11%formaldehyde(froma37%stockequilibratedwithmethanol)100mMNaCl1mMEDTA0.5mMEGTA50mMHEPES(pH8)100mMPMSF(phenylmethylsulfonylfluoride)(inisopropanol;stableatroomtemperature)

5mMPIPES(pH8)85mMKCl0.5%NP-401mMPMSFproteinaseinhibitors(leupeptin,aprotinin,pepstatin;eachfinalconcentration2µg/ml)

50mMTris-HCl(pH8)10mMEDTA0.8%SDS(sodiumdodecylsulfate)1mMPMSFproteaseinhibitors(leupeptin,aprotinin,pepstatin;eachfinalconcentration2µg/ml)

10mMTris-HCl(pH8.0)0.5mMEGTA1%TritonX-100140mMNaCl1mMPMSFproteaseinhibitors(leupeptin,aprotinin,pepstatin;eachfinalconcentration2µg/ml)

10mMTris-HCl(pH8.0)1mMEDTA0.5mMEGTA1%TritonX-1000.1%sodiumdeoxycholate0.1%SDS140mMNaCl1mMPMSF

0.25MLiCl0.5%NP-400.5%sodiumdeoxycholate1mMEDTA10mMTris-HCl(pH8.0)

1mMEDTA10mMTris-HCl(pH8.0)

0.25%bromophenolblue0.25%xylenecyanol30%glycerol

12.5mMMgCl₂25mMdithiothreitol(DTT)1.25mMATP50mMTris-HCl(pH7.6)

7%SDS1mMEDTA1%bovineserumalbumin(BSA)0.5MNaHPO₄(pH7.2),thisis0.25MNa₂HPO₄withthepHadjustedto7.2withortho-phosphoricacid(seeOrlandoetal.1997)

5%SDS1mMEDTA0.5%BSA40mMNaHPO₄(pH7.2)

SonicatorHybridizationoven,presetto65°CHeatingblocks,presetto50°C,65°CGlassbeads(150-200µm,acid-washed)Falcontubes,15-mland50-mlShaker,at4°CRotator,at4°COven,presetto80°CvCentrifuge,precooledto4°C

