TITLE: Ultra-Long Sequencing of Yeast Cells (S. cerevisiae) on ONT Sequencers – A Modified FindingNemo Protocol
AUTHORS: Inswasti Cahyani

[DESCRIPTION]
This protocol is a bespoke modification of the FindingNemo protocol (LongRead Club) to enable ultra-long (UL) sequencing of yeast on Nanopore sequencers. 
We have tested and optimised this in Saccharomyces cerevisae using the obsolete SQK-ULK001 kit.
It's not tested yet but we think the new ULK114 can be used to replace ULK001, using exact volumes of the corresponding reagents. 
Let us know if you have tried it with the new kit!
Our best read N50 so far was ~67 kb (look at profile pic). This is not (yet) ultra-long, but may also be due to the distribution of chromosome sizes which is generally smaller than more complex eukaryotes, e.g. mammals.

[STEPS]
SECTION: Spheroplasting
1. In a 2 ml DNA LoBind tube, pellet ~200 million cells (50 µL volume of pellet per reaction).

SECTION: Spheroplasting
2. Wash cells in cold PBS and centrifuge at 10.000 x g, 1 min 

SECTION: Spheroplasting
3. Remove supernatant.
SECTION: Spheroplasting
4. Repeat step 2-3 once more.
SECTION: Spheroplasting
5. Resuspend pellet in 480 µL SpheroBuffer.

SECTION: Spheroplasting
6. Add 20 µL lyticase (5U/μl), mix well with a wide-bore pipette tip and incubate at 30 °C30 min. Check spheroplast formation from time to time (if this has not yet been optimised before).  

SECTION: Cell Lysis
7. Add 480 µL lysis buffer (LB) and 20 µL Proteinase K, carefully mix with wide-bore pipette tip while avoiding any bubbles.

SECTION: Cell Lysis
8. Incubate at 56 °C for 15 min whilst shaking at  . The solution should clear up when protein digestion takes place.

SECTION: Cell Lysis
9. Add 4 µL RNaseA and mix with a wide-bore pipette tip while avoiding any bubbles.

SECTION: Cell Lysis
10. Incubate at 56 °Cfor 10 min.

SECTION: Cell Lysis
11. Add 400 µL 5M Ammonium acetate and 100 µL 5M NaCl
SECTION: Cell Lysis
13. Centrifuge at 16.000 x g, 10 min.

SECTION: Cell Lysis
14. Transfer the supernatant to a new 2 ml tube, careful not to transfer protein layer/precipitate at the bottom. If lysate is too thick to have layers, move as much clear lysate as possible.
SECTION: DNA Precipitation
15. Add 3 glass beads (diameter = 3mm) to the cell lysate and 500 µL isopropanol. 
 


SECTION: DNA Precipitation
16. Mix on a Hula mixer at  .

SECTION: DNA Precipitation
17. Check that the DNA has bound tightly around the beads and let sit for another minute.
SECTION: DNA Precipitation
18. Remove liquid by pipetting or carefully tipping it over while guarding the beads with the pipette tip.
SECTION: DNA Precipitation
19. Wash bound DNA with 1 mL of 70% ethanol, rotate the tube 2-3 times then discard the wash buffer.

SECTION: DNA Precipitation
20. Repeat wash once more.
SECTION: DNA Precipitation
21. Remove ethanol as much as possible, pour beads into a retainer tube on a collection column. Quickly spin (less than 1 second) on a table-top minicentrifuge.

 

SECTION: DNA Precipitation
22. Quickly pour beads to a new 2 ml tube previously aliquoted with 100-120 µL 10mM Tris-HCl pH 9.0
 


SECTION: DNA Precipitation
23. Incubate at 37 °C for at least 30 min with regular pipetting every 10-15 min using a wide-bore tip.

SECTION: DNA Precipitation
24. Pour the beads into a bead retainer on a new 1.5 ml tube and spin at maximum speed for 1 min.

SECTION: DNA Precipitation
25. Incubate at Room temperature for a few hours or 120 min (slow rotation may aid elution).

SECTION: Tagmentation and Adapter Ligation
31. In another tube make up FRA dilution (1.2 µl FRA per microgram of yeast DNA):

 

SECTION: Tagmentation and Adapter Ligation
35. Incubate the reaction as follows:
22 °C  - 10 min 
70 °C  - 5 min 
22 °C  - 5 min 
SECTION: Tagmentation and Adapter Ligation
36. Add 5 µL  of RAP to the pooled sample with a regular pipette tip. Use a wide-bore tip to pipette mix. Visually check to ensure the reaction is thoroughly mixed. 

