染色质免疫共沉淀 ChIP Protocol及crosslink 的原理.docVIP

ChIP Protocol I. Formaldehyde cross-linking of cells Use 5x107 – 1x108 cells (70-80% confluency for adhesion cells of 8-12 15 cm2 plates or 175 cm2 flasks) for each immunoprecipitation. Adherent cells: 1. Add 1/10 volume of fresh 11% Formaldehyde Solution to plates. 2. Swirl plates briefly and let them sit at room temperature for 10 minutes. 3. Add 1/20 volume of 2.5 M glycine to plates to quench formaldehyde. 4. Rinse cells twice with 5 ml 1x PBS. Harvest cells using silicon scraper. 5. Pool cells in 50 ml conical tubes and spin at 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant and resuspend pellet in 10 ml 1x PBS per 108 cells. 6. Transfer 5x107 – 1x108 cells to 15ml conical tube and spin at 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant. 7. Flash freeze cells in liquid nitrogen and store pellets at –80°C. Suspension cells: 1. Add 1/10 volume of fresh 11% Formaldehyde Solution to flasks. 2. Swirl flasks briefly and let them sit at room temperature for 20 minutes. 3. Add 1/20 volume of 2.5 M glycine to flasks to quench formaldehyde. 4. Spin cells at 1,350 x g for 5 minutes at 4°C. 5. Pool cells in required number of 50 ml conical tubes and spin at 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Dump supernatant. 6. Resuspend cells in 50 ml 1X PBS, spin at 1,350 x g for 5 minutes at 4°C. Discard supernatant. Repeat once. 7. Resuspend in 10 ml per 108 cells. Aliquot 5x107 – 1x108 cells to 15ml conical tubes and spin 1,350 x g for 5 minutes at 4°C (Sorvall Legend RT centrifuge with swinging bucket rotor). Discard supernatant. 8. Flash freeze cells in liquid nitrogen and store pellets at –80°C. Formaldehyde Solution Final Conc. Stock For 50 ml 50 mM 1M Hepes-KOH, pH 7.5 2.5 ml 100 mM 5M NaCl 1.0 ml 1 mM 0.5M EDTA 50.0 μl 0.5 mM 0.5M EGTA 100.0 μl 11% 37% Formaldehyde 14.9 ml ddH2O 31.5 ml II.