Direct RNA sequencing (SQK-RNA004-XL) (DRS_9219_v4_revA_19Feb2025)

Oxford Nanopore Technologies
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MinION: Protocol

V DRS_9219_v4_revA_19Feb2025

FOR RESEARCH USE ONLY

1. Overview of the protocol

IMPORTANTE

This is an Early Access product.

For more information about our Early Access programmes, please refer to this article on product release phases.

Please ensure you always use the most recent version of the protocol.

Direct RNA Sequencing Kit XL features

This kit is highly recommended for:

  • Exploring attributes of native RNA, such as modified bases
  • Removing RT or PCR bias
  • Transcripts that are difficult to reverse transcribe

Introduction to the Direct RNA sequencing XL protocol

This protocol describes how to carry out sequencing of native RNA using the Direct RNA Sequencing Kit XL (SQK-RNA004-XL). Starting from either poly(A) tailed RNA or total RNA, a second complementary cDNA strand is synthesised for stability by reverse transcription. Sequencing adapters are then attached to the RNA-cDNA hybrid for sequencing on either MinION™/GridION™ or PromethION™ Flow Cells RNA (FLO-MIN004RA or FLO-PRO004RA respectively). Please note, the complementary cDNA strand is not sequenced, but improves the RNA sequencing output.

It is recommended that a control experiment using the RNA Control Strand (RCS) is completed first to become familiar with the technology.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

  • Extract your RNA and check its length, quantity, and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
  • Ensure you have your sequencing kit, the correct flow cells and equipment, and third-party reagents.
  • Download the software for acquiring and analysing your data.
  • Check your flow cell(s) to ensure it has sufficient pores for a good sequencing run.

Library preparation

The Table below is an overview of the steps required in the library preparation, including timings and stopping points.

Library preparation Process Time Stop option
Reverse transcription Synthesise the complementary strand of the RNA ~85 minutes At this stage the RT-RNA can be stored at -80°C for later use.

Please note, this is the only pause point in this protocol.
Adapter ligation and clean-up Attach the sequencing adapters to the RNA-cDNA hybrid ends 45 minutes We strongly recommend sequencing your library as soon as it is adapted.
Priming and loading the flow cell Prime the flow cell and load the prepared library for sequencing 5 minutes

Direct RNA004-XL workflow

Sequencing and analysis

You will need to:

  • Start a sequencing run using the MinKNOW™ software, which will collect raw data from the device and basecall the reads.
IMPORTANTE

Unlike DNA, RNA is translocated through the nanopore in the 3'-5' direction. However, the basecalling algorithms automatically flip the data, and the reads are displayed 5'-3'.

IMPORTANTE

Compatibility of this protocol

This protocol should only be used in combination with:

2. Equipment and consumables

Material
  • 300 ng de ARN con apéndice de poli(A) o 1 µg de ARN total en 8 µl
  • Direct RNA Sequencing Kit (SQK-RNA004-XL)
Consumibles
  • Celdas de flujo ARN MinION/GridION (FLO-MIN004RA) o celdas de flujo ARN PromethION (FLO-PRO004RA)
  • Flow Cell Wash Kit (EXP-WSH004) or Flow Cell Wash Kit XL (EXP-WSH004-XL)
  • 0.2 ml PCR strip tubes
  • Eppendorf twin.tec® PCR Plate 96 LoBind®, semi-skirted (Eppendorf, 0030129504) with heat seals
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Pipetting troughs
  • Induro® Reverse Transcriptase and 5x Induro® RT Reaction Buffer (NEB, M0681)
  • 10 mM dNTP solution (e.g. NEB N0447)
  • NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
  • T4 DNA Ligase 2M U/ml (NEB, M0202M)
  • Murine RNase Inhibitor (NEB, M0314)
  • Agencourt RNAClean XP beads (Beckman Coulter®, A63987)
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • Qubit™ RNA HS Assay Kit (ThermoFisher, Q32852)
  • Qubit™ dsDNA HS Assay Kit (ThermoFisher, Q32851)
  • Tubos de ensayo Qubit™ (Invitrogen Q32856)
Instrumental
  • Dispositivos MinION, GridION o PromethION
  • Magnetic separation rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (ThermoFisher, 12027) or Magnetic separator suitable for 0.2 ml PCR tube strips, e.g. DynaMag™-PCR Magnet (ThermoFisher, 492025) or DynaMag™-96 Side Magnet (ThermoFisher, 12331D)
  • Microcentrífuga
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Termociclador
  • Mezclador vórtex
  • Temporizador
  • Cubeta con hielo
  • Pipeta y puntas P1000
  • Pipeta y puntas P200
  • Pipeta y puntas P100
  • Pipeta y puntas P20
  • Pipeta y puntas P10
  • Pipeta y puntas P2
  • Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
  • Qubit™ fluorometer (or equivalent for QC check)
Equipo opcional
  • Centrifuga Eppendorf 5424 (o equivalente)

