This kit is highly recommended for:

• Multiplexing up to 24 native RNA samples
• Exploring attributes of native RNA, such as modified bases
• Removing RT or PCR bias
• Transcripts that are difficult to reverse transcribe

This protocol describes how to carry out multiplex sequencing of native RNA using the Direct RNA Barcoding Kit 24 (SQK-DRB004.24). Starting from poly(A)+ enriched RNA or from poly(A)-tailed IVT transcripts, reverse transcription barcodes are ligated to your samples, and your samples are pooled together. The barcoded samples are then reverse transcribed to perform first-strand synthesis, providing stability to the native RNA strand. A USER digestion reaction is performed to digest the overhangs from the excess Reverse Transcription Barcodes, preventing barcode cross-contamination. 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.

A control experiment following this method and using the RNA Control Strand (RCS) as your sample input can be 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.

Note: Please ensure your extracted RNA concentration is sufficient to enable 450 fmol of RNA in 5 µl per sample input.

Tip: If using the NEBNext® High Input Poly(A) mRNA Isolation Module, we suggest reducing the elution volume of your sample to 7 µl to increase the concentration. This will allow sufficient volume for 1 µl to be used in the fragment analyser, 1 µl for quantification, and 5 µl to be taken forward for library preparation.

Note: The quantity of RNA required will vary depending on your RNA length. We suggest using a tool like the NEBioCalculator or equivalent to calculate the correct input mass for your sample(s).

• Perform poly(A)+ selection or transcript enrichment on your RNA samples.
• Ensure you have your sequencing kit, the correct 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 Barcode ligation	Ligate the Reverse Transcription Barcodes, quench the reaction with EDTA, pool your samples together and perform a sample clean-up	~90 minutes	No pause at this stage.
Reverse transcription	Perform first-strand synthesis of the RNA for stability, and perform a sample clean-up	~90 minutes	No pause at this stage.
Excess barcode inactivation	Perform USER digestion reaction to digest the overhangs from the excess Reverse Transcription Barcodes (RTB), and perform a sample clean-up	~90 minutes	At this stage the RT-RNA can be stored at -80°C for later use.
Sequencing Adapter ligation and clean-up	Attach the sequencing adapters to the RNA-cDNA hybrid ends	~90 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	10 minutes

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.

Note: Please ensure your extracted RNA concentration is sufficient to enable 450 fmol of RNA in 5 µl per sample input.

Tip: If using the NEBNext® High Input Poly(A) mRNA Isolation Module, we suggest reducing the elution volume of your sample to 7 µl to increase the concentration. This will allow sufficient volume for 1 µl to be used in the fragment analyser, 1 µl for quantification, and 5 µl to be taken forward for library preparation.

The quantity of RNA required will vary depending on your RNA length. It is possible to start the protocol from a lower sample input, however this will likely yield a lower output.

We suggest using a tool like the NEBioCalculator or equivalent to calculate the correct input mass for your sample(s).

Please note: This method has not been validated for total RNA inputs. Using total RNA as your starting material can lead to variable results, lower yields, and decreased sequencing outputs.

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 quality control of your RNA sample, please read the Input DNA/RNA QC protocol.

For further information on using RNA as input, please read the links below.

• RNA contaminants
• RNA stability
• RNA Integrity Number (RIN)

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

We recommend using the NEBNext® High Input Poly(A) mRNA Isolation Module (E3370S) to isolate your poly(A)+ RNA samples. Alternative methods may be suitable, but these have not been tested by our internal teams.

We have validated and recommend the use of all the 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.

We highly recommend that you check the number of 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 unused flow cell with fewer than the number of pores listed 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

The Direct RNA Barcoding Kit 24 contains 24 unique barcodes and sufficient reagents for up to 6 library preparation reactions.

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
RNA Barcode Adapter	RBA	Green	1	45
RNA Elution Buffer	REB	Brown	1	300
Displacement Strand	DS	Orange	1	10
Sequencing Buffer	SB	Red	1	700
Library Solution	LIS	White cap, pink label	1	600
EDTA	EDTA	Blue	1	700
RNA Flush Tether	RFT	Purple	1	200
S Fragment Buffer	SFB	Clear bottle	1	13,000
Flow Cell Flush	FCF	Clear cap, light blue label	1	8,000
Reverse Transcription Barcode plate	RTB01-24	-	2 plates, 3 sets of barcodes per plate	5 µl per well

• For poly(A) enriched RNA samples, our internal input testing was performed on ~2.4 kb poly(A) selected UHRR.
• For poly(A) tailed IVT transcript samples, our internal input testing was performed on 1–1.2 kb IVT transcripts.

We recommend following the input guidance below for your initial experiments. Further input optimisation may be beneficial for samples of different lengths.

This greatly improves pore occupancy and output on your flow cell.

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 see the device user manuals:

• MinION Mk1D user manual
• GridION user manual

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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 Barcoding Kit 24 (SQK-DRB004.24) on the Kit selection page.

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.

Note: Run settings will have POD5 file generation OFF by default. If you would like to look into RNA modifications using Dorado in your downstream analysis, please ensure you turn ON the POD5 setting in the run configuration setup.

5. Click Start to initiate the sequencing run.

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.

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.

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.

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.

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.

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 videos for how to load a MinION Flow Cell and how to load a PromethION Flow Cell.
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 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.

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.

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.

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 how to load a MinION Flow Cell or how to load a PromethION Flow Cell videos 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.

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.

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.