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1、ChIP protocol Fixation type: Type I:1% formaldehyde /PBS(100ml): 36.5%formaldehyde (F8775-500ml) 2.5ml diluted in 1XPBS 88.75 mlRoom temperature, 15minType II:2-5mM DSG (stock 50mM: 10mg in 540ul of dry DMSO), Room temperature, 45min1% formaldehyde /PBS, Room temperature, 15minType III:1% Glutaralde
2、hyde solution(Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative) sigma G5882-100ml, Room temperature, 15minStock buffer: 20%SDS; 0.5M, pH8.0 EDTA; 1M, pH7.8 Tris-Cl; 10% TritonX100; 5M NaCl, Buffer recipe:Lysis Buffer10ml2ml20%SDS0.5ml100ul0.5M, pH8.0 EDTA0.2ml50ul1M
3、, pH7.8 Tris-Cl0.5ml100ul50Xcocktail(Roche)0.2ml(add freshly)40ul(add freshly)Double Distilled water8.6ml1720ulDilution Buffer500ml100ml10% TritonX10050ml10ml0.5M, pH8.0 EDTA2ml0.4ml5M NaCl15ml3ml1M, pH7.8 Tris-Cl10ml2ml50Xcocktail(Roche)10ml(add freshly)2ml(add freshly)Double Distilled water413ml82
4、.6ml100mlTSE1TSE220%SDS0.5ml0.5ml10% TritonX10010ml10ml0.5M, pH8.0 EDTA0.4ml0.4ml5M NaCl3ml8ml1M, pH7.8 Tris-Cl2ml2ml50Xcocktail(Roche)1ml(add freshly)1ml(add freshly)Double Distilled water83.1ml78.1ml1.25M Glycine(MW: 75.07): 18.77g plus 200ml ddw 5M NaCl(MW58.45): 292.2g plus 1L ddw, autoclave20%
5、SDS(MW288.38): 20g plus 100ml ddw1M, pH7.8 Tris-Cl: 121.1g plus 1L ddw, autoclaveOption: Drug treatment：10-7 M E2 treat 1hrDay1: cell fixation, cell lysis, sonication and antibody incubationCollecting cells1. After fixation, cold PBS wash twice2. 1ml PBS to scrape cells and 0.5ml PBS scrape cells 3.
6、 Transfer cells into 1.5ml tubes, max speed 30sec1min, centrifugation at 44. 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 5Cell lysis and sonication5. Add 300ul lysis buffer plus complete cockta
7、il (avoid creating bubbles)6. Sonication（声波降解法）: we use bioruptor for sonication: 30sec/30sec, the cycles of sonication dependent on cell type, cell numbers and crosslinking methods. (for example: 293T, 1%formaldehyde, cells did not freeze; 20cyces can obtain acceptable DNA fragment)7. max speed, 10
8、15min 4 to precipitate cell lysate and aspirate 10ul supernatant for sonication efficiency test (100-500bp)8. 95,10min to decrosslink protein from DNA fragment , and 1%agarose electrophoretic analysis (actually, we will get the size from 100bp, or 100-200bp)(Tips: during test, samples should be kept
9、 at 4 instead of ice bath)9. If the size of DNA fragment met ChIP grade requirement, then continue to incubation specific antibody as following; if not, resuspend samples for further sonication10. Cell lysate should be diluted 10-fold by dilution buffer plus cocktail (150ul cell lysate plus 1350ul d
10、ilution buffer) and left 5ul cell lysate as input. 11. 150ul+1350ul dilution buffer +2.5ug specific antibody (2-5ug), 4rotation overnightDay2: DecrosslinkingIncubating Dynabeads (Dynabeads Protein G for Immunoprecipitation, 10004D)12. Add Dynabeads 30ul per sample, 4, 3-4hrsWashing beads13. 1.5ml pe
11、r sample TSE1 plus 0.5Xcocktail(Roche) ,4, rotation 15min20min14. 1.5ml per sample TSE2 plus 0.5Xcocktail(Roche), 4, rotation 15min20min15. 1.0ml TE Buffer wash twice R.T, using vacuum to remove supernatant at the first time, and using 1ml pipettor to remove TE buffer instead of vacuum because Dynab
12、eads become incompact at the second washing stepDecrosslinking 16. 300ul per sample of TE/SDS buffer (decrosslinking buffer) at 65 , 1,400 rpm mixing 1min every 1 hour overnight using Eppendorf Thermomixer. if sample fixed by 1% Glutaraldehyde(irreversible crosslinking), protease K(Ambion, AM2546) s
13、hould be used to digest first at 50 (1,400 rpm mixing 15sec every 30min) in TE/SDS buffer and then 65(1,400 rpm mixing 1min every 1 hour) overnight.Day3: DNA fragment Purification（QIAGEN PCR product purification kit）17. Quickly short centrifugation and put samples on DynaMag-2 Magnet(12321D), and tr
14、ansfer supernatant to a new 2ml tube which contains 1.2ml PB Buffer in advance18. Mix up and down gently by pipettor, then transfer samples to QIAquick spin column19. maxi speed, 1 min20. 750ul PE wash column, maxi speed, 1 min21. Remove PE, maxi speed 1 min to remove residual ethanol completely22. 60ul Elution buffer to elute DNA fragmentq-PCR test before sequencing
