TITLE: HMW gDNA purification and ONT ultra-long-read data generation
AUTHORS: Glennis Logsdon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the purification of high-molecular-weight genomic DNA from mammalian cells and the generation of ultra-long (N50 >100 kbp) Oxford Nanopore data. It is based on the Sambrook and Russell protocol and Josh Quick's protocol with additional modifications. This protocol improves upon previous protocols developed for ultra-long read sequencing, as it gives longer reads with greater yield. In our hands, we obtain about 1-2 Gb of ultra-long-read ONT data with an N50 ~150 kbp on one MinION flow cell.</div></div>

[STEPS]
?. [Cell collection and lysis]
Freeze down 2-7 x 10^7 cells as a cell pellet, and store at -80C.
?. [Cell collection and lysis]
When you are ready to purify the DNA, thaw the cell pellet on ice (usually takes ~30 mins).
?. [Cell collection and lysis]
Resuspend thawed cells in ice-cold TE (pH 8.0) at a concentration of 5 x 10^7 cells/mL on ice.
?. [Cell collection and lysis]
Transfer the cell suspension to a glass Erlenmeyer flask.- For 1 mL of cell suspension, use a 50-mL flask; for 2 mL of cell suspension, use a 125-mL flask.- Make sure that the cells are well-dispersed over the inner surface of the Erlenmeyer flask. This dispersal minimizes the formation of intractable clumps of cells.
?. [Cell collection and lysis]
Quickly add 10 mL of lysis buffer + RNase A for each mL of cell suspension, drop-wise in a circular motion.
?. [Cell collection and lysis]
Incubate the cell suspension for 1 hr at 37°C.
?. [Cell collection and lysis]
Add proteinase K to a final concentration of 200 ug/mL in a drop-wise manner.- For 10 mLs of cell suspension, add 100 uL Proteinase K.
?. [Cell collection and lysis]
Swirl the flask to mix the enzyme gently into the viscous cell lysate.
?. [Cell collection and lysis]
Incubate the lysate in a water bath for 2 hours at 50°C. Swirl the viscous solution once per hour.
?. [Cell collection and lysis]
Cool the solution to RT.
?. [Phenol-chloroform extraction]
Add light phase-lock gel to 4 15-mL polypropylene tubes.- Phase-lock gels typically come in 2-mL tubes. Transfer the gel by cutting the lid off 3 2-mL phase-lock tubes, placing them upside down into the 15 mL tube, and spinning it at 4000 rpm for 1 min each.
?. [Phenol-chloroform extraction]
Split the viscous lysate into the 2 15-mL Falcon tubes prepared with phase-lock gel.- This is easiest using a 10 mL serological pipette at low speed.
?. [Phenol-chloroform extraction]
Add an equal volume of ultra-pure phenol:chloroform:isoamyl alcohol (~5-6 mLs) to each Falcon tube containing lysate.
?. [Phenol-chloroform extraction]
Gently mix the two phases by slowly turning the tube end-over-end for 20 mins on a tube mixer. If the two phases have not formed an emulsion at this stage, place the tube on a roller apparatus for 1 hr.
?. [Phenol-chloroform extraction]
Spin in a centrifuge at 4500 rpm for 10 mins.
?. [Phenol-chloroform extraction]
Pour the aqueous phases into two new 15-mL Falcon tubes containing phase-lock gel.
?. [Phenol-chloroform extraction]
Add an equal volume of ultra-pure phenol:chloroform:isoamyl alcohol (~5-6 mLs) to each Falcon tube containing lysate.
?. [Phenol-chloroform extraction]
Gently mix the two phases by slowly turning the tube end-over-end for 20 mins on a tube mixer. If the two phases have not formed an emulsion at this stage, place the tube on a roller apparatus for 1 hr.
?. [Phenol-chloroform extraction]
Spin in a centrifuge at 4500 rpm for 10 mins.
?. [Phenol-chloroform extraction]
Combine the aqueous phases from the two tubes by slowly pouring them into a new 50-mL Falcon tube.
?. [Ethanol precipitation]
Add 0.4 volume of 5 M ammonium acetate to the purified DNA, and gently swirl to mix (usually takes several minutes).- It is very important to mix gently here. The gentler you mix, the longer your DNA will be.
?. [Ethanol precipitation]
Add 2 volumes of ethanol at RT and gently swirl to mix (usually takes ~30 mins).- Swirl gently, sometimes rocking back and forth slowly to get the DNA in solution with the salt and ethanol.
?. [Ethanol precipitation]
Store the solution O/N at 4°C.- We have found that storing the DNA overnight results in purer DNA that sequences better and gives more yield.
?. [Ethanol precipitation]
The DNA forms a precipitate. Remove the precipitate in one piece from the ethanolic solution with a disposable inoculating needle shaped into a U.- If the DNA precipitate becomes fragmented, abandon the needle and collect the precipitate by centrifugation 4500 rpm for 5 mins at RT.
