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ChIP实验步骤 - 百度文库
2025-11-16
I.SonicationviaDiagenodesonicatorEnsureDiagenodeChillerisfilledwithdH20.Turnsonicatoronandsetchillerto4C.Resuspend1proteaseinhibitorcocktail(PIC)tabletin1mlofultrapurewatertoobtaina50Xconcentrate.PICisstablefor1daybutcanbeusedupto1weekifsampleisspikedwithphenylmethanesulfonylfluoride(PMSF).A.Nucleipreparation1.Thawcross-linkedcellsonicefor~10min.2.Addnucleilysisbuffer(NLB)tocellstoachieveaconcentrationof~20X106cellsper0.4mlofbuffer,takingintoconsiderationtheinitialvolumeofthecellpellet.3.Add50XPICtoafinalconcentrationof1X(andifneeded200XPMSF).Add1Msodiumbutyratetoafinalconcentrationof10mM.4.Resuspendandbreakupanycellclumpsbygentlypipeting.Incubatefor10minonice.5.Aliquot0.4mlofsampleintoone15mlFalconpolypropylenetubes.B.ChromatinShearing1.CleanDiagenode\"tipprobes”wellwith70%ethanolanddH20beforeandaftereachuse.2.Screwtheprobesintothe15mltubecontainingthesampletosonicate.Ensureprobesarecenteredwithintubesandincubateonicefor~3min.3.Setmetal\"O”ringsinplaceonsonicatormachineandsetsamplesinposition.4.Sonicatetwicefor15min,usingsettingsbelow.Ensurecapshavenotcomelooseaftereachshearinginterval.On=0.5minOff=0.5minSetting=\"H”5.Sonicatedsampleshouldappeartransparentincolor.Samplescanbefurthersonicatedanadditional5-15minifneeded.6.Aliquotsamplesintoa1.7mltube.Centrifugeatmaxspeedfor10minat4C.Transfersupernatanttoalabeledconicaltube,takingcarenottodisturbthepellet.7.Per0.1mlofsonicatedsampleinNLB,addthefollowingbuffersperratiobelow:0.9mlChIPDilutionBuffer(CDB)0.5mlRIPA-15028ul50XPIC7ulof200XPMSF(ifneeded)14ulof1Msodiumbutyrate8.Reserve100ulofsampleforQCpurposesandstoretherestat-80C.Afterchromatinshearingefficiencyischecked,samplescanbestoredas1.4mlaliquotsat-80C.9.Ifneeded,reservea200ulaliquotforinputcontrollibraryuse.Addthefollowingtoinputcontrolandincubatefor4hrsat65C:275ulof1xTEbuffer,30ul5MNaCl,25ulof20%SDSand1ulofRNAseA.Allowsamplestocoolslightlyandadd5ulofProteinseK.IncubateO/Nat55C.Cleanusingphenol/chloroformextractionandphase-locktubes(seebelowforprocedure).EtOHprecipitateandresuspendin100ulofQiagenEB.QuantifyusingQubitHSkit.
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