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Split-PoolRecognitionofInteractionsbyTagExtension(SPRITE)forDNA:ExperimentalProtocolsSofiaQuinodozsquinodo@lncrna.caltech.eduMitchellGuttmanLaboratoryLastedited:August9,2018ThisdocumentdescribestheexperimentalproceduresforourSPRITE(Split-PoolRecognitionofInteractionsbyTagExtension)methodtobeusedformappinggenome-widehigherorderinteractionsbetweenDNAmolecules.

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TableofContents:1 Materials

1.1 Equipment1.2 Solutions1.3 AdditionalMaterialsandReagents

2 CellCulture3 AdaptorandBarcodeDesign

3.1 DNAPhosphateModified(DPM)Adaptor3.2 OddandEvenTags3.3 TerminalTag3.4 FinalLibraryAmplification3.5 DPMprimersforQCofDPMligation

4 SamplePreparation

4.1Formaldehyde-DSGCrosslinking4.2CellLysis4.3DNAFragmentation

5 LibraryPreparationPt.1

5.1NHSCoupling5.2EndRepair5.3DPMAdaptorLigation5.4QC:ChecktoDetermineLigationEfficiencyoftheDPMAdaptor

6 SPRITEandLibraryPreparationPt.2

6.1SPRITE6.2LibraryPreparationPt.2

7 SequencingandDataAnalysis

7.1ComputationalPipeline7.2DNAcontactmaps

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1 Materials1.1 SolutionsDSGCrosslinkingSolution1xPBS2mMDSGinDMSOScrapingBuffer1xPBSpH7.50.5%BSAStoreat4°CCellLysisBufferA50mMHepespH7.41mMEDTA1mMEGTA140mMNaCl0.25%Triton-X0.5%NP-4010%GlycerolCellLysisBufferB50mMHepespH7.41.5mMEDTA1.5mMEGTA200mMNaCl

CellLysisBufferC50mMHepespH7.41.5mMEDTA1.5mMEGTA100mMNaCl0.1%DOC0.5%NLS10xSPRITEDNaseBuffer200mMHepespH7.41MNaCl0.5%NP-405mMCaCl225mMMnCl225xDNaseStopSolution250mMEDTA125mMEGTAMyRNKBuffer20mMTrispH7.5100mMNaCl10mMEDTA10mMEGTA0.5%Triton-X

0.2%SDSCouplingBuffer1xPBS0.1%SDSModifiedRLTBuffer1xBufferRLTsuppliedbyQiagen10mMTrispH7.51mMEDTA1mMEGTA0.2%NLS0.1%Triton-X0.1%NP-40SPRITEWashBuffer20mMTrispH7.550mMNaCl0.2%Triton-X0.2%NP-400.2%DOC10XAnnealingBuffer100mMTris-HClpH7.52MLiCl1.5mMEDTA0.5mMEGTA

Note1:ModifiedRLTBuffercontainsguanidinethiocyanatewhichwhenmixedwithbleachproduceshydrogencyanidegasandhydrogenchloridegas.BecarefultoensurethatallliquidModifiedRLTBufferwasteisdisposedofinitsownwastecontainer.SolidsthathavetouchedModifiedRLTBuffersuchastipsandreservoirsshouldalsobediscardedinaseparatesolidModifiedRLTBuffercontainer.Note2:DTThasashorthalf-lifeatpH7.4at20C.ItisimportanttokeepPBLSD+Bufferoniceduringtheprocedureandfrozenat-20Cifnotinuse.

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1.2 EquipmentMicrocentrifugePlateCentrifugeSonicationinstrumentandchillerGelElectrophoresisEquipmentQubitFluorometerEppendorfThermomixerEppendorfSmartBlock1.5mLthermoblockEppendorfSmartBloackPCR96thermoblockMagneticrackfor1.5mLtubes(e.g.InvitrogenDynaMag-2)Magneticrackfor15mLconicaltubesMagneticrackfor96wellplatePCRmachineAgilentBioanalyzer

