FindingNemo Extraction 1: Phenol-based Method
Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose
ultra-long sequencing
cohex
glass bead
nanopore
MinION
UHMW DNA
Monarch
Circulomics
phenol
SDS
CTAB
GM12878
Whatman
PromethION
Nanobind
Abstract
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.
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.
Kit-free, phenol-based method is a scaled-down version of Josh Quick's protocol (dx.doi.org/10.17504/protocols.io.mrxc57n) with additional glass bead step for DNA precipitation.
We tested this sub-protocol listed here in human cell line , with input cells of 5 millions. As a rule of thumb, a million cells will suffice for one load on a MinION.
Before start
Things to observe at all times:
- Excessive and vigorous pipetting and vortexing should be avoided as these may shear the DNA.
- Make up buffers with nuclease-free water to avoid introducing nucleases to solutions.
- Avoid unnecessary heating and freezing; isolated DNA should be stable for storage in the fridge for months.
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).
Cell Lysis
Pellet 5 million cells in a 2 ml tube by centrifuging at 500 x g for 5 min at 4oC.
500x g,4°C
Wash with 500 μl cold PBS and centrifuge at 500 x g for 3 min at 4oC. Discard supernatant.
500x g,4°C
Resuspend well by pipette mixing in 20 μl cold PBS.
Add 1 ml TLB-SDS and 100 μg RNase A (1 μl) and vortex at full speed for 5 seconds.
2000rpm
Incubate at 37°C for 1 hour, mix by inversion every 15 minutes.
37°C 1h 0m 0s
Add 200 μg Proteinase K (10 μl). Mix by slow inversion 5 times.
Incubate at 50°C for 2 hours, mix every 30 minutes by slow inversion 3 times.
50°C 2h 0m 0s
Phenol Separation
Split the lysate into 2 phase-lock gel tubes (ca. 550 ul per tube).
Add 550 μl buffer-saturated phenol to each tube containing lysate.
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.
0h 10m 0s
Centrifuge at 4500 rpm for 10 minutes.
4500rpm
Transfer the aqueous phase to another phase-lock gel tube by pouring or using a wide-bore P1000 tip.
Add 250 μl buffer-saturated phenol and 250 μl chloroform-isoamyl alcohol to each tube.
Repeat step 11-12 and continue to step 15.
Transfer and combine the aqueous phase to a 2 ml tube (sample will be ca. 1 ml).
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.
Add 0.4x volume of 5M Ammonium Acetate (ca. 400 μl). Mix by slow inversion of tube.
Add 3 clean glass beads.
Add 3 ml absolute ethanol.
Rotate the tube with a vertical rotator at 9 rpm for 5 minutes.
0h 5m 0s
Remove solution, taking care not to disturb bound DNA on the glass beads.
Wash bound DNA with 1 ml of 70% ethanol. Invert tube for 2-3 times and discard the ethanol.
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.
Quickly pour the beads into a new 2 ml low-bind tube and immediately add 250 µl of elution buffer.
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 0h 30m 0s
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.
12000rpm
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.
Room temperature
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.
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) ".
4°C