For this protocol, you will need 300 ng of poly(A) tailed RNA or 1 µg of total RNA in 8 µl.

It is possible to start the protocol with a lower sample input however, this will likely yield a lower output.

Please refer to the following input titration graphs for guidance:


Poly(A) tailed RNA

Input titration RNA004 Poly A

Total RNA

Input titration RNA004 Total

Input RNA

It is important that the input RNA meets the quantity and quality requirements. Using too little or too much RNA, or RNA of poor quality (e.g. fragmented or containing chemical contaminants) can affect your library preparation.

For instructions on how to perform a quality control check on your RNA sample, please read the Input DNA/RNA QC protocol.

For further information on using RNA as an input, please refer to the links below.

These documents can also be found in the DNA/RNA Handling page.

Multichannel pipettes

For scaling up library preparation using the Direct RNA Sequencing Kit XL, you will need multichannel pipettes and appropriate pipette tips. Although the choice of brand is left to your discretion, our R&D team can recommend Rainin Pipet-Lite LTS L200-XLS+ (20–200 μl) and Pipet-Lit LTS L20-XLS+ (2–20 μl) pipettes and Rainin LTS pipette tips.

Third-party reagents

We have validated and recommend the use of all third-party reagents for this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.

For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

Check your flow cell

We highly recommend that you check the number of available pores in your flow cell, prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing your MinION/GridION or PromethION Flow Cells RNA. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores cited in the Table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To perform the flow cell check, please follow the instructions in the Flow Cell Check document.

Flow cell Minimum number of active pores covered by warranty
MinION/GridION Flow Cell RNA 800
PromethION Flow Cell RNA 5000

Direct RNA Sequencing Kit XL (SQK-RNA004-XL) contents:

RNA004-XL kit contents

Name Acronym Cap colour No. of vials/ bottles Fill volume per vial/ bottle (μl)
RT Adapter RTA Blue 1 vial 150
RNA Ligation Adapter RLA Green 1 vial 400
RNA CS RCS Yellow 3 vials 25
Wash Buffer WSB White 1 bottle 24,000
RNA Elution Buffer REB White 1 bottle 10,000
Library Solution LIS White cap, pink label 2 vials 1,800
Sequencing Buffer SB Red 3 vials 1,700
RNA Flush Tether RFT Violet 1 vial 1,600
Flow Cell Flush FCF White 4 bottles 15,500

Note: The RNA CS (RCS) is the control strand and contains the Enolase II from YHR174W, extracted from the yeast Saccharomyces cerevisiae. The reference FASTA files for the yeast is available here.

3. Library preparation

Material
  • 300 ng de ARN con apéndice de poli(A) o 1 µg de ARN total en 8 µl
  • RT Adapter (RTA)
  • RNA CS (RCS)
  • Wash Buffer (WSB)
  • RNA Ligation Adapter (RLA)
  • RNA Elution Buffer (REB)
Consumibles
  • Induro® Reverse Transcriptase and 5x Induro® RT Reaction Buffer (NEB, M0681)
  • 10 mM dNTP solution (e.g. NEB N0447)
  • NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
  • T4 DNA Ligase 2M U/ml (NEB, M0202M)
  • Murine RNase Inhibitor (NEB, M0314)
  • Agencourt RNAClean XP beads (Beckman Coulter®, A63987)
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 0.2 ml PCR strip tubes
  • Eppendorf twin.tec® PCR Plate 96 LoBind®, semi-skirted (Eppendorf, 0030129504) with heat seals
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851)
  • Qubit™ RNA HS Assay Kit (ThermoFisher, Q32852)
  • Tubos de ensayo Qubit™ (Invitrogen Q32856)
  • Pipetting troughs
Instrumental
  • Magnetic separation rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (ThermoFisher, 12027) or Magnetic separator suitable for 0.2 ml PCR tube strips, e.g. DynaMag™-PCR Magnet (ThermoFisher, 492025) or DynaMag™-96 Side Magnet (ThermoFisher, 12331D)
  • Microcentrífuga
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
  • Termociclador
  • Mezclador vórtex
  • Temporizador
  • Cubeta con hielo
  • Pipeta y puntas P1000
  • Pipeta y puntas P200
  • Pipeta y puntas P100
  • Pipeta y puntas P20
  • Pipeta y puntas P10
  • Pipeta y puntas P2
  • Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
  • Qubit™ fluorometer (or equivalent for QC check)
VERIFICACIÓN

