This protocol accurately detects chromosome folding features across multiple length scales with a low background signal. For instance, promoter enhancer interactions can be analyzed in the context of chromosome compartments. The use of restriction endonucleases circumvents optimizing the digestion level of input material.
No selection by gel isolation is required to capture ligation products. The most common problem is the loss of cells after DSG fixation and capturing enough DNA to produce high-quality libraries. To begin, aspirate the medium with a Pasteur pipette coupled to a vacuum trap from the 150-millimeter plate containing 5 by 10 to the sixth cells.
Wash the cells twice with 10 milliliters of HBSS. To cross-link the cells, pour 23.125 milliliters of the 1%formaldehyde solution into the 15-centimeter plate. Incubate at room temperature for 10 minutes, gently rocking the plate by hand every two minutes.
Next, add 1.25 milliliters of 2.5-molar glycine and gently swirl the plate to quench the cross-linking reaction, before incubating at room temperature for five minutes. Continue incubation on ice for 15 minutes to stop cross-linking. Scrape the cells from the plate with a cell scraper or rubber policeman and transfer the cell suspension to a 50-milliliter conical tube with a pipette.
Centrifuge the suspension at 1, 000 times G for 10 minutes at room temperature and discard the supernatant by aspiration. Wash the pellet once with 10 milliliters of Dulbecco's phosphate-buffered saline, or DPBS. Then, centrifuge again before proceeding to DSG cross-linking.
For cross-linking with DSG, resuspend the pelleted cells in 9.9 milliliters of DPBS. Then, add 100 microliters of 300-millimolar DSG. Mix the tube by inversion and cross-link the cells at room temperature for 40 minutes on a rotator.
After cross-linking, add 1.925 milliliters of 2.5-molar glycine, invert to mix, and incubate at room temperature for five minutes. Next, centrifuge the cells at 2, 000 times G for 15 minutes and resuspend the pellet in one milliliter of 0.05%bovine serum albumin DPBS before transferring it to a 1.7-milliliter tube. Centrifuge the cells at 2, 000 times G for 15 minutes at four degrees Celsius and discard the supernatant.
Snap-freeze the pellet in liquid nitrogen before proceeding to the chromosome confirmation capture. Resuspend the cross-linked cell aliquot in one milliliter of ice-cold lysis buffer containing 10 microliters of protease inhibitor cocktail and transfer to a Dounce homogenizer for 15 minutes of incubation on ice. Slowly move pestle A up and down 30 times to homogenize the cells and incubate for one minute to allow the cells to cool down before another 30 strokes.
After the last stroke, transfer the lysate to a 1.7-milliliter tube. Centrifuge the lysed suspension at 2, 500 times G for five minutes. Discard the supernatant and flick or vortex to resuspend the wet pellet.
Remove as much of the supernatant as possible to obtain a yogurt-like substance with minimal clumps. Resuspend the pellet in 500 microliters of ice-cold restriction buffer before centrifugation. After the second wash, resuspend the pellet in 360 microliters of restriction buffer by pipetting after adding approximately 340 microliters to the carryover volume of the pellet, depending on the cell size.
Set aside 18 microliters of lysate for testing the chromatin integrity, or CI.To the remaining lysate, add 38 microliters of 1%sodium dodecyl sulfate to hi-C tube to make a total volume of 380 microliters and mix carefully by pipetting without introducing bubbles. Incubate the samples at 65 degrees Celsius without shaking for 10 minutes to open up the chromatin. Then, immediately place the tube on ice.
After preparing the digestion mixture with Triton X-100, add 107 microliters of this mixture to the hi-C tube to make a total volume of 487 microliters and digest the chromatin overnight at 37 degrees Celsius in a thermomixer with interval shaking. The next day, transfer the samples to 65 degrees Celsius for 20 minutes to deactivate the remaining endonuclease activity. After placing the sample on ice, set aside 10 microliters of digestion control, or DC, and store them at four degrees Celsius.
Remove the condensation from the lid with a pipette or by spinning. Then, add 58 microliters of biotin fill-in mix to make a total volume of 535 microliters. Pipette gently without forming bubbles before incubating at 23 degrees Celsius for four hours in a thermomixer.
After incubation, add 665 microliters of ligation mix, making the sample volume 1, 200 microliters. Mix the sample by pipetting and incubate at 16 degrees Celsius for four hours in a thermomixer. Next, add 50 microliters of proteinase-K in two fractions to the hi-C sample tube and incubate overnight at 65 degrees Celsius with interval shaking.
For DNA purification, add 100%ice-cold ethanol and centrifuge the tubes at 18, 000 times G for 30 minutes at four degrees Celsius. Remove the supernatant completely from the non-pellet side and air-dry the sample for 10 minutes or until the pellets are visibly dry. Once the pellets are dry, solubilize them in 450 microliters of Tris low-EDTA by pipetting or swirling.
Transfer the solubilized suspension to 0.5-millimeter centrifugal filter unit, or CFU, with a three-kilodalton molecular weight cutoff. Centrifuge the CFU at maximum speed for 10 minutes and discard the flow-through. After the second wash, add 80 microliters of Tris low-EDTA to the column.
Turn the column into a new collection tube for two minutes of centrifugation at maximum speed. Finally, load the samples onto a 0.8%agarose gel. Agarose gel with PCR titration results demonstrated that most libraries require five to eight cycles of final PCR amplification.
Cell digestion of the final amplified PCR library, as observed by smaller fragments on an agarose gel, confirms the presence of proper ligation junctions and serves as a quality check before sequencing. After sequencing, the mapped data showed that the combination of DpnII and DdeI significantly increases short-range contacts, as adding DSG to the conventional formaldehyde cross-linking. An improved signal could also be observed at long and short distances directly from 2D interaction matrices.
Compartment signals are CRISPR at long distances and dots formed by chromatin loops are more prominent at short distances. In addition to formaldehyde, cross-linking with DSG decreases random ligations, improving the relative coverage in cis. It is important not to lose cells during and after crosslinking.
Cell pellets can be loose or invisible, and cells can stick to pipette tips in some of the buffers.
