TITLE: Protocol to use for direct RNA sequencing of plants with Oxford Nanopore Technologies
AUTHORS: Nivedha Nataraj

[DESCRIPTION]
Transcriptomic analysis is a key component of understanding gene expression and regulatory mechanisms. Oxford Nanopore’s direct RNA sequencing kit is optimized to sequence native RNA without converting RNA samples into cDNA. The kit has been developed to sequence strands ranging from 70 to 26,000 nucleotides. Nanopore Direct RNA Sequencing, DRS, has been used to analyze cellular mRNA, noncoding RNA, and RNA viruses. (Jain et al, 2022)

Oxford Nanopore Technologies, ONT, recommends that the RNA sample has an absorbance 260/280 ~2.0 and a 260/230 ~2.0-2.2. 
If the RNA has a 260/280 less than ~2.0, it indicates the presence of DNA. A 260/280 less than ~1.8 presence of protein or phenol in the RNA sample.


[STEPS]
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
1. Add 10 μL of 2M DTT for each 500 μL of Plant RNA Lysis Solution.
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
2. Add 500 μL of the Lysis+DTT Solution is added to a 1.5 mL Eppendorf Microcentrifuge tube.
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
3. Weigh 100 g of plant tissue into the microcentrifuge tub and vortex to mix.
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
4. Incubate the sample for 3 minutes at 56℃.
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
5. Centrifuge for 5 minutes at 20,000 xg (14,000 rpm).
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
6. Transfer the supernantant to a clean Eppendorf Microcentrifuge tube.
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
7. Add 250 μL of 96% ethanol.
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
8. Transfer the samples to a GeneJET Purification Column and centrifuge the samples for 1 minute at 12,000 xg (11,000 rpm). Discard the flow-through solution and reassemble the column.
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
9. Add 700 μL of Wash Buffer WB 1 to the purification column (ensure the wash buffer has been prepped to the manufacturer’s specifications). Centrifuge for 1 minute at 12,000 xg (11,000 rpm). Discard the flow-through and collection tube, 
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
10. Place the purification column into a clean 2 mL collection tube.
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
11. Add 500 μL of Wash Buffer WB 2 to the purification column. Centrifuge for 1 minute at 12,000 xg (11,000 rpm). Discard the flow-through. Reassemble the column and collection tube.
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
12. Repeat step 11 and re-spin the column for 1 minute at a maximum speed ≥20,000 xg (≥ 14,000 rpm). Discard the collection tube and flow through. Transfer the purification column to an RNase-free 1.5 mL collection tube.
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
13. To elute the RNA, add 50 μL of nuclease-free water to the center of the purification column membrane and centrifuge for 1 minute at 12,000 xg (11,000 rpm).
SECTION: RNA Extraction - ThermoScientific GeneJET Plant RNA Purification Mini Kit
14. Discard the purification column. The purified RNA can be stored at -20℃ until use. 
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
15. Before Starting Library Preparation: 1 μL of total RNA in 7μL of nuclease-free water to make 8 μL of total sample.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
16. Spin down and RT Adapter (RTA, Direct RNA Sequencing Kit ) and RNA Ligation Adapter (RLA, Direct RNA Sequencing Kit).
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
17. Thaw the Wash Buffer (WSB, Direct RNA Sequencing Kit) and RNA Elution Buffer (REB, Direct RNA Sequencing Kit) at room temperature and mix by vortexing before placing them on ice.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
18. Dilute 1 μL of RNA sample using nuclease-free water (ThermoFisher, AM9937) in a 0.2 mL thin-walled PCR tube. Adjust the final volume to 8 μL.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
19. To the 0.2 mL thin-walled PCR tube, add:
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
19.1. 
 


SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
20. Mix by pipetting and spin down.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
21. Incubate at room temperature for 10 minutes.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
22. In a clean 1.5 mL Eppendorf tube, combine:
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
22.1. 
 

SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
23. Transfer the reverse transcriptase master mix to the 0.2 mL PCR tube with the adapter-ligated RNA.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
24. Add 2 μL of the SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, 18080044) to the reaction.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
25. Place the tube in a thermal cycler and incubate at 50℃ for 50 minutes and then 70℃ for 10 minutes, and bring the sample to 4℃.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
26. Transfer the sample to a clean 1.5 mL Eppendorf DNA LoBind tube.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
27. Add 72 μL of resuspended Agencourt RNAClean XP beads to the reaction.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
28. Incubate on a Hula mixer for 5 minutes at room temperature.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
29. Spin down the sample and pellet on a magnet. Keep the tube on the magnet and pipette off the supernatant when the sample is clear.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
30. Wash the beads with 150 μL of freshly prepared 70% ethanol. Carefully remove the 70% ethanol and discard.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
31. Spin down the tube and place it back on the magnet. Remove any residual ethanol.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
32. Remove the tube from the magnet and resuspend the pellet with 23 μL nuclease-free water. Incubate for 5 minutes at room temperature.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
33. Pellet the beads on a magnet until the elute is clear.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
34. Remove and retain the 23 μL of elute in a clean 1.5 mL Eppendorf DNA LoBind tube.
SECTION: Library Preparation - ONT Direct RNA Sequencing Kit
35. Store at -80℃ for later use.
SECTION: Addition of Adapters
36. In the sample tube the sample is stored in, add:
SECTION: Addition of Adapters
36.1. 
 

SECTION: Addition of Adapters
37. Incubate the reaction for 10 minutes at room temperature.
SECTION: Addition of Adapters
38. Add 16 μL of Agencourt RNAClean XP beads to the reaction and mix by pipetting.
SECTION: Addition of Adapters
39. Incubate on a hula mixer for 5 minutes at room temperature.
SECTION: Addition of Adapters
40. Spin down the sample and pellet on a magnet. Pipette off the supernatant.
SECTION: Addition of Adapters
41. Add 150 μL of the Wash Buffer (WSB, Direct RNA Sequencing Kit), resuspend the pellet, and return to the magnet.
SECTION: Addition of Adapters
42. Repeat step 41.
SECTION: Addition of Adapters
43. Spin the sample, replace it with the magnet, and pipette off the remaining Wash Buffer.
SECTION: Addition of Adapters
44. Add 13 μL of RNA Elution Buffer (REB, Direct RNA Sequencing Kit) and pellet the beads on a magnet for 5 minutes until the elute is clear.
SECTION: Addition of Adapters
45. Retain the 13 μL of elute in a clean 1.5 mL Eppendorf tube.
SECTION: Addition of Adapters
46. Quantity 1 μL of adapted RNA using the Qubit fluorometer DNA HS assay.
SECTION: Addition of Adapters
47. RNA must immediately be sequenced and cannot be stored.
SECTION: Priming and Loading the SpotON Flow Cell
48. Combine the following reagents in a clean 1.5 mL Eppendorf DNA LoBind Tube. Mix by vortexing at room temperature.
SECTION: Priming and Loading the SpotON Flow Cell
48.1. 
 

SECTION: Priming and Loading the SpotON Flow Cell
49. Open the GridION device lid and slide the flow cell under the clip. Press firmly to ensure correct thermal and electrical contact.
SECTION: Priming and Loading the SpotON Flow Cell
50. Slide the flow cell priming port cover clockwise to open and check for small air bubbles under the cover. Use a P1000 pipette that is set at 200 μL, insert it into the priming port, and turn the wheel to 220-230 μL to draw back 20-30 μL, or until a small volume of buffer enters the pipette tip.
SECTION: Priming and Loading the SpotON Flow Cell
51. Load 800 μL of the priming mix into the flow cell, ensuring to avoid any air bubbles. Wait for 5 minutes to prepare the library.
SECTION: Priming and Loading the SpotON Flow Cell
52. Prepare the library in a new 1.5 mL Eppendorf DNA LoBind tube by adding:
SECTION: Priming and Loading the SpotON Flow Cell
52.1. 
 

SECTION: Priming and Loading the SpotON Flow Cell
53. Load 200 μL of the priming mix to complete the flow cell priming.
SECTION: Priming and Loading the SpotON Flow Cell
54. Mix the prepared library before loading, and add 75 μL of the prepared library into the flow cell dropwise. 
SECTION: Priming and Loading the SpotON Flow Cell
55. Gently replace the sample port cover and place the light shield onto the flow cell.
SECTION: Priming and Loading the SpotON Flow Cell
56. Close the lid and set up a sequencing run.