ChIP protocol实验室成熟的CHIP-seq及CHIP-qPCR操作流程.docVIP

ChIP protocol Fixation type: Type I: 1% formaldehyde /PBS(100ml): 36.5%formaldehyde (F8775-500ml) 2.5ml diluted in 1XPBS 88.75 ml Room temperature, 15min Type II: 2-5mM DSG (stock 50mM: 10mg in 540ul of dry DMSO), Room temperature, 45min 1% formaldehyde /PBS, Room temperature, 15min Type III: 1% Glutaraldehyde solution (Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative) sigma G5882-100ml, Room temperature, 15min Stock buffer: 20%SDS; 0.5M, pH8.0 EDTA; 1M, pH7.8 Tris-Cl; 10% TritonX100; 5M NaCl, Buffer recipe: Lysis Buffer 10ml 2ml 20%SDS 0.5ml 100ul 0.5M, pH8.0 EDTA 0.2ml 50ul 1M, pH7.8 Tris-Cl 0.5ml 100ul 50Xcocktail(Roche) 0.2ml(add freshly) 40ul(add freshly) Double Distilled water 8.6ml 1720ul Dilution Buffer 500ml 100ml 10% TritonX100 50ml 10ml 0.5M, pH8.0 EDTA 2ml 0.4ml 5M NaCl 15ml 3ml 1M, pH7.8 Tris-Cl 10ml 2ml 50Xcocktail(Roche) 10ml(add freshly) 2ml(add freshly) Double Distilled water 413ml 82.6ml 100ml TSE1 TSE2 20%SDS 0.5ml 0.5ml 10% TritonX100 10ml 10ml 0.5M, pH8.0 EDTA 0.4ml 0.4ml 5M NaCl 3ml 8ml 1M, pH7.8 Tris-Cl 2ml 2ml 50Xcocktail(Roche) 1ml(add freshly) 1ml(add freshly) Double Distilled water 83.1ml 78.1ml 1.25M Glycine(MW: 75.07): 18.77g plus 200ml ddw 5M NaCl(MW58.45): 292.2g plus 1L ddw, autoclave 20% SDS(MW288.38): 20g plus 100ml ddw 1M, pH7.8 Tris-Cl: 121.1g plus 1L ddw, autoclave Option: Drug treatment：10-7 M E2 treat 1hr Day1: cell fixation, cell lysis, sonication and antibody incubation Collecting cells After fixation, cold PBS wash twice 1ml PBS to scrape cells and 0.5ml PBS scrape cells Transfer cells into 1.5ml tubes, max speed 30sec~1min, centrifugation at 4℃ If you want to stop at step3, you can freeze the samples by liquid nitrogen quickly, then store at -80℃ for long time storage. If not, continue to step 5 Cell lysis and sonication Add 300ul lysis buffer plus complete cocktail (avoid creating bubbles) Sonication（声波降解法）: we use bioruptor for sonication: 30sec/30sec, the cycles of sonication dep