Before start checklistDirect RNA Sequencing Kit (SQK-RNA001) SpotON Flow Cell FLO-MIN106 Agencourt RNAClean XP beads SuperScript III Reverse Transcriptase RTA / RCS / ELB / WSB on ice RRB / RMX in freezer until needed Freshly prepared 70% EtOH Nuclease-free water (NFW)

10 mM dNTP solutionNEB Blunt / TA Ligase Master MixT4 DNA ligaseNEBNext® Quick Ligation Reaction BufferQubit RNA HS Assay KitQubit dsDNA HS Assay KitMicrofugeVortexer and Hula mixerMagnet for bead separation

Approx 20 DNA LoBind Eppendorf tubes0.2 ml thin-walled PCR tubesThermal cyclerPipettes P2, P20, P100/200, P1000 Pipette tips P2, P20, P100/200, P1000Update checks for MinKNOW™ completed Notebook sleep timer / update offRun Platform QC prior to library prep

Reverse transcription adapter ligationMix in a 0.2 ml thin-walled PCR tube:

Direct RNA Sequencing Protocol for the MinIONTM

using SQK-RNA001 (1/2) Flow Cell NumberDNA Samples

0.2 ml PCR tube 15 µl

Quantify 1 µl of reverse-transcribed and adapted RNA using a Qubit DNA HS assay.

0.2 ml PCR tube 38 µl

MASSFLOW INSTRUCTIONS NOTES / OBSERVATIONS TIME / DATE

2 µl

RNA Adapter Mix ligationMix in a clean 1.5 ml DNA LoBind tube:

20 µl reverse-transcribed RNA8 µl NEBNext Quick Ligation Reaction Buffer6 µl RMX 3 µl NFW3 µl T4 DNA Ligase

Mix by pipetting + spin downIncubate at RT for 10 minutes

Add 72 µl resuspended RNAClean XP beads at RT Incubate on rotator for 5 minutes, spin down and pellet on magnet. Discard the supernatantKeep on magnet, wash 1x with 150 µl fresh 70% EtOH, by twisting the tube in the magnetic rack 2x 180°, waiting for the pellet to migrate to the other side of the tube each timeBriefly spin down, replace on magnet, pipette off residual EtOH Resuspend pellet in 20 µl NFW, incubate at RT for 5 minutes Pellet beads on a magnet, remove eluate of reverse-transcribed RNA and transfer to fresh DNA LoBind tube

1.5 ml DNA LoBind tube 40 µl

9 µl (500 ng) full-length polyA RNA0.5 µl RNA CS3 µl NEBNext Quick Ligation Reaction Buffer 1 µl RTA1.5 µl T4 DNA Ligase

Mix by pipetting + spin downIncubate at RT for 10 minutes

Make the reverse transcription master mix:9 µl NFW2 µl 10 mM dNTPs8 µl 5X first strand buffer (from SuperScript III Reverse Transcriptase kit)4 µl 0.1 M DTT

Add master mix to the adapter ligation reaction

Reverse transcriptionMix together:

38 µl RNA in reverse transcription master mix2 µl SuperScript Reverse Transcriptase

Incubate in a thermal cycler: 50 °C for 50 min 70 °C for 10 min bring the sample to 4 °CTransfer to a 1.5 ml DNA LoBind tube

Add 40 µl resuspended RNAClean XP beads at RT Incubate on rotator for 5 minutes, spin down and pellet on magnet. Discard the supernatantKeep on magnet, wash 2x with 150 µl WSB. Resuspend beads by flicking the tube, then pellet on a magnet and remove the supernatantBriefly spin down, replace on magnet, pipette off residual wash Resuspend pellet in 21 µl ELB, incubate at RT for 10 minutes Pellet beads on a magnet, remove eluate of adapter-ligated RNA and transfer to fresh DNA LoBind tube

23 µl

0.2 mlPCR tube

DNA LoBind tube

72 µl

20 µl

DNA LoBind tube 20 µl

Wash 1x 150 µl

20 µl

DNA LoBind tube 40 µl

DNA LoBind tube

40 µl

21 µl

DNA LoBind tube 20 µl

Wash 2x 150 µl

1 µl

EXAMPLE PROTOCOL

MASSFLOW INSTRUCTIONS NOTES / OBSERVATIONS TIME / DATE

Prepare the MinION for sequencing protocolThe platform QC should be run prior to library preparation beginning

Assemble the MinION and MinION Flow CellSet up MinKNOW to run the Platform QC – name the run and start the protocol script – NC_Platform_QC.pyAllow the script to run to completion; the number of active pores are reported

Direct RNA Sequencing Protocol for the MinIONTM

using SQK-RNA001 (2/2) Flow Cell NumberDNA Samples

Prime the Flow Cell ready for the library to be loaded when library preparation is completePrepare priming buffer

500 µl RRB500 µl Nuclease-free water

Prime the Flow CellOpen the priming port. Draw back a few µls of buffer to make

sure there is continuous buffer flow from the priming port across the sensor array.

Load 800 µl of the priming buffer. Wait 5 minutesGently lift the activator to make the sample port accessible Load 200 µl of the priming buffer as before.

Priming cover Activator

Priming Port

800 µl

5 minutes

DNA LoBind

20 µl

17.5 µl

DNA LoBind 75 µl

Prepare the library for loading20 µl RNA library17.5 µl NFW37.5 µl RRB kept on ice

Mix by inversion and spin down

Loading the prepared libraryAdd 75 µl of sample to the Flow Cell via the sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.Gently replace the activator, making sure the bung enters the sample portClose the sample port cover and replace the MinION lid.

Starting the sequencing script in MinKNOW and the workflow in the Metrichor Agent

Return to MinKNOW, name the run, select the NC_48Hr_Sequencing_Run_FLO-MIN106_SQK-RNA001.py and start using the start in the MinKNOW dialogue boxMinKNOW will report the number of pores available for sequencing before data collection begins. These may differ from those reported in the Platform QC.Allow the protocol to proceed until MinKNOW reports Finished Successfully System Ready. Use the Stop in the Control Panel to finish the protocol.Run the Albacore software to basecall the reads

After sequencing checklistStore washed Flow Cell at 4 °C or complete the returns form in the Nanopore Community Store MinION at RTReturn reagents to the freezer

Priming and loading the library

200 µl

37.5 µl

EXAMPLE PROTOCOL