Random-primedDNAsynthesiskitPCRproductpurificationkitDNAquantificationsoftware

1. Grow100mlofDrosophilaSchneiderSL2tissueculturecellsinanappropriatemedium(e.g.,Schneider"sDrosophilamediumsupplementedwith12.5%fetalbovineserumorinserum-freeinsectculturemedium),incellculturebottles,toadensityof3x10⁶to6x10⁶perml.
2. Addthefixationsolution(1/10thofvolumeofcells;e.g.,11mlinto100mlofmedium-thefinalformaldehydeconcentrationshouldbe1%)directlytotheflaskandmix.Incubatefixationreactionfor10minat4°Conashaker.
3. Stopthefixationbyaddingglycinepowdertoafinalconcentrationof125mM.Mixwell.Transfercellstoa50-mlFalcontubeandcollectbycentrifugingat800gfor5minat4°C.Washthecellsoncewithice-coldPBS.
4. Resuspendthecellpelletin15mlofice-coldcelllysisbuffer,pipettingupanddownuntilallthecellshavebeenresuspended.Standthemonicefor10min.Collectthenucleibycentrifugingat2000gfor5minat4°C.Carefullydiscardthesupernatantandresuspendthepelletin2mlofice-coldnuclearlysisbufferbypipettingupanddown.Transferthesuspensiontoa15-mlFalcontubethathasbeencutdowntothe10-mlmarktoallowthesonicatortiptoreachthesuspension.Leaveonicefor10min.
5. Add~0.5mlofglassbeadstothecellsuspension.Storeoniceorsonicateimmediately.
6. Sonicatethesamplewithsix30-secpulses(outputnearmicrotiplimit),usingahigh-powersonicator(e.g.,SanyoSoniprep150,exponentialmicroprobe,10amplitudemicrons).Keepthetubecoolbyholdingitinabeakercontaininganice/watermix.Thesonicatortipshouldbeimmersedroughly1/4intotheliquid.Avoidfoaming.Iffoamingoccurs,centrifugethetubebrieflytoreducethefoamlayer.Leaveoniceforsomeminutesandsonicateagain.Forinitialtrialexperiments,takeanaliquotfromthechromatinsuspensionaftereachsonicationpulse(e.g.,10µl).Increasethesamplevolumeto100µlwithTEandprocessasdescribedinstep8.
7. Transferthesonicatedsuspensiontotwo15-mlFalcontubes(leavingmostoftheglassbeadsbehind)andcentrifugefor10minat12,000-14,000gat4°C.Dilutethesupernatantwithdilutionbuffertoafinalvolumeof8ml(i.e.,4timesdilution).Rotatethetubesonawheelfor10minat4°C.Takea50-µlaliquottochecktheaveragesizeoftheDNAfragments(steps8,9,and10).Fromtheremainingsample,prepare600-mlaliquotsandstoreat-80°C,orusethechromatindirectlyforimmunoprecipitation.
8. Tothe50-µlaliquottakeninstep7,add50µlofTE.Incubateovernightat65°C(ifnotusingsafelock-tubes,sealtubeswithParafilm).AddproteinaseKto500mg/mlandSDSto0.5%(w/v).Incubateat50°Cfor3hr.Centrifugebriefly.
9. Addonevolumeofphenol-chloroform-isoamylalcohol,vortexfor2min,andcentrifugeat12,000-14,000gfor8min.Transfertheaqueoussupernatanttoanewtube.Addonevolumeofchloroform-isoamylalcohol,vortexfor2min,andcentrifugeat12,000-14,000gfor8min.Tothesecondaqueoussupernatant,add1/10volumeof3Msodiumacetate(pH5.2)and2.5volumesof100%ethanolandmixwell.Leaveat-20°Cforatleast30min.Centrifugeat12,000-14,000gfor15minat4°C.Carefullydiscardthesupernatantandwashthepelletin800µlof70%ethanol.Centrifugeagainandallowthepellettoair-dryfor5-10min.Dissolvethepelletin10µlofTE.
10. Addtoeachsample0.5µgofDNase-freeRNase,andincubatefor30minat37°C.Add3µlofgelloadingsolution.Runthesampleona0.8%agarosegel(~15-20cmlongforbestseparation).Whenthebromophenolbluedyehasmigratedalong2/3ofthegel,staingelwith0.5µg/mlethidiumbromideandviewonaUVtransilluminator.IftheaveragelengthoftheDNAisnotshortenough(thereshouldbeasmearofthemolecularweightof~300-1000bp),thestoredaliquotscanberesonicated(step6).

11. Foreachimmunoprecipitation(IP),themockcontrolandtheinputcontrol,take300µlofchromatin(obtainedinstep7)andaddanequalvolumeofRIPAbuffer.Add20µlofproteinA/Gagarosebeadsusingacutoff(wideaperture)pipettetip.Incubatefor1-2hrat4°Cforpreclearing,andcentrifugeinamicrofugeat13,000rpmfor10minat4°C.
12. Transfertheresultingsupernatanttoanewtubeandaddtheappropriateamountofantibody(usually1µgofanaffinity-purifiedantibody;dilutionsof1:100to1:500).Usethesameamountofpreclearedchromatininthecontrols,withouttheadditionofantibody(formockandinputcontrol),orwithpreimmuneserumoranappropriatenonspecificantibody.Fordetailsanddiscussionsonantibodyproduction,affinitypurification,typesofantibodies(polyclonals,monoclonals),andsubclassesofimmunoglobulins,seeHarlowandLane(1988).Incubatethesamplesfrom2-3hrtoovernightat4°Conarotator.
13. Centrifugethesamplesinamicrocentrifugeat13,000gfor10minat4°C.TransfertheIPstonewtubes.Add20µlofthe50%proteinA/Gagarosebeadsolutionandincubateforafurther2-4hr.Pelletthebeadswithashortcentrifugation(20secatmaximumspeed)inabenchtopcentrifuge.Transferthesupernatantoftheno-antibodycontroltoanewtubeandleaveonice.Thismaterialwillserveastotalinputcontrol.Discardtheothersupernatants.Washthebeadsfivetimeswith600µlofRIPAbuffer,oncewith600µlofLiClbuffer,andoncewith600µlofTE(pH8.0),collectingthebeadsbetweenwasheswithbriefcentrifugations.Finally,resuspendthebeadsin100µlofTE.
14. Add1mgofDNase-freeRNase(alsototheinputcontrol)andincubatesamplesovernightat65°C.Thenextday,adjustsamplesto0.5%SDSand0.5mg/mlproteinaseKandincubateforafurther3hrat50°C.Phenol-chloroform-extractthesamplesasdescribedinstep9.Back-extractthephenolphasebyaddinganequalvolumeofTE(pH8.0)andvortex.Combinetheaqueousphasesandperformonemorechloroformextraction.PrecipitatetheDNAbyaddingglycogento100µg/mlascarrier,1/10volumeof3Msodiumacetate(pH5.2),and2.5volumesof100%ethanol.Incubateat-20°Cfor2hrtoovernight.CollecttheDNAbycentrifugingat12,000-14,000gfor15minat4°C,andwashthepelletin800µlof70%ethanol.Repeatcentrifugationanddiscardthesupernatant.Allowthepellettoair-dryfor5-10min.RedissolvetheprecipitatedDNAin30µlofTE(PCRanalysis)or9µlofwater(Southernanalysis)andstoreat4°C(toavoidDNAprecipitation,donotfreeze).

