TITLE: Nanopore Sequencing with Flongle Flow Cells
AUTHORS: NUS iGEM

[DESCRIPTION]
The 2023 NUS-Singapore iGEM Team followed this protocol to sequence their STAR oligo variants. The original protocol was released by Oxford Nanopore Technologies, but due to the use of a new type of flow cell in the experiment, some minor modifications to the steps were made to tailor it to our project.

[STEPS]
SECTION: End-prep
1. Mix   and DNA Control Sample (DCS) at room temperature by vortexing. Keep the beads at room temperature and store the DNA Control Sample (DCS) on ice.

SECTION: End-prep
2. Prepare the    in accordance with the manufacturer's instructions, and place on ice. (  do not vortex,
  vortex for at least 30 s until no precipitate is visible.)


SECTION: End-prep
3. Dilute the DNA Control Sample (DCS) by adding 105 µL Elution Buffer directly to one DCS tube. Mix gently by pipetting and spin down. (excess storage at -20 °C)


SECTION: End-prep
4. In clean 0.2 ml thin-walled PCR tubes (or a clean 96-well plate), aliquot 200 fmol (130 ng for 1 kb amplicons) of DNA per sample.

SECTION: End-prep
5. Make up each sample to 11.5 µL using nuclease-free water. Mix gently by pipetting and spin down.

SECTION: End-prep
6. Combine the following components per tube/well:
Between each addition, pipette mix 10 - 20 times.
Recommend to prepare a master mix and add 2.5 µL to each well.
     200 fmol (130 ng for 1 kb amplicons) amplicon DNA      11.5μL        Diluted DNA Control Sample (DCS)      1μL        Ultra II End-prep Reaction Buffer      1.75μL        Ultra II End-prep Enzyme Mix      0.75μL   


SECTION: End-prep
7. Ensure the components are thoroughly mixed by pipetting and spin down in a centrifuge.
SECTION: End-prep
8. Using a thermal cycler, incubate at 20 °C for 5 min and 65 °C for 5 min.

SECTION: End-prep
9. Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: End-prep
10. Resuspend   by vortexing.


SECTION: End-prep
11. Add 15 µL of resuspended   to each end-prep reaction and mix by flicking the tube.

SECTION: End-prep
12. Incubate on a Hula mixer (rotator mixer) for 5 min at room temperature.

SECTION: End-prep
13. Prepare 500 µL of fresh 80% ethanol in nuclease-free water.

SECTION: End-prep
14. Spin down the samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.

SECTION: End-prep
15.  Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.


SECTION: End-prep
16. Repeat step 15.
SECTION: End-prep
17. Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual ethanol. Allow to dry for 30 s, but do not dry the pellets to the point
of cracking.


SECTION: End-prep
18. Remove the tubes from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Spin down and incubate for 2 min at room temperature.


SECTION: End-prep
19. Pellet the beads on a magnet until the eluate is clear and colourless.

SECTION: End-prep
20. Remove and retain 10 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube. Sample storage at 4 °C.


SECTION: Native Barcode Ligation
21. Prepare   according to the manufacturer's instructions, and place on ice.
SECTION: Native Barcode Ligation
22. Thaw the EDTA at room temperature and mix by vortexing. Then spin down and place on ice.
SECTION: Native Barcode Ligation
23. Thaw the Native Barcodes (NB01-24) required for the number of samples at room temperature. Individually mix the barcodes by pipetting, spin down, and place them on ice.
SECTION: Native Barcode Ligation
24. Select a unique barcode for each sample to be run together on the same flow cell. Up to 24 samples can be barcoded and combined in one experiment. (only use one barcode per sample)
SECTION: Native Barcode Ligation
25. In clean 0.2 ml PCR-tubes or a 96-well plate, add the reagents in the following order per well:
     End-prepped DNA      7.5μL        Native Barcode (NB01-24)      2.5μL        Blunt/TA Ligase Master Mix      10μL   

SECTION: Native Barcode Ligation
26. Ensure the reaction is thoroughly mixed by gently pipetting and spin down briefly.
SECTION: Native Barcode Ligation
27. Incubate for 20 min at room temperature.
SECTION: Native Barcode Ligation
28. Add the 2 µL of clear cap EDTA to each well and mix thoroughly by pipetting and spin down briefly.
SECTION: Native Barcode Ligation
29. Pool all the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube; 22 µL per sample
SECTION: Native Barcode Ligation
30. Resuspend   by vortexing.
SECTION: Native Barcode Ligation
31. Add 9 µL   per sample to the pooled reaction, and mix by pipetting for a 0.4X clean.
SECTION: Native Barcode Ligation
32. Incubate on a Hula mixer (rotator mixer) for 10 min at room temperature.
SECTION: Native Barcode Ligation
33. Prepare 2 mL of fresh 80% ethanol in nuclease-free water.
SECTION: Native Barcode Ligation
34. Spin down the sample and pellet on a magnet for 5 min. Keep the plate on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.
SECTION: Native Barcode Ligation
35. Keep the tube on the magnetic rack and wash the beads with 700 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: Native Barcode Ligation
36. Repeat step 35.
SECTION: Native Barcode Ligation
37. Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol.
Allow the pellet to dry for 30 s, but do not dry the pellet to the point of cracking.
SECTION: Native Barcode Ligation
38. Remove the tube from the magnetic rack and resuspend the pellet in 35 µL nuclease-free water by gently flicking.
SECTION: Native Barcode Ligation
39. Incubate for 10 min at 37 °C. Every 2 min, agitate the sample by gently flicking for 10 s to encourage DNA elution.
SECTION: Native Barcode Ligation
40. Pellet the beads on a magnetic rack until the eluate is clear and colourless. Remove and retain 35 µLof eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Adaptor Ligation and Clean-up
41. Prepare the NEBNext Quick Ligation Reaction Module according to the manufacturer's instructions, and place on ice. (Do NOT vortex  .)
SECTION: Adaptor Ligation and Clean-up
42. Spin down the Native Adapter (NA) and  , pipette mix and place on ice.
SECTION: Adaptor Ligation and Clean-up
43. Thaw Elution Buffer at room temperature and mix by vortexing. Then spin down and place on ice.
SECTION: Adaptor Ligation and Clean-up
44. Thaw either Long Fragment Buffer (LFB) >3kb or Short Fragment Buffer (SFB) <3kb at room temperature and mix by vortexing. Then spin down and place on ice.
SECTION: Adaptor Ligation and Clean-up
45. In a 1.5 ml Eppendorf LoBind tube, mix in the following order:
     Pooled barcoded sample      30μL        Native Adapter (NA)      5μL        NEBNext Quick Ligation Reaction Buffer(5X)      10μL        Quick T4 DNA Ligase      5μL   

