TITLE: Nanopore Library Preparation for R10 Ligation Sequencing Kit
AUTHORS: Guan Jie Phang

[DESCRIPTION]
Protocols for Nanopore R10 flow cell Ligation Sequencing library construction.

[STEPS]
SECTION: Step1. End-prep
1.  In a 200 µL PCR tube, mix the following chemicals:
 

 


 

SECTION: Step1. End-prep
1.1. Incubate in a thermocycler at 20 °C for 30 min and 65 °C for 30 min. Hold at 4 °C 
SECTION: Step2. Adapter ligation
2. Thaw the Ligation Adapter (LA) and Ligation Buffer (LNB). Vortex and spin down the tubes, and place on ice immediately afterwards.
 

 


SECTION: Step2. Adapter ligation
2.2. In a 1.5 mL tube, add the following chemicals:
 

 

SECTION: Step2. Adapter ligation
2.3. Incubate overnight at 20 Room temperature .

SECTION: Step3. Cleanup
8. Wash the beads by adding either 250 µL Long Fragment Buffer (LFB) or 250 µL Short Fragment Buffer (SFB).  Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. 

 

 

SECTION: Step3. Cleanup
4. Resuspend the AMPure XP beads by vortexing.
SECTION: Step3. Cleanup
8.1. Remove the supernatant using a pipette and discard.
SECTION: Step4. Priming and loading the SpotON flow cell
14. Thaw the flow cell, Sequencing Buffer (SQB), Loading Solution (LIS), and one tube of Flush mixture (FB & FLT) at room temperature on cooling block.

 

SECTION: Step5. Flow Cell Wash and Storage
26. Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.
Thaw one tube of Wash Diluent (DIL) at room temperature on a cooling block.
 

SECTION: Step5. Flow Cell Wash and Storage
26.1. In a clean 1.5 mL microtube, prepare the following Flow Cell Wash Mix:
 

 

SECTION: Step5. Flow Cell Wash and Storage
26.2. Lift the sensory cover.

 

SECTION: Step5. Flow Cell Wash and Storage
26.3. Rotate the flow cell priming port cover clockwise so that the priming port is visible.
SECTION: Step5. Flow Cell Wash and Storage
26.4. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
1. Set a P1000 pipette to 800 μl.
2. Insert the tip into the priming port.
3. Turn the wheel until the dial shows 820–830 μl, or until you can see a small volume of liquid entering the pipette tip.
 

SECTION: Step5. Flow Cell Wash and Storage
26.5. Load 200 μl of the prepared Flow Cell Wash Mix into the flow cell via the priming port, avoiding the introduction of air.
SECTION: Step5. Flow Cell Wash and Storage
26.6. Close the priming port and keep into 4°C for 2 days.
SECTION: Step3. Cleanup
3. Thaw either Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) at room temperature on a cooling block, mix by vortexing, spin down and place on ice.
SECTION: Step3. Cleanup
7. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

 

SECTION: Step3. Cleanup
6. Incubate for 5 minutes at Room temperature.
SECTION: Step3. Cleanup
5. Add 0.45X AMPure XP Beads (AXP) to the tube and mix by flicking the tube.

SECTION: Step3. Cleanup
10. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for 5~30 seconds.
 

SECTION: Step3. Cleanup
11. Remove the tube from the magnetic rack and resuspend the pellet in 15 µL Elution Buffer (EB). Spin down and incubate for 10 minutes at 37 °C.

SECTION: Step3. Cleanup
9.  . 
SECTION: Step3. Cleanup
12. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
SECTION: Step3. Cleanup
13. Leave the tube aside, and go to the priming steps.

 

SECTION: Step2. Adapter ligation
2.1. Spin down the KAPA HyperPrep Ligase and place on ice immediately.

SECTION: Step4. Priming and loading the SpotON flow cell
15. Open the MinION device lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.
 

 

SECTION: Step4. Priming and loading the SpotON flow cell
16. Slide the flow cell priming port cover clockwise to open the priming port.
 

SECTION: Step4. Priming and loading the SpotON flow cell
17. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles: 
1. Set a P1000 pipette to 800 μl.
2. Insert the tip into the priming port.
3. Turn the wheel until the dial shows 820–230 µl, or until you can see a small volume of buffer entering the pipette tip.
 

SECTION: Step4. Priming and loading the SpotON flow cell
18. Load 800 μl of the priming mix into the flow cell via the priming port by turning the pipet wheel, avoiding the introduction of air bubbles. Wait for 5 minutes. During this time, prepare the library for loading by following the steps below.
SECTION: Step4. Priming and loading the SpotON flow cell
20. Complete the flow cell priming:
1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
2. Load 200 μl of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
 

SECTION: Step4. Priming and loading the SpotON flow cell
19. Aspirate 12 µL of the eluted prepared library into a new 1.5 mL microtube.
SECTION: Step4. Priming and loading the SpotON flow cell
19.1. In that new tube, prepare the library for loading as follows:
 

 

SECTION: Step4. Priming and loading the SpotON flow cell
21. Mix the prepared library gently by pipetting up and down in the tip using a P200 pipet just prior to loading.
SECTION: Step4. Priming and loading the SpotON flow cell
22. Add the library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
SECTION: Step4. Priming and loading the SpotON flow cell
23. Gently close the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port, cover the sensor array with the sensory cover, and close the MinION device lid.
 

SECTION: Step5. Flow Cell Wash and Storage
25. Stop or pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.
 

SECTION: Step5. Flow Cell Wash and Storage
26.7.   

SECTION: Step5. Flow Cell Wash and Storage
28. Thaw one tube of Storage Buffer (S) at room temperature on a cooling block.
SECTION: Step5. Flow Cell Wash and Storage
28.1. Mix contents thoroughly by pipetting and spin down briefly.
SECTION: Step5. Flow Cell Wash and Storage
27. Take out the MinION device from 4°C fridge.
SECTION: Step5. Flow Cell Wash and Storage
29. Rotate the flow cell priming port cover clockwise so that the priming port is visible. Open the device lid.
SECTION: Step5. Flow Cell Wash and Storage
30. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
1. Set a P1000 pipette to 800 μl.
2. Insert the tip into the priming port.
3. Turn the wheel until the dial shows 820–830 μl, or until you can see a small volume of liquid entering the pipette tip.
 

SECTION: Step5. Flow Cell Wash and Storage
31. Add 500 μl of Storage Buffer (S) through the priming port of the flow cell by turning the pipet wheel.
SECTION: Step5. Flow Cell Wash and Storage
31.1. Close the priming port.
SECTION: Step5. Flow Cell Wash and Storage
32. Using a P1000, remove all fluids from the waste channel through Waste port 2. As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.

 

 

SECTION: Step5. Flow Cell Wash and Storage
33. Perform a Flow Cell Check.
SECTION: Step5. Flow Cell Wash and Storage
34. The flow cell can now be stored at 4°C.
SECTION: Step4. Priming and loading the SpotON flow cell
24. Quantify 1 µL of eluted sample and the original DNA library using Qubit.