This kit is recommended for users who:

• Want to sequence native RNA
• Want to use total RNA or an enriched (e.g. poly(A)-tailed) sample as a starting input material

This protocol describes how to carry out sequencing of native RNA using the Q-Line Direct RNA Sequencing Kit (Q-SQK-RNA004). 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 the Q-Line MinION Flow Cell RNA (Q-FLO-MIN004R). This kit requires no fragmentation and provides enough material for six reactions.

Note: The complementary cDNA strand is not sequenced, but improves the RNA sequencing output.

Reverse transcription adapters are annealed to the poly(A) tails of full-length RNA, followed by synthesis of the complementary strand by reverse transcription. Sequencing adapters are then ligated to the RNA–cDNA hybrid ends, and the prepared library is loaded onto a flow cell for sequencing.

Prepare for your experiment

Note: We recommend following RNA handling guidelines and Good Laboratory Practice when carrying out this protocol to prevent degradation of the RNA substrate. Key practices include wearing PPE and changing gloves frequently, decontaminating work surfaces and equipment, using clean nuclease-free consumables/reagents and keeping samples on ice.

You will need to:

• Extract your RNA, and check its length, quantity, and purity.
• Ensure you have a Q-Line Direct RNA Sequencing Kit (Q-SQK-RNA004), the correct equipment and third-party reagents.
• Check your flow cell(s) to ensure they have sufficient health to run the assay. A failed flow cell check will prevent the start of a run.

Library preparation

You will need to:

• Synthesise the complementary strand of the RNA.
• Attach sequencing adapters to the ends of the RNA–cDNA hybrid.
• Prime the flow cell, and load your RNA library onto the flow cell.

Sequencing and analysis

You will need to:

• Start a sequencing run using the Sequencing Software, which will collect raw data from the device and basecall the reads.

Warnings

• Follow Good Laboratory Practice in relation to personal safety, including but not limited to the use of personal protective equipment when handling the samples and test reagents. Refer to Safety Data Sheets provided with the product for specific safety requirements.
• Take care to prevent the reagents from contacting your skin, eyes, and mucous membranes.
• If contact occurs, immediately wash the affected area with soap and water. Thoroughly wash hands after handling samples and test reagents.
• In the event of a serious incident, contact the Manufacturer of the device. The user may also wish to contact the competent authority of the Member State in which the device was used.
• The Q-Line GridION as supplied provides only raw sequence data, which requires validated downstream analysis and the inclusion of assay-specific controls.
• Using the Q-Line GridION outside of the intended use is not recommended and is at the user’s risk.

Precautions

• Refer to local or WHO health and safety guidance for handling samples containing hazard group (HG)/BioSafety Level (BSL) 3 organisms.
• Decontaminate surfaces with 0.5% sodium hypochlorite (bleach) diluted with deionised or purified water.
• Prevent contamination of reagents by following standard contamination prevention procedures including but not limited to the use of personal protective equipment and using filter pipette tips for handling nucleic acids.
• Reagents must be stored under the indicated storage conditions before and after use.
• Waste should be disposed of in accordance with local waste disposal regulations.
• Do not mix different lots of reagents.
• We recommend using DNase-free pipette tips.
• Never reuse the consumables intended for single use (for example, tips, gloves, and test tubes).
• When using a pipette, use correct volumes and adhere to manufacturer’s instructions. The use of multichannel pipettes is recommended where possible. Mouth pipettes must not be used.
• Do not use the reagents if the package is damaged or reagent tubes are leaking (reagents may be compromised, leading to an incorrect result). Please contact technical support.
• Do not use test reagents after their expiration date.
• Do not use the flow cell if the package is damaged or the flow cell is leaking as the flow cell may be compromised, leading to an incorrect result. If leakage occurs, refer to the Flow Cell Safety Data Sheet for handling guidance, and contact technical support.
• After use, the flow cell must be prepared for return and disposal as described in the Q-Line GridION user guide.

Library description	Sequencing run time	Accuracy	Output
Poly(A)-tailed RNA (RCS)	24 hours	~98.1% modal single-read accuracy	>2.6 x106 reads
QPCR human reference total RNA	24 hours	~97.4% modal single-read accuracy	>2.0 x106 reads

Statistics are representative of average observed performance in verification tests. Values vary depending on sample quality, library preparation method, flow cell type, and basecalling model. Values provided assume high-quality samples that are prepared following our optimised protocols.

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.

We recommend using the NEBNext® Companion Module for Oxford Nanopore Technologies® Direct RNA Sequencing (NEB, E3440S), which contains all the NEB reagents needed for use with the Direct RNA Sequencing Kit.

The reagents contained in the companion module are also available to purchase separately through NEB.

Name	Abbreviation	Cap colour	Quantity	Minimum fill volume per container (µl)
RT Adapter	RTA	Blue	1	6
RNA Ligation Adapter	RLA	Green	1	36
Wash Buffer	WSB	Orange	2	900
RNA Elution Buffer	REB	Amber	1	78
Library Solution	LIS	White cap with pink label	1	153
Sequencing Buffer	SB	Red	1	225
RNA Flush Tether	RFT	Violet	1	180
Flow Cell Flush	FCF	N/A	1	7,020

The Q-Line GridION device contains all the hardware required to control up to five sequencing experiments simultaneously and acquire the data. The device is further enhanced with high-performance GPU technology for real-time basecalling. Read more in the Q-Line GridION configuration guide.

The Sequencing Software controls the Q-Line GridION, collects sequencing data in real-time and processes it into basecalled reads. The software can also demultiplex reads by barcode, and basecall/demultiplex data after a sequencing run has completed. You will be using the Sequencing Software for every assay you run.

For instructions on how to run the Sequencing Software on the Q-Line GridION, please refer to the Q-Line GridION user guide.

Once an RNA library has been prepared using this protocol, we do not recommend storing it; the library should be sequenced immediately. Therefore, a flow cell check is recommended before starting library preparation.

Note: This protocol describes the process of starting a sequencing run using the default Q-SQK-RNA004 assay supplied with Q-Line GridION. For more information on setting up a sequencing run, see the Q-Line GridION user guide. For more information on configuring an assay definition file for your own assay, see the Q-Line GridION configuration guide. Ensure you select the correct assay for your experiment.