Table 1.

Troubleshooting guide for mapping histone repositioning by chromatin remodeling enzymes via OP-directed DNA cleavage reactions

Problem	Possible cause	Solution
Loss of sample (Basic Protocol 1)	Dialysis-related sample loss	Check dialysis tubing for possible leakage
		Avoid vigorous mixing of (micro)dialysis setup to prevent disassembly of (micro)dialysis setup (e.g., bag or chambers)
		Avoid overloading microdialysis chambers (≤ 150 μL per tube)
		Do not use dialysis tubing with large molecular weight cutoff value (> 3.5 kDa) to prevent passing the labeled histone core through the membrane
	Miscellaneous sample loss	While concentrating the labeled histone core for long-term storage, use the centrifuge speeds that are less than the maximum g force of the concentrators.
Low intensity or an absence of cleavage bands (Basic Protocol 3)	Inadequate OP-labeling of the histone core (Basic Protocol 1)	Use a fresh OP solution (less than 6 months old)
		Check the pH, concentration, and volume of TCEP solutions to assure ensure complete reduction of disulfide bonds
		Check the concentration and volumes of OP solution added to labeling reaction mixture
	High amounts of EDTA in sliding/quenching mixtures containing OP-labeled nucleosomes	If possible, replace EDTA. For instance, use hexokinase instead of EDTA to quench sliding reactions, as described in Basic Protocol 2
High background in cleavage pattern obtained from scanning the denaturing sequencing gel (Support Protocol 2)	Large amounts of non-target DNA in buffer used to quench reactions	Avoid using competitive DNA, and instead use hexokinase to quench sliding reactions
	Unclean gel plates	Thoroughly clean glass plates, as described in Support Protocol 2.
Gel solution polymerized while pouring gels	High amount of added APS and TEMED	If the room temperature is ≥ 25°C, pour the gel with 10% lower amounts of APS and TEMED
	Warmed-up gel solution	Keep the acrylamide solution at 4°C until ready to add APS and TEMED, and pour the gel immediately
Malformed wells in sequencing gel (Support Protocol 2)	Leakage of the gel solution from in between the gel sandwich	Leakage of the gel equally affects all the lanes. If the wells are too small to hold the individual sample solutions, or are not formed at all, prepare a new gel. Use the wells if the leakage is not severe and the volume of the formed wells is enough to hold the loaded sample
	Disturbing the comb before the gel is fully polymerized or inappropriate removal of the comb after polymerization	Remove the comb by slowly pulling it from the two ends simultaneously, to prevent disruption of the wells. If the wells are formed properly but are bent, use a P10 or P20 pipette tip to straighten the lanes.
Running buffer leaking between the short plate and aluminum plate, or from the contact sites between the silicon gasket and sealing foam blocks of the spacers (Support Protocol 2)	Sequencing gel is not completely sealed against the S2 apparatus	Slightly loosen the clamps by unscrewing the knobs at the top of the gel by half a turn. This may release an over-pressed and wrinkled silicon gasket. If the leakage does not stop, tighten the screw knobs.
		Apply grease to the side of the gel sandwich where the solution is leaking. Normally, the leakage happens at the border of aluminum plate, silicon gasket and edge of gel at the top.
		If the leakage does not stop, remove the gel sandwich. Inspect the silicon gasket and sealing foam blocks for any tearing or damage, apply extra grease to previously leaking sites and put the gel back on S2 apparatus.
Smeared or blurred bands on sequencing gel (Support Protocol 2)	Debris, particles, or bubbles inside the wells or within the polymerized gel	Filter (0.22 μm) and degas the gel solution. Check the gel immediately after placing the comb for the bubbles in the vicinity of the comb teeth. If there are bubbles and extra gel solution is available, take out the comb, immediately add more gel solution, and replace the comb
	Urea crystals inside the wells while loading the DNA sample	Flush the wells twice (before pre-run and immediately before loading).
	Small fragment formamide loading dye is too old or not properly stored	To avoid formamide degradation, store aliquots with fragment formamide loading dye at −20 °C. Avoid freeze/thawing
	High salt content from precipitated DNA	Avoid using NaCl instead of 3M NaOAC/HOAC to precipitate DNA
	Overloaded DNA	Keep the amount of nucleosome/hexasome per lane below 2 pmol.
	Overheated gel sandwich	Run the gel at constant 65 W, not constant voltage.
In the sequencing gel, different migration of the bands in the middle compared with the sides of the gel	Uneven pressure applied to the sides of the gel during polymerization	Place binder clips symmetrically on both sides of the gel and have equal spacing between the placed clips.
	Uneven pressure applied to the sides of the gel during electrophoresis	Do not overtighten the internal gel clamps on S2 apparatus.
Appearance of dark spots after gel scan with excitation at wavelengths corresponding to one or both fluorophores (Basic Protocol 3, Support Protocol 2)	Contamination in Ultrapure water (e.g., mili-Q) used to prepare the gel solutions or running buffers	Change the filter on ultrapure water source or use a different ultrapure water source