Chromatin Over-Fragmentation to Mono-Nucleosome Length (ChIP (CST Guide))

Question

Accepted Answer

More than 80% of DNA fragments are shorter than 500 bp, resulting in diminished PCR signal especially for amplicons >150 bp, and potential disruption of chromatin integrity and antibody epitopes. Common causes: 1) Enzymatic protocol: insufficient starting cells or tissue for the amount of micrococcal nuclease used 2) Enzymatic protocol: excessive micrococcal nuclease added to digestion reaction 3) Sonication protocol: excessive sonication power or duration causing chromatin damage 4) Enzymatic digestion time too long relative to material amount Solutions: 1) Enzymatic protocol: weigh tissue or count cells accurately prior to cross-linking to ensure correct cell/enzyme ratio 2) Enzymatic protocol: reduce micrococcal nuclease volume to achieve 150-900 bp range (use 10× dilution optimization) 3) Sonication protocol: use minimal sonication cycles required for desired fragment length; reduce power setting if >80% <500 bp 4) Design primers for amplicons <150 bp if mono-nucleosome fragmentation cannot be avoided