Capture Hi-C allows deciphering the 3D chromatin organization of megabase-size genomic region of interest at high resolution. With Capture Hi-C, higher resolution and allele specificity are achieved while improving the time effectiveness and affordability of the technique. The application of Capture IC to different model system, cell types, and developmentally regulated chromatin landscapes in health and pathological conditions is likely to facilitate our understanding of the interplay between genome topology and gene regulation.
To begin, tryphtonise and count cells from the control plate prepared specifically for cell counting using an automated cell counter. Include viability staining to determine the percentage of viable cells. From this cell count, estimate the total number of cells in the plate prepared for crosslinking.
Remove the culture medium from the plates prepared for crosslinking and replace it with the fixation solution. Fix for 10 minutes at room temperature under gentle mixing on a shaker. Quench the fixed reaction by adding 2.5 molar glycine PBS to a final concentration of 0.125 molars.
Add 530 microliters of 2.5 molar glycine PBS to 10 milliliters in a 10 centimeter plate. Incubate for five minutes at room temperature, gently mixing on a shaker. Transfer the plates to ice and incubate for an additional 15 minutes on ice while gently mixing on a shaker.
Remove the fixation solution from the cells by pouring it into a beaker to ensure fast handling. Rinse the 10 centimeter plate quickly two times with five milliliters of cold 0.125 molar glycine PBS to wash off the debris and dead cells. Remove the liquid from the plate by pouring it into a beaker to ensure fast handling.
Add five milliliters of cold 0.125 molar glycine PBS to the 10 centimeter plate. And quickly scrape the cells from the plate using a plastic cell scraper. Transfer the cell suspension into a pre-cool 50 milliliter conical centrifuge tube using a serological pipette.
Rinse the plate twice with five milliliters of cold 0.125 molar glycine PBS and add the cell suspension to the conical centrifuge tube. Spin down at 480 G for 10 minutes at four degrees Celsius. Remove the supernatant by aspirating with a benchtop aspiration system.
Resuspend the cells and aliquot 500 microliters of the cell suspension into the calculated number of 1.5 milliliter micro centrifuge tubes. Spin down at 480 times G for 10 minutes at four degrees Celsius and store the dry cell pellets at 80 degrees Celsius. For cell lysis, thaw the frozen pellets on ice.
Prepare 1.5 milliliters of lysis buffer in water. Add 600 microliters of the cold lysis buffer and resuspend well on ice. Spin down at 2, 655 G for five minutes at four degrees Celsius and remove the supernatant using a benchtop aspiration system.
Resuspend in 100 microliters of 0.5%SDS. After incubation at 62 degrees Celsius swirling at 1400 RPM for 10 minutes, add 290 microliters of water plus 50 microliters of 10%Triton X 100, and mix well, avoiding air bubbles. Incubate at 37 degrees Celsius with swirling at 1400 RPM for 10 minutes before adding 50 microliters of 10 X DPN tube buffer and invert the tube to mix.
Take 50 microliters of undigested DNA for quality control into a separate tube. Do not forget to take the undigested control sample. For DPN2 digestion, add 10 microliters of DPN2 high concentration and mix by inverting.
Incubate the samples and the undigested control in a thermal mixer at 37 degrees Celsius, swirling at 1400 rotations per minute for more than four hours. For ligation and reversal of cross-linking, take 50 microliters of the ligated digested DNA for quality control in a separate tube. Store the undigested and unligated controls at 20 degrees Celsius.
Add 800 microliters of ligation cocktail. Incubate at 16 degrees Celsius, swirling at 1000 rotations per minute overnight. For DNA purification, transfer the samples on ice to pre-cool 15 milliliter conical centrifuge tubes and add two milliliters of water, 10.5 milliliters of ice cold ethanol, and 583 microliters of three molar sodium acetate.
Add 200 microliters of ice cold ethanol, 10.8 microliters of sodium acetate, and one microliter of the coprecipitant to the undigested and ligated quality controls. Incubate at minus 80 degrees Celsius for at least four hours up to overnight. Spin the 15 milliliter tubes at 2200 G at four degrees Celsius for 45 minutes.
Carefully remove ethanol and air dry the samples and controls at room temperature for 10 to 15 minutes. Do not over-dry. Resuspend the samples and controls in 100 microliters and 20 microliters of water, respectively.
For quality control of 3C template preparation, load 100 to 200 nanograms of each sample and each control on a 1%agros 1XTBE gel. Then to perform target enrichment for pared end multiplex sequencing, take four micrograms of 3C template, resuspend it in 130 microliters of water for each capture reaction. Sonicate the sample and continue the preparation with three micrograms of the sheared DNA.
To hybridize the prepared to DNA samples to the target-specific RNA probes, use 750 nanograms of DNA samples in a final volume of 3.4 microliters, resulting in an initial concentration of 221 nanograms per microliter. Use speed vacuum concentration if required to achieve the final volume of 3.4 microliters. For samples re suspended in 10 microliters, 15 to 20 minutes of concentration is sufficient.
Incubate the hybridization mixture for 16 to 18 hours at 65 degrees Celsius with a heated lid at 105 degrees Celsius, according to the manufacturer's instructions. To capture the targeted ligation fragments, add streptivide in coupled beads to the probe hybridized sample. Finally, to prepare the samples for multiplex sequencing, continue the protocol according to the target enrichment system used in this study for paired and multiplexed sequencing.
The resolution of the Capture Hi-C interaction map allows the identification of two smaller domains in the Tsix-tad which contains the promoter of the Tsix locus. This was not previously achieved with 5C. Moreover, other sub-tad structures, such as contact loops, are clearly visible from the Capture Hi-C data, such as the loop between Xist and FTX, previously identified with Capture C and the loop between Xist and Xert recently identified using a similar protocol for Capture Hi-C.
Other contacts can also be mapped more precisely due to the increased resolution of the Capture Hi-C profiles, such as those forming the known contact hotspots within the Tsix-tad between the Linx, Chic1, and Xite loci. In comparison with the shown Hi-C data, Capture Hi-C allowed for a fourfold increase in resolution, yet it required only one fourth of sequencing depth. It is very important to not forget to take 50 microliters of undigested and unligated DNA samples as the quality control for the 3C template preparation.
