TITLE: FindingNemo Extraction 2: Phenol-free Method
AUTHORS: Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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 a </span><span style = "font-weight:bold;">phenol-free extraction </span><span>method. </span></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-free protocol </span><span>is a modification of NEB's Monarch HMW DNA Extraction Kit for Cells & Blood, with the option to use SDS or CTAB in the lysis buffer. This protocol also uses glass beads for DNA precipitation matrix.</span></div><br/><div class = "text-block"><span>We tested this sub-protocol in </span><span style = "font-weight:bold;">human cell line</span><span>, with input cells of 3 millions but can be varied from 1-5 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 adapted from Monarch® HMW DNA Extraction Kit for Cells & Blood.
Either SDS (anionic surfactant) or CTAB (cationic surfactant) can be used in the lysis buffer. Providing alternative surfactants in the lysis buffer may help with different cell systems that require different biochemistry.
?. [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 15 into a clean 2 ml DNA low-bind tube. Pour the beads from step 17 and centrifuge at 12,000 x g for 1 minute.
Centrifuge: 12000 33, 1 min
?. [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.
?. [DNA Elution]
Store at 4oC or continue to UL Library Preparation as per "Modified ULK001". If only SQK-RAD004 is available, follow library preparation in "Modified RAD004"  or "KrazyStarFish (KSF)".
[for storage]
?. [Cell Lysis]
Pellet 3 million cells in 1.5 ml tube by centrifuging at 1000 x g for 1 min at 4oC.
Centrifuge: 1000 34, 1 min, 4 10
?. [Cell Lysis]
Wash with PBS (make sure all media and serum are rinsed off), spin at 1000 x g for 1 min at 4oC.
Centrifuge: 1000 34, 1 min, 4 10
?. [Cell Lysis]
Add 149 μl of TLB-SDS or TLB-CTAB which has been added with 100 μg RNaseA (1 μl) and vortex at full speed for 3 seconds.
Make master mix of the TLB and RNaseA if handling more than one sample.
?. [Cell Lysis]
Incubate at 37°C for 10 minutes.
37 °C
?. [Cell Lysis]
Add 140 μl TLB and 200 μg Proteinase K (10 μl). Mix by slow inversion 5 times or with a P1000 wide-bore pipette tip.
?. [Cell Lysis]
Incubate at 55°C for 20 minutes.
55 °C
Shaking in a thermomixer at 300-700 rpm can help lysis and homogenization, especially when more than 3 million cells are used.
?. [DNA Precipitation]
Add 75 μl of 5M Ammonium acetate, mix well with wide-bore P1000 pipette tip.
?. [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.
[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 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 step 13 once with 500 μl. Discard the ethanol, taking care not to disturb the DNA precipitate.