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1.3 AdditionalMaterialsandReagentsBarcodesandadaptorsLow-retentionpipettetipsLow-bind96-wellplateReservoirsPCRStriptubesProteinLo-bind1.5mLeppendorftubes15mLFalconconicals50mLFalconconicalsTrypsinVersenePhosphateBuffer(TVP),(1mMEDTA,0.025%Trypsin,1%SigmaChickenSerum)PBSSterileDisuccinimidylglutarate(DSG),50mgbottlefromPierce,broughtto0.5MwiththeadditionofDMSO16%FormaldehydeSolutionAmpulesfromPierce2.5MGlycineProteaseCocktailInhibitorTURBODNasefromThermoFisherScientificProteinaseKfromNewEnglandBiolabsRNACleanandConcentrator-5KitwithCappedColumnsfromZymoResearchGelElectrophoresisSystemPierceNHS-ActivatedMagneticBeadsNEBNextEndRepairEnzymeMixfromNewEnglandBiolabsNEBNextEndRepairReactionBufferfromNewEnglandBiolabsKelnowFragment(3’to5’exo-)FromNewEnglandBiolabsNEBNextdA-TailingReactionBufferFromNewEnglandBiolabsInstantSticky-endLigaseMasterMixfromNewEnglandBiolabsNEBNextQuickLigationReactionBufferSigma1,2-Propanediol#398039-25mLQ5HotStartHigh-Fidelity2XMasterMixfromNewEnglandBiolabsAgencourtAMPureXPMagneticBeadsfromBeckmanCoulterQubitdsDNAHSAssayKitHighSensitivityDNAKitfortheAgilentBioanalyzerD1000ScreentapefortheAgilent2200TapeStation

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2 CellCulture

MaterialsandMethodsMouseEScellcultureandXistinductionAllmouseEScelllineswereculturedinserum-free2i/LIFmediumaspreviouslydescribed(7,11,13).FemaleEScells(F12-1line,generouslyprovidedbyK.Plath)areanF1hybridwild-typemouseEScelllinederivedfroma129×CAST(castaneous)cross.Maintenanceof2XchromosomesinthislinewasmonitoredbyXchromosomepaintimaging,restrictionlengthpolymorphismanalysis,aswellasSangersequencingofSNPsontheXchromosome.ThepSM33EScellline(kindlyprovidedbyK.Plath)isamaleEScellline,derivedfromtheV6.5EScellline,expressingthelncRNAXistfromtheendogenouslocusunderthetranscriptionalcontrolofatet-induciblepromoterandtheTettransactivator(SPRITEWASHBUFFERrtTA)fromtheRosa26locus.ToinduceXist,doxycycline(Sigma,D9891)wasaddedtoculturesatafinalconcentrationof2ug/mlfor6-24hrs.HumanlymphoblastcellcultureGM12878cells(CoriellCellRepositories),ahumanlymphoblastoidcellline,wasculturedinRPMI1640(Gibco,LifeTechnologies),2mML-glutamine,15%fetalbovineserum,and1xpenicillin-streptomycinandmaintainedat37°Cunder5%CO2.Cellswereseededevery3-4daysat200,000cells/mlinT25flasksandpassagedorharvestedbeforereaching1,000,000cells/ml.

References

1. R.Galupa,E.Heard,X-chromosomeinactivation:newinsightsintocisandtransregulation.Curr.Opin.Genet.Dev.31,57–66(2015).

2. E.Splinteretal.,TheinactiveXchromosomeadoptsauniquethree-dimensionalconformationthatisdependentonXistRNA.GenesDev.25,1371–1383(2011).

3. S.S.Raoetal.,A3Dmapofthehumangenomeatkilobaseresolutionrevealsprinciplesofchromatinlooping.Cell.159,1665–1680(2014).

4. A.Rego,P.B.Sinclair,W.Tao,I.Kireev,A.S.Belmont,ThefacultativeheterochromatinoftheinactiveXchromosomehasadistinctivecondensedultrastructure.JCellSci.121,1119–1127(2008).

5. A.Wutz,GenesilencinginX-chromosomeinactivation:advancesinunderstandingfacultativeheterochromatinformation.Nat.Rev.Genet.12,542–553(2011).

6. C.M.Clemson,L.L.Hall,M.Byron,J.McNeil,J.B.Lawrence,TheXchromosomeisorganizedintoagene-richouterrimandaninternalcorecontainingsilencednongenicsequences.Proc.Natl.Acad.Sci.U.S.A.103,7688–7693(2006).

