Identification of m6A RNA Modifications by Nanopore Sequencing Protocol

Direct RNA Sequencing Library Preparation

Preparing Input RNA

1. Pool each curlcake (for unmodified and m6A modified separately) into a DNA LoBind tube that will contain 200 ng from each (800 ng total).
2. Adjust the volume to 9μL with nuclease-free water and mix thoroughly by inversion.
3. Spin down briefly in a microfuge.
Adapter Ligation
1. In a 0.2 mL thin-walled PCR tube, mix the reagents from the Direct RNA sequencing kit.
2. Mix by pipetting and spin down.
3. Incubate the reaction for 10 min at RT. In the meantime, proceed to the reverse transcription step.

Reverse Transcription and Cleanup

1. Mix the reagents together to make the reverse transcription master mix.
2. Add the master mix to the 0.2 mL PCR tube containing the RT Adapter-ligated RNA from the "RT Adapter ligation" step above. Mix by pipetting.
3. Add 2μl SuperScript III reverse transcriptase to the reaction and mix by pipetting.
4. Place the tube in a thermal cycler, incubate at 50 °C for 50 min and 70 °C for 10 min, and bring the sample to 4 °C before proceeding to the next step.
5. Transfer the sample to a 1.5 mL DNA LoBind Eppendorf tube.
6. Resuspend the stock of Agencourt RNAClean XP beads by vortexing, add 72μL beads to the reverse transcription reaction, and mix by pipetting.
7. Incubate on a Hula Mixer for 5 min at RT.
8. Prepare 200μL of fresh 70% ethanol in nuclease-free water.
9. Spin down the sample and pellet on a magnet.
10. Keep the tube on the magnet and wash the beads with 150μL 70% ethanol without disturbing the pellet.
11. Remove the ethanol and discard. Spin down tubes, place back on the magnetic rack, and remove any residual ethanol.
12. Remove the tube from the magnetic rack and resuspend the pellet in 20μL nuclease-free water. Incubate for 5 min at RT.
13. Pellet the beads on the magnet until the eluate is clear and colorless.
14. Pipette 20μL of the eluate into a clean 1.5 mL Eppendorf DNA LoBind tube.
15. Measure cDNA and RNA on a Qubit or similar.

RMX Adapter Ligation and Cleanup

1. In a clean 1.5 mL Eppendorf DNA LoBind tube, mix the reagents.
2. Mix by pipetting and incubate for 10 min at RT.
3. Resuspend the stock of Agencourt RNAClean XP beads by vortexing, add 40μL of beads to the adaptor ligation reaction, and mix by pipetting.
4. Incubate on a Hula Mixer for 5 min at RT.
5. Spin down the sample and pellet on a magnet. Keep the tube on a magnet and pipette off the supernatant.
6. Add 150μL of the wash buffer (WSB) provided in the Direct RNA sequencing kit to the beads. Resuspend the beads by flicking the tube. Return the tube on the magnetic rack, allow beads to pellet, and pipette off the supernatant. Repeat. Allow to air-dry for 2 min.
7. Remove the tube from the magnetic rack and resuspend the pellet in 21μl elution buffer. Incubate for 10 min at RT.
8. Pellet the beads on magnet until eluate is clear and colorless.
9. Remove and retain 21μL of eluate into a clear 1.5 mL Eppendorf DNA LoBind tube.
10. Measure cDNA and RNA on Qubit or similar.
11. Mix the library with 17.5μl water.
12. Add 37.5μL RRB buffer (mix RRB by vortexing before using) and mix well. Library is now ready to be loaded to the flow cell.

Reference:

1. Liu H, Begik O, Novoa E M. EpiNano: Detection of m6A RNA Modifications Using Oxford Nanopore Direct RNA Sequencing[M]//RNA Modifications. Humana, New York, NY, 2021: 31-52.