ChIP-on-chip PROTOCOL

ChIP-on-chipPROTOCOLChIP-on-chipProtocol                                             DAY1I.ChromatinImmunoprecipitationProtocolPartA.OptimizationofDNAShearingNote:Steps3-7shouldbedoneonice.Stimulateortreat1x106cellsona10cmdishasappropriate.(Cellsshouldbetreatedunderconditionsforwhichtranscriptionalactivationofthegeneofinteresthasbeendemonstrated).Includeoneextradish(1X106cells)tobeusedsolelyforestimationofcellnumber.*AdherentCells:1.CrosslinkhistonestoDNAbyaddingformaldehydedirectlytoculturemediumtoafinalconcentrationof1%andincubatefor10minutesat37ºC.(Forexample,add270μl37%formaldehydeinto10mLofgrowthmediumonplate).2.Aspiratemedium,removingasmuchmediumaspossible.WashcellstwiceusingicecoldPBScontainingproteaseinhibitors(1mMphenylmethylsulfonylfluoride(PMSF),1µg/mLlaprotininand1µg/mLpepstatinA).3.Scrapecellsintoconicaltube.*Suspensioncells:1.Centrifugethecellsfor4minutesat2000xg@RT.2.CrosslinkhistonestoDNAbyresuspendingcellpelletin200μl1%formaldehydeandincubatefor10minutesat37ºC.3.Centrifugethecellsfor4minutesat2000xg@RT.Aspiratetheformaldehyde,removingasmuchaspossible.WashcellstwiceusingicecoldPBScontainingproteaseinhibitorsasabove.*Tissue:1.Dounceoniceusingahomogenizer,transfertoatubewithascrewcaplidandresuspendinPBS(10mL/30gtissue).2.CrosslinkhistonestoDNAbyresuspendingcellpelletin200μl1%formaldehydeandincubatefor10minutesat37ºC.3.Centrifugethecellsfor4minutesat2000xg@RT.Aspiratetheformaldehyde,removingasmuchaspossible.WashcellstwiceusingicecoldPBScontainingproteaseinhibitorsasabove.4.Pelletcellsfor4minutesat2000xgat4ºC.WarmSDSLysisBuffertoroomtemperaturetodissolveprecipitatedSDSandaddproteaseinhibitors(inhibitors:1mMPMSF,1μg/mLaprotininand1μg/mLlpepstatinA).5.Resuspendcellpelletin200μLofSDSLysisBufferandincubatefor10minutesonice.Note:The200µLofSDSLysisBufferisper1X106cells;ifmorecellsareused,theresuspendedcellpelletshouldbedividedinto200µLaliquotssothateach200µLaliquotcontains~1X106cells.6.SonicatelysatetoshearDNAtolengthsbetween200and1000bpbeingsuretokeepsamplesicecold(Note:Oncesonicationconditionshavebeenoptimizedfollowingsteps1to9,proceedtoPartB,step1below).Varythepowersettingand/orthenumberof10-secondpulsesduringsonicationofthesamples.Besuretokeepthesampleoniceatalltimes(thesonicationgeneratesheatwhichwilldenaturetheDNA).CheckthesizeofsonicatedDNAbygelelectrophoresisafterreversionofcross-links.OurexperienceshowsDNAisshearedtotheappropriatelengthwith3-4setsof10-secondpulsesusingaColeParmer,HighIntensityUltrasonicProcessor/Sonicator,50-wattmodelequippedwitha2mmtipandsetto30%ofmaximumpower.7.Centrifugesamples(partA,step7)for10minutesat13,000rpmat4°C,andadd200μLofthesonicatedcellsupernatanttoanew1.5mLmicrocentrifugetube.Discardpellet.8.Take3µLofsupernatant,runitina1%agarosegeltovisualizeshearingefficiency(200-1000bp).PartB.Experimentalprotocol.1.Dilutethesonicatedcellsupernatant10foldinChIPDilutionBuffer,addingproteaseinhibitorsasabove.Thisisdonebyadding1800μLChIPDilutionBuffertothe200μLsonicatedcellsupernatantforafinalvolumeof2mLineachimmunoprecipitationcondition.Note:Save30μLasWCEandstore@-20°C.ItneedstohavetheHistone-DNAcrosslinksreversedbyheatingat65°Cfor4hours(seePartC,step3.).2.Toreducenonspecificbackground,pre-clearthe2mLdilutedcellsupernatantwith80μLofSalmonSpermDNA/ProteinAAgarose-50%Slurryfor30minutesat4°Cwithagitation.4.Pelletagarosebybriefcentrifugationandcollectthesupernatantfraction.5.Addtheimmunoprecipitatingantibody(theamountwillvaryperantibody)tothe2mLsupernatantfractionandincubateovernightat4°Cwithrotation.DAY26.Add60μLofSalmonSpermDNA/ProteinAAgaroseSlurryforonehourat4ºCwithrotationtocollecttheantibody/histonecomplex.7.Pelletagarosebygentlecentrifugation(700to1000rpmat4ºC,~1min).Carefullyremovethesupernatantthatcontainsunbound,non-specificDNA.WashtheproteinAagarose/antibody/histonecomplexfor3-5minutesonarotatingplatformwith1mlofeachofthebufferslistedintheorderasgivenbelow:a)LowSaltImmuneComplexWashBuffer,onewashb)HighSaltImmuneComplexWashBuffer,onewashc)LiClImmuneComplexWashBuffer,onewashd)TEBuffer,twowashesII.SamplepreparationforCHIP-on-chipPartC.DigestthecellularproteinandRNA.1.Freshlyprepareelutionbuffer(1%SDS,0.1MNaHCO3).2.Elutethehistonecomplexfromtheantibodybyadding250μLelutionbuffertothepelletedproteinAagarose/antibody/histonecomplexfromstep7dabove.Vortexbrieflytomixandincubate@RTfor15minuteswithrotation.Spindownagarose@8000/2min/RT,andcarefullytransferthesupernatantfraction(eluate)toanothertubeandrepeatelution.Combineeluates(totalvolume=~500μL).3.Add20μL5MNaCltothecombinedeluates(500μL);Add1.2μL5MNaCltothe30μLinput/startingmaterialDNAandreversehistone-DNAcrosslinksbyheatingat65ºCfor4hours.Atthisstepthesamplecanbestoredand-20°Candtheprotocolcontinuedthenextday.NOTE:Handlethesamplescarefully,someDNAmaybelostduringthepurificationsteps.DAY34.Add10μLof10mg/mLRNaseA(0.2mg/mLfinalconcentration);asforWCE:add0.6μLRNaseA.Mixandincubateat37ºCfor2hours.5.Add10μLof0.5MEDTA20μL1MTris-HCl,pH6.52μLof10mg/mLProteinaseKtothecombinedeluatesandWCE,thenincubatefor1hourat45ºC.(ForWCEsample,addTEbufferto500μLfirst).6.RecoverDNAbyphenol/chloroformextractionandethanolprecipitation—Add:•400μLphenol:chloroform:isoamylalcoholtoeachtube,mixonavortexmixer,spinat12,000to16,000xgfor5minutes@RT.•Transfertheaqueouslayertoanew1.5mLmicrocentrifugetube.•1.5μLof20μg/μLglycogen(30μgtotal)•880μLEtOHCoolthemixturefor30minutesat-80°C.Spinthemixtureat14,000xgfor15minutesat4°CtocreateDNApellets.Washthepelletswith500μLof70%EtOH.Drythepelletsfor10minuteswithaSpeedVac,andresuspendeachpelletin70μLof10mMTris-HCl,pH8.0.7.Save15μLoftheIPsampleforfuturecheckpointsorverification.8.MeasuretheDNAconcentrationofWCEwithNanoDrop(NanoDropTechnologies)anddilutetheWCEDNAto100ng/μL.DAY4PartD.BlunttheDNAendsandligatethelinkers.UseT4polymerasetoblunttheDNAendsKeepthesampleonicewhileyoudothefirst7steps.1.Put2μL(200ng)WCEDNAintoaPCRtube(0.2to0.5mL)andadd53μLddH2O.SetuponeWCEforeachIPsampleyouhave.2.Put55μLofeachIPsampleintoseparatePCRtubes(0.2to0.5mL)onice.3.Makebluntingmixonice(55μLofmixperreaction):IfyouareusingaMasterMixformultiplesamples,include10%extravolume.Table1.BluntingMixStock1xMixFinalConcentration*10XNEBuffer211.0μL1x10μg/μLBSA(NEB)0.5μL5μg10mMeachdNTP1.1μL100μM3U/μLT4DNApolymerase(NEB)0.5μL1.5UddH2O41.9μLTotal55μL*TheFinalConcentrationisthereagentconcentrationinthefinalreactionandnotthemastermix.4.Add55μLofbluntingmixtoallsamples.5.Coolfor20minutesat12°Cinathermalcycler.6.Placetubesonice.7.Add110μLphenol.Thoroughlymixthesamplebyusingapipettetomovethesampleupanddownonice. 8.Centrifugeat14,000rpmfor5min@4°C.Removetheaqueoussupernatantandtransfertoasterilemicrocentrifugetube.9.Add110μLchloroform:isoamylalcohol.Thoroughlymixthesamplebyusingapipettetomovethesampleupanddownonice. 10.Centrifugeat14,000rpmfor5min@4°C.Removetheaqueoussupernatantandtransfertoasterilemicrocentrifugetube.11.Add11.5μLofcold3Msodiumacetateand0.5μLglycogen(20μg/μL)andmixwell.Keeponice.12.Add500μLEtOHandmixwell.13.Precipitatein-80°Cfreezerfor30min.14.Centrifugeat14,000rpmfor15min@4°C.Carefullyremovethesupernatant,whilenotdisturbingthe pellet.15.Washthepelletwith500μLcold70%EtOH.16.Centrifugeat14,000rpmfor15min@4°C.Carefullyremovethesupernatant,whilenotdisturbingthe pellet.17.Drythepelletsfor10minuteswithavacuumdessicator,suchasaSpeedVac,andresuspendeachpelletin25μLddH2O.Chillonice.Ligatetheblunt-end1.Makeligasemixonice(25μLofmixperreaction):IfyouareusingaMasterMixformultiplesamples,include10%extravolume.Table2.LigaseMixStock1xMixFinalConcentration*10xligasebuffer(Invitrogen)5.0μL1x15μMlinkers**6.7μL2μM400U/μLT4DNAligase(NEB)0.5μL200UddH2O12.8μLTotal25.0μL*TheFinalConcentrationisthereagentconcentrationinthefinalreactionandnotthemastermix.**TopreparelinkersforLM-PCR:i.Mixthefollowing250μL       Tris-HCl(1M)pH7.9375μL       oligoJW102(40μMstock)375μL       oligoJW103(40μMstock)oligoJW1025’-GCGGTGACCCGGGAGATCTGAATTC-3‘oligoJW1035’-GAATTCAGATC-3‘Ordertheseoligosdesiccated,thenresuspendinddH20to40μM.ii.Put100μLofthemixtureintoPCRtubes.iii.Placethetubesinathermalcyclerandrunthisprogram:Step1:95°C5minutesStep2:70°C 1minuteStep3:Rampdownto4°C(0.4°C/min)Step4:4°C HOLDiv.Storethelinkersat-20°C.2.Add25μLofligasemixto25μLofsample.3.Coolfor16hoursin16°Cwaterbathorathermalcycler.DAY54.Add6μLof3Msodiumacetateand130μLEtOH.5.Chillthesamplefor30minutes@-80°C.6.Spinat20,000xgfor10minutes@4°CtopelletDNA.7.Washthepelletswith500μLof70%EtOH.8.Drythepelletsfor10minutesinavacuumdessicator,suchasaSpeedVac,andresuspendeachpelletin25μLddH2O.PartE.AmplifytheIPandWCEsamplesThisprotocolenableslarge-scaleamplificationofIPandWCEsamples.After15cyclesofPCR-basedamplification,thereactionisdilutedandusedastemplateforasecondroundof25cycles.Remainingtemplatecanbestoredlong-termat-20°C.1.Put25μLeachofIPandWCEDNAintoseparatePCRtubes(0.2to0.5mL).2.Maketwobuffermixes:Table3.MixAStock1xMixFinalConcentration*10XThermopolbuffer(NEB)4.00μL1xdNTPmix(2.5mMeach)5.00μL250μMoligoJW102(40μM)1.25μL1μMddH2O4.75μLTotal15.0μL*TheFinalConcentrationisthereagentconcentrationinthefinalreactionandnotthemastermix.Table4.MixBStock1xMixFinalConcentration*10XThermopolbuffer(NEB)1.0μL1xTaqpolymerase(5U/μL)0.5μL2.5UddH2O8.5μLTotal10.0μL*TheFinalConcentrationisthereagentconcentrationinthefinalreactionandnotthemastermix.3.Add15μLofMixAtoeachsample.4.RunanLM-PCRprograminathermocycler:a.Starttheprogrambelow.b.MidwaythroughStep1,pausetheprogram.c.Add10μLMixBtoeachtubetohotstartthereactions.Maintainthetubesat55°CwhileaddingMixB.d.Continuetheprogram.Step1:  55°C  4minutesStep2:  72°C  3minutesStep3:  95°C  2minutesStep4:  95°C  30secondsStep5:  60°C  30secondsStep6:  72°C  1minuteStep7:  GOTOStep4x14timesStep8:  72°C  5minutesStep9:  4°CHOLD5.Transfertheproducttoa1.5mLmicrofugetubeandadd475μLddH20(totalvolumeapproximately525μL).6.Put5μLoftheresultingPCRproductintoaPCRtube(0.2to0.5mL)forasecondexpansion.7.MakethePCRmixture:Table5.PCRMixtureStock1xMixFinalConcentration*10XThermopolbuffer(NEB)5.0μL1xdNTPmix(2.5mMeach)5.0μL250μMoligoJW102(40μM)1.25μL1μMTaqpolymerase(5U/μL)0.25μL1.25UddH2O33.5μLTotal45.0μL*TheFinalConcentrationisthereagentconcentrationinthefinalreactionandnotthemastermix.8.Put45μLofPCRmixtoindividualPCRtubes.9.RuntheLM-PCRprogrambelowinathermocycler:Step1:95°C2minutesStep2:95°C30secondsStep3:60°C30secondsStep4:72°C1minuteStep5:GOTOStep2x24timesStep6:72°C5minutesStep7:4°CHOLD10.Maketheprecipitationmix:Table6.PrecipitationMixStock1xMixFinalConcentration*7.5MAmmoniumacetate25.0μL625mMEtOH225.0μL75%Total250.0μL*TheFinalConcentrationisthereagentconcentrationinthefinalreactionandnotthemastermix.11.Transfertheproducttoa1.5mLmicrofugetube.12.Add250μLprecipitationmixtoeachtube.13.Coolfor30minutesat-80°C.14.Spinat20,000xgfor10minutesat4°CtopelletDNA.15.Washthepelletswith500μLof70%EtOH.16.Drythepelletsfor10minuteswithavacuumdessicator,suchasaSpeedVac,andresuspendeachpelletin50μLddH2O.17.MeasureDNAconcentrationwithNanoDrop(NanoDropTechnologies)(use10-folddilutions,ifnecessary)andnormalizeallsamplesto100ng/μL.DAY6DARK!!!!!!!!PartF.LabeltheIPandWCEInthisstep,youusetherandom-primed,Klenow-basedextensionprotocolthatisusedwithInvitrogen’sCGHLabelingkit.ThisprotocolvariesfromtheinstructionsprovidedbyInvitrogeninbothreactionvolumeandreagentconcentrations(yielding20reactionsper“30reaction”Invitrogenkit,p/n18095-011).Apairofreactions,oneforeachdye,yieldsenoughmaterialfor1to2hybridizations.Toscaleupformorearrays,increasethenumber,notthevolume,ofindividualreactions.1OpentherequirednumberofInvitrogenCGHkitsandconsolidatethestocksof2.5xRandomPrimerSolution,10xdUTPNucleotideMix,Klenow,andStopBuffer.2PuttheLM-PCRproductintoaPCRtube(0.2to0.5mL)andaddrandomprimersolutionandwaterasfollows:Table7.Stock1xMixFinalConcentration*LM-PCRproduct(100ng/μL)20.0μL2μg2.5xRandomPrimersolution35.0μL1xddH2O20.0μLTotal75.0μL*TheFinalConcentrationisthereagentconcentrationinthefinalreactionandnotthemastermix.3.Mixonavortexmixerfor30seconds.4.Placethetubesinathermalcyclerpreheatedto95°Candincubatefor5minutes.5.Immediatelytransferthetubestoanice-waterbathandcoolfor5minutes.6.Whilethereactionsarecooling,createtheLabelingMix.TypicallyCy5mixisusedforIPDNAandCy3forWCEDNA.Table8.LabelingMixStock1xMixFinalConcentration*10xdUTPNucleotideMix8.2μL112/56nMCy5-orCy3-dUTP(1mM)1.5μLKlenow(40U/μL)1.5μL1xddH2O1.8μLTotal13.0μLKeepthereactionsinDARK!!!asmuchaspossibletominimizedegradationofCydyes.7.Mixthemixtureonavortexmixerfor30seconds.8.Put13μLofthelabelmixineachtube.Mixbypipettingupanddownmultipletimes.9.Incubatefor3hoursat37°C.KeepthesamplesintheDARK.10.Add9μLstopbuffertoeachtubeandmix.11.Transfereachsampletoa1.5mLmicrofugetube.12.CleanupthesamplesusingInvitrogen’sCGHcolumnasfollows:a.Add0.4mLofPurificationBufferAtoeachtubeandmixwithavortexmixerfor30seconds.b.PlacethePurificationColumnintoa2mLcollectiontube.c.UseapipettetotransferthesampletothePurificationColumn.d.Spinthecolumninacentrifugeat8,000×gfor1minute@RT.e.Add0.6mLofPurificationBufferBtothecolumn(makesureEthanolhasbeenaddedtoBufferB).f.Spinthesampleinacentrifugeat8,000×gfor1minute@RT.Discardtheflow-throughfromthecollectiontube,andplacethecolumnbackinthetube.g.Add0.2mLofPurificationBufferBtothecolumn.h.Spinthesampleinacentrifugeat8,000×gfor1minute@RT.Discardtheflow-through.i.PlacethePurificationColumninanew,sterile1.5-mLcollectiontube.j.Add50μLofsterilewaterandincubateatroomtemperaturefor1minute.k.Spinthesampleinacentrifugeat8,000×gfor1minute@RT.Theflow-throughcontainsthepurifiedlabeledDNAsample.13.MeasureCylabelandtotalDNAyieldusingtheNanoDrop.Expect>3.5pmol/μLCy3labelincorporation,>2.5pmol/μLCy5labelincorporation,and>5μgtotalDNAperreaction(approximately100ng/μL).II.HybridizationandWashPartG.Hybridizationthemicroarray1.CombineCy5-andCy3-labeledsampleswithddH2Oina1.5mLmicrocentrifugetubeforatotalvolumeasindicatedintable9.Table9.1×244KArrayFormatCy5-labledsamples5μgCy3-labledsamples5μgTotalvolumewithddH2O150μL2.Addthefollowingintheorderindicatedtothemicrocentrifugetube:Table10.1×244KArrayFormatFinalConcentrationHumanCot-1DNA(1.0mg/mL)50μL0.1mg/mLAgilentBlockingAgent(10×)50μL1×AgilentHybridizationBuffer(2×)250μL1×3.Mixcontentsandquickspintocollect.4.Heatsamplesfor3minutes@95°C.5.Immediatelytransferthesampletubestoacirculatingwaterbathorheatblock@37°Candincubatefor30minutes.6.Spinat17,900×gfor1minute@RTtocollectthesample.7.LoadacleangasketslideintotheAgilentSureHybchamberbasewiththelabelfacingupandalignedwiththerectangularsectionofthechamberbase.Ensurethatthegasketslideisflushwiththechamberbaseandisnotmisaligned.8.Slowlydispensehybridizationsampleontothegasketwellina“draganddispense”mannerinthisamount:1x244Kformat490μL9.Placeamicroarray“activeside”downontotheSureHybgasketslide,sothenumericbarcodesideisfacingupandthe“Agilent”barcodeisfacingdown.Verifythatthesandwich-pairisproperlyaligned.10.PlacetheSureHybchambercoverontothesandwichedslidesandslideontheclampassembly.Hand-tightentheclampontothechamber.11.Verticallyrotatetheassembledchambertowetthegasketandassessthemobilityofthebubbles.Taptheassemblyonahardsurfaceifnecessarytomovestationarybubbles.12.Placeassembledslidechamberinrotisseriehybridizationovensetto65°C.Hybridizeat10rpmfor40hours.Youcanuseahigherrotationspeed(upto20rpm)toenhancetheoverallassaysignalintensity.DAY8PartH.WashthemicroarrayTheStabilizationandDryingSolutionmustbeset-upinafumehood.Forpracticalreasons,OligoaCGH/ChIP-on-chipWashBuffers1and2set-upareasshouldbeplacedcloseto,orpreferablyin,thesamefumehood.Glovesandeye/faceprotectionshouldbeusedineverystepofthewarmingprocedure.Table11.WashConditionsDishWashBufferTemperatureTimeDisassembly1OligoaCGH/ChIP-on-chipWashBuffer1RT1stwash2OligoaCGH/ChIP-on-chipWashBuffer1RT5min2ndwash3OligoaCGH/ChIP-on-chipWashbuffer231°C5min3rdwash4AcetonitrileRT1min4thwash5StabilizationandDryingSolutionRT30sec1.Completelyfillslide-stainingdish#1withOligoaCGH/ChIP-on-chipWashBuffer1atroomtemperature.2.Placeasliderackintoslide-stainingdish#2.Addamagneticstirbar.Fillslide-stainingdish#2withenoughOligoaCGH/ChIP-on-chipWashBuffer1atroomtemperaturetocoverthesliderack.Placethisdishonamagneticstirplate.3.Fillslide-stainingdish#3approximatelythree-fourthsfullwithOligoaCGH/ChIP-on-chipWashBuffer2(prewarmedto31°C).Addamagneticstirbarandplacethisdishonamagneticstirplatewithheatingelement.Adjustheatingelementtomaintainwashbuffertemperatureat31°C;monitorusingathermometer.4.Fillslide-stainingdish#4approximatelythree-fourthsfullwithacetonitrile.Addamagneticstirbarandplacethisdishonamagneticstirplate.5.Fillslide-stainingdish#5approximatelythree-fourthsfullwithStabilizationandDryingSolution.Addamagneticstirbarandplacethisdishonamagneticstirplate.6.Removeonehybridizationchamberfromincubatorandrecordtime.Recordwhetherbubblesformedduringhybridization,andifallbubblesarerotatingfreely.7.Preparethehybridizationchamberdisassembly.aPlacethehybridizationchamberassemblyonaflatsurfaceandloosenthethumbscrew,turningcounterclockwise.bSlideofftheclampassemblyandremovethechambercover.cWithglovedfingers,removethemicroarray-gasketsandwichfromthechamberbasebygrabbingtheslidesfromtheirends.Keepthemicroarrayslidenumericbarcodefacingupasyouquicklytransferthesandwichtoslide-stainingdish#1.dWithoutlettinggooftheslides,submergethemicroarray-gasketsandwichintoslide-stainingdish#1containingOligoaCGH/ChIP-on-chipWashBuffer1.8.WiththesandwichcompletelysubmergedinOligoaCGH/ChIP-on-chipWashBuffer1,prythesandwichopenfromthebarcodeendonly:aSliponeofthebluntendsoftheforcepsbetweentheslidesbGentlyturntheforcepsupwardsordownwardstoseparatetheslides.cLetthegasketslidedroptothebottomofthestainingdish.dRemovethemicroarrayslideandplaceintosliderackintheslide-stainingdish#2containingOligoaCGH/ChIP-on-chipWashBuffer1atroomtemperature.Minimizeexposureoftheslidetoair.Touchonlythebarcodeportionofthemicroarrayslideoritsedges!9.Repeatstep6throughstep8foruptofouradditionalslidesinthegroup.Amaximumoffivedisassemblyproceduresyieldingfivemicroarrayslidesisadvisedatonetimeinordertofacilitateuniformwashing.10.Whenallslidesinthegroupareplacedintothesliderackinslide-stainingdish#2,stirusingsetting4(or250to300rpmfordigitalstirplates)for5minutes.11.Transferslideracktoslide-stainingdish#3containingOligoaCGH/ChIP-on-chipWashBuffer2at31°C,andstirusingsetting4(or250to300rpmfordigitalstirplates)for5minutes.12.Transferslideracktoslide-stainingdish#4filledwithAcetonitrile,andstirusingsetting4(250to300rpmfordigitalstirplates)for1minute.13.Immediatelytransfertheslideracktoslide-stainingdish#5containingStabilizationandDryingSolution,andstirusingsetting4(or250to300rpmfordigitalstirplates)for30seconds.14.Slowlyremovetheslideracktryingtominimizedropletsontheslides.Itshouldtake5to10secondstoremovethesliderack.15.DiscardusedOligoaCGH/ChIP-on-chipWashBuffer1andWashBuffer2.CAUTIONTouchonlythebarcodeportionofthemicroarrayslideoritsedges!NOTE:TheacetonitrileandtheStabilizationandDryingSolutionmaybereusedforwashingofuptofourgroupsofslides(thatis,atotalof20microarrayslides).Aftereachuse,rinsethesliderackandtheslide-stainingdishthatwereincontactwiththeStabilizationandDryingSolutionwithacetonitrilefollowedbyarinseinMilli-Qwater.16.Repeatstep1throughstep15forthenextgroupoffiveslidesusingfreshOligoaCGH/ChIP-on-chipWashBuffer1andOligoaCGH/ChIP-on-chipWashBuffer2prewarmedto31°C.17.Scanslidesimmediatelytominimizeimpactofenvironmentaloxidantsonsignalintensities.Ifnecessary,storeslidesinorangeslideboxesinaN2purgebox,inthedark.18.DisposeofacetonitrileandStabilizationandDryingSolutionasaflammablesolvents.