Perform a flow cell check

We recommend performing a flow cell check before starting your library preparation to ensure you have a flow cell with sufficient pores for a good sequencing run.

To perform a flow cell check, please follow the instructions in the Flow Cell Check document.

Prepare the NEBNext® Quick Ligation Reaction Buffer and T4 DNA Ligase according to the manufacturer's instructions, and place on ice:

  1. Thaw the Quick Ligation Reaction Buffer at room temperature and place the T4 DNA Ligase on ice.

  2. Spin down the reagent tubes for 5 seconds.

  3. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes. Note: Do NOT vortex the T4 DNA Ligase.

The NEBNext Quick Ligation Reaction Buffer may have some precipitate. Allow the buffer to come to room temperature and then 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.

IMPORTANTE

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).

Thaw and spin down the RT Adapter (RTA), RNA CS (RCS) (if using), and RNA Ligation Adapter (RLA). Mix by pipetting and place on ice.

Thaw the Wash Buffer (WSB) and RNA Elution Buffer (REB) at room temperature and mix by vortexing. Then place on ice.

Prepare each RNA sample in nuclease-free water as follows:

  • Transfer 300 ng of poly(A) tailed RNA or 1 µg of total RNA into a tube of a 0.2 ml PCR tube strip or to a separate well of a 96 well PCR plate.

  • Adjust the volume to 8 μl with nuclease-free water.

  • Close the lids on the tube strip and mix by gently flicking the tubes to avoid unwanted shearing or if using a 96 well PCR plate, mix by gently pipetting.

  • Briefly spin down the tube strip or sealed PCR plate in a microplate centrifuge.

MEDIDA OPCIONAL

The use of the RNA CS (RCS) in this preparation is an optional control measure for library preparation QC and troubleshooting.

We recommend the inclusion of the RNA CS (RCS) in the library preparation for troubleshooting purposes.

  1. If using the RCS to prepare the RT Adapter (RTA) reaction mix in Step 5 below, dilute the RCS with nuclease-free water in a clean 0.2 ml PCR tube as follows:
Reagent Volume
RNA CS (RCS) 1 µl
Nuclease-free water 2 µl
Total 3 µl
  1. Mix thoroughly by pipette mixing 10 times.
  • You will require 0.5 µl of diluted RCS for each RNA sample when preparing the RTA reaction mix in Step 5.

Prepare the RT Adapter (RTA) reaction as follows:

For each RNA sample in the 0.2 ml PCR tube strip or 96 well PCR plate, combine the reagents in the following order as shown in the Table below.

(If you choose not to include the RNA CS (RCS) in this reaction mix, replace the volume of RCS with nuclease-free water.)

Reagent Volume
RNA from previous step 8 µl
NEBNext Quick Ligation Reaction Buffer 3 µl
Diluted RNA CS (RCS) or nuclease-free water 0.5 µl
Murine RNase Inhibitor 1 µl
RT Adapter (RTA) 1 µl
T4 DNA Ligase 1.5 µl
Total 15 µl
CONSEJO

For ease, prepare a master mix of these reagents prior to adding to the RNA samples:

  • Combine the NEBNext Quick Ligation Reaction Buffer, diluted RCS (or nuclease-free water), Murine RNase Inhibitor, and RTA in the correct ratio (with an excess volume to allow for pipetting losses) and mix well by gently pipetting the entire volume and spin down.
  • Add 5.5 µl of the master mix to each 8 µl RNA sample using a multichannel pipette.
  • Then add 1.5 µl of T4 DNA Ligase to each RNA sample using a multichannel pipette.

Mix well by gently pipetting the entire volume within each tube/well, or by flicking the tube strip. Then spin down the tube strip or sealed plate.

Incubate the reactions for 10 minutes at room temperature.

