TITLE: HMW gDNA purification and ONT ultra-long-read data generation
AUTHORS: Glennis Logsdon, Shu-Cheng Chuang

[DESCRIPTION]
This protocol describes the purification of high-molecular-weight genomic DNA from mammalian cells and the generation of ultra-long (N50 >100 kbp) Oxford Nanopore data using the PromethION. It is based on the Sambrook and Russell protocol and Josh Quick's protocol with additional modifications. This protocol improves upon previous protocols developed for ultra-long read sequencing, as it gives longer reads with greater yield. In our hands, we obtain about 40-80 Gb of ultra-long-read ONT data with a read N50 of ~110-150 kbp on one PromethION flow cell.

[STEPS]
SECTION: Cell collection and lysis
1. Freeze down ~2 x 10^7 cells as a cell pellet, and store at -80°C.
SECTION: Cell collection and lysis
2. When you are ready to purify the DNA, thaw the cell pellet on ice (usually takes ~30 mins).
SECTION: Cell collection and lysis
3. While the cells are thawing, add RNase A to the lysis buffer at a final concentration of 20 ug/mL. This must be done fresh each time. Keep the lysis buffer + RNase A solution at RT until ready to use.
SECTION: Cell collection and lysis
4. Resuspend thawed cells in 500 uL ice-cold TE (pH 8.0) on ice.

SECTION: Cell collection and lysis
5. Transfer the cell suspension to a 125-mL glass Erlenmeyer flask.
- Make sure that the cells are well-dispersed over the inner surface of the Erlenmeyer flask. This dispersal minimizes the formation of intractable clumps of cells.
SECTION: Cell collection and lysis
6. Quickly add 5 mL of lysis buffer + RNase A to the cell suspension, drop-wise in a circular motion. Swirl to mix.
SECTION: Cell collection and lysis
7. Cover the flask with a parafilm lid and incubate the cell suspension for 1 hr at 37°C in a water bath.
SECTION: Cell collection and lysis
8. Add proteinase K to a final concentration of 200 ug/mL in a drop-wise manner.
- For 5 mLs of cell suspension, add 50 uL Proteinase K.
SECTION: Cell collection and lysis
9. Swirl the flask to mix the enzyme gently into the viscous cell lysate.
SECTION: Cell collection and lysis
10. Incubate the lysate in a water bath for 2 hours at 50°C. Swirl the viscous solution once per hour.
SECTION: Cell collection and lysis
11. Cool the solution to RT.
SECTION: Phenol-chloroform extraction
12. Add the viscous lysate to a 15-mL conical tube.
- This is easiest using a 10-mL serological pipette at low speed.
SECTION: Phenol-chloroform extraction
13. Add an equal volume of ultra-pure phenol:chloroform:isoamyl alcohol (~5-5.5 mLs) to the tube containing lysate.
SECTION: Phenol-chloroform extraction
14. Gently mix the two phases by slowly rotating the tube end-over-end for 10 mins at 18 rpm on a Hulamixer in a fume hood.
SECTION: Phenol-chloroform extraction
15. Pour the lysate into a 15-mL MaXtract tube containing a high-density gel.
SECTION: Phenol-chloroform extraction
16. Spin in a centrifuge at 4000 rpm for 10 mins.
SECTION: Phenol-chloroform extraction
17. Pour the aqueous phase into a new 15-mL conical tube.
SECTION: Phenol-chloroform extraction
18. Add an equal volume of ultra-pure phenol:chloroform:isoamyl alcohol (~5-5.5 mLs) to the tube containing lysate.
SECTION: Phenol-chloroform extraction
19. Gently mix the two phases by slowly rotating the tube end-over-end for 10 mins at 18 rpm on a Hulamixer in a fume hood.
SECTION: Phenol-chloroform extraction
20. Pour the lysate into a 15-mL MaXtract tube containing a high-density gel.
SECTION: Phenol-chloroform extraction
21. Spin in a centrifuge at 4000 rpm for 10 mins.
SECTION: Phenol-chloroform extraction
22. Pour the aqueous phase into a new 50-mL conical tube.
SECTION: Ethanol precipitation
23. Add 2 mL of 5M ammonium acetate (0.4 volume of lysate) to the purified DNA, and gently swirl to mix (~20 mins).
- It is very important to mix gently here. The gentler you mix, the longer your DNA will be.
SECTION: Ethanol precipitation
24. Add 10 mL of 100% ethanol (2 volume of lysate) at RT and gently swirl to mix (usually takes ~1 hr).
- Swirl gently, sometimes rocking back and forth slowly to get the DNA in solution with the salt and ethanol.
SECTION: Ethanol precipitation
25. Store the precipitating DNA solution overnight at 4°C.
- We have found that storing the DNA overnight results in purer DNA that sequences better and gives more yield.
SECTION: Ethanol precipitation
26. Remove the precipitate in one piece from the ethanolic solution with a disposable inoculating needle shaped into a U and place it into an Eppendorf 2-mL DNA LoBind tube.
SECTION: Ethanol precipitation
27. Wash the DNA precipitate 2x with 1 mL 70% ethanol, and collect the DNA by centrifuging at max speed (~15,000 rpm) for 15 secs.
SECTION: Ethanol precipitation
29. Store the pellet of DNA in an open tube at RT until the last visible traces of ethanol have evaporated (1-2 mins).
- Do not allow the pellet of DNA to dry completely. Desiccated DNA is very difficult to dissolve.
SECTION: Ethanol precipitation
30. Add 760 uL EB and incubate without mixing at RT for 2 days to allow the pellet to fully resuspend into a translucent viscous gel.
SECTION: ONT library preparation with the SQK-ULK114 kit
31. Pre-warm a heat block to 75°C.
SECTION: ONT library preparation with the SQK-ULK114 kit
32. Thaw the Fragmentation Mix (FRA), FRA Dilution Buffer (FDB) and Rapid Adapter (RA). Spin down briefly and keep on ice.
SECTION: ONT library preparation with the SQK-ULK114 kit
33. Add the following reagents to a 1.5 mL Eppendorf DNA LoBind tube on ice:

