This protocol is significant as it shows high resolution chromosome loops and other short-range interaction features. The main advantage of this technique is the mapping of the 3D genome organization with nucleosome resolution with high signal-to-noise ratio. To perform the MNase titration, thaw one pellet of one times 10 to the sixth cells on ice for 10 minutes, and resuspend the cells in 500 microliters of DPBS, and incubate the cell suspension on ice for 20 minutes.
Collect the cells by centrifugation at 10, 000 G for five minutes at room temperature and remove the supernatant. Resuspend the pelleted cells in 500 microliters of MB Number one buffer. Thaw one vial of 20 units per microliter MNase, and dilute it with 10 millimolar Tris for the digestion conditions as described in the text.
With appropriate time intervals of 10 to 20 seconds, add one microliter of the first MNase digestion solution to one of the four samples'tubes, vortex and incubate in a ThermoMixer at 37 degrees Celsius for 10 minutes at 800 RPM. Continue adding one microliter from the remaining MNase dilutions to the other cell aliquots. Stop the MNase digestion by adding 200 microliters of freshly prepared stop buffer to each tube in the same order and the same time interval in which the MNase was added.
Incubate at 65 degrees Celsius for two hours. Add 500 microliters of PCI to each sample, and mix thoroughly by vortexing. Centrifuge at 19, 800 G for five minutes at room temperature to separate the phases.
And transfer the aqueous phase to new tubes. Purify the DNA using a commercial DNA purification kit according to the manufacturer's instructions, and elute the samples in 12 microliters of elution buffer. Add two to five microliters of loading dye to the purified DNA sample.
Run the samples on a 1.5%agarose gel, and choose the best degree of digestion for the experiment. An optimal digestion degree displays little to no subnucleosomal fragments, and a 70 to 90%mono to dinucleosome ratio. In this representative sample, an intermediate digestion degree between lanes three and four was selected for follow-up experiments.
Based on the MNase titration, digest the chromatin by adding the appropriate amount of MNase to each sample aliquot. Mix well and incubate in the ThermoMixer at 37 degrees Celsius, 800 RPM for 10 minutes. Stop the MNase digestion by adding 1.6 microliters of 0.5 molar EGTA to each aliquot, and incubate in a ThermoMixer at 65 degrees Celsius for 10 minutes with 800 RPM shaking.
Collect the sample by centrifugation at 10, 000 G for five minutes at room temperature, and discard the supernatant. Resuspend the cell pellet in 500 microliters of 1X NEB buffer 2.1. Pool samples equivalent to an input of five times 10 to the sixth cells or less for further processing.
Before proceeding to the proximity ligation, transfer 10%of the sample as an input control to monitor the MNase digestion level. Add 150 microliters of 10 millimolar Tris, 25 microliters of 10%SDS, and 25 microliters of 20 milligrams per milliliter proteinase K to the digestion control, and incubate overnight at 65 degrees Celsius. Collect the remaining sample by centrifugation at 10, 000 G for five minutes at four degrees Celsius and discard the supernatant.
Resuspend the pellet in 90 microliters of freshly prepared Micro-C master mix one, and incubate in a ThermoMixer at 37 degrees Celsius, 800 RPM for 15 minutes. Add 10 microliters of five units per microliter Klenow fragment and incubate in a ThermoMixer. Then add 100 microliters of freshly prepared Micro-C master mix two.
Incubate for 45 minutes at 25 degrees Celsius, 800 RPM, and quench the enzymatic reaction with EDTA as described in the text. Incubate in a Thermo Mixer for 20 minutes at 65 degrees Celsius, 800 RPM. Collect the sample by centrifugation at 10, 000 G for five minutes at four degrees Celsius, and discard the supernatant.
Resuspend the sample in 500 microliters of freshly prepared Micro-C master mix three, and incubate for 2.5 hours at room temperature at 15 to 20 RPM. Repeat centrifugation and remove the supernatant. Then resuspend the sample in 200 microliters of freshly prepared Micro-C master mix four, and incubate in a ThermoMixer set at 37 degrees Celsius for 15 minutes at 800 RPM.
For reverse cross-linking and deproteination, add 25 microliters of 20 milligrams per microliter proteinase K and 25 microliters of 10%SDS to the sample, and incubate at 65 degrees Celsius overnight with intermittent mixing. Add 500 microliters of PCI to the samples and input control, then mix by vortexing. Separate the phases by centrifugation at 19, 800 G for five minutes, and transfer the upper aqueous phase to a fresh tube.
Concentrate the DNA and elute the samples in 30 microliters, and the input controls in 15 microliters. Run a 1.5%agarose gel to separate the mononucleosomes and dinucleosomes. Excise the DNA fragments that have a dinucleosomal size, and extract the DNA as described in the text.
Add 150 microliters of pre-prepared beads to 150 microliters of the sample, and incubate at room temperature. Place the tubes in an appropriate magnet and wait until the solution clears. Remove the supernatant and resuspend the beads in 300 microliters of 1X TBW, and repeat this step.
Repeat the magnetic separation, and after the solution clears, remove the supernatant and resuspend the beads in 100 microliters of 0.1X TE.Repeat and resuspend in 50 microliters of 0.1X TE.Transfer the solution to PCR tubes and perform the DNA manipulation as described in the text. After adapter ligation is completed, wash the sample in 1X TBW, discard the supernatant and resuspend the beads in 20 microliters of 0.1X TE.Optimize the number of PCR cycles and amplify the sequence libraries as described in the text. Chromatin from 250, 000 cells per reaction was digested with a fourfold dilution of MNase.
The highest concentration, 10 units, showed overdigested chromatin almost exclusively consisting of mononucleosomal DNA. The digestion of 250, 000 mouse ES cells with 0.625 units of MNase offered the most promising starting point for preparative digestions in Micro-C experiments. However, an intermediate MNase concentration between the conditions shown in lanes three and four, corresponding to five units per 1 million cells, should be considered.
The proximity ligated mononucleosome band had an approximate size of 300 base pairs, similar to that of dinucleosomes. Therefore, the mono to dinucleosomal band signal ratio shifted from predominantly mononucleosomes toward dinucleosomes. The quality and quantity of the prepared sequencing library were assessed via minimal PCR.
Visualization showed one distinct band at 420 base pairs, and no bands for adapter dimers. While both samples in this study displayed high map rates, the good sample had a higher fraction of cis reads. A high rate of transchromosomal interactions indicates random ligation events, although some transchromosomal interactions might be important.
Additionally, the good sample had a moderate rate of forward-reverse mapping read pairs, with distances lesser than 500 base pairs. This indicated a low rate of dinucleosomes not cleaved by MNase having a similar size to two proximity ligated nucleosomes. A proper MNase digestion degree is crucial for this experiment.
Overdigested nucleosomes are inefficiently ligated, and underdigested chromatin has a large fraction of undigested dinucleosomes that can contaminate the sequencing library. Micro-C can be combined with a capture approach to map locus-specific genome organization with enormously high resolution.
