ChIPprotocolBasiconEZ-ChIP?ChromatinImmunoprecipitationKitUpstateCatalog#17-?1、.(25K、5Kfor4h)?2、CrosslinkproteintoDNAbyaddingformaldehydedirectlytoculturemediumtoafinalconcentrationof1%andincubate10minutesatroomtemperature.(add270ul37%formaldehydeinto10mlofgrowthmediumonplate)?.?4、Aspiratemedium,washcellstwiceusing10mlicecoldPBS.?5、Add1mlcoldPBScontainingProteaseInhibitortodish.?6、℃for2-5minutestopelletcells.?7、Removesupernatant.?8、Resuspendeachcellpelletin600ulofSDSLysisBuffer(1%SDS,10mMEDTA,50mMTris,).(Remove5ulofcelllysateforunshearedDNAcontrol).(Lysatecanbefrozenat-80℃atthisstep.)?1、Sonicatechromatintoanaveragelengthofabout500bpwhilekeepingsamplesonice.(60%output,10s/6timesrecall#20program)?2、Microfugeat13,000rpmfor10minutesat4℃.Carefullyremovethesupernatantandtransfertoanewtubein100ulaliquots.(Shearedcrosslinkedchromatincanbestoredat-80°Cforupto2months.)?3、Toallthesamples(unshearedandsheared),(1%SDS,)toafinalvolumeof30ul.?4、Add1ulofRNaseA(10mg/ml)andincubatefor30minutesat37℃.?5、Add1ulProteinaseK(10mg/ml)andincubateat62℃for2hour.?6、L
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