TABLE 2│.

Troubleshooting table

Step	Problem	Possible reason	Solution
68	Trapped air bubbles	Sudden injection of IP buffer into the hydrophobic PDMS device	Set the flow rate at 50 μl/min. Close the sieve valve. Wait for 5 s, and then open the valve to flush the air bubble out with the help of pressure generated in the chamber. Alternatively, press the chamber with a pair of tweezers to push the bubble out.
78	Air bubbles in the chamber after chromatin solution loading	The air bubble that separates IP buffer and chromatin solution is too large.	C-Flex tubing in long-tubing assembly needs to be changed periodically (roughly after 20 experiments) because it may expand over time and hold large air bubble during solution loading.
100	No visible pellet at the bottom of the microtube	Low concentration of ammonium acetate or ethanol	Be sure to prepare 5 M ammonium acetate solution in ultrapure water accurately. Use high quality glycogen and ethanol.
107	Low enrichment (<10 when primers in Table 1 are used) for ChIP DNA detected by qPCR	Chromatin fragmentation is not optimal	Adjust conditions for cross-linking and sonication to achieve 200–500 bp fragment size. Keep the temperature of the sample around 4 °C during sonication. For MNase digestion-based fragmentation, try different concentrations of MNase to achieve 170–540 bp fragment size.
		The quality of antibody is not good.	Test antibodies from other batches or suppliers.
		Problem with the reagents used in the experiment.	Make sure to add protease inhibitors as instructed in the protocol.
		Oscillatory washing conditions are not optimal.	Adjust the time and pressure associated with washing. Generally, increased washing improves enrichment but decreases DNA amount. Ideal percent input is lower than 1% for negative loci, and 10%-40% for positive loci.
		Primer design is not optimal	Design alternative primer sets.
		Selected loci are not optimal	Target other loci in the genome. Order commercial primer sets if they are available.
137	More than 16 cycles are needed in library amplification when the amount of ChIP DNA is > 10 pg	Too much DNA is lost during SPRI beads cleanup steps.	Perform the SPRI cleanup with extra attention and use correct buffer ratio. Be sure to dry the beads after 80% (vol/vol) ethanol washing and also avoid over-drying the beads.
143	A large amount of adapter dimer in the library (~120 bp)	Insufficient size-selection step	Do the size-selection cleanup with accurate SPRI-to-buffer ratio.