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1、ChIPprotocolFixationtype:TypeI:1%formaldehyde/PBS(100ml):36.5%formaldehyde(F8775-500ml)2.5mldilutedin1XPBS88.75mlRoomtemperature,15minTypeII:2-5mMDSG(stock50mM:10mgin540ulofdryDMSO),Roomtemperature,45min1%formaldehyde/PBS,Roomtemperature,15minTypeIII:1%Glutaraldehyd
2、esolution(GradeI,25%inH2O,speciallypurifiedforuseasanelectronmicroscopyfixative)sigmaG5882-100ml,Roomtemperature,15minStockbuffer:20%SDS;0.5M,pH8.0EDTA;1M,pH7.8Tris-Cl;10%TritonX100;5MNaCl,Bufferrecipe:LysisBuffer10ml2ml20%SDS0.5ml100ul0.5M,pH8.0EDTA0.2ml50ul1M,pH7.
3、8Tris-Cl0.5ml100ul50Xcocktail(Roche)0.2ml(addfreshly)40ul(addfreshly)ChIPprotocolDoubleDistilledwater8.6ml1720ulDilutionBuffer500ml100ml10%TritonX10050ml10ml0.5M,pH8.0EDTA2ml0.4ml5MNaCl15ml3ml1M,pH7.8Tris-Cl10ml2ml50Xcocktail(Roche)10ml(addfreshly)2ml(addfreshly)Dou
4、bleDistilledwater413ml82.6ml100mlTSE1TSE220%SDS0.5ml0.5ml10%TritonX10010ml10ml0.5M,pH8.0EDTA0.4ml0.4ml5MNaCl3ml8ml1M,pH7.8Tris-Cl2ml2ml50Xcocktail(Roche)1ml(addfreshly)1ml(addfreshly)ChIPprotocolDoubleDistilledwater83.1ml78.1ml1.25MGlycine(MW:75.07):18.77gplus200mld
5、dw5MNaCl(MW58.45):292.2gplus1Lddw,autoclave20%SDS(MW288.38):20gplus100mlddw1M,pH7.8Tris-Cl:121.1gplus1Lddw,autoclaveOption:Drugtreatment：10-7ME2treat1hrDay1:cellfixation,celllysis,sonicationandantibodyincubationCollectingcells1.Afterfixation,coldPBSwashtwice2.1mlPBS
6、toscrapecellsand0.5mlPBSscrapecells3.Transfercellsinto1.5mltubes,maxspeed30sec~1min,centrifugationat4℃4.Ifyouwanttostopatstep3,youcanfreezethesamplesbyliquidnitrogenquickly,thenstoreat-80℃forlongtimestorage.Ifnot,continuetostep5Celllysisandsonication5.Add300ullysisb
7、ufferpluscompletecocktail(avoidcreatingbubbles)6.Sonication（声波降解法）:weusebioruptorforsonication:30sec/30sec,thecyclesofsonicationdependentoncelltype,cellnumbersandcrosslinkingmethods.(forexample:293T,1%formaldehyde,cellsdidnotfreeze;20cycescanobtainacceptableDNAfragm
8、ent)ChIPprotocol1.maxspeed,10~15min4℃toprecipitatecelllysateandaspirate10ulsupernatantforsonicationefficiencytest(100-500bp)2.95℃,10mintod
