TITLE: Plant nuclei enrichment for chromatin capture-based Hi-C library protocols
AUTHORS: Elena Hilario

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Chromatin capture-based protocols to produce Hi-C libraries start with a crude nuclei extract or a total cell extract. Either way, plants can be challenging due to the high content of contaminants that could interfere at the chromatin capture step.</div><div class = "text-block">This protocol will help clean up the starting material needed for preparing Hi-C libraries. The nuclei yield varies, but should be enough for at least two preps. This protocol has been tested on blueberry, bilberry, pepino, Gillenia and rewarewa.</div></div>

[STEPS]
?. [Nuclei isolation]
Weight 1-2 g of young leaves, either fresh or frozen.
?. [Nuclei isolation]
Pre-cool the mortar and pestle by pouring liquid nitrogen to the rim of the mortar. Once the liquid nitrogen is evaporated, repeat the pre-cooling step.
?. [Nuclei isolation]
Add the leaves to the mortar and pour liquid nitrogen over them to freeze them immediately. You can crush the leaves slightly to make them fit.
?. [Nuclei isolation]
Transfer the powder to the beaker containing 50 mL of NEB-complete +Triton X100. Stir at moderate speed until all the powder is dissolved. Any large clumps can be broken with the spatula against the beaker's internal wall. .
[Organellar lysis]
[Ice box on magnetic stirring plate]
If you go overtime, do not worry. The amount of Triton-X100 will not lyse the nuclei, but keep in mind that the crude lysate has large cell wall debris that might break them.
?. [Nuclei isolation]
When the liquid nitrogen has evaporated, grind the leaves with circular motion, and also by pushing from the rim to the center of the mortar. Retrieve the powder from the pestle with a pre-cooled spatula. Add more liquid nitrogen and repeat this step until a fine powder is obtained. For most plants it takes 3 rounds of grinding. The powder should resemble icing sugar.
?. [Nuclei isolation]
Filtrate the sample through the Miracloth sheet. Very carefully pipette the extract in the funnel to detach the residue stuck in the Miracloth.
?. [Nuclei isolation]
Collect the nuclei by centrifugation  . Discard the supernatant and dispose it according to your institution's waste disposal guidelines.
Centrifuge: 1800 34
[Nuclei pellet]
[Centrifugation]
The centrifugal force depends on the genome size but also on the medium's density. The osmoticum used in this buffer (mannitol) is at 0.5 M and seems to work well with genomes upto 3 Gb (N. benthamiana), at 1800 x g, but please see recommendations here. Other cellular material such as starch might contribute to the sedimentation of nuclei of large genomes such as the N. benthamiana.
?. [Nuclei isolation]
Add 30 mL NEB-βME to the pellet and resuspend it gently by inverting the tube, in a rocking movement. Collect the nuclei by centrifugation
[Nuclei pellet]
?. [Nuclei isolation]
Add 7 mL of NEB-A buffer to the pellet and resuspend it gently. Transfer the mixture to the Dounce homogenizer and place it on the ice box. Use the "B" pestle to homogenize the nuclei sample by pulling and pushing the pestle very gently. Take care not to lift it out of the liquid or creating too many bubbles.
?. [Percoll gradient]
Carefully overlay  over each 75% Percoll® solution. The loaded tubes should weigh exactly the same.
[filtrated nuclei suspension]
?. [Nuclei isolation]
Assemble the 100 μm strainer on a 50 mL Falcon tube. Transfer the filtrated extract to the strainer. This filtration step should flow more or less quickly.
?. [Nuclei isolation]
Assemble the 40 μm strainer on a 50 mL Falcon tube and transfer the filtrated extract to the strainer. This step might take about 10 min. Place the assembled tube on ice.You can help the filtration by pipetting the filtrate in the strainer very gently with a disposable Pasteur pipette (cut the tip to make it wide bore). If the strainer becomes clogged half way through the filtration, replace it with a new one.
?. [Percoll gradient]
Collect the nuclei at the Percoll® gradient interphase by centrifugation . Adjust the acceleration = 9 (max), and deceleration = 1 (min).
[Percoll gradient]
[Percoll gradient]
?. [Percoll gradient]
With a light source behind the gradient, collect the sample found at the interphase of the Percoll® gradient using a glass Pasteur pipette. Do this step very slowly. Transfer sample to a new 15 mL Falcon tube and keep on ice. You will obtain about 4 mL of nuclei solution.
The interphase can form as a tight band between the two solutions of different density, or as a fluffy band. In either case, collect as much as possible. Most contaminants will show as a dense pellet at the bottom of the gradient, or as a colored solution on the top layer.
?. [Percoll gradient]
Add enough NEB-A solution to the nuclei to have a final volume of 10 mL. Collect nuclei by centrifugation
?. [Percoll gradient]
Discard the supernatant . Retrieve the nuclei pellet by adding ~ 500 μL NEB-A and gently resuspend it with a wide bore pipette tip. Transfer it to a 1.5 mL screw-capped tube. Add another ~ 500 μL NEB-A to the Falcon tube to retrieve any remaining nuclei and transfer it to the screw-capped tube. Make sure the solution is homogeneous. Transfer half of it to another 1.5 mL screw-capped tube and add enough NEB-A to each tube to have a final volume of 1 mL. Collect the nuclei by centrifugation  . Discard the supernatant.
Centrifuge: 5200 33
[Enriched nuclei sample]
10 Room temperature
?. [Percoll gradient]
Estimate the yield by weighting the tubes (subtract the empty tube weight). Store the enriched nuclei sample   unitl ready to start the crosslinking steps of the Hi-C protocol.
[Storage]