15. 15.Performthetest,negativecontrol,andtheinput-control(dilutionsof1/10,1/100,and1/1000oftheinput)PCRsin25-µlvolumes,usingtheoptimummagnesiumconcentrationforeachprimerpair.Startbyusing1mloftheimmunoprecipitatedDNAasatemplate(in1xreactionbuffer,0.25µMNTPs,1mMprimer,0.5unitsofTaqpolymerase).

    NumberofCycles	Denaturation	Annealing	Polymerization
    1	94°Cfor2min
    35	94°Cfor1min	60-65°Cfor1min	72°Cfor1min
    1			72°Cfor6min

    Adjusttheannealingtemperatureandnumberofcyclesforeachprimerpairuntilnosignalisdetectedforthenegativecontrol-IPDNA.Signalsobtainedfromthetestreactionsundertheseconditionscanbeconsideredsignificant.
16. Aftertheamplification,add6µlofgelloadingsolutiontoeachPCR,loadhalfofthereactionontoa1.5%agarosegel,andvisualizeamplifiedDNAwithethidiumbromide.Toincreasesignalintensities,theamountoftemplateforthePCR(ofallthesamples,includingthenegativecontrols)canbeincreased.
17. Quantifytheresultingbands(e.g.,withtheQuantityOnesoftwarebyBio-Rad)andplotthemaspercentageoftheinput(thetotalofchromatin-DNAusedforoneIP,fromstep13).

18. Preparetheoligonucleotidelinkerbyannealingtwooligonucleotides,a24-merofsequence5´AGAAGCTTGAATTCGAGCAGTCAG-3´,anda20-merofsequence5´CTGCTCGAATTCAAGCTTCT(whenorderingthesyntheticoligonucleotides,takecarethatonlythe24-merisphosphorylatedatthe5´end).MixequimolaramountsoftheseoligonucleotidesinTE(e.g.,10µlofa100µMstockofbotholigonucleotidesin100µlofTE),boilfor5min,andallowthereactiontocoolslowlytoroomtemperature.
19. Resuspendtheimmunopurified-chromatinDNAin9µlofligasebuffercontainingthelinkeradapteratafinalconcentrationof0.8µM.Add4unitsofT4DNAligase(Roche)andincubateat4°Cfor24hr.
20. Usetheligatedmixturedirectlyasatemplateina100-mlPCRusing1unitofTaqpolymerase,1xcorrespondingbuffer,and2mMMg2+.Theprimerusedisthe20-meroligonucleotidedescribedabove,addedtoafinalconcentrationof1mM.Amplificationisperformedasfollows.

