Table 1.

Troubleshooting

Step	Problem	Possible reason	Solution
2	Tagged protein not expressed	Cloning and mutation issues	Verify the construct by sequencing
4	Expression level of the tagged protein is too high relative to the endogenous protein	Inefficient viral infection	Titer the virus prior to infection, use a GFP control virus
		Viral infection with high multiplicity of infection (MOI)	Infect with low MOI, use a vector with weaker promoter
14	Under/over shearing of DNA	Inappropriate sonication	Extend/reduce sonication time, keep the concentration of input sample constant
27	Inefficient elution from M2 beads	Inefficient TEV cleavage	Extend incubation with TEV to overnight at 4 °C
		Inefficient 3xFLAG peptide competition	Increase the concentration of 3xFLAG peptide to ~400 µg/ml
28	Weak binding to Ni-NTA	Presence of reducing agents, such as DTT	Remove interfering compounds with dialysis prior to purification with Ni-NTA.
40– 43	Inefficient elution from Ni-NTA beads	Short elution and/or low imidazole concentration	Perform elution with imidazole for extended time (>3 h) at room temperature
44– 46	Insufficient DNA recovery	Tagged protein non- Functional Protein folding masks TAP tag	Functional validation and correct cellular localization of the tagged protein Assay the efficiency of IP with anti FLAG antibody
		Washes were too stringent or too extended	Each washing step should be ~5 min. The ratio of wash buffer to beads should be ~10:1
			Use molecular biology grade and nuclease- free reagents
		Cross-linking sub optimal	Make fresh fixation buffer prior to ChIP.
47	ChIP-seq data is suboptimal	Chromatin fragmentation not appropriate	Size exclusion during ChIP library construction results in chromatin fragment between 100–300 base pairs. Assess the efficiency of chromatin fragmentation prior to ChIP experiment
		Insufficient antibody quantity used	Increase the amount of antibody. We typically use 150 µl of M2 agarose beads slurry for ChIP with 100–150 million cells
		Insufficient sequencing depth	Run additional lane on the flow cell or switch to another platform such as HI-SEQ2000
		No enrichment on the expected genomic regions	TAP tag epitope may have been masked due to cross-linking or protein folding. Place TAP tag on the opposite end of protein or switch to a different tag

TIMING

Steps 1–4 Generation of stable cell lines expressing TAP tagged construct ~1 week

Steps 5–14 Cell harvesting, lysis and chromatin shearing ~3 h

Steps 16–22 Chromatin tandem affinity purification: ~3–4 days.

Step 27 Elution of chromatin from anti FLAG M2 affinity beads: option1, 3–4 h; option 2, 1 d

Steps 28–42 Final purification of chromatin by Ni-NTA affinity chromatography: 0.5 days.

Steps 43–46 Reverse cross-linking of chromatin and ChIP DNA purification: 1d

Step 47 ChTAP library construction and sequencing: ~2–3 days.