TITLE: Typha (Cattail) Crosslinking and Nuclei Isolation
AUTHORS: Kelly Colt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Typha Crosslinking and Nuclei Isolation protcol</div><div class = "text-block">Key notes:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Allow for extra time if working with pre-ground samples. Crosslinking steps and subsequent washes will take longer to complete.</li><li style = "counter-reset:ol0;list-style-type:disc;">1% Triton X-100</li><li style = "counter-reset:ol0;list-style-type:disc;">600g pelleting</li><li style = "counter-reset:ol0;list-style-type:disc;">Optional supernatant recovery steps</li></ul></div></div>

[STEPS]
?. [Buffers]
Formaldehyde Buffer1% FormaldehydeWaterFinal Volume 50mL
?. [Buffers]
125mM Glycine125mM GlycineWaterFinal Volume 50mL
?. [Buffers]
Nuclei Isolation Buffer (NIB)Prepare this buffer the same day that you will perform the nuclei isolation. Prepare 30mL of NIB, chilled to 4C, per sample:250mM Sucrose20mM HEPES pH 8.05mM Potassium Chloride1mM Magnesium Chloride40% GlycerolJust before use, add the following:0.1mM PMSF1% Protease Inhibitor Cocktail0.1% 2-mercaptoethanol1% Triton X-100Bring the final volume to 30mL with water.
Resuspend anhydrous Magnesiem Chloride very slowly and carefully. Anhydrous MgCl2 is very hygroscopic (absorbs water readily) and can flash boil.
?. [Equipment]
?. [Crosslinking]
In a chemical hood, prepare a vacuum dessication setup. Ensure that the valve on the dessication chamber is open and attach the hose coming from the flask to the nozzle. On the outside of the hood, attach the hose coming from the erlenmeyer catch flast to the vaccum pump.
?. [Crosslinking]
Weigh out 2-6g of tissue. Using a sterile razor blade cut the tissue into small, approximately 1 cm X 1 cm pieces. If you are working with pre-ground tissue, use more if possible. You will lose more tissue in the initial crosslinking steps.
?. [Crosslinking]
Once all buffers and equipment are prepared, put the sample in a cotton bag, and place in a 50mL conical. The cotton bag helps to keep all of the tissue uniformly submerged in the buffers. Though not ideal, if you do not have a cotton bag,  use weigh paper to keep all tissue submerged in the buffers.For pre-ground tissue; Do not put pre-ground tissue in a cloth bag.
?. [Crosslinking]
Add 50 mL crosslinking buffer, ensuring the tissue is fully submerged. Use the screened cap to allow air pressure to change in the tube, but keep the original 50mL tube cap, you will use it in the wash steps.
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Put the tube in the tube rack, and place in the dessicator + replace the lid. Vacuum incubate for 10 min at 30cm Hg (400milibar) pressure. A small amount of bubbleing should be visible. Stay and watch the pressure on the pump the entire time, or until pressure stabilizes.
?. [Crosslinking]
Release vacuum pressure by turning the pump off. Remove the dessicator lid and pour out the crosslinking buffer though the screened cap into the appropriate hazardouse liquid waste container.
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Add 50mL 125mM glycine, make sure the tissue is fully submerged, and replace the screened cap. Make sure the original cap is not on the tube, air pressure needs to change in this step. Put the tube in the tube rack, and place in the dessicator + replace the lid. Vacuum incubate for 5 min at 30cm Hg (400milibar) pressure. A small amount of bubbling should be visible.
?. [Crosslinking]
Release vacuum pressure by turning the pump off. Remove the dessicator lid and pour out the 125mM glycine in the appropriate hazardous liquid waste container.
?. [Crosslinking]
Add 50mL milliq/di water, make sure the tissue is fully submerged, replace the original cap on top of the screened cap, and mix. Repeat this process three times to fully wash the tissue. Change gloves if processing multiple samples to prevent cross contamination.
?. [Crosslinking]
Transfer the plant tissue to aluminum foil and fold it up. Try to allow any remaining water to run off of the sample. Snap-freeze the sample by submerging the aluminum foil containing the tissue in liquid nitrogen.
?. [Safe Stop]
Store at -80C until ready to proceed to nuclei isolation.
?. [Nuclei Isolation]
Before Starting, aquire:Liquid NitrogenDry IceCold Resistant GlovesCeramic Mortar and PestleMiracloth, cut into ~7cm x 7cm piecesCool a large centrifuge to 4CEnsure the sucrose in the nuclei isolation buffer is fully resuspended, and the solution has been chilled on ice.
?. [Nuclei Isolation]
Place a mortar and a 50mL conical tube onto a bed of dry ice, and place a pestle into the mortar. Pour liquid nitrogen into the mortar until the entire pestle tip is submerged. Allow liquid nitrogen to evaporate completely.Cool a spatula at -20°C or colder for later use.