SECTION: Adaptor Ligation and Clean-up
46. Ensure the reaction is thoroughly mixed by gently pipetting and spin down briefly.
SECTION: Adaptor Ligation and Clean-up
47. Incubate the reaction for 20 min at room temperature.

SECTION: Adaptor Ligation and Clean-up
48. Resuspend   by vortexing.

SECTION: Adaptor Ligation and Clean-up
49. Add 20 µL of resuspended   to the reaction and mix by pipetting.
SECTION: Adaptor Ligation and Clean-up
50. Incubate on a Hula mixer (rotator mixer) for 10 min at room temperature.
SECTION: Adaptor Ligation and Clean-up
51. Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
SECTION: Adaptor Ligation and Clean-up
52. Wash the beads by adding either 125 µL Long Fragment Buffer (LFB) or 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. Remove the supernatant using a pipette and discard.
SECTION: Adaptor Ligation and Clean-up
53. Repeat Step 52.
SECTION: Adaptor Ligation and Clean-up
54. Spin down and place the tube back on the magnet. Pipette off any residual supernatant.
SECTION: Adaptor Ligation and Clean-up
55. Remove the tube from the magnetic rack and resuspend pellet in 7 µL Elution Buffer (EB).

SECTION: Adaptor Ligation and Clean-up
56. Spin down and incubate for 10 min at 37 °C. Every 2 min, agitate the sample by gently flicking for 10 s to encourage DNA elution.
SECTION: Adaptor Ligation and Clean-up
57. Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 min.
SECTION: Adaptor Ligation and Clean-up
58. Remove and retain 7 µL of EB containing the DNA library into a clean 1.5 ml Eppendorf SNA LoBind tubes.
SECTION: Adaptor Ligation and Clean-up
59. Quantify 1 µL of eluted sample using Nanodrop.
SECTION: Adaptor Ligation and Clean-up
60. Make up the library to 5 µL at 5-10 fmol.
SECTION: Loading the Flongle Flow Cell
61. Thaw the Sequencing Buffer(SB), Library Beads(LIB), Flow Cell Tether(FCT) and one tube of Flow Cell Flush(FCF) at room temperature before mixing by vortexing. Then spin down and
store on ice.
SECTION: Loading the Flongle Flow Cell
62. In a fresh 1.5 ml Eppendorf DNA LoBind tube, mix 117 µL of Flow Cell Flush(FCF) with 3 µL of Flow Cell Tether(FCT) and mix by pipetting.
SECTION: Loading the Flongle Flow Cell
63. Place the Flongle adaptor into the MinION or one of the five GridION positions.
SECTION: Loading the Flongle Flow Cell
64. Place the flow cell into Flonge adapter, and press the flow cell down until hearing a click.
SECTION: Loading the Flongle Flow Cell
65. Peel back the seal tab from the Flongle flow cell, up to a point where the sample port is exposed. Lift up the seal tab, pull the seal tab to open access to the sample port, then hold the seal tab open by using adhesive on the tab to stick to the MinION Mk1B lid.
SECTION: Loading the Flongle Flow Cell
66. To prime the flow cell with the mix of Flow Cell Flush(FSF) and Flow Cell Tether(FCT) that was prepared earlier, ensure that there is no air gap in the sample port or the pipette tip.
Place the P200 pipette tip inside the sample port and slowly dispense the priming fluid into the Flonge flow cell by slowly pipetting down. Recommend twisting the pipette plunger down to avoid flushing the flow cell too vigorously.

SECTION: Loading the Flongle Flow Cell
67. Vortex the vial of Library Beads(LIB). Immediately prepare the sequencing Mix in a fresh 1.5 ml        Eppendorf DNA LoBind tube for loading the Flongle as follows:
     Sequencing Buffer(SB)      15μL        Library Beads(LIB) mixed immediately before use      10μL        DNA Library      5μL   

SECTION: Loading the Flongle Flow Cell
68. To add the sequencing mix to the flow cell, ensure that there is no air gap in the sample port or the
pipette tip. 
Place the P200 tip inside the sample port and slowly dispense the sequencing mix into the flow cell by slowly pipetting down. Recommend twisting the pipette plunger down to avoid flushing the flow cell too vigorously.
SECTION: Loading the Flongle Flow Cell
69. Seal the Flonge flow cell using the adhesive on the seal tab: stick the transparent adhesive tape to the sample port, replace the top(wheel icon section) of the seal tab to its original position.
SECTION: Loading the Flongle Flow Cell
70. Replace the sequencing platform lid and start sequencing.