For each RT-adapter ligated RNA sample, combine the following reagents together to prepare the reverse transcription mix:

Reagent Volume
Nuclease-free water 13 µl
10 mM dNTPs 2 µl
5x Induro® RT reaction buffer 8 µl
Total 23 µl

  • Mix well by gently pipetting the entire volume and spin down.

  • Then add 23 µl of reverse transcription mix to the RT-adapter ligated RNA sample and mix well by pipetting.

CONSEJO

For ease, prepare a master mix of these reagents prior to adding to the RNA samples:

  • Combine the regents in the correct ratio (with an excess volume to allow for pipetting losses) and mix well by gently pipetting the entire volume and spin down.
  • Using a multichannel pipette, transfer 23 µl of the reverse transcription master mix to each tube/well containing your RT-adapter ligated RNA and mix well by pipetting.

Add 2 µl of Induro® Reverse Transcriptase to each reaction. Mix well by pipetting. Close the tube lids or seal the plate and spin down.

CONSEJO

Use a multichannel pipette to add 2 µl of Induro® Reverse Transcriptase to each reaction in a tube/well. Mix well by pipetting and spin down.

Place the tube strip or 96 well PCR plate in a thermal cycler and incubate at 60°C for 30 minutes, then 70°C for 10 minutes. Bring the samples to 4°C before proceeding to the next step.

Spin down the tube strip or 96 well PCR plate.

Resuspend the stock of Agencourt RNAClean XP beads by vortexing and transfer to a pipetting trough. Ensure that the volume transferred is sufficient for 72 µl to be added to each sample, with an excess to allow for dead volume within the pipetting trough.

IMPORTANTE

Resuspend the beads in the pipetting trough immediately before use to ensure the beads do not settle.

Keep the reverse transcribed RNA samples in their original tubes/wells. Add 72 µl of resuspended RNAClean XP beads to each sample using a multichannel pipette and mix by pipetting the entire volume up and down ten times. Retain any unused beads.

Incubate for 5 minutes at room temperature.

Freshly prepare 70% ethanol in nuclease-free water and pour into a pipetting trough. Allow 250 µl for each sample, with an excess to allow for dead volume within the pipetting trough.

Pellet the beads on a magnet for at least 2 minutes, or until the supernatant is clear. Keep the tube strip/plate on the magnet and pipette off the supernatant.

Keeping the tube strip/plate on the magnet, wash each pellet of beads with 150 µl of freshly prepared 70% ethanol, without disturbing the pellets.

Carefully remove the 70% ethanol from each tube/well using a pipette and discard.

Close the tube lids or seal the plate and spin down. Place the tube strip/plate back on the magnet and wait until the eluate is clear and colourless. Keeping the tube strip/plate on the magnet, pipette off any residual ethanol.

Remove the tube strip/plate from the magnet and resuspend each pellet in 23 µl nuclease-free water. Incubate for 5 minutes at room temperature

Pellet the beads on the magnet until the eluate is clear and colourless.

Remove and retain 23 µl of eluate from each sample and transfer into a clean 0.2 ml PCR tube strip or 96 well PCR plate.

MEDIDA OPCIONAL

At this stage the RT-RNA samples can be stored at -80°C for later use.

Please note, this is the only pause point in this protocol.

In the same 0.2 ml tube strip or 96 well PCR plate, combine the reagents in the following order:

Reagent Volume
RT-RNA sample 23 µl
NEBNext Quick Ligation Reaction Buffer 8 µl
RNA Ligation Adapter (RLA) 6 µl
T4 DNA Ligase 3 µl
Total 40 µl

Mix well by pipetting.

Incubate the reactions for 10 minutes at room temperature.

Resuspend the stock of Agencourt RNAClean XP beads by vortexing and transfer to a pipetting trough. Ensure that the volume transferred is sufficient for 16 µl to be added to each sample, with an excess to allow for dead volume within the pipetting trough.

IMPORTANTE

Resuspend the beads in the pipetting trough immediately before use to ensure the beads do not settle.

Add 16 µl of resuspended Agencourt RNAClean XP beads to each reaction using a multichannel pipette and mix well by pipetting.

Incubate for 5 minutes at room temperature.

Pellet the samples on the magnet. Keep the tube strip/plate on the magnet for 5 minutes for the beads to pellet and then pipette off the supernatant when clear and colourless.