SECTION: Tagmentation and Adapter Ligation
37. Incubate for 30 min at 22 °C.

SECTION: Nemo Clean-up
38. Add 3 glass beads into the tube with the adapted DNA.
SECTION: Nemo Clean-up
39. Add 1:1 volume (240 µL) of 10 mM CoHex.

SECTION: Nemo Clean-up
40. Rotate tube at  . Make sure there is no bead stuck at the bottom or at the cap, flick if this is the case.

SECTION: Nemo Clean-up
41. Invert the tube again 2-3 times to ensure all DNA has precipitated and is tightly bound to the beads.
SECTION: Nemo Clean-up
42. Discard supernatant. Take care not to disturb the DNA precipitated onto the beads.
SECTION: Nemo Clean-up
43. Wash the glass beads by gently adding 750 µL PEGW buffer and gently invert 2-3 times.

SECTION: Nemo Clean-up
44. Incubate for 3 min  at room temperature.

SECTION: Nemo Clean-up
45. Aspirate and discard the supernatant, taking care not to disturb the DNA precipitate.
SECTION: Nemo Clean-up
46. Repeat wash once more.
SECTION: Nemo Clean-up
47. Pipette 120 µL of EB into a clean DNA LoBind 2 ml tube.

 

SECTION: Nemo Clean-up
48. Discard the second wash buffer as much as possible.
SECTION: Nemo Clean-up
49. Quick spin the tube and use a 10 µl tip to discard the rest of the buffer at the bottom of the tube (move the beads aside with the tip while doing it). There will be a few microliters buffer left as a dead volume. This will not affect sequencing.
SECTION: Nemo Clean-up
50. Immediately pour the beads into the 2 ml tube with the elution buffer.
SECTION: Nemo Clean-up
51. Incubate the library at 37 °C for 30 min. Gently aspirate and dispense the eluate over the glass beads at regular intervals with a wide-bore pipette tip to aid elution.

SECTION: Nemo Clean-up
52. Continue with overnight incubation at room temperature.
SECTION: Nemo Clean-up
53. The next day, insert a bead retainer into a clean 1.5-ml tube.
SECTION: Nemo Clean-up
54. Pour the beads and spin at 10.000 x g, 1 min.

SECTION: Nemo Clean-up
55. Incubate for at least 30 min at Room temperature with regular pipette mixing.

SECTION: Nemo Clean-up
56. Quantify the library
SECTION: Nemo Clean-up
57. Load 30-40 µL library with SQB in 1:1 volume ratio. 
 


SECTION: Nemo Clean-up
58. Incubate for 30 min at Room temperature  

SECTION: Cell Lysis
12. Mix by vertical rotation (Hula mixer)   

SECTION: Nemo Clean-up
59. Load the library on a primed MinION flow cell.

SECTION: Tagmentation and Adapter Ligation
30. Take 5 µg DNA and dilute with 10 mM Tris-HCl pH 9.0 to a total volume of 180 µL. Mix well or rotate and incubate for 10 min. Cool sample on ice.
 


SECTION: Tagmentation and Adapter Ligation
32. Mix the diluted FRA by vortexing. Cool on ice.
SECTION: Tagmentation and Adapter Ligation
33. On ice, add 60 µL of the diluted FRA to the extracted DNA. Stir the reaction with the pipette tip whilst expelling the diluted FRA to ensure an even distribution.

	

SECTION: Tagmentation and Adapter Ligation
34. Mix by gentle pipetting with a wide-bore pipette tip. 
 


	
SECTION: HMW DNA QC
26. An accurate measurement of DNA concentration is important as this will determine the
optimum ratio of transposase (FRA) to DNA molecules at the library prep step. Also, the
viscous nature of UHMW DNA requires that sample measurement represents all parts of the
DNA solution.
Two nucleic acid quantification methods, i.e., fluorometric (Qubit) and spectrophotometric
(Nanodrop), can be used in parallel to assess both the quantity and quality of the extracted
DNA. 
SECTION: HMW DNA QC
28. For Nanodrop measurement, measure 3 positions from top, middle, and bottom part of the sample. Percent CV can then be calculated from these concentrations to gauge DNA homegeneity.
 

SECTION: HMW DNA QC
27. Measure DNA concentration with Qubit.
 

SECTION: HMW DNA QC
29. Whenever possible, the quality of extracted DNA sample should be analysed by method(s) that
enable visual inspection of molecule length distribution such as:
Regular agarose gel electrophoresis
Pulsed-Field Gel Electrophoresis, e.g., using Pippin Pulse (Sage Science)
Agilent Bioanalyzer DNA
Agilent TapeStation DNA