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Pour liquid nitrogen into the mortar until the entire pestle tip is submerged. Transfer frozen plant tissue into mortar with liquid nitrogen.
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Pulverize plant tissue in the mortar with the pestle until the sample resembles a fine powder. Ensure the plant tissue is always submerged in liquid nitrogen. Carefully re-fill the mortar with liquid nitrogen as necessary. The pulverization process should take at least 5 min per sample. The key is to pulverize until the tissue resembles a fine powder without visible chunks.
Put on protective cryo gloves. Eye protection is also recommended.
?. [Nuclei Isolation]
Once the sample resembles a fine powder, allow liquid nitrogen in the mortar to evaporate just enough for sample to stay submerged. Carefully pour pulverized plant tissue and remaining liquid nitrogen from the mortar into the pre-chilled 50mL conical tube. Ensure the tube does not overflow with liquid nitrogen.
?. [Nuclei Isolation]
Using the pre-cooled spatula, transfer any remaining pulverized plant tissue from the mortar into the 50mL tube. Place 50mL conical tube on dry ice to keep the pulverized plant tissue frozen. DO NOT CAP the 50mL conical until all of the liquid nitrogen has evaporated.
?. [Safe Stop]
Crosslinked, ground tissue may be stored at -80C until ready to proceed to continue nuclei isolation
?. [Nuceli Isolation]
Add the PMSF, Protease Inhibitor Cocktail, Triton X-100, and 2-mercaptoethanol to the chilled nuclei isolation buffer, and bring the volume to 30mL with water.
?. [Nuceli Isolation]
Add 20mL Nuclei Isolation Buffer to the pulverized plant tissue and mix gently by inversion
?. [Nuceli Isolation]
Place a 100μm cell strainer or 10cm X 10cm section of Miracloth over a new 50mL conical tube and insert the miracloth down into the tube, creating a small basket.
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Pour the pulverized plant tissue over the miracloth or cell strainer. The Nuclei will pass through the cloth while the larger plant debris will be caught.Optional: Pipette 1mL of NIB over the tissue caught by the miracloth to wash more nuclei through.
?. [Nuceli Isolation]
Place another 10cm X 10cm section of Miracloth or a 100μm cell strainer over a new 50mL conical tube, and pour the filtrate over the miracloth/strainer. Repeat this step once more for a total of three miracloth/cell strainer filtrations.
?. [Nuceli Isolation]
Discard the Miracloth/cell strainer and place a cap on the 50mL tube containing the nuclei. Wipe down the tube(s) and then pellet by centrifugation at 600 g at 4C for 15 min.
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OPTIONAL: Save the supernatant for each step! Nuclei can be lost in the process of pouring supernatant off or if that sample needs a different centrifugal force. It's possible to recover a small amount of nuclei if the main pellet yeild looks very low.Pour off supernatant, into new 50mL conical or into the appropriate hazardous waste container.
?. [Nuceli Isolation]
Resuspend pellet in 2mL NIB Buffer. Pellet sample by centrifugation at 600 g at 4C for 10 min.
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Pour off supernatant into designated supernatant reservoir or into the appropriate hazardous waste container.
?. [Nuceli Isolation]
Resuspend pellet in 1mL NIB.
?. [Nuceli Isolation]
OPTIONAL: If the nuclei pellet is still green, spike in a few μl of triton X-100 to lyse chloroplasts and re-filter the sample through a 100μm cell strainer or miracloth and pellet at 600 g at 4C for 10 minIf you are concerned with yield and want to recover any possible lost nuclei from the combined supernatants, centrifuge supernatant at 600 g at 4C for 10 min. Pour off and discard the supernatant. Resuspend the pellet (if there is one) in 500 uL NIB and move into a 1.5mL tube. Centrifuge the tube at 600g for 10 minutes. Pour off and discard the supernatant. Resuspend the pellet (if there is one) in 500 uL NIB, and centrifuge the tube at 600g for 10 minutes. Pour off and discard the supernatant. Now resuspend the pellet in up to 1mL NIB and combine with the main nuclei pellet.
?. [Nuceli Isolation]
Optional: Make aliquots of the resuspended nuclei:Make a small aliquot of the pellet for the to determine the yield - 100 to 200uL should work. Be conscious of the size of your pellet, you don't want to use too much for QC if possible. Record the volume of this aliquot and stock volume, you will need it to accurately back calculate to determine total yield.If you have a very large pellet, you may also make multiple "stock" aliquots. Record the number and volume of each aliquot so you can determine the amount of nuclei in each
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Pellet the nuclei at 600 g at 4C for 10 min.
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Discard supernatant and resuspend each aliquot in 500uL NIB for the final wash step.
?. [Nuceli Isolation]
Pellet the nuclei at 600 g at 4C for 10 min. Discard the supernatant leaving behind only a nuclei pellet and minimal residual liquid. Freeze samples in glycerol nuclei storage buffer or as pellets on dry ice or liquid nitrogen and store at -80C.