Remove the tube strip/plate from the magnet and add 150 μl of Wash Buffer (WSB) to the beads using a multichannel pipette. Resuspend each pellet thoroughly by pipetting the entire volume of buffer up and down ten times. Fully resuspending the beads at this step ensures optimal kit performance. Return the tube strip/plate to the magnet and allow the beads to pellet and the supernatant to become clear. Remove the supernatant using a pipette and discard.

Repeat the previous step.

IMPORTANTE

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

Spin down the tube strip/plate and replace onto the magnet. Wait until the beads have pelleted to pipette off any remaining Wash Buffer (WSB).

Remove the tube strip/plate from the magnet and resuspend each pellet in 13 µl RNA Elution Buffer (REB). Mix by gently flicking the tube strip or gently pipetting the beads in the wells. Incubate for 10 minutes at room temperature .

Pellet the beads on the magnet for 5 minutes, until the eluate is clear and colourless.

Remove and retain 13 µl of eluate from each sample and transfer into a clean tube or plate.

Quantify 1 µl of reverse-transcribed and adapted RNA on a Qubit™ fluorometer using the Qubit™ dsDNA HS assay.

The recovery aim in the final eluate is >30 ng.

Recovery quantities can vary between different inputs and library preparations. However, we always recommend taking forward the full volume of RNA library for the best sequencing results.

FIN DEL PROCESO

The reverse-transcribed and adapted RNA samples are now ready for loading into the flow cells.

IMPORTANTE

The RNA libraries must be sequenced immediately and cannot be stored for later use.

4. Priming and loading a MinION/GridION Flow Cell

Material
  • Flow Cell Flush (FCF)
  • RNA Flush Tether (RFT)
  • Sequencing Buffer (SB)
  • Library Solution (LIS)
Consumibles
  • MinION/GridION Flow Cells RNA (FLO-MIN004RA)
  • Tubos de 1,5 ml Eppendorf DNA LoBind
Instrumental
  • Dispositivo MinION o GridION
  • MinION/GridION Flow Cell Light Shields
  • Mezclador vórtex
  • Microcentrífuga
  • Pipeta y puntas P1000
  • Pipeta y puntas P200
  • Pipeta y puntas P100
  • Pipeta y puntas P20
IMPORTANTE

Please note, this kit is only compatible with RNA flow cells (FLO-MIN004RA).

CONSEJO

Priming and loading a flow cell

We recommend that you watch the 'Priming and loading your flow cell' video before your first run.

Thaw the Sequencing Buffer (SB), Library Solution (LIS), RNA Flush Tether (RFT) and Flow Cell Flush (FCF) at room temperature. Mix by vortexing and spin down where applicable.

To prepare the flow cell priming mix, combine the following reagents in a clean 1.5 ml Eppendorf DNA LoBind tube. Mix by vortexing and spin down.

  • Increase the reagent volumes accordingly, depending on the number of flow cells you are planning to run.
Reagent Volume per flow cell
RNA Flush Tether (RFT) 30 µl
Flow Cell Flush (FCF) 1,170 µl
Total 1,200 µl

Open the MinION or GridION device lid and slide the flow cell under the clip. Press down firmly on the priming port cover to ensure correct thermal and electrical contact.

Flow Cell Loading Diagram Step 1a

Flow Cell Loading Diagram Step 1b

MEDIDA OPCIONAL

Complete a flow cell check to assess the number of pores available before loading the library.

This step can be omitted if the flow cell has been checked previously.

See the flow cell check instructions in the MinKNOW protocol for more information.

Slide the flow cell priming port cover clockwise to open the priming port.

Flow Cell Loading Diagram Step 2

IMPORTANTE

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 the pores.

After opening the priming port, check for small air bubbles under the cover. Draw back a small volume to remove any bubbles:

  1. Set a P1000 pipette with a tip to 200 µl.
  2. Insert the tip into the priming port.
  3. Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, or until you can see a small volume of buffer entering the pipette tip.

Note: Visually check that there is continuous buffer from the priming port, across the sensor array.

Flow Cell Loading Diagram Step 3

Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time, prepare the library for loading by following the steps below.

Flow Cell Loading Diagram Step 4

In a clean 1.5 ml Eppendorf DNA LoBind tube, prepare each library for loading as follows:

Reagent Volume per flow cell
Sequencing Buffer (SB) 37.5 µl
Library Solution (LIS) 25.5 µl
RNA library 12 µl
Total 75 µl

Complete the flow cell priming:

  1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
  2. Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

Flow Cell Loading Diagram Step 5

Flow Cell Loading Diagram Step 6

Mix the prepared library by gently pipetting up and down, just prior to loading.

Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

Flow Cell Loading Diagram Step 7

Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and closes the port.

Flow Cell Loading Diagram Step 8

Flow Cell Loading Diagram Step 9

IMPORTANTE

For optimal sequencing output, install the light shield on your flow cell as soon as the library has been loaded.

We recommend leaving the light shield on the flow cell after the library is loaded, including during any washing and reloading steps. The shield can be removed when the library has been removed from the flow cell.

Place the light shield onto the flow cell, as follows:

  1. Carefully place the leading edge of the light shield against the clip. Note: Do not force the light shield underneath the clip.

  2. Gently lower the light shield onto the flow cell. The light shield should sit around the SpotON cover, covering the entire top section of the flow cell.

Light shield insertion

ATENCIÓN

The MinION Flow Cell Light Shield is not secured to the flow cell. Therefore, careful handling is required after installation.

FIN DEL PROCESO

Close the device lid and set up a sequencing run on MinKNOW™.

5. Data acquisition and basecalling

How to start sequencing

Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition, and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

MinKNOW can be used and set up to sequence in multiple ways:

  • On a computer either directly or remotely connected to a sequencing device.
  • Directly on a GridION or PromethION 24/48 sequencing device.

For more information on using MinKNOW on a sequencing device, please refer to the device user manuals:

To start a sequencing run on MinKNOW:

1. Navigate to the start page and click Start sequencing.

2. Fill in your experiment details, such as name and flow cell position and sample ID.

3. Select the Direct RNA Sequencing Kit (SQK-RNA004) on the Kit selection page.

(Please note: There is no specific script for the XL kit.)

4. Configure the sequencing and output parameters for your sequencing run or keep to the default settings on the Run configuration tab.

Note: If basecalling was turned off when a sequencing run was set up, basecalling can be performed post-run on MinKNOW. For more information, please see the MinKNOW protocol.

5. Click Start to initiate the sequencing run.

Data analysis after sequencing

After sequencing has completed on MinKNOW, the flow cell can be reused or returned, as outlined in the Flow cell reuse and returns section.

After sequencing and basecalling, the data can be analysed. For further information about options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.

In the Downstream analysis section, we outline further options for analysing your data.

6. Flow cell reuse and returns

Material
  • Flow Cell Wash Kit (EXP-WSH004) or Flow Cell Wash Kit XL (EXP-WSH004-XL)
IMPORTANTE

Our Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) is compatible with RNA flow cells and the Direct RNA Sequencing Kit XL (SQK-RNA004-XL).

However, please be aware that:

  • The DNase I present in the wash kit will not help to recover blocked pores in your flow cell following Direct RNA sequencing.
  • Instead, it will wash off most of the library from the array and remove all adapter from the remaining sample, preventing it from being recaptured and sequenced. This will allow a subsequent library to be loaded.

If you would like to reuse the flow cell after your sequencing experiment is complete, please follow the Flow Cell Wash Kit protocol and store the washed flow cell at +2°C to +8°C.

The Flow Cell Wash Kit protocol is available on the Nanopore Community.

CONSEJO

We recommend you to wash the flow cell as soon as possible, after you stop the run. However, if this is not possible, leave the flow cell on the device and wash it the next day.

Alternatively, follow the returns procedure to send the flow cell back to Oxford Nanopore.

Instructions for returning flow cells can be found here.

IMPORTANTE

If you encounter issues or have questions about your sequencing experiment, please refer to the Troubleshooting Guide in this protocol.

7. Downstream analysis

Post-basecalling analysis

There are several options for further analysing your basecalled data:

EPI2ME workflows

For in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in EPI2ME. The platform provides a vehicle where workflows deposited in GitHub by our Research and Applications teams can be showcased with descriptive texts, functional bioinformatics code and example data.

Research analysis tools

The Research division at Oxford Nanopore Technologies has created a number of analysis tools, which are available in the Oxford Nanopore GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are provided as-is, with minimal support.

Community-developed analysis tools

If a data analysis method for your research question is not provided in any of the resources above, please refer to the resource centre and search for bioinformatics tools for your application. Numerous members of the Nanopore Community have developed their own tools and pipelines for analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.

