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

In a 2 ml DNA LoBind tube, pellet ~200 million cells (50µL volume of pellet per reaction).

Wash cells in cold PBS and centrifuge at 10.000x g

Remove supernatant.

Repeat step 2-3 once more.

Resuspend pellet in 480µL SpheroBuffer .

Add 20µL lyticase (5U/μl), mix well with a wide-bore pipette tip and incubate at 30°C``0h 30m 0s. Check spheroplast formation from time to time (if this has not yet been optimised before).$$

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

Incubate at 56°C for 0h 15m 0s whilst shaking at 700rpm. The solution should clear up when protein digestion takes place.

Add 4µL RNaseA and mix with a wide-bore pipette tip while avoiding any bubbles.

Incubate at 56°Cfor 0h 10m 0s.

Add 400µL 5M Ammonium acetate and 100µL 5M NaCl

Mix by vertical rotation (Hula mixer) 9rpm

Centrifuge at 16.000x g.

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.

Add 3 glass beads (diameter = 3mm) to the cell lysate and 500µL isopropanol.

Mix on a Hula mixer at 9rpm.

Check that the DNA has bound tightly around the beads and let sit for another minute.

Remove liquid by pipetting or carefully tipping it over while guarding the beads with the pipette tip.

Wash bound DNA with 1mL of 70% ethanol , rotate the tube 2-3 times then discard the wash buffer.

Repeat wash once more.

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.

Quickly pour beads to a new 2 ml tube previously aliquoted with 100-120µL 10mM Tris-HCl pH 9.0

Incubate at 37°C for at least 0h 30m 0s with regular pipetting every 10-15 min using a wide-bore tip.

Pour the beads into a bead retainer on a new 1.5 ml tube and spin at maximum speed for 0h 1m 0s.

Incubate at Room temperature for a few hours or 2h 0m 0s (slow rotation may aid elution).

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.

Measure DNA concentration with Qubit.

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.

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

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

In another tube make up FRA dilution ( 1.2 µl FRA per microgram of yeast DNA ):

A	B
	Volume (µL)
FRA	6
FDB (FRA Dilution Buffer)	55
Total volume	60

Mix the diluted FRA by vortexing. Cool on ice.

On ice, add 60µL 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.

Mix by gentle pipetting with a wide-bore pipette tip.

Incubate the reaction as follows:

22°C - 0h 10m 0s

70°C - 0h 5m 0s

22°C - 0h 5m 0s

Add 5µL 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.

Incubate for 0h 30m 0s at 22°C.

Add 3 glass beads into the tube with the adapted DNA.

Add 1:1 volume (240µL) of 10 mM CoHex .

Rotate tube at 9rpm. Make sure there is no bead stuck at the bottom or at the cap, flick if this is the case.

Invert the tube again 2-3 times to ensure all DNA has precipitated and is tightly bound to the beads.

Discard supernatant. Take care not to disturb the DNA precipitated onto the beads.

Wash the glass beads by gently adding 750µL PEGW buffer and gently invert 2-3 times.

Incubate for 0h 3m 0s at room temperature.

Aspirate and discard the supernatant, taking care not to disturb the DNA precipitate.

Repeat wash once more.

Pipette 120µL of EB into a clean DNA LoBind 2 ml tube.

Discard the second wash buffer as much as possible.

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.

Immediately pour the beads into the 2 ml tube with the elution buffer.

Incubate the library at 37°C for 0h 30m 0s. Gently aspirate and dispense the eluate over the glass beads at regular intervals with a wide-bore pipette tip to aid elution.

Continue with overnight incubation at room temperature.

The next day, insert a bead retainer into a clean 1.5-ml tube.

Pour the beads and spin at 10.000x g.

Incubate for at least 0h 30m 0s at Room temperature with regular pipette mixing.

Quantify the library

Load 30-40µL library with SQB in 1:1 volume ratio.

Incubate for 0h 30m 0s at Room temperature

Load the library on a primed MinION flow cell.