246 uL HMW gDNA
4 uL FRA Buffer 

Total = 250 uL
SECTION: ONT library preparation with the SQK-ULK114 kit
34. Mix the diluted FRA by pipetting.
SECTION: ONT library preparation with the SQK-ULK114 kit
35. Add 250 uL of diluted FRA to 750 uL of extracted DNA on ice. Stir the reaction with the pipette tip whilst expelling the diluted FRA to ensure an even distribution.
SECTION: ONT library preparation with the SQK-ULK114 kit
36. Immediately mix the reaction by slowly pipetting 10x with a wide-bore pipette tip while on ice.
- Make sure that the solution is well-mixed. The enzyme needs to interact with the DNA in order to tagment it and add an adapter.
SECTION: ONT library preparation with the SQK-ULK114 kit
37. Incubate the reaction as follows:

1. RT for 10 mins
2. 75°C for 10 mins
3. On ice for >10 mins
SECTION: ONT library preparation with the SQK-ULK114 kit
38. Add 5 uL of RA to the reaction with a regular pipette tip. Use a P1000 wide-bore tip to mix by pipetting 5x. Visually check to ensure the reaction is thoroughly mixed.
SECTION: ONT library preparation with the SQK-ULK114 kit
39. Incubate for 1 hr at RT.
SECTION: Library clean-up
41. Add 500 uL of PTB to the tube with your gDNA.
SECTION: Library clean-up
42. Mix the reaction by rotating end-over-end 10 mins at 3 rpm on a Hulamixer. Check to see if a glassy white mass is visible.
SECTION: Library clean-up
43. Spin in a centrifuge at 1,000g for 1 min.

SECTION: Library clean-up
44. Discard the supernatant, taking care not to disturb the DNA precipitate.
SECTION: Library clean-up
45. Pulse-spin the tube for 1 second and remove the last traces of supernatant.
SECTION: Library clean-up
46. Add 200 uL of EB to the DNA precipitate.
SECTION: Library clean-up
47. Incubate O/N at RT.
SECTION: Library clean-up
48. The next day, gently mix the DNA library by slow pipetting 5-10 times with a P1000 wide-bore tip.
SECTION: Flow cell priming and loading
51. Let a PromethION flow cell equilibrate to RT for ~20 mins. Make sure a light shield is on the flow cell.
SECTION: Flow cell priming and loading
52. Place the flow cell onto the PromethION and run platform QC.
SECTION: Flow cell priming and loading
53. Thaw the Sequencing Buffer (SQB), Loading Solution UL (LSU), Flush Tether UL (FTU), and Flow Cell Flush (FCF) at RT. Mix by inverting and spin down.
SECTION: Flow cell priming and loading
54. In a new 2 mL tube, aliquot 100 uL of DNA library using a P1000 wide-bore tip.
SECTION: Flow cell priming and loading
58. Incubate the reaction for 30-60 mins at RT.
SECTION: Flow cell priming and loading
59. Prepare the flow cell priming mix in a 1.5 mL Eppendorf tube, and mix by pipetting.

30 uL FTU
1170 uL FCF

Total = 1200 uL
SECTION: Flow cell priming and loading
60. Using a P1000 set to ~500 µl, place the pipette tip in the inlet port of the flow cell and turn the wheel to the right to remove the air and a small amount of storage buffer. This will make the storage buffer flush with the inlet port opening.
SECTION: Flow cell priming and loading
61. Load 500 µl priming mix (FTU + FCF) via the inlet port slowly. Close the cover and wait 5 minutes. 
SECTION: Flow cell priming and loading
62. Pipette up 200 uL of your DNA library with a P200 with a 200 uL wide-bore tip so that it's ready to load. Load another 500 µl Priming Mix (FTU + FCF) via the inlet port slowly.
SECTION: Flow cell priming and loading
63. Immediately load 200 uL of the DNA library into the inlet port. To aid in this process, you can use the P1000 pipette to draw the DNA into the flow cell by sucking it from the waste port.
SECTION: Flow cell priming and loading
65. Ensure there are no gaps between subsequent DNA library loads and avoid introducing air bubbles that can damage the nanopores.
SECTION: Flow cell priming and loading
64. Let the flow cell sit for at least 30 minutes before sequencing.
SECTION: Flow cell priming and loading
66. Wash the flow cell and reload a fresh library every 24 hours for a total of three or four loads per flow cell. You can recover the DNA from the first two loads, store them at 4°C, and reload them for the third and fourth runs to maximize output.
SECTION: Library clean-up
40. Thaw PTB and EB at RT. Spin down briefly and keep on ice.
SECTION: Library clean-up
49. Measure the DNA library concentration using a Nanodrop.
SECTION: Library clean-up
50. Keep the DNA library on ice until you're read to load. Alternatively, store the library at 4°C.
SECTION: Flow cell priming and loading
55. In a new 1.5 mL tube, add the following and mix thoroughly by pipetting:

100 uL SBU
10 uL of LSU

Total = 110 uL
SECTION: Flow cell priming and loading
56. Add 110 uL of buffer mixture to the DNA library.
SECTION: Flow cell priming and loading
57. Gently mix the reaction by slowly pipetting 5-10 times with a P1000 wide-bore tip. Visually inspect to ensure the sample is homogenous.
SECTION: Ethanol precipitation
28. Pulse-spin the tube for 1 second and remove the last traces of supernatant.