7. J.M.Engreitzetal.,TheXistlncRNAexploitsthree-dimensionalgenomearchitecturetospreadacrosstheXchromosome.Science(80-89).341,1237973(2013).

8. M.D.Simonetal.,High-resolutionXistbindingmapsrevealtwo-stepspreadingduringX-chromosomeinactivation.Nature.504,465–469(2013).

9. J.Chaumeil,P.LeBaccon,A.Wutz,E.Heard,AnovelroleforXistRNAintheformationofarepressivenuclearcompartmentintowhichgenesarerecruitedwhensilenced.GenesDev.20,2223–2237(2006).

10. A.Wutz,T.P.Rasmussen,R.Jaenisch,ChromosomalsilencingandlocalizationaremediatedbydifferentdomainsofXistRNA.Nat.Genet.30,167–174(2002).

11. C.A.McHughetal.,TheXistlncRNAinteractsdirectlywithSHARPtosilencetranscriptionthroughHDAC3.Nature.521,232–236(2015).

12. C.Chuetal.,SystematicdiscoveryofXistRNAbindingproteins.Cell.161,404–416(2015).13. C.Chenetal.,XistrecruitstheXchromosometothenuclearlaminatoenablechromosome-widesilencing.Science.

354,468-472(2016).

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3 AdaptorandBarcodeDesign

TheabovefiguredemonstratestheadaptorandtagschemethatiscentraltotheSPRITEprocess.SPRITEusesasplit-and-poolstrategytouniquelybarcodeallmoleculeswithinacrosslinkedcomplexbyrepeatedlysplittingallcomplexesintoa96-wellplate,ligatingaspecifictagsequencewithineachwell,followedbypoolingofthesecomplexessuchthatthefinalproductcontainsaseriesoftagsligatedtoeachmolecule,whichwerefertoasabarcode.3.1 DNAPhosphateModified(DPM)Adaptor5'Phos AAACACCCAAGATCGGAAGAGCGTCGTGTA 3’ Spcr |||||||||||||||||||| 3' TTTTGTGGGTTCTAGCCTTCTGTACTGTTCAGT 5’Phos TheabovedsDNAmoleculeisanexampleofoneofthe96DPMadaptorsusedduringourprocess.The5’endofthemoleculehasamodifiedphosphategroupthatallowsfortheligationbetweenDPMandthetargetDNAmoleculesaswellasthesubsequenttag.ThehighlightedregionsonDPMhavethefollowingfunctions:

1. TheyellowToverhangisasticky-endthatligatestoourtargetDNAmolecules,whicharegivena5’Aoverhangfollowingendrepair.

2. Thepinkregionisthe9-nucleotidesequenceuniquetoeachofthe96DPMadaptors.Theseuniquesequenceshelptoidentifypost-sequencingDNAmoleculesthatareinacomplex.

3. Thegreensequenceisastickyendthatligatestothefirsttag.4. ThegreysequenceiscomplementarytotheFirstPrimerusedforlibrary

amplification.Partofthegreysequencemakesupa3’spacertopreventthetopstrandoftheOddtagfromligating,andonlythebottom5’phosphorylatedstickyendoftheOddtagwillligatetothegreentag.Itspurposeisdiscussedinsection3.4.

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3.2 OddandEvenTagsOddandEventagsaresonamedbecausetheOddtagisligated1st,3rd,5th,etc…duringtheSPRITEprocessandtheEventagisligated2nd,4th,6th,etc…duringSPRITEforhowevermanyroundsoftaggingandpoolingarecompleted.ItisnotnecessarytoligateonlyanevennumberoftagsoronlyanoddnumberoftagssolongastherearetwosetsofTerminaltags;onethatcanligatetoOddtagsandonethatcanligatetoEventags.5'Phos CAAGTCAAGCTAGATTCCACGAAGAGTTGTCACGTCAGCCGCAGTATC 3’ ||||||||||||||||||||||||||||||||||||||||| 3' TCGATCTAAGGTGCTTCTCAACAGTGCAGTCGGCGTCATAGGTTCAGT 5’Phos TheabovedsDNAmoleculeisanOddtagandanEventagligatedtogether.Thefollowingpointsareimportanttonote:

1. The5’overhangonthetopstrandligateseithertotheDPMadaptor(greensequenceinsection3.1)orthe5’overhangonthebottomstrandoftheEventag.

