Procedures | Our optimized N-ChIP protocol | |
---|---|---|
Starting material | Non-fruit tissues or suspension cells [13, 19,20,21, 43,44,45,46,47] | Fleshy fruit tissues |
Nuclei isolation | No particular steps for homogenization [20, 21, 47]. Homogenize in an all-glass homogenizer using a tight pestle [13, 45, 46]. Homogenize in a roller stirrer [19] or through syringe [44]. Homogenize in an undescribed way [43] | The powder was washed in PBS-PSV twice firstly. Homogenize in a glass homogenizer followed by lysis on ice with gentle agitation for 10Â min |
Chromatin extraction | Extraction buffer contains 0.4–0.8% NP40 [45, 46], 0.1% Triton X-100 [43, 47], 0.5% Triton X-100 [21], 1% Triton X-100 [44], 0.25% Tween 40 [19], 1% Tween 40 [13] as the detergent, or no detergent [20] | Extraction buffer contains 0.5–1% Triton X-100 Wash at least four times |
Fragmentation | Digestion at 37 °C for 10–12 min [19,20,21, 43, 46] or 4–7 min [19, 44, 45, 47] | Digestion for 10 min at 37 °C (30U/400 μl). Recommend optimizing the enzyme dosage and digestion time for each preparation |
Immunoprecipitation Antibody Protease inhibitors Reducing reagents | Protein A–Sepharose [13, 19, 20, 45], Dynabeads Protein G [46], Dynabeads Protein A [21, 47] or Dynabeads Protein A/G beads [43, 44] ChIP-grade or only tested by western blot [13, 19,20,21, 43,44,45,46,47] Buffers contain protease inhibitor cocktail [20, 43,44,45,46,47], 0.1–0.2 mM PMSF [13, 19, 20, 43,44,45] Buffers contain 0.5 μM–0.5 mM DTT [45, 46], 10 mM β-mercaptoethanol [21, 43, 47] | Dynabeads Protein A beads Passed ChIP-grade antibodies validated by the ENCODE project Buffer contains 1 × protease inhibitor cocktail tablets, 1 mM PMSF Buffers contain 1 mM DTT and 11 mM β-mercaptoethanol |