Table 3 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
10, Box 1	Low yield of annealed linker	Oligonucleotide was incompletely dissolved or problems in oligo synthesis	Check the quality of the single-stranded oligonucleotide by measuring concentrations and/or resolve a 5-pmol sample on a denaturing polyacrylamide gel
		Incomplete annealing	Double-check the sequences of oligonucleotides, salt concentration of annealing reaction and the ramping speed of the thermal cycler; HPLC-purify oligos
		Incomplete precipitation	All precipitations require at least 30 min incubation at <4 °C. Longer time of incubation may improve yield for short DNA molecules
26	Loss of cells (>50% loss, visually estimated by cell pellet size)	Incomplete pelleting of cells	Increase the starting number of cultured cells; use a swinging-bucket rotor for centrifuging cells instead of a fixed-angle rotor; centrifugation time and speed can be increased up to 5 min at 2,300g
		Fixed cells stick to the tube during centrifugation	Try different types of conical tubes; use PBST instead of PBS
34	Loss of nuclei (>50% loss, visually estimated by nuclei pellet size)	Incomplete pelleting of nuclei	Increase the starting number of cultured cells; put the 1.5-ml LoBind tube inside a 50-ml conical tube and then use swinging-bucket rotors for centrifuging; centrifugation time and speed can be increased up to 5 min at 2,300g
		SDS concentration was too high	Incubating fixed cells with SDS at 62 °C may dissolve the cell membrane as well as the fixed nuclear matrix, leaving only debris in the solution. The SDS concentration should be decreased in such cases
	>50% of cells with cytoplasm intact around the nuclei	Cells are not in a single-cell suspension	Separate the cells by pipetting with smaller pipette tips (e.g., 20- to 200-µl tips) or by passing them through a syringe and checking under a microscope before adding SDS
		The SDS concentration is too low to efficiently lyse cell membranes	SDS concentration can be increased up to 0.5% (wt/vol)
113	No band visible and/or band obscured by a strong background	DNA impurity	Perform DNA extraction and use ethanol to wash and precipitate the DNA again at −20 °C overnight after Step 107
		Too much input DNA	DNA input can be further decreased to 500 ng after Step 93
		Incomplete ssDNA release from Dynabeads MyOne Streptavidin C1	Use fresh Dynabeads MyOne Streptavidin C1 and 150 mM NaOH; pool samples if the concentration in Step 93 is <200 ng
		Insufficient washing of Dynabeads MyOne Streptavidin C1	Increase the washing time and strength by vortex or rotation
		Low efficiency of MmeI digestion	Add more MmeI by repeating Step 102 before adding proteinase K in Step 103; it is recommended to estimate the molar amount of restriction sites in the DNA sample and adjust MmeI amount to close to a 1:1 molar ratio; replace and use fresh SAM
		DNA ligation and/or RNA ligation failed	Replace relevant reagents; check linker pre-adenylation efficiency
	No 85-bp band, but the 65-bp band is present	DNA ligation failed	Replace relevant reagents. Note that DTT may be degraded in old ligase buffer
	Nothing on gel	Low amount of input DNA	Pool samples if concentration in Step 93 is <200 ng
138	Specific band in 175- to 190-bp range is not missing in the mock sample	Oligo A1 and A2 were not annealed	Check annealed adaptors in an 18% native polyacrylamide gel
		Incomplete precipitation of input DNA	Increase isopropanol precipitation time to 3 h; detergent sometimes affects low-quantity DNA precipitation steps after gel extraction. Remove the detergent by phenol–chloroform extraction before precipitation, if necessary
		Desired band migrates out of the 175- to 190-bp range	This can happen if the electrophoresis conditions are different. Any band that is clearly missing in the mock control should be purified for PCR
	Strong background smear and/or multiple undesired bands	Impurity of the paired-end tags	Cut the 85-bp band as close as possible in Step 114 to avoid gDNA contamination
		Low paired-end tag input	Multiple 85-bp bands in Step 114 from the same sample could be pooled to increase input DNA amount for adaptor ligation
150	Low yield of the amplified 188-bp DNA	Incomplete precipitation of input DNA	Increase isopropanol precipitation time to 3 h
		Low amount of input DNA	Pool parallel adaptor-ligated samples to increase the input template amount for PCR
	Strong background smear and/or multiple unspecific bands	Impurity of the adaptor-ligated library	Cut the adaptor-ligated band as close as possible in Step 138 to avoid adaptor-ligated gDNA contamination
		Usage of a different polymerase	Use Phusion polymerase from Thermo Fisher Scientific (cat. no. F530S). Phusion polymerase from other manufactures generated a much higher background with the same pre-PCR library
158	High level of duplicated mate-pair reads (>10%)	Low complexity of the library due to excessive PCR amplification	Reduce the number of PCR cycles
	Low PCC at 1 kb/bin (<0.5)	Low complexity of the library	Pool parallel adaptor-ligated samples to increase the input template amount for PCR
159	Low number of chromatin-associated RNA species (<500)	Low complexity of the library	Pool parallel adaptor-ligated samples to increase the input template amount for PCR
Box 1, step 6	The RNA stretch is missing on the polyacrylamide gel	RNA degradation	The RNA stretch of oligo L1 is labile. Repeated freeze–thaw cycles and nuclease contamination should be avoided
		Nuclease contamination in the polyacrylamide gel	Use fresh reagents and nuclease-free water to make gels and staining solutions
Box 2, step 15	Too many DNA fragments >1 kb	Insufficient permeabilization of the nuclei	Increase the SDS concentration in Step 30 up to 0.5% (wt/vol)
		Low AluI activity	Use no more than 2.5 × 106 mammalian or 1.3 × 107 Drosophila nuclei for each digestion reaction; use fresh enzymes; increase the time for AluI digestion