8. Issues during RNA extraction and library preparation

Below is a list of the most commonly encountered issues, with some suggested causes and solutions.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

Low sample quality

Observation Possible cause Comments and actions
Low RNA integrity (RNA integrity number <9.5 RIN, or the rRNA band is shown as a smear on the gel) The RNA degraded during extraction Try a different RNA extraction method. For more info on RIN, please see the RNA Integrity Number document. Further information can be found in the DNA/RNA Handling page.
RNA has a shorter than expected fragment length The RNA degraded during extraction Try a different RNA extraction method. For more info on RIN, please see the RNA Integrity Number document. Further information can be found in the DNA/RNA Handling page.

We recommend working in an RNase-free environment, and to keep your lab equipment RNase-free when working with RNA.

9. Issues during an RNA sequencing run

Below is a list of the most commonly encountered issues, with some suggested causes and solutions.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

Fewer pores at the start of sequencing than after Flow Cell Check

Observation Possible cause Comments and actions
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check An air bubble was introduced into the nanopore array After the Flow Cell Check it is essential to remove any air bubbles near the priming port before priming the flow cell. If not removed, the air bubble can travel to the nanopore array and irreversibly damage the nanopores that have been exposed to air. The best practice to prevent this from happening is demonstrated in this video.
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check The flow cell is not correctly inserted into the device Stop the sequencing run, remove the flow cell from the sequencing device and insert it again, checking that the flow cell is firmly seated in the device and that it has reached the target temperature. If applicable, try a different position on the device (GridION/PromethION).
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check Contaminations in the library damaged or blocked the pores The pore count during the Flow Cell Check is performed using the QC DNA molecules present in the flow cell storage buffer. At the start of sequencing, the library itself is used to estimate the number of active pores. Because of this, variability of about 10% in the number of pores is expected. A significantly lower pore count reported at the start of sequencing can be due to contaminants in the library that have damaged the membranes or blocked the pores. Alternative RNA extraction or purification methods may be needed to improve the purity of the input material. The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

MinKNOW script failed

Observation Possible cause Comments and actions
MinKNOW shows "Script failed"
Restart the computer and then restart MinKNOW. If the issue persists, please collect the MinKNOW log files and contact Technical Support. If you do not have another sequencing device available, we recommend storing the flow cell and the loaded library at 4°C and contact Technical Support for further storage guidance.

Pore occupancy below 40%

Observation Possible cause Comments and actions
Pore occupancy <40% Not enough library was loaded on the flow cell Ensure you load the recommended amount of good quality library in the relevant library prep protocol onto your flow cell. Please quantify the library before loading and calculate mols using tools like the NEBio Calculator, choosing "RNA ss: mass to moles"
Pore occupancy close to 0 No tether on the flow cell Tethers are adding during flow cell priming (FCT tube). Make sure Flow Cell Tether (FCT) was added to Flow Cell Flush (FCF) before priming.

Large proportion of inactive pores

Observation Possible cause Comments and actions
Large proportion of inactive/unavailable pores (shown as light blue in the channels panel and pore activity plot. Pores or membranes are irreversibly damaged) Air bubbles have been introduced into the flow cell Air bubbles introduced through flow cell priming and library loading can irreversibly damage the pores. Watch the Priming and loading your flow cell video for best practice
Large proportion of inactive/unavailable pores Contaminants are present in the sample The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

Temperature fluctuation

Observation Possible cause Comments and actions
Temperature fluctuation The flow cell has lost contact with the device Check that there is a heat pad covering the metal plate on the back of the flow cell. Re-insert the flow cell and press it down to make sure the connector pins are firmly in contact with the device. If the problem persists, please contact Technical Services.

Failed to reach target temperature

Observation Possible cause Comments and actions
MinKNOW shows "Failed to reach target temperature" The instrument was placed in a location that is colder than normal room temperature, or a location with poor ventilation (which leads to the flow cells overheating) MinKNOW has a default timeframe for the flow cell to reach the target temperature. Once the timeframe is exceeded, an error message will appear and the sequencing experiment will continue. However, sequencing at an incorrect temperature may lead to a decrease in throughput and lower q-scores. Please adjust the location of the sequencing device to ensure that it is placed at room temperature with good ventilation, then re-start the process in MinKNOW. Please refer to this link for more information on MinION temperature control.

Last updated: 2/21/2025

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