    NumberofCycles	Denaturation	Annealing	Polymerization
    1	94°Cfor2min
    35	94°Cfor1min	55°Cfor1min	72°Cfor1min
    1			72°Cfor6min

21. Extractthesamplesoncewithphenol-chloroform-isoamylalcoholandoncewithchloroform-isoamylalcohol;ethanol-precipitateasdescribedinstep9.RemovelinkersbydigestingtheDNAwithHindIIIandseparatethemfromtheamplifiedDNAbygelfiltration(e.g.,withtheQiaQuickPCRpurificationkit,QIAGEN).Analiquotwithlinkersmaybestoredat-20°Casreservoir.
22. LabeltheamplifiedDNAwith[α-³²P]dCTP(specificactivity3000Ci/mmole)usingarandom-primedDNAsynthesiskitaccordingtotheinstructionsofthemanufacturer.
23. SeparatethedigestedtargetDNA(e.g.,bacterialclones,lambdaclones)onanagarosegelandtransfertheDNAtonylonmembranesusingstandardtechniques(SambrookandRussell2001).Bakethemembraneat80°Cfor2hr.Hybridizationisbestperformedinglassbottlesandahybridizationoven.Prehybridizethemembranefor3hrat65°Cin10mlofhybridizationbuffer.Addtheheat-denaturedprobe,fromthepreviousstep,directlytothehybridizationsolution,andincubatethefilterovernightat65°C.
24. Washfiltersoncewithwashbufferfor10minat65°C,andatleastfourtimes,for5mineach,at65°Cinthesamebuffer,butcontaining1%SDS.Afterwashing,sealthefiltersintoplasticbagsandexposetoX-rayfilmor,foramoresensitiveandquantitativeanalysisofthehybridizationsignals,analyzeusingaphosphorimager.

CaoR.,WangL.,WangH.,XiaL.,Erdjument-BromageH.,TempstP.,JonesR.S.,andZhangY.2002.RoleofhistoneH3lysine27methylationinPolycomb-groupsilencing.Science298:1039-1043.

ChuaY.L.,MottE.,BrownA.P.,MacLeanD.,andGrayJ.C.2004.Microarrayanalysisofchromatin-immunoprecipitatedDNAidentifiesspecificregionsoftobaccogenesassociatedwithacetylatedhistones.PlantJ.37:789-800.

HarlowE.andLaneD.1988.Antibodies:Alaboratorymanual.ColdSpringHarborLaboratory,ColdSpringHarbor,NewYork.

OrlandoV.,StruttH.,andParoR.1997.Analysisofchromatinstructurebyinvivoformaldehydecross-linking.Methods11:205-214.

OrlandoV.,JaneE.P.,ChinwallaV.,HarteP.J.,andParoR.1998.BindingoftrithoraxandPolycombproteinstothebithoraxcomplex:DynamicchangesduringearlyDrosophilaembryogenesis.EMBOJ.17:5141-5150.

SambrookJ.andRussellD.2001.Molecularcloning:Alaboratorymanual,3rdedition.ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork.

Strahl-BolsingerS.,HechtA.,LuoK.,andGrunsteinM.1997.SIR2andSIR4interactionsdifferincoreandextendedtelomericheterochromatininyeast.GenesDev.11:83-93.

http://inside.wi.mit.edu/young/pub/locationanalysis.htmlGenome-widelocationanalysis,YoungLab,WhiteheadInstitute

http://genomics.ucdavis.edu/farnham/protocol.htmlProtocols(ChIP-CpGislandmicroarraybindinganalysis;chromatinimmunoprecipitationassay(ChIPs);ChIPsintissues;ChIPscloningprotocol;preparationoftotalRNAfromtissue;preparationoftargetcRNAforAffymetrixGeneChipanalysis)

http://www.fhcrc.org/labs/hahn/methods/mol_bio_meth/hahnlab_ChIP_method.htmlYeastchromatinimmunoprecipitation(ChIP),HahnLab,FredHutchinsonCancerResearchCenter

http://www.igh.cnrs.fr/equip/cavalli/link.labgoodies.htmlProtocols,CavalliLab,InstitutdeGénetiqueHumaine

http://www.protocol-online.org/cgi-bin/prot/search.cgi?query=ChIP&submit2=SearchProtocolOnline,ChIP