mixer
- Ice bucket with ice
- Timer
- Thermal cycler
- Qubit fluorometer (or equivalent for QC check)
- Eppendorf 5424 centrifuge (or equivalent)
- Pipettes and pipette tips P2, P10, P20, P100, P200, P1000
Troubleshooting
Library preparation
Prepare the NEBNext Quick Ligation Reaction Buffer and T4 DNA Ligase according to the manufacturer's
instructions, and place on ice:
Thaw the reagents at RT.
Spin down the reagent tubes for 5 seconds
Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
Note: Do NOT vortex the T4 DNA Ligase.
Important: We do not recommend using the Quick T4 Ligase for this protocol. We have found that the T4 DNA Ligase (2M U/ml - NEB M0202M) works better. It needs to be used in combination with the Quick Ligation Reaction Buffer (NEB B6058).
Spin down the RT Adapter (RTA), RNA CS (RCS) (if using), and RNA Ligation Adapter (RLA), pipette mix and place on ice.
Thaw the Wash Buffer (WSB) and RNA Elution Buffer (REB) at RT and mix by vortexing. Then spin down and place on ice.
Prepare the RNA in Nuclease-free water:
Transfer 300 ng of poly(A) tailed RNA or 1 μg of total RNA into a 0.2 ml thin-walled PCR 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.
In the same 0.2 ml thin-walled PCR tube, combine the reagents in the following order:
| Reagent | Volume (uL) | |
| RNA | 8 | |
| NEBNext Quick Ligation Reaction Buffer | 3 | |
| RNA CS (RCS) | 0.5 | |
| Murine RNase Inhibitor | 1 | |
| RT Adapter (RTA) | 1 | |
| T4 DNA Ligase | 1.5 |
Mix by pipetting and spin down.
Incubate the reaction for 10 minutes at RT.
In a clean 1.5 ml DNA LoBind Eppendorf tube, combine the following reagents together to make the reverse transcription master mix:
| Reagent | Volume (uL) | |
| Nuclease-free water | 13 | |
| 10 mM dNTPs | 2 | |
| 5x Induro RT reaction buffer | 8 |
Transfer the reverse transcriptase master mix to the 0.2 ml PCR tube containing your adapter-ligated RNA and mix by pipetting.
Add 2 μl of Induro Reverse Transcriptase to the reaction and mix by pipetting.
Place the tube in a thermal cycler and incubate at 60°C for 30 minutes, then 70°C for 10 minutes, and bring the sample to 4°C before proceeding to the next step.
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 RT.
Prepare 200 μl of fresh 70% ethanol in Nuclease-free water.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
Keep the tube on magnet until the supernatant is clear and colourless before washing the beads with 150 μl of freshly prepared 70% ethanol, as described below:
Keeping the magnetic rack on the benchtop, rotate the tube by 180°. Wait for the beads to migrate towards the magnet and to form a pellet.
Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet again.
Carefully 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.
Remove the tube from the magnetic rack and resuspend the pellet in 23 μl Nuclease-free water. Incubate for 5 minutes at RT.
Pellet the beads on a magnet until the eluate is clear and colourless.
Remove and retain 23 μl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
In the same 1.5 ml Eppendorf DNA LoBind tube, combine the reagents in the following order:
| Reagent | Volume (uL) | |
| RT-RNA sample | 23 | |
| NEBNext Quick Ligation Reaction Buffer | 8 | |
| RNA Ligation Adapter (RLA) | 6 | |
| T4 DNA Ligase | 3 |
Mix by pipetting.
Incubate the reaction for 10 minutes at RT.
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.
Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet for 5 minutes, and pipette off the supernatant when clear and colourless.
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 for 5 minutes and pipette off the supernatant when clear and colourless.
Repeat the previous step.
Spin down the tube and replace onto the magnetic rack until the beads have pelleted to pipette off any remaining Wash Buffer (WSB).
Remove the tube from the magnetic rack and resuspend pellet in 33 μl RNA Elution Buffer (REB) by gently flicking the tube. Incubate for 10 minutes at RT.
Pellet the beads on a magnet for 5 minutes until the eluate is clear and colourless.
Remove and retain 33 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.
The reverse-transcribed and adapted RNA is now ready for loading into the flow cell.
Important: The RNA library must be sequenced immediately and cannot be stored for later use.
Priming and loading the PromethION flow cell
Important: This kit is only compatible with RNA flow cells (FLO-PRO004RA).
Thaw the Sequencing Buffer (SB), Library Solution (LIS), RNA Flush Tether (RFT) and Flow Cell Flush (FCF)
at RT. Mix by vortexing and spin down.
To prepare the flow cell priming mix in a clean 1.5 ml Eppendorf DNA LoBind tube, combine the following
reagents. Mix by vortexing and spin down at RT.
| Reagent | Volume (uL) | |
| RNA Flush Tether (RFT) | 30 | |
| Flow Cell Flush | 1,170 |
Important: After taking flow cells out of the fridge, wait 20 minutes before inserting the flow cell into the PromethION for the flow cell to come to RT. Condensation can form on the flow cell in humid environments. Inspect the gold connector pins on the top and underside of the flow cell for condensation and wipe off with a lint-free wipe if any is observed. Ensure the heat pad (black pad) is present on the underside of the flow cell.
For PromethION 2 Solo, load the flow cell(s) as follows:
- Place the flow cell flat on the metal plate.
- Slide the flow cell into the docking port until the gold pins or green board cannot be seen.
For the PromethION 24/48, load the flow cell(s) into the docking ports:
- Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position.
- Press down firmly onto the flow cell and ensure the latch engages and clicks into place.
Important: Insertion of the flow cells at the wrong angle can cause damage to the pins on the PromethION and affect your sequencing results. If you find the pins on a PromethION position are damaged, please contact [email protected] for assistance.
Slide the inlet port cover clockwise to open.
Important: Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 μl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
After opening the inlet port, draw back a small volume to remove any air bubbles:
Set a P1000 pipette tip to 200 μl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 μl, or until you see a small volume of buffer entering the pipette tip.
Load 500 μl of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles. Wait five minutes. During this time, prepare the library for loading using the next steps in the protocol.
In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:
| Reagent | Volume (uL) | |
| Sequencing Buffer (SB) | 100 | |
| Library Solution (LS) | 68 | |
| RNA library | 32 |
Complete the flow cell priming by slowly loading 500 μl of the priming mix into the inlet port.
Mix the prepared library gently by pipetting up and down just prior to loading.
Load 200 μl of library into the inlet port using a P1000 pipette.
Install the light shield on your flow cell as soon as library has been loaded for optimal sequencing output.
If the light shield has been removed from the flow cell, install the light shield as follows:
Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.
Close the PromethION lid when ready to start a sequencing run on MinKNOW.
Flow cell reuse and returns
Important: Our Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) is compatible with RNA Flow Cells and the Direct RNA Sequencing Kit (SQK-RNA004).
The wash kit doesn’t work as nuclease flush for Direct RNA sequencing: it won’t recover blocked pores.
It works as a flush to reload new sample: it will wash off most of the library from the array and remove all adapter from the remaining sample, preventing it from being captured and sequencing. This will allow subsequent library loads.
After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the
Flow Cell Wash Kit protocol and store the washed flow cell at 2-8°C.
Alternatively, follow the returns procedure to flush out the flow cell ready to send back to Oxford Nanopore.
Important: If you encounter issues or have questions about your sequencing experiment, please refer to the Troubleshooting Guide that can be found in the online version of this protocol.
Protocol references
Acknowledgements
The protocol reproduced here is originally provided by Oxford Nanopore Technologies along with the Direct RNA sequencing kit (cat. #SQK-RNA004). Original protocol is attached as a pdf, document version DRS_9195_v4_revD_20Sep2023.