Farnham Lab Chromatin Immunoprecipitation (ChIPs) Protocol for Tissues

Day0

BlockStaphAcells(Pansorbin,CalBiochem507862)

1. Thaw1tube(200μL)ofStaphAcellsforapproximately5experimentalChIPs.
2. Add25μLofherringspermDNA(PromegaD1815),10mg/ml);previouslyboiledfor5minutesthenchilledinice.
3. Add25μLofBSA(NEBB9001S;10mg/ml).
4. Incubateontherotatingplatformat4^oCovernight.
5. Microfugefor5minutesat14,000rpmat4^oC.
6. Removesupernatantandwashpellettwicewith1.4mLdialysisbuffer.
7. Resuspendcellsinavolumeofdialysisbufferwithoutsarkosylequaltotheoriginalstartingvolume(200μLfor1tube).AddPMSFata1:100dilution(seebelow).

Day1

Wegenerallyuse30mgoftissueperantibodyineveryChIP.Theexactamountoftissuedependsuponhowabundanttheproteinofinterestis,howstronglytheantibodybindsandhowefficientthecrosslinkingis.

1. Weightfrozenorfreshtissue.
2. Choptissueintosmallpiecesusing2razorblades(between1-3mm3).
3. Transfertissueintoatubewithascrewcaplidandadd10mLPBS(plusproteaseinhibitors)pergramoftissue.
4. Addformaldehydetoafinalconcentrationof1%androtatetubeatroomtemperaturefor15minutes.
5. Stopthecrosslinkingreactionbyaddingfreshglycinetoafinalconcentrationof0.125M.Continuetorotateatroomtempfor5minutes.
6. Centrifugetissuesamplesatlowspeed(100gor707rpm)at4⁰C.
7. AspiratemediaandwashoncewithcoldPBS(plusproteaseinhibitors).Centrifuge.
8. Resuspendtissuein10mLcoldPBS(plusproteaseinhibitors)pergramofstartingmaterial.Putonice.

   TissuedisaggregationForthisstep,weuseaMedimachinefromBectonDickinsontoachieveasinglecellsuspension.Use2medicones(50μm)pergramoftissuetoprocess.

   1. Cuta1000μLpipettetiptomaketheorificelarger.
   2. Putbetween50-100mgoftissueresuspendedin1mLofPBSineachmedicone.
   3. Grindtissuefor2minutes.
   4. Collectcellsfrommediconebyinsertingan18gbluntneedleanda1mLsyringe.Putthemonice.
   5. Keepputtingbetween50-100mgoftissueresuspendedin1mLofPBSineachmediconeuntilallthetissueisprocessed.
   6. Ifnecessary,addmorePBS(plusproteaseinhibitors)totissueforahomogeneoussuspension.
   7. Checkcellsuspensionbymicroscope.
   8. Centrifugecellsat1000rpmat4^oC.Measurecellpelletvolume.

   ChromatinPreparation

   1. Resuspendcellpelletin6XvolumeofcelllysisbufferplusproteaseinhibitorsPMSF(10μL/mL),aprotinin(1μL/mL)andleupeptin(1μL/mL).Thefinalvolumeofcelllysisbuffershouldbesufficientsothattherearenoclumpsofcells.Incubateonicefor10-15minutes.
   2. LysecellsusingaBdounceseveraltimestoaidinnucleirelease.
   3. Centrifugenucleiat1000g(2235rpm)at4^oC.Discardsupernatant.
   4. Resuspendnucleiin5Xvolumeofnucleilysisbufferplusthesameproteaseinhibitorsasthecelllysisbuffer.Incubateonicefor20minutes.
   5. Flashfreezeandthawnucleiinliquidnitrogen2timestoaidinnuclearlysis.
   6. Dividethesamplematerialinasuitablevolumeforsonication.2mLmaximumvolumeina15mLPolystyreneConicaltube(FALCON2099).
   7. Sonicatechromatin.Bioruptormaximumpower.30secondsONfollowedby1minOFF.Totaltime=10minutes.
   8. Chromatinconfirmation:Takeanaliquotequivalentto10mgofstartingtissuematerial(approx.25μL).AddH2Ouptoa100μLvolume.Add10μLNaCl5M.Boilfor15minutes.Add1μLDNase-freeRNase.Incubate30minutesat37^oC.Add1μLProteinaseK.Incubate15minutesat45^oC.Centrifugeatfullspeed.CollectsupernatantandpurifychromatinusingaQIAquickcolumn(Qiagencat#28104).Elutein50μL.MeasureDNAconcentrationbyNanodrop.Run2-3μgofchromatiningel.Sonicatedchromatinshouldhaveanaveragelengthbetween500-1000bp.
   9. Whileperformingstep8,transfersonicatedchromatinfromstep7to2mLtubes.Centrifugeat14,000rpmfor10minutesat4^oC.Collectandcombinesupernatants.Measurevolume.
   10. Aliquotchromatinintubes.Putineachtubetheequivalentto200mgofstartingtissuematerial(approx.500μL).Flashfreezeinliquidnitrogen.Storeat-70^oC.

   Day2

   1. Thawonetubeofchromatininthecoldroomusingtherotatingwheel.
   2. Preclearchromatinbyadding50μLofblockedStaphAcells.
   3. Incubateonarotatingplatformat4^oCfor15minutes,nolonger.
   4. Microfugeat14,000rmpfor10minutes.
   5. Transfersupernatanttoacleantubeandmeasurethevolume.
   6. Dividechromatinequallyamongyoursamples.
   7. BesuretoincludeanIgGsample.Youcanalsoincludea"mock"samplewhichcontains1Xdialysisbufferinsteadofchromatin(IgGandmockarecriticaltocontrolfornonspecificinteractionsandDNAcontaminationofIPandwashsolutions).
   8. Remove10%oftheamountofchromatinusedforoneIP.ThisistheTotalINPUTDNA.Saveitforlater.
   9. AdjustthefinalvolumeofeachsamplewithIPdilutionbufferplusproteaseinhibitorsifnecessary.Samplesvolumesshouldbebetween500and600μL.
   10. Add3μgofantibodytoeachsample.
   11. Incubateontherotatingplatformat4^oCovernight.Ifyouareusingmonoclonalantibodies,thenextdayyoushouldadd3μgofanappropriatesecondaryantibodyandincubateforanadditional1hour.

   Day3

   1. Add20μLofblockedStaphAcellstoeachsample.
   2. Incubateontherotatingplatformatroomtempfor15minutes,nolonger.
   3. Microfugesamplesat14,000rpmat4^oC.
   4. Discardthesupernatantusingapipette.
   5. Washpelletstwicewith1.4mLof1Xdialysisbuffer(ifusingamonoclonalantibody,omitthesarkosyl).
   6. Washfourtimeswith1.4mLofIPbuffer(pH8.0formonoclonalantibodies).
   7. Foreachwash,dissolvethepelletin700μLofbuffer.Usea200pipettetiptoresuspendthepellet.Thenaddtheremaining700μLofbuffer.Foreachwash,incubatesamplesonarotatingplatformfor3minutesthenmicrofugeat14,000rmpfor3minutesat4^oC.TrytoremoveasmuchbufferaspossibleaftereachwashwithoutaspiratingtheStaphAcellsusingapipettetip.
   8. Afterthelastwash,microfugeat14,000rpmfor3minutesat4^oCandremovethelasttracesofbuffer(besuretoorientthepelletsinthemicrofuge).
   9. Eluteantibody/protein/DNAcomplexesbyadding100μLofIPelutionbuffer.
   10. Shakeonvortexerforatleast15minutesatsetting"vortex3".
   11. Microfugeat14,000rpmfor5minutesat4^oC.
   12. Transfersupernatantstocleantubes.
   13. Repeatelutionstepandcombinebothelutionsinthesametube.
   14. Microfugesamplesat14,000rpmfor5minutesat4^oCtoremoveanytracesofStaphAcells.Transfersupernatantstocleantubes.
   15. Add20μLNaCl5Mtoafinalconcentrationof0.45M.
   16. Boilsamplesinthewaterbathfor15minutestoreverseformaldehydecrosslinks.
   17. PurifyDNAinaQIAquickcolumn.

   TotalINPUTDNA

   1. AddH2OtoeachTotalINPUTDNAsampletoafinalvolumeof1mL.
   2. Add100μLNaCl5M.
   3. Boilsamplesinthewaterbathfor15minutestoreverseformaldehydecrosslinks.
   4. Add2.75μLofDNase-freeRNase.Finalconcentrationof25μgenzyme/mL.
   5. Incubate30minutesat37^oC.
   6. Add3μLofProteinaseK.
   7. Incubate15minutesat45^oC.
   8. PurifyDNAinaQIAquickcolumn.

   SolutionsCellLysisbuffer5mMPIPESpH8.085mMKCL0.5%Igepal(addfresh)proteaseinhibitors

   NucleiLysisbuffer50mMTris-ClpH8.110mMEDTA1%SDSproteaseinhibitors

   IPDilutionbuffer0.01%SDS1.1%TritionX1001.2mMEDTA16.7mMTris-ClpH8.1167mMNaCl

   1XDialysisbuffer2mMEDTA50mMTris-ClpH8.00.2%Sarkosyl(omitformonoclonalantibodies)

   IPWashbuffer100mMTris-ClpH9.0(8.0formonoclonalantibodies)500mMLiCl1%Igepal1%deoxycholicacidElutionbuffer50mMNaHCO31%SDS

   ProteaseInhibitors100mMPMSF(SigmaP-7626)inethanol,useat1:10010mgpermlaprotinin(SigmaA-1153)in0.01MHEPESpH8.0,useat1:1,00010mgpermlleupeptin(SigmaL-2884)inwater,useat1:1,000

   StaphACellsResuspend1gramoflyophilizedStaphAcells(Pansorbin,fromCalBiochem#507862)in10mLof1Xdialysisbuffer.Centifugeat10,000rpmfor5minutesat4C.Repeat.Resuspendin3mLof1XPBSplus3%SDSand10%BME.Boilfor30minutes.Centifugeat10,000rpmfor5minutes.Washin1Xdialysisbufferandcentrifugeat10,000rpmfor5minutes.Repeat.Resuspendin4mLof1Xdialysisbuffer.Divideinto200μLaliquots,snapfreezeandstoreinliquidnitrogen.