TITLE: FindingNemo Extraction 3: NEB Monarch Kit
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 </span><span style = "font-weight:bold;">New England Biolabs (NEB) Monarch HMW DNA Extraction Kit for Cells & Blood</span><span>. </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>The </span><span style = "font-weight:bold;">New England Biolabs (NEB) Monarch HMW DNA Extraction Kit for Cells & Blood </span><span>is a quick, tweakable extraction protocol fitting for one-day library prep.</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 varied between 1-6 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 using Monarch® HMW DNA Extraction Kit for Cells & Blood.
?. [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]
We have obtained optimal extractions using the NEB Monarch kit. This combines speed with high quality UHMW DNA. Follow the manufacturer’s instructions as described here, BUT incorporate the following changes as described below.
Our most homogeneous extracted DNA samples were obtained by lysis at 600-700 rpm speed using the Monarch kit. This is one area that can be optimised depending on the input sample.
?. [One-day Protocol]
To complete DNA extraction from cell to library prep and sequencing in one day, start early!
One million human cells are sufficient for a single library load on the MinION. At least two million human cells are required for a single load on the PromethION. Other samples can be scaled according to total amount of DNA recovered. So for a 1 gigabase genome you would require 3 million cells etc.
?. [One-day Protocol]
Dilute the eluted DNA with 150 μl of NEB Elution Buffer II.
This is following the NEB UHMW Monarch kit protocol where DNA is first eluted with 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.
?. [One-day Protocol]
Incubate the eluted DNA at 37oC for about 2-3 hours with regular pipette mixing.
37 °C
[max (2-3 hours)]
During mixing, observe by eye that the viscous DNA 'blob' has been more or less dissolved to the different parts of the tube (i.e., less heterogeneous). This is usually observed after 2 hours of incubation. Otherwise, continue the incubation to 3 hours and proceed to the next step.
?. [One-day Protocol]
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.
?. [One-day Protocol]
Store at 4oC or continue to UL Library Preparation as per "Modified ULK001". If only SQK-RAD004 is available, follow library preparation as in "Modified RAD004" or "KrazyStarFish (KSF)".
[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]
?. [Cell Lysis]