TITLE: FindingNemo Extraction 1: Phenol-based Method
AUTHORS: Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a sub-protocol designed to extract/isolate ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers using phenol-based method. </div><div class = "text-block">A DNA extraction protocol that yields clean and homogeneous UHMW DNA is important for a good UL sequencing output. The choice of protocol should be based on achieving these parameters.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Kit-free, phenol-based method</span><span> is a scaled-down version of Josh Quick's protocol (</span><a href="https://dx.doi.org/10.17504/protocols.io.mrxc57n" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">dx.doi.org/10.17504/protocols.io.mrxc57n</span></a><span>) with additional glass bead step for DNA precipitation.</span></div><br/><div class = "text-block"><span>We tested this sub-protocol listed here in </span><span style = "font-weight:bold;">human cell line</span><span>, with input cells of 5 millions. As a rule of thumb, a million cells will suffice for one load on a MinION.</span></div><br/></div>

[STEPS]
?. [UHMW DNA Extraction]
This protocol is a scaled-down and modified version of the “Ultra-long read sequencing protocol for RAD004 V.3” by Josh Quick (https://dx.doi.org/10.17504/protocols.io.mrxc57n).
Elution volume is adjusted for downstream application of preparing ultra-long DNA library following the new ONT protocol (SQK-ULK001).
?. [Cell Lysis]
Pellet 5 million cells in a 2 ml tube by centrifuging at 500 x g for 5 min at 4oC.
Centrifuge: 500 34, 5 min, 4 10
?. [Cell Lysis]
Wash with 500 μl cold PBS and centrifuge at 500 x g for 3 min at 4oC. Discard supernatant.
Centrifuge: 500 34, 3 min, 4 10
?. [Cell Lysis]
Resuspend well by pipette mixing in 20 μl cold PBS.
Thorough resuspension is important for next lysis step.
?. [Cell Lysis]
Add 1 ml TLB-SDS and 100 μg RNase A (1 μl) and vortex at full speed for 5 seconds.
33
?. [Cell Lysis]
Incubate at 37°C for 1 hour, mix by inversion every 15 minutes.
37 °C
[mix per 15 min]
?. [Cell Lysis]
Add 200 μg Proteinase K (10 μl). Mix by slow inversion 5 times.
?. [Cell Lysis]
Incubate at 50°C for 2 hours, mix every 30 minutes by slow inversion 3 times.
50 °C
[mix per 30 min (3x inversion)]
?. [Phenol Separation]
Split the lysate into 2 phase-lock gel tubes (ca. 550 ul per tube).
?. [Phenol Separation]
Add 550 μl buffer-saturated phenol to each tube containing lysate.
?. [Phenol Separation]
Place on a HulaMixer or any vertical rotator at 20 rpm for 10 minutes. If a fine emulsion has not formed after a minute gradually increase the rotation speed.
[vertical rotator]
?. [Phenol Separation]
Centrifuge at 4500 rpm for 10 minutes.
Centrifuge: 4500 33, 10 min
?. [Phenol Separation]
Transfer the aqueous phase to another phase-lock gel tube by pouring or using a wide-bore P1000 tip.
?. [Phenol Separation]
Add 250 μl buffer-saturated phenol and 250 μl chloroform-isoamyl alcohol to each tube.Repeat step 11-12 and continue to step 15.
?. [Phenol Separation]
Transfer and combine the aqueous phase to a 2 ml tube (sample will be ca. 1 ml).
?. [Phenol Separation]
Add 1 ml chloroform-isoamyl alcohol.Repeat step 11-12 and continue to 17.
?. [DNA Precipitation]
Using a wide-bore P1000 tip, transfer the aqueous phase to a 5 ml conical tube.
Do not bring any liquid from the interface of the chloroform phase, as this will affect DNA purity in the downstream steps.
?. [DNA Precipitation]
Add 0.4x volume of 5M Ammonium Acetate (ca. 400 μl). Mix by slow inversion of tube.
?. [DNA Precipitation]
Add 3 clean glass beads.
?. [DNA Precipitation]
Add 3 ml absolute ethanol.
?. [DNA Precipitation]
Rotate the tube with a vertical rotator at 9 rpm for 5 minutes.
[vertical rotator]
If a rotator is not available, hand inversion for 30-40 times can be used. Invert the tube slowly by hand so that a full inversion cycle takes 5-6 seconds.
?. [DNA Precipitation]
Remove solution, taking care not to disturb bound DNA on the glass beads.
?. [DNA Precipitation]
Wash bound DNA with 1 ml of 70% ethanol. Invert tube for 2-3 times and discard the ethanol.
?. [DNA Precipitation]
Repeat step 23.
?. [DNA Elution]
Insert a bead retainer to a collection tube. Pour the beads into the bead retainer and spin for 1 s in a mini centrifuge (or the shortest time possible) to remove residual wash buffer. Keep the bead retainer.
?. [DNA Elution]
Quickly pour the beads into a new 2 ml low-bind tube and immediately add 250 µl of elution buffer.
Do not let the beads with DNA dry out. (As an alternative, 250 μl of elution buffer can be aliquoted into a 2 ml tube prior to this step.)
?. [DNA Elution]
Incubate at 37oC for 30 min. Gently aspirate and dispense the eluate over the glass beads at regular intervals with a wide-bore P1000 tip to aid elution.
37 °C
[mix per 10 min]
?. [DNA Elution]
Insert the bead retainer from step 25 into a clean 2 ml DNA low-bind tube. Pour the beads from step 27 and centrifuge at 12,000 x g for 1 minute.
Centrifuge: 12000 33, 1 min
?. [DNA Elution]
Add another 510 μl of elution buffer to the eluate from step 28 and mix with a wide-bore P1000 tip.Leave overnight at room temperature.
0 Room temperature
DNA sample will be viscous, but it is important to pipette thoroughly with a wide-bore pipette tip to ensure homogenization. Homogeneous DNA sample will ensure consistent output length and yield.
?. [DNA Elution]
Store at 4oC or continue to UL Library Preparation as per Section "Modified ULK001". If only SQK-RAD004 is available, follow library preparation in Section "Modified RAD004" or "KrazyStarFish (KSF)".
[for storage]
?. [Cell Lysis]
Pellet 3 million cells in a 1.5 ml tube by centrifuging at 1000 x g for 1 min.
Centrifuge: 1000 34, 1 min
?. [Cell Lysis]
Wash with cold PBS (make sure all media and serum are rinsed off), spin at 1000 x g for 1 min
Centrifuge: 1000 33, 1 min
?. [Cell Lysis]
Add 149 μl of TLB-SDS or TLB-CTAB which has been added with 100 ug RNaseA (1 μl) and vortex at full speed for 5 seconds.
33
Make a master mix of the TLB if handling more than one sample.
?. [Cell Lysis]
Incubate at 37°C for 10 minutes
37 °C
?. [Cell Lysis]
Add 140 μl TLB-SDS or TLB-CTAB and 200 μg Proteinase K (10 μl). Mix by slow inversion 5 times or with a P1000 wide-bore pipette tip.  Incubate at 55°C for 20 minutes.
55 °C
Shaking in a thermomixer at 300-700 rpm can help lysis and homogenization, especially when using more than 3 million cells.
?. [DNA Precipitation]
Add 75 μl of 5M Ammonium acetate, mix well with wide-bore P1000 pipette tip or by tube inversions.
?. [DNA Precipitation]
Add 3 clean glass beads to the cell lysate.
?. [DNA Precipitation]
Add 275 μl of Isopropanol
?. [DNA Precipitation]
Rotate the tube with a vertical rotator at 9 rpm for 5 minutes
If using 5 million cells as input, rotate for 10 minutes. If a rotator is not available, hand inversion for 30-40 times can be used. Invert the tube slowly by hand so that a full inversion cycle takes 4-5 seconds.
?. [DNA Precipitation]
Remove and discard liquid by pipetting.
?. [DNA Precipitation]
Wash bound DNA with 1 ml of 70% ethanol, invert tube 3 times, remove and discard ethanol.
?. [DNA Precipitation]
Repeat wash step no. 12.
?. [DNA Elution]
Discard the supernatant, taking care not to disturb the DNA precipitate. It is OK to leave a small volume of liquid behind.
?. [DNA Elution]
Insert a bead retainer to a collection tube. Pour the beads into the bead retainer and spin for 1 s in a mini centrifuge (or the shortest time possible) to remove residual wash buffer. Keep the bead retainer.
?. [DNA Elution]
Quickly pour the beads into a new 2 ml low-bind tube and immediately add 200 µl of elution buffer.
Do not let the beads with DNA dry out. As an alternative, 200 μl of elution buffer can be aliquoted into a 2 ml tube prior to this step.
?. [DNA Elution]
Incubate at 37oC for 30 min. Gently aspirate and dispense the eluate over the glass beads at regular intervals with a wide-bore P1000 tip to aid elution.
37 °C
[pipette per 10 min]
?. [DNA Elution]
Insert the bead retainer from step 15 into a clean 2 ml DNA low-bind tube. Pour the beads and centrifuge at 12,000 x g for 30 seconds.
Centrifuge: 12000 34, 30 s
For cell input of more than 3 millions, centrifuge for 1 min.
?. [DNA Elution]
Incubate the DNA at 37oC 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.
37 °C
[pipette per 10 min]
?. [DNA Elution]
Add another 200 μl of elution buffer into the eluted DNA, pipette mix. Incubate overnight at room temperature.
0 Room temperature
?. [DNA Elution]
Store at 4oC or continue to UL Library Preparation as per Section "Modified ULK001". If only SQK-RAD004 is available, follow library preparation in Section "Modified RAD004".
[for storage]
?. [UHMW DNA Extraction]
Follow the manufacturer’s instructions as descried here, BUT for one-day prep (optimized for human cells GM12878) add the changes as described in the following steps.
Our most homogeneous extracted DNA samples were obtained by lysis at 600-700 rpm speed from human cell lines. This step can be further optimised for other sample types.
?. [One-day Protocol]
Start the extraction early in the day and follow the manufacturer’s instructions.
[max (ca. 30-60 min)]
One million cells are sufficient for one-time library load on MinION and at least two million for PromethION.
?. [One-day Protocol]
Incubate the eluted DNA at 37oC for about 2-3 hours with regular pipette mixing.
37 °C
[max (2-3 hours)]
?. [One-day Protocol]
Quantify DNA as per section "UHMW DNA QC" and check homogeneity by calculating %CV values.
?. [One-day Protocol]
Store at 4oC or continue to UL Library Preparation as per Section "Modified ULK001". If only SQK-RAD004 is available, follow library preparation as in Section "Modified RAD004".
[for storage]
?. [UHMW DNA Extraction]
Follow the manufacturer’s instructions as described here.
?. [UHMW DNA Extraction]
Store at 4oC or continue to UL Library Preparation as per Section "Modified ULK001". If only SQK-RAD004 is available, follow library preparation as in Section "Modified RAD004".
[for storage]
?. [One-day Protocol]
Dilute the eluted DNA with 150 μl of NEB Elution Buffer II.
This is following the standard protocol where DNA is eluted in 100 μl buffer. After this step, sample volume will be 250 μl total. Quantification of a very viscous UHMW DNA is problematic and will not produce accurate results, hence the dilution. Gradual dilution is recommended to achieve homogeneous concentration of 50-100 ng/μl.
?. [DNA Elution]
Quantify DNA as per "UHMW DNA QC" and check homogeneity by calculating %CV values. If the DNA is not sufficiently homegeneous, incubate the DNA for longer.