Chromatin Under-Fragmentation with Excessive Large Fragments

Symptom

Chromatin fragments are too large (>900 bp for enzymatic, >1 kb for sonication), leading to increased background signal and lower resolution in ChIP results.

1. 1 Over-crosslinking: fixation time exceeded 30 minutes
2. 2 Too much input material (cells or tissue) per sonication volume
3. 3 Enzymatic protocol: insufficient micrococcal nuclease added to chromatin digestion
4. 4 Sonication protocol: insufficient sonication duration or power setting too low

1. 1 Shorten crosslinking time to 10-30 minute range
2. 2 Reduce amount of cells/tissue per sonication to 100-150 mg tissue or 1×10⁷-2×10⁷ cells per 1 ml buffer
3. 3 Enzymatic protocol: increase amount of micrococcal nuclease or perform time-course optimization (test 0, 2.5, 5, 7.5, 10 µl diluted enzyme)
4. 4 Sonication protocol: conduct sonication time course to determine optimal duration and verify >60% fragments <1 kb

Related Video (3)

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