Table 3. Troubleshooting table.

Step	Problem	Cause	Solution
Reagent Setup	Excess Lysis buffer	Small number of samples.	To make smaller volumes of lysis buffer, one cOmplete Protease Inhibitor Cocktail tablet can be dissolved in 2 mL of PCR grade water to generate a 25× stock. This can be aliquoted and stored at - 20°C for several months.
9	Fewer than 5 x 106 cells	Working with a rare cell population or limited number of cells following cell sort.	For fixation, PBS wash and lysis the volumes can be scaled down to accommodate fewer cells (down to 2 x 104 cells). Maintain cells at ~1 x 106 cells per 1 mL of growth media except for ≤1 x 106 cells where 1 mL of media should be used and fixation and lysis performed in a 1.5 mL tube. Perform digestion reactions in 200 μL for between 2 x 104 and 5 x 106 cells.
	More than 5 x 106 cells	Working with a cell line	For fixation, PBS wash and lysis the volumes can be scaled up to accommodate more cells. Maintain cells at ~1 x 106 cells per 1 mL of growth media. For greater numbers of cells, perform multiple, parallel digestions and combine material in 300 μL of TE buffer after nuclear isolation.
37	Phenol use is not desireable or prohibited	Phenol is a dangerous chemical	Use of column extraction is possible and is considerably faster, e.g. Qiagen DNeasy Mini kit can used from the point where nuclei are pelleted, step 32. However, also pellet Control 1 and Control 2, then increase Proteinase K treatment to 4 hours at 65°C, and elute the DNA from the columns using the volumes outlined at step 45, before preceding to Quality Control.
46	DNA not visible in agarose gel	Low amount of DNA because of low-cell contraints	Run 1 μL of each sample on a Genomic DNA ScreenTape.
47	Low DNA yield	Loss of nuclear pellet	The nuclear pellet can be hard to see and may accidentally be disturbed. If suffering low DNA yields, retain the supernatant and perform Phenolchloroform isoamyl alcohol DNA extraction. A good Nuclear 3C library should have >90% of DNA within the nuclear pellet. The combined DNA from the nuclear pellet and the supernatant is equivalent to an in situ 3C library.
		Incomplete de-crosslinking	Perform decrosslinking overnight
		Incomplete precipitation	Freezing at -80°C overnight may be beneficial for DNA yield, particularly for low-input samples.
48	No control DNA	Working with low cell numbers	For low-input samples (≤150,000 cells), where very little DNA is available for controls, digestion efficiency can be directly calculated from re-ligated 3C libraries against a genomic DNA input control. Note that due to re-ligation into the original fragment configuration, lower values for digestion will be observed than for a true digestion control.
49	Low digestion efficiency	Short digestion period or sub-optimal enzyme activity	The total digest time should be 20-24 hours. Additional restriction enzyme can be added at each optimal enzyme activity of the three timepoints (steps 21-23) for cells generating low digestion efficiency.
	Non-exponential amplifiction	Reaction conditions for primers not optimized to thermocycler	Perform a dilution series analysis wigh genomic DNA and include a melt curve to ensure no primer dimers are being produced.
61	DNA not at correct size	Sonicator settings not optimized	Each sonicator may vary and should be set accordingly. Settings for sonication should be first determined by testing with high molecular weight genomic DNA rather than wasting 3C library. It is important to take into account the mass of DNA being sheared.
77	Vacuum centrifuge is not available	Specific equitment may not available	DNA may be purified by AMPure XP SPRI bead clean-up (e.g. steps 53-59) with elution into 40.2 μL of Universal Enhancing Oligonucleotides (6.7 μL per library).
86	Beads stick to plastic	High affinity of streptavidin beads for plastic tubes	Streptavidin dynabeads tend to stick to plastics. We find this effect is minimised by using high-quality, non-sticky tubes, from Sorenson BioScience (39640T).
116	Loss of DNA after capture	Failed PCR reaction, user error during DNA bead clean-up.	Captured material is amplified off the beads in two batches. Although these reactions can be performed simultaneously, it is prudent to do each individually to protect against error or misfortune and to ensure adequate amplification has occurred.
	Low DNA yield post capture	Incomplete hybridization	A longer hybridization time of 68-72 h may increase capture yield.
138	Beads stick to plastic	High affinity of streptavidin beads for plastic tubes	Streptavidin beads tend to stick to plastics. We find this effect is minimised by using high-quality, non-sticky tubes, from Sorenson BioScience (39640T).
159	Loss of DNA after capture	Failed PCR reaction, user error during DNA bead clean-up	Captured material is amplified off the beads in four PCR reactions (two per hybridisation reaction). Here, these reactions are performed simultaneously, though it is possible to do these in two batches to protect against error or misfortune and to determine if adequate amplification has occurred.
172	Tiled-C matrix not generated	Using coodinates for a single viewpoint not a region	Change the bed file coordinates to match the Tiled-C targeted region including the start of the first targeted fragment and the end of the and last targeted fragment.
	Interaction matrix not generated	Using Capture-C configuration settings	Set analysis method in config.xml to either “tiled” for Tiled-C or “tri” for Tri-C.