2. BoththeOddtagsandEventagshavemodified5’phosphategroupstoallowfortagelongation.

3. Theboldedregionsofcomplementarityoneachtagarethesequencesuniquetoeachofthe96tags(192total,accountingtoOddtagsandEventags).

3.3 TerminalTagTheterminaltagbelowligatestoOddtags,thoughaterminaltaghasalsobeenmadetoligatetoEventags.Thekeyfeatureoftheterminaltagisthatthereisnomodified5’phosphateonthebottomstrand. 5' Phos AGTTGTCACCATAATAAGATCGGAAGA 3’ |||||||||||||||||||| 3' TGGTATTATTCTAGCCTTCTCGTGTGCAGAC 5’

1. ThegreysequenceiscomplementarytotheSecondPrimerusedforlibraryamplification.

2. HoweversinceDNAcannotbesynthesizedina3’to5’direction,theSecondPrimerannealstoadaughterstrandsynthesizedfromtheFirstPrimer,asexplainedinsection3.4.Thetopstrandisnotprimedbecausethereisabreakinthesequencegeneratedbythe3’spacerontheDPMmoleculeandthereforeprimingthetopstrandoftheterminaltagwouldterminateatthebarcodesandwouldnotPCRthroughtothegDNAsequenceligatedtothebarcodes.

3. TheboldedsequenceontheTerminaltagisuniquetoeachofthe96tags.

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3.4FinalLibraryAmplification

TheDPMadaptorisdesignedwitha3’spacertoaidinfinallibraryamplification.Ifthe3’spacerisabsent,eachstrandwillformahairpinloopduringtheinitialdenaturationduetoreversecomplementarityofthesequencesoneithersideofthetargetDNAmolecule.Instead,the3’spacerallowsthebarcodestoonlyligatetothe5’endofeachsingle-strandedDNAsequence,andnotthe3’end,preventingthesehairpinfromforming.2P_universal(Fprimer)5’ AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 3’ 2P_barcoded_85(Rprimer)5’ CAAGCAGAAGACGGCATACGAGATGCCTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 3’

Duetoreversecomplementarityofthesequences,onlyoneprimeramplifiesthetaggedDNAinthefirstPCRcycle.ThisFirstPrimerannealstoasequenceintheDPMadaptorandextends,synthesizingtwodaughterstrandswithreversesequences.ThisfirstprimerservesastheRead1primerduringIlluminasequencing.Tosynthesizethecomplement,theSecondPrimerannealstothedaughterstrandextendedfromtheFirstPrimerinthesecondPCRcycle.The2P_barcodedprimercontainsan8nucleotidebarcodewithintheprimer.Thisbarcodeisreadfromtheilluminasequencerduringtheindexingprimingstep.ThisbarcodeeffectivelyservesasanadditionalroundoftagadditionduringSPRITE.DilutionofthesampleintomultiplewellsisperformedatthefinalstepofSPRITEpriortoproteinaseKelutionfromNHSbeads.EachdilutionofthesamplepriortoproteinaseKelutionisolatesasubsetofthetaggedcomplexesintodifferentwells.Eachdilutionofcomplexesareamplifiedwithadifferent2P_barcodedprimer.BoththeFirstandSecondprimersarearound30nucleotideseach.Yetthesequencestheyannealtoinitiallyare~20nucleotides.Forthisreason,wesettwodifferentannealingtemperaturesduringthefinallibraryPCR.Thefirstannealingtemperatureisforthefirstfourcyclesuntilenoughcopiesaremadewithfullyextendedprimerregions.Afterthesefourcycles,theannealingtemperatureisraisedforaremainingfivecycles.3.5 DPMprimersforQCofDPMligation TheseprimersareusedtoensurethattheDPMadaptorhasbeensuccessfullyligatedtoDNAofthelysate.Ifnolibrariesareobtainedatthisstepafter14-16cyclesofPCR,westronglyrecommendtonotproceedassubsequentligationoftagsandamplificationoftaggedDNAduringtheSPRITEprotocolwillbeunsuccessful. DPMQCprimerF 5’ TACACGACGCTCTTCCGATCT 3’DPMQCprimerR 5’ TGACTTGTCATGTCTTCCGATCT 3’

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TheForwardandReverseprimersamplifythetopstrandandbottomstrandoftheDPMadaptor,respectively(section3.1).

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4 SamplePreparationGoal: CrosslinkcellstofixinvivoDNAcomplexes.LysecellsandfragmentDNAtoappropriatesizesviasonicationandDNase.Optimizationoflysisconditions(amountofsonication,amount/timingofDNase)isacriticalstepinestablishingtheprotocolforthefirsttime.Thelengthofsonicationmightvaryfrom30secondstoseveralminutesandDNasetreatmentmightvaryfrom10-30%DNaseconcentration,dependingoncellnumber,ploidy,crosslinkingstrength,andthedesiredDNAfragmentsize.TooptimizeDNasetimingandconditions,DNasesampleswithvaryingenzymeconcentrationfor20minutes,quenchwithEDTAandEGTAonice,andassayDNAsizesforconcentrationasdescribedintheprotocol.IfanappropriatecombinationofsolubilizationandDNAfragmentsizescannotbeobtainedbyvaryingtheamountofsonicationorDNase,thenreducingthestrengthofthecrosslinkingmaybenecessary.(1)4.1Formaldehyde-DSGCrosslinking

1. Growadherentcellson15cmplates.Countthecellsononeplatebeforeproceedingwithcrosslinking.Thisprotocoldetailscrosslinkingmultipleplatesofcellsinonesuspension,butitisimportanttomaintainconsistencyinlysatebatches.Wetypicallystore5-10millioncellsperpelletina1.7mLmicrocentrifugetube.

2. Anhourbeforestarting,warmTVPandwashsolutionat37°C.Chillonebottleof1xPBSat4°C,keeponebottleof1xPBSatroomtemperature.Storescrapingbufferat4°C.

3. Liftcellsfromplateandwash:Removemediafromplates.Add5mLTVPto

each15cmplateandrockgentlyfor3-4minutes✔.Afterwards,add25mLwashsolutiontoeachplate.Vigorouslysuspendcellsinthewashsolutionandtransferfromplatetoa50mLconicaltube.Rinsetheplatewithextrawashsolutionandaddtothe50mLconical.Thesamecellsfromdifferentplatescanbepooledintothesameconical.Pelletinacentrifugefor3minutesat3300xGatroomtemperature.Washcellsbyresuspendingin4mLroomtemperature1xPBSper10millioncellsandtransfertoa15mLconical.Pelletagain.

✔ Note:Cellsshouldliftfromtheplatewithinthistime.If5minuteshavepassedandcellshavenotstartedtoliftfromtheplate,quenchthetrypsinimmediatelywithwashsolutiontopreventover-trypsinizationandprematurelysisofcells.Breakcellsofftheplatebywashingvigorouslywithwashsolution.

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4. CrosslinkcellsinDSG:Makea0.5MsolutionofDSGbyadding306uLDMSOtoonebottlecontaining50mgDSG.MakeanappropriateamountofDSGcrosslinkingsolutionbyadding16uLofDSGper4mLofroomtemperaturePBS.ResuspendcellsinDSGcrosslinkingsolution(4mLper10millioncells)✖.Rotategentlyatroomtemperaturefor45minutes.✖CriticalPoint:Itisvitalthatatthebeginningofthecrosslinkingprocessthepelletisuniformlyinsuspension.Toachievethis,completelyresuspendthepelletin1mLofDSGcrosslinkingsolutionusingaP-1000micropipette.Afterthepelletiscompletelydissolved,addtheremainingvolumeofDSGCrosslinkingSolution.IfyouaddthefullvolumeofDSGCrosslinkingSolutionwithoutfirstresuspendingthepellet,itwillbealmostimpossibletocompletelybreakupthepelletandwillresultincellclumpsbeingcrosslinkedtogether.

5. Pelletcellsfor4minutesat1000xGatroomtemperature.Discard

supernatant.

6. Washcellswith4mLroomtemperature1xPBSper10millioncells✖.Pelletasbefore,discardingsupernatant.

✖ CriticalPoint:Asbefore,eachtimeyouresuspendthecellpellet,whethertowashortoputinformaldehydeorscrapingbuffer,ensurethatthepelletiscompletelyresuspendinthesolution.Achievethisbyfirstresuspendingthepelletin1mLoftheappropriatesolutionusingaP-1000micropipette,thenaddingtheremainingvolume.Ifyouforgettodothisandaddthefullvolumewithoutfirstresuspending,vortexthepelletuntilitisfullyresuspended.

7. Resuspendcellpelletin3%formaldehydeinroomtemperaturePBS.Rock

gentlyatroomtemperaturefor10minutes.

8. Add200uLof2.5Mglycinestopsolutionper1mLofcellsuspension.Rockgentlyatroomtemperaturefor5minutes.

9. Pelletcellsat4°Cfor4minutesat1000xG.Discardformaldehyde

supernatantinanappropriatewastecontainer.Fromhere,keepcellsat4°C.

10. Resuspendcellpelletin4°Cscrapingbufferandgentlyrockfor1-2minutes.

11. Pelletcellsat4°Cfor4minutesat1000xG.Discardsupernatantinformaldehydewastecontainer.

12. Resuspendcellpelletincoldscrapingbufferagainandgentlyrockfor1-2

minutes.Pelletasbeforeanddiscardsupernatant.

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13. Resuspendpelletin1mLofscrapingbufferper10millioncells.

14. Aliquot10millioncellseachinto1.7mLmicrocentrifugetubesandpelletat4°Cfor5minutesat2000xG.Removesupernatant.

15. Flashfreezepelletsinliquidnitrogenandstorepelletsat-80C.

(1) Engreitz,Jesse“RNAAntisensePurification(RAP):ExperimentalProtocols”

4.2CellLysis

1. ChillLysisBuffersA,B,andConice.

2. Ifusinganelectronicchillerforthesonicationchamber,pre-chillto4°C.

3. Thawacellpellet(10millioncells)onicefortwominutes.

4. Add700uLofLysisBufferAsupplementedwith1xProteinaseCocktailInhibitor(PIC)toeachpelletandresuspendfully✖.

✖ CriticalPoint:Ensurethatthepelletisfullyresuspendedinthelysisbuffer.Anypelletthatdoesnotresuspendandremainsclumpy,evenaftervigorouspipetting,maybeover-crosslinkedand/ormaybedifficulttoDNase.

5. Incubatemixturesonicefor10minutes.

6. Pelletcellsat4°Cfor8minutesat850xG.

7. Discardthesupernatant,takingcarenottodisturbthepellet.

8. Add700uLofLysisBufferBsupplementedwith1xPICtoeachcellpelletand

resuspendfully.

9. Incubatemixturesonicefor10minutes.

10. Pelletcellsat4°Cfor8minutesat850xG.

11. Discardthesupernatant,takingcarenottodisturbthepellet.

12. Add550uLofLysisBufferCsupplementedwith1xPICtoeach10millionnucleipelletandresuspend.

13. Incubatemixtureonicefor8minutes.

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14. Sonicateeachsampleat4-5wattsfor1minute:1pulsefor0.7secondsON,3.3secondsOFF.Duringandaftersonication,keeplysateat4°C.ABransonneedle-tipsonicatorkeptat4°Cwasusedforthisprotocol.

15. Ifsonicatingmorethanonepelletofthesamecellandconditionsandyoudo

notwishtokeepthemasbiologicalreplicates,poolalllysatestogetherandsplitagaininto10millioncellaliquots.ThisensuresthatallsamplesineachtubeareequallyprocessedandDNasedinthesubsequentsteps.

16. Flashfreezelysateandstoreat-80°C,ormovedirectlyintoDNA

fragmentation.4.3DNAFragmentation

1. Thawonetubeoflysateonice.

2. FragmenttheDNAwithDNase.ToobtainadesiredDNAsizedistribution,performseveralreactionswithvaryingDNaseconcentrations✔.

✔ Note:WetypicallypreformSPRITEonDNAthathasasizedistributionfrom50-1000basepairswithanaveragesizebetween200-300basepairs.Ingeneral,a20%DNasereactionachievesthis.

StockSolution Volume10XSPRITEDNaseBuffer 2uLLysate 10uLTurboDNasefromThermoFisher 3/4/5uLH2O 5/4/3uLTotal 20uLNOTE:DoNOTuse10xTurboDNAseBufferprovidedwiththeEnzyme.Usethe10xSPRITEDNAseBuffercomponentslistedabove(page3).

3. Incubateat37°Cfor20minutes.

4. Add1uLof25XDNaseStopSolutiontoeachsampletoterminatethereaction.

5. Reversethecrosslinksforhalfofeachsample.Flashfreezetheremaininghalf

oftheDNasedsampleandstoreat-80°C✔.

✔ Note:ReversingthecrosslinksononlyhalftheDNasedsampleensuresthatthereisremainingcrosslinkedsamplereadyforSPRITE.ThisnegatestheneedtoDNasetheentiretubeoflysate(10millioncells)whichcouldyieldadifferentsizedistributionthanintended.

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StockSolution VolumeDNasedLysate 10uLMyRNKBuffer 82uLProteinaseK 8uLTotal 100uL

6. Incubateforat65°Cfor2hoursatminimum.

7. CleanupsamplesbyfollowingtheprotocolprovidedintheRNACleanand

Concentrator-5Kit,bindingin6volumesofDNABindingBuffer.Elutein10uLofH2O.

8. DetermineconcentrationofDNAineachsamplebyfollowingdirections

providedwiththeQubitdsDNAHSAssayKit.

9. DeterminethesizedistributionofDNAineachsamplebyfollowingdirectionsprovidedwitheithertheHighSensitivityDNAKitfortheAgilentBioanalyzerortheD1000ScreentapefortheAgilent2200TapeStation.

10. IfnoneoftheseconcentrationsofTURBODNaseledtoidealfragmentation,

adjustconcentrationsandrepeattheDNasinguntiloptimalfragmentationisachieved.DNAforSPRITEgenerallyhasasizedistributionfrom50-1000basepairswithanaveragesizebetween200-300basepairs.

11. OPTIONAL:DNasetheentirebatchofcrosslinkedlysateattheidentified

optimalDNAaseconcentration✔.

✔ Note:DNasingthebatchofcrosslinkedlysateisnotnecessaryforSPRITEifhalfoftheDNasedmaterialwassavedfromstep5intheDNAfragmentationprocess.

StockSolution Volume10XDNaseBuffer 110uLLysate 550uLTurboDNasefromThermoFisher XuLH2O XuLtoreachfinalvolumeTotal 1100uL

a. Incubateat37°Cfor20minutes.b. Add44uLof25xDNaseStopSolutiontoeachsampletoterminatethe

reaction.c. FlashfreezeDNasedlysateandstoreat-80°C.

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5 LibraryPreparationPt.1Goal:LysateiscoupledtoPierceNHS-ActivatedMagneticBeadstoallowforeasyDNAlibrarypreparation.DNAoverhangscausedbyfragmentationarerepairedandbluntedtheNEBNextEndRepairModule,whichcontainsenzymeswith5’to3’polymeraseactivityaswellas3’to5’exonucleaseactivity.Klenowfragment(-exo)isusedtoaddanadeninedNTPto3’endsofeachDNAmolecule.ThisaidsinligationoftheDPMadaptor,whichhasa3’thymineoverhang,withoutcreatingspuriousligationproducts.ItiscrucialtohaveanoptimalbeadtomoleculeratioforthelibrarypreparationandSPRITEprocesses.Weaimtobindata1:4to3:4ratioofDNAmoleculestobeads;generallywebindaround50billionmoleculesto75billionbeads.Assumingthatwehave50%bindingefficiency,couplingratioisthen1:8to1:2.7moleculesperbead.Weassumethattherearefarmoremoleculesthancrosslinkedcomplexesmakingthecomplextobeadratioevenlowerthanstatedabove,butusemoleculesasanover-estimatetoreducenoiseperbead.TodeterminethemicroliteramountoflysatetocouplewecalculatetheDNAmolarityfromtheconcentrationandaveragesizemeasurementsobtainedintheDNAFragmentationstepoftheprotocol.Molarityismultipliedbythelysatevolume(10uLofcrosslinked,DNasedlysateremainsinthetube)toobtainDNAmoleculenumber.5.1 NHSCoupