This method can help answer key questions in the chromatin architecture field such as how the 3D structure of chromatin can influence gene expression and development. The main advantage of this technique is that it provides a high resolution view of chromatin architecture and precisely staged fly embryos. Though this method can provide insight into chromatin organization and development, one can also use existing lines from transgenic fly libraries to test their effect in chromatin organization.
To begin the protocol, adjust the total volume of previously collected embryos to two milliliters with PBST. Add six milliliters of heptane and 100 microliters of 37%formaldehyde in water. After adding formaldehyde, vigorously shake the tube up and down for one minute.
The aqueous and organic phase will combine to form a shampoo-like consistency. Agitate the mixture on a rotary mixer for 10 minutes. Centrifuge the tube at 500 times g for one minute at room temperature to collect embryos at the bottom of the tube.
Aspirate the entire shampoo-like liquid and discard it, taking care not to aspirate any embryos. 15 minutes after the addition of formaldehyde, resuspend the embryos in five milliliters of PBST with 125 millimolar glycine. Shake the embryos up and down vigorously for one minute.
After centrifuging, aspirate the supernatant. Using a 1, 000 microliter pipette, transfer a batch of 100 embryos to a small glass vessel suitable for sorting, preferably on a dark background and place it on ice. Sort embryos by nuclear density and cell cycle status using a needle or syringe tip.
Remove all mitotic embryos recognizable by their dispersed, non-nuclear distribution of EGFP PCNA and embryos that partially show a non-nuclear GFP signal. To aid the sorting, line up reference embryos at nuclear cycles 12, 13, and 14 in each batch to match embryos with one of the reference embryos. Pipette up the desired embryos using a 1, 000 microliter pipette.
Transfer them to a fresh tube and place them on ice. Continue until enough embryos are sorted for the planned experiment. Spin tubes briefly at 100 times g at room temperature.
Remove the supernatant and ensure the embryos are as dry as possible for freezing. Flash freeze embryos by submerging the tubes in liquid nitrogen and store at minus 80 degrees Celsius. Place the tubes with frozen embryos on ice.
Resuspend the embryos in 500 microliters of ice cold lysis buffer. Wait one minute to allow the embryo to settle at the bottom of the tube. Next, grind the embryos with a metal micro pestle pre-cooled on ice that is designed to tightly fit a 1.5 milliliter microcentrifuge tube.
To avoid agitating the embryos, insert the pestle slowly until it touches the bottom of the tube. Push down and then grind by rotating the pestle twice in both directions. Lift the pestle very slightly.
Push to the bottom of the tube again and repeat the grinding procedure 10 times or until the embryos are completely lysed. The solution should be homogenous and no residual large pieces of embryos should remain. Incubate the homogenized suspension on ice for 15 minutes.
Spin at 1, 000 times g four degrees Celsius for five minutes. Discard the supernatant and wash the pellet by resuspending in 500 microliter ice cold lysis buffer and pipetting up and down. After another spin, discard the supernatant.
Resuspend the washed pellet in 100 microliters of 0.5%Sodium Dodecyl Sulfate or SDS. Then permeabilize the nuclei by incubating the sample for 10 minutes at 65 degrees Celsius in a heating block. Add 50 microliters of 10%Triton X100 and 120 microliters of water.
Flick the tube to mix the contents and incubate the tube at 37 degrees Celsius for 15 minutes in a heat block. Add 25 microliters of 10X restriction enzyme buffer and 20 units of five units per microliter MBO1. Flick the tube to mix the contents and incubate the tube in a heat block to digest the DNA.
Add another 20 units of MBO1. Continue the incubation for 90 minutes. After the second 90-minute incubation, incubate the sample at 62 degrees Celsius for 20 minutes to inactive the MBO1.
Next, add 18 microliters of 0.4 millimolar biotin 14-dATP, 2.25 microliters of an unmodified 3.3 millimolar dCTP-dGTP-dTTP mix, and eight microliters of five units per microliter DNA polymerase I Klenow fragment. Flick the tube to mix the contents and incubate the sample at 37 degrees Celsius for 90 minutes. Next, add 657 microliters of water, 120 microliters of 10X T4 DNA ligase buffer, 100 microliters of 10%Triton X100, six microliters of 20 milligrams per milliliter bovine serum albumin, and finally five microliters of five units per microliter T4 DNA ligase.
Next, rotate the tube gently at 20 RPM at room temperature for two hours. Add another five microliters of five units per microliter T4 DNA ligase. Continue rotating for two more hours, then spin down the nuclei at 2, 500 times g for five minutes.
After centrifugation, discard the supernatant. Resuspend the pellet in 500 microliters of extraction buffer and add 20 microliters of 20 milligrams per milliliter proteinase K.Flick the tube and incubate the tube to digest the protein. Add 130 microliters of five molar sodium chloride and incubate overnight to de-crosslink.
The next day, pipette the sample into a new two milliliter tube with low DNA binding characteristics. Add 0.1X volume of three molar sodium acetate and two microliters of 15 milligrams per milliliter GlycoBlue. Add 1.6 volumes of pure absolute ethanol and invert the contents, then incubate the sample at minus 80 degrees Celsius for 15 minutes.
After incubation, centrifuge the contents. Locate the DNA pellet which can only be spotted by the GlycoBlue. Remove the supernatant carefully, moving the pipette tip into the tube along the opposite wall from the DNA pellet.
Wash the pellet with 800 microliters of 70%ethanol. Mix the sample by inverting and centrifuge it at 20, 000 times g at room temperature for five minutes. Remove remaining droplets during this step and the following washes by pushing them out of the tubes with a P10 tip, then open lid to air dry for up to five minutes.
Once no liquid is remaining, add 50 microliters of 10 millimolar tris chloride. Repeatedly pipette the solution over the area where the pellet was located to solubilize the DNA. Add one microliter of 20 milligrams per milliliter RNase A.Mix by flicking the tube.
Incubate the sample at 37 degrees Celsius for 15 minutes to digest RNA. Finally, store the sample in the freezer or fridge until library preparation. Images of the EGFP PCNA signal of each sorted embryo batch reveal precise stage and cell cycle statuses of every single embryo for downstream experiments.
Bioanalyzer traces show that the size distribution of DNA fragments after size selection is between 300 to 700 base pairs with a maximum at around 450 base pairs. Hi-C interaction maps show that at nuclear cycle 12, few Topologically Associated Domains or TADs are detected which changes dramatically at nuclear cycles 13 and 14 when TADs are increasingly prominent and unspecific long range contacts are depleted. While attempting this procedure, it's important to remember to wash the beads thoroughly and not to prolong incubation times unnecessarily especially at high temperatures since this can lead to low quality Hi-C libraries.
This technique combining accurate staging and high-resolution chromatin confirmation mapping paved the way for researchers in the field of epigenetics to explore the role of three-dimensional chromatin organization in an in vivo developmental setting.
