Step	Problem	Possible reasons	Solution
3	Low percentage of long-range contacts in sequencing library or BamHI digest does not shift library	Inefficient or incomplete formaldehyde crosslinking	For new cell types, optimizing the amount of formaldehyde used for crosslinking may be necessary.
7	Nuclear pellet disappears during in situ enzymatic treatments	Overtreatment of fixed nuclei with SDS	Reduce the amount of SDS used in the cell lysis.
14	gDNA digestion efficiency is poor	Undertreatment of fixed nuclei with SDS; inadequate amount of DNase I used for digestion	Optimization of the appropriate SDS and DNase I amounts may be necessary. We recommend performing the protocol through Step 38 for a variety of SDS concentrations (i.e. 0.1% – 0.5%) and DNase I amounts (i.e. 1U – 8U).
111	FastQC metrics are poor	High duplication rate in library (e.g. Fewer than 60% unique sequences); low quality sequencing run (e.g. total percentage of bases with q > 30 is less than 85%)	To maximize library complexity, make sure to set up several PCR reactions in Step 114. Issues with sequencing runs themselves may be difficult to diagnose and may require outside help.