TITLE: Algal nuclei isolation for Nanopore sequencing of HMW DNA
AUTHORS: Robert Auber, Jennifer Wisecaver

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol was developed for extraction of high molecular weight (HMW) DNA from </span><span style = "font-style:italic;">Prymnesium parvum, </span><span> a unicellular haptophyte alga, for the purpose of whole genome sequencing using Oxford Nanopore Technology (ONT) long reads. </span><span style = "font-style:italic;">P. parvum</span><span> is known to produce several specialized metabolic compounds that may compromise isolated DNA, leading to decreased sequencing yield. We found that separating intact nuclei from cellular debris prior to DNA isolation, improved read length and throughput. Isolated nuclei were processed using a Circulomics NanoBind kit to extract HMW DNA. </span></div></div>

[STEPS]
?. [Prepare equipment and reagents]
Chill centrifuge to
4 °C
?. [Prepare equipment and reagents]
Chill 50 ml conical tubes (4 per sample) and NIB buffers on ice.
?. [Cell Lysis]
Transfer  to   of culture to a pre-chilled 50 ml conical tube and centrifuge   at  for   to pellet cells.The amount of culture spun down as well as the centrifugation speed at which cultures are pelleted should be optimized for each organism. In our experience, the pellet size should be at least the size of a lentil for small (5 μm) for our alga.
20 ml
50 ml
4 °C
?. [Cell Lysis]
Discard the supernatant and snap freeze the conical tube in liquid nitrogen for at least  .Some organisms will require some type of tissue disruption to lyse cells.
?. [Cell Lysis]
Place cell pellet on ice and add   of ice cold NIB.If you know your pellet is not very soluble, only add 1mL of NIB at first and mix by pipetting up and down with a 1mL pipette. Add remaining 9mL NIB after clumps are broken up.
10 ml
?. [Cell Lysis]
Mix by slowly pipetting up and down with a 10mL serological pipettor until the mixture is smooth and not clumpy.
?. [Filter to remove particulate material]
Place a 100 μm filter on top of a pre-chilled 50 ml conical tube.
?. [Filter to remove particulate material]
Slowly pour the sample through the filter into the clean tube.
?. [Filter to remove particulate material]
Use a serological pipettor to wash the filter with  of ice cold NIB.
10 ml
?. [Filter to remove particulate material]
Place a 70 μm filter on top of a second pre-chilled 50 ml conical tube.
?. [Filter to remove particulate material]
Slowly pour the sample through the filter into the clean tube.
?. [Filter to remove particulate material]
Use a serological pipettor to wash the filter with  of ice cold NIB.
10 ml
?. [Filter to remove particulate material]
Place a 40 μm filter on top of a third pre-chilled 50 ml conical tube.
?. [Pellet nuclei]
Add  of ice cold NIB + TritonX solution to filtrate.
1.5 ml
?. [Pellet nuclei]
Centrifuge   at  for  to pellet nuclei.The speed at which the sample is spun down should be reevaluated for different organims based on genome size.
4 °C
?. [Confirm isolation via microscopy]
Aliquot  of the pellet into a clean 1.5ml eppendorf tube. Add  of fresh NIB and   of propidium iodide to the tube.
1 µl
18 µl
1 µl
?. [Extract DNA from Nuclei]
Extract DNA by referring to Circulomics Plant Nuclei Big DNA Kit protocol.https://www.circulomics.com/store/Nanobind-Plant-Nuclei-Big-DNA-Kit-Alpha-Version-p99924200
?. [Filter to remove particulate material]
Slowly pour the sample through the filter into the clean tube. (Do not wash with NIB)
?. [Confirm isolation via microscopy]
Transfer the stained nuclei to a microscope and confirm presence of fluorescing nuclei under an epifluorescent light.Image was taken on a Nikon Ts2R-FL microscope with a CFP LP cube (Excitation 448/23nm (436.5-459.5nm), Emission 472nm Long Pass)
?. [Pellet nuclei]
Discard the supernatant.
?. [Prepare equipment and reagents]
Add BME to premade NIB. Per sample add  BME into  premade NIB.
35 µl
35 ml
?. [Prepare equipment and reagents]
Set thermomixer to
55 °C
?. [Prepare equipment and reagents]
Cool solutions to  on ice.
0 °C
?. [Prepare equipment and reagents]
Make 10% Triton X-100 NIB solution. Per sample, aliquot  NIB prepared in previous step and add  Triton X-100.
1.8 ml
200 µl