Establishing a ChIP-seq protocol in Aiptasia is the first step in investigating the epigenetic landmarks in symbiotic cnidarians, and how these may regulate an organism's interaction with its environment. The protocol is optimized to address challenging cnidarian features, such as high mucus production and a high water-to-tissue ratio, by increasing the cross-linking time and minimizing tissue loss. Collect the anemones in a 15-milliliter tube and let them settle to the bottom of the tube.
Alternatively, spin them in a centrifuge at room temperature for a few seconds, going up to 3, 500 g. Then, remove the excess sea water with a vacuum pump. Wash the anemones by suspending them in DPBS.
And remove the DPBS after washing. Using tweezers or a plastic spatula, transfer the anemones, without the superfluous buffer, into the 0.5%cross-linking buffer. Incubate them for one hour on a rotator at a speed of 12 rotations per minute and four degrees Celsius.
After incubation, remove the 0.5%cross-linking buffer from the tube containing the anemones. Refill the tube with the 1%cross-linking buffer. And incubate it on a rotator overnight at 12 rotations per minute and four degrees Celsius.
On the next day, after removing the 1%cross-linking buffer, quench the cross-linking reaction by suspending the anemones in the quenching buffer and incubating the suspension on a rotator for 20 minutes at 12 rotations per minute and four degrees Celsius. Then, remove the quenching buffer and wash the anemones twice in DPBS. Decant the DPBS.
And empty the anemones onto a paper tissue to remove as much liquid as possible. Pour some liquid nitrogen into the mortar and transfer the anemones using tweezers. Using the pestle, start by carefully breaking up the tissue.
Keep topping up with liquid nitrogen, as required, to prevent defrosting. Continue grinding until the sample becomes a fine powder. Using a spatula, collect the sample and transfer it into the lysis buffer, ensuring the sample dissolves in the buffer.
Let the sample rest on ice for one minute before mixing the same by inversion. Next, after washing a dounce tissue grinder with lysis buffer, transfer the sample and dounce it 20 to 30 times with a tight pestle. Transfer the sample back into the tube.
And incubate it overnight on a rotator at 14 rotations per minute and four degrees Celsius. Place the sample in the sonciator with the needle one centimeter above the bottom of the tube and not touching the walls. Start the sonciator and ensure no foam is being produced in the sample.
Slowly increase the output power to two and sonicate for two more minutes. After that, switch off the sonciator, set the output power back to zero, and let the sample rest and cool down for two minutes. Depending on the sample volume and the number of immunoprecipitations, or IPs, planned, increase the total sample volume by adding 10%Triton X-100 and 100X Protease Inhibitor Cocktail, or PIC, to final concentrations of 1%and 1X respectively.
Transfer the volume for each IP into a separate, low-retention, 1.5 milliliter-tube for chromatin immunoprecipitation, or ChIP, followed by quantitative PCR, or qPCR. Place the equivalent volume of the sample into another 1.5-milliliter tube as a mock control. Take 10%of the volume of the IPs as the input control and store it at minus 20 degrees Celsius.
Add four micrograms of antibody histone 3 lysine 4 trimethylation, or H3K4 trimethylation, to each IP, but not to the mock. Incubate the IP reactions and the mock on the tube rotator overnight at 12 rotations per minute and four degrees Celsius. Cut off the tip of a pipette tip to increase its diameter.
And transfer 50 microliters of magnetic beads into separate tubes, one tube per IP and one for the mock. Add one milliliter of blocking solution to each tube. And incubate them on a rotator for 30 minutes, as before.
Using a pipette, remove and discard the blocking solution supernatant and flush the beads off the wall with the IP or mock reaction. Place the mix back into the respective low-retention tubes containing the samples. Incubate all the tubes, as before, for three hours.
After the incubation, place the IPs and mock on the magnetic rack and wait for about 10 to 20 seconds for the magnets to separate. Discard the supernatant and add one milliliter of wash buffer with low salt. After incubating the tubes, as before, for five minutes, place them back on the magnetic rack.
Remove the wash buffer with low salt. Add one milliliter of wash buffer with high salt and incubate for five more minutes. Remove and discard the supernatant using a pipette and wash with one milliliter of TE salt buffer.
Repeat the wash once more and flush out any beads from the tube cap. After discarding the second TE salt buffer wash, add 210 microliters of elution buffer to each reaction. Suspend the magnetic beads and elute at 65 degrees Celsius, shaking at 700 rotations per minute for 15 minutes.
Place the reactions on the magnetic rack, collect the eluent, and place it in a fresh 1.5-milliliter tube. Next, remove the input from the freezer and add elution buffer to a total volume of 420 microliters. Reverse cross-link all the eluents and the input by incubating them at 65 degrees Celsius and 700 rotations per minute overnight.
For RNA digestion, add 10 microliters of RNase cocktail and incubate at 42 degrees Celsius and 700 rotations per minute for 30 minutes. For protein digestion, add eight microliters of Proteinase K and incubate at 55 degrees Celsius and 700 rotations per minute for one hour. Under a fume hood, add one sample and the same volume of phenol-chloroform-isoamyl alcohol mixture to each phase-lock gel tube.
Shake the tubes vigorously and vortex them briefly, until they form a frothy white layer. Spin for 10 minutes at four degrees Celsius and 20, 000 g. Check that the aqueous phase is clear.
Transfer the aqueous phase into a fresh tube using a pipette. Add 100%ethanol, equivalent to two times the sample volume in it, and shake. Incubate at minus 20 degrees Celsius overnight, or at minus 80 degrees Celsius for 30 minutes.
Pellet down the DNA by spinning the samples for 30 minutes at 15, 000 g. Carefully, decant the supernatant and wash the pellets with one milliliter of 70%ethanol. Spin at maximum speed for five minutes at four degrees Celsius.
Carefully decant before removing all the ethanol by pipetting. Resuspend the pellets in 30 microliters of nuclease-free water. DNA, associated with the trimethylation of histone 3 lysine 4, or H3K4, trimethylation was immunoprecipitated, and the E.diaphana tissue sections were stained by immunofluorescent staining.
The obtained DNA fragments were analyzed by sequencing and qPCR. The model-based analysis of the ChIP-seq data identified 19, 107 peaks. As expected for H3K4 trimethylation, most peaks were located near the transcriptional start site, or TSS, and the peak count frequency declined sharply on both sides of the TSS, especially toward the gene body.
qPCR primers were designed to target several loci near the respective TSS of three genes. High enrichment of H3K4 trimethylation relative to the input and mock controls was observed. The percentage of input varied between 2.7 to 10.7%with enrichment differing across genes and the loci within the same gene.
Hopefully, an understanding of the epigenetic regulation of cnidarians in response to environmental changes caused by the climate crisis could inform coral reef conservation and restoration efforts.
