Nanopore Direct RNA Sequencing Protocol

Poly(A) Tailing Reaction

1. 17 μl RNA in PCR tube is incubated at 65 °C for 2 min and immediately placed on ice to avoid secondary structure formation.
2. Add 3.5 μl 10× E. coli Poly(A) Polymerase Reaction Buffer into 17 μl RNA in PCR tube, bring to room temperature.
3. Add 4.5 μl ATP into the PCR tube, followed by 4 μl E. coli poly(A) polymerase and 1 μl RNase inhibitor into the PCR tube.
4. Mix gently with P200 pipette and incubate reaction at 37 °C for 10 min.
5. Stop the reaction by directly proceed to the bead cleanup step. Mix the Agencourt RNAClean XP bead well so that the reagent appears homogeneous and consistent in color. Add 60 μl homogeneous-Agencourt RNAClean XP bead into PCR tube and mix well using P200 pipette. Incubate for 5 min.
6. Put the PCR tube into the magnetic stand for 2 min.
7. Gently remove supernatant.
8. Wash RNA twice with 80% ethanol by adding 180 μl of 80% ethanol and incubate for 1 min. Then remove the ethanol without disturbing the pellet.
9. Air-dry at room temperature for 2 min (until dry but not crack).
10. Elute by 12 μl nuclease-free water and flick the tube. Incubate for 2 min at room temperature.
11. Put PCR tube into the magnetic stand for 2 min.
12. Pipet 11 μl poly(A) tailed-RNA into a new PCR tube and put the tube on ice.
13. Measure the final RNA concentration (optional).

dRNA-seq

1. Adapter ligation: Mix 3.0 μl NEBNext Quick Ligation Reaction Buffer into poly(A) tailed-RNA followed by 1.0 μl RT Adapter (RTA) and 1.5 μl T4 DNA Ligase. Mix by pipetting and spin down. Incubate the reaction for 10 min at room temperature.
2. Reverse transcription: Prepare the reverse transcription master mix by mixing 9.0 μl nuclease-free water, 2.0 μl 10 mM dNTPs, 8.0 μl 5x first-strand buffer, 4.0 μl 0.1 M DTT. Add the master mix to the PCR tube containing the RT adapter-ligated RNA. Mix by 200 μl pipette. Add 2 μl of SuperScript IV reverse transcriptase to the reaction. Place the tube in a thermal cycler and incubate at 50 °C for 10 min, then 70 °C for 10 min, and bring the reaction to 4 °C.
3. RNA cleanup using 80% ethanol: Vortex the Agencourt RNAClean XP beads to resuspend. Add 72 μl of resuspended Agencourt RNAClean XP beads to the reverse transcription reaction and mix by pipetting. Incubate for 5 min at RT. Spin down the sample and pellet on a magnetic stand for 3 min. Keep the tube on the magnet and pipet off the supernatant. Wash RNA twice with 180 μl of 80% ethanol. Remove the 80% ethanol using a pipette and discard. Spin down and place the tube back on the magnet. Pipet off any residual 70% ethanol. Air-dry at room temperature for 1 min. Remove the tube from the magnetic stand and resuspend the pellet in 20 μl nuclease-free water and flick the tube. Incubate for 5 min at room temperature. Place the tube back on the magnet for 1 min and pipet 20 μl of eluate into a new 0.2 PCR tube.
4. Motor protein ligation: Add 8.0 μl NEBNext Quick Ligation Reaction Buffer into the 20 μl eluted reverse-transcribed RNA. Then add 4.5 μl - 6.0 μl RNA Adapter (RMX) followed by 3.0 μl nuclease-free water and 3.0 μl T4 DNA Ligase. Mix by 200 μl pipette. Incubate the reaction for 10 min at room temperature.
5. RNA cleanup using Wash Buffer (WSB): Vortex the Agencourt RNAClean XP beads to resuspend. Add 40 μl of resuspended Agencourt RNAClean XP beads to the reaction tube and mix by pipetting. Incubate for 5 min at RT. Spin down the sample and pellet on a magnetic stand for 3 min. Keep the tube on the magnet and pipet off the supernatant. Add 150 μl of the WSB to the beads. Close the tube lid and gently flick the tube. Spin down and place the tube back on the magnet. Remove the WSB using a pipette, and discard. Repeat adding and removing WSB again. Pipet off any residual WSB. Remove the tube from the magnetic stand and resuspend pellet in 20 μl Elution Buffer and gently flick the tube. Incubate for 10 min at room temperature. Place the tube back on the magnet for 2 min or until the eluate is clear. Pipet 20 μl of eluate into a new 0.2 PCR tube. Add 17.5 μl of nuclease-free water to the prepared RNA library and mix with 200 μl pipette. The total volume is 37.5 μl.
6. Priming and loading the SpotON flow cell: Mix the RNA Running Buffer (RRB), Flush Buffer (FB), and Flush Tether (FLT) tubes thoroughly by vortexing and spin down and keep all solutions at room temperature. To prepare the flow cell priming mix, add 30 μl FLT directly to the tube of FB, and vortex. Slide the priming port cover clockwise to open the priming port. Set a P1000 pipette to 200 μl and insert the tip into the priming port. Turn the wheel until you can see a small volume (20 μl of yellow color) of buffer entering the pipette tip. Load 800 μl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 min. During this time, prepare the library for loading.
(a) In a new PCR tube, mix 37.5 μl RRB and 37.5 μl RNA library in nuclease-free water. Gently open the SpotON port cover. Load 200 μl of the priming mix into the priming port (not the SpotON sample port). Set 200 μl pipette to 75 μl. Mix the prepared library by pipetting up and down prior to loading to the sample port. Load all 75 μl of sample to SpotON sample port in a dropwise fashion. Close the SpotON port, close the priming port and close the MinION Mk1B lid.
7. Sequencing: Double–click the MinKNOW icon located on the desktop to open the MinKNOW GUI. Click the "New Experiment" button and fill in the information based on your experiment, such as sample ID/name. Choose the RNA002 kit. Click "Start run." In case of real-time base calling option, ensure the computer has enough resource to run a MinION without deterioration of performance.

Reference:

1. Wongsurawat T, Jenjaroenpun P, Nookaew I. Direct Sequencing of RNA and RNA Modification RNA modifications Identification Using Nanopore[M]//Yeast Functional Genomics: Methods and Protocols. New York, NY: Springer US, 2022: 71-77.