?. [Ethanol precipitation]
Wash the DNA precipitate 2x with 70% ethanol, and collect the DNA by centrifuging at max speed (~15000 rpm) for 15 secs.
?. [Ethanol precipitation]
Remove as much of the 70% ethanol as possible. Store the pellet of DNA in an open tube at RT until the last visible traces of ethanol have evaporated (~30 mins to 1 hr).- Do not allow the pellet of DNA to dry completely. Desiccated DNA is very difficult to dissolve.
?. [Ethanol precipitation]
Add 125-250 uL EB + 0.02% Triton-X100 and incubate without mixing at 4°C for 2 days to allow the pellet to fully resuspend into a translucent viscous gel.
?. [Cell collection and lysis]
While the cells are thawing, add RNAse A to the lysis buffer at a final concentration of 20 ug/mL. This must be done fresh each time. Keep the lysis buffer + RNase A solution at RT until ready to use.
?. [ONT library preparation]
Pre-set a thermocycler to the following program:a. 30°C for 1.5 minb. 80°C for 1 minc. 4°C forever
?. [ONT library preparation]
Pre-warm the thermocycler by starting the program and then immediately pressing “Pause”.
?. [ONT library preparation]
Add the following reagents to a PCR tube on ice (in order). Note: Use a wide-bore pipette tip for the gDNA to avoid shearing.  10 uL HMW gDNA (2-3 ug)5 uL dH2O3 uL FRA Buffer Total = 18 uL
?. [ONT library preparation]
Pipette the gDNA mixture up and down slowly >20 times on ice with a wide-bore pipette tip. Do not worry about shearing the DNA. Try to avoid creating bubbles.
?. [ONT library preparation]
In a lo-bind tube on ice, dilute FRA 1:12 in FRA buffer on ice- FRA buffer composition is proprietary, but ONT will send you free FRA buffer if you ask them.- This dilution can be modified depending on the needs of the sequencing experiment. In our hands, increased FRA concentration gives shorter reads but greater throughput.
?. [ONT library preparation]
Add 1.5 µl FRA (diluted) to the gDNA mixture on ice.
?. [ONT library preparation]
Pipette the gDNA mixture up and down slowly >20 times with a wide-bore pipette tip while keeping the tube on ice. Try to avoid creating bubbles.
?. [ONT library preparation]
Place the tube in the thermocycler and let the program continue.
?. [ONT library preparation]
After the thermocycler is finished, move the tube to ice, and bury it in the ice to cool it down from all sides.
?. [ONT library preparation]
In a lo-bind tube on ice, dilute RAP 1:12 in RAP buffer.- RAP buffer composition is proprietary, but ONT will send you free RAP buffer if you ask them.
?. [ONT library preparation]
Add 0.5 µl RAP (diluted) to the gDNA mixture on ice .
?. [ONT library preparation]
Pipette the gDNA mixture up and down slowly >20 times with a wide-bore pipette tip while keeping the tube on ice. Try to avoid creating bubbles.
?. [Flow cell priming and loading]
Place the flow cell on a MinION/GridION and run platform QC.
?. [Flow cell priming and loading]
Remove the flow cell from the GridION and place in its holder to let it cool down for at least 10 mins.
?. [Flow cell priming and loading]
Add 30 µl FLT to the tube of FB. Invert to mix and spin down.
?. [Flow cell priming and loading]
Using a P1000 set to ~800 µl, place the pipette tip in the inlet port of the flow cell and turn the wheel to the right to remove the air and a small amount of storage buffer. This will make the storage buffer flush with the inlet port opening.
?. [Flow cell priming and loading]
Load 800 µl FLB + FLT via the inlet port slowly. Wait 5 minutes.
?. [Flow cell priming and loading]
Meanwhile, thoroughly mix SQB by inverting.
?. [Flow cell priming and loading]
Add 20 µl dH2O and 34 µl SQB to the gDNA mixture.
?. [Flow cell priming and loading]
Pipette the gDNA mixture up and down slowly >20 times. Try to avoid creating bubbles.
?. [Flow cell priming and loading]
Lift the cover off the SpotON port. Load 200 µl FLB + FLT via the inlet port slowly using a P1000 pipette. Try to dispense at a speed where a bead of liquid becomes visible over the SpotON port which then gets siphoned back in.
?. [Flow cell priming and loading]
Using a P200 set to 75 µl with a wide-bore tip, pipette the diluted library onto the SpotON port as it gets siphoned in.
?. [Flow cell priming and loading]
Close the SpotON sample port cover, and close the priming port.
?. [Flow cell priming and loading]
Let incubate for 10 min before placing on the MinION/GridION and starting the run.
?. [ONT library preparation]
Incubate the gDNA mixture at room temperature for 2 hours. - This incubation time can be reduced to 30 min, but we obtain greater yield with an extended incubation.
?. [ONT library preparation]
Equilibrate an ONT MinION/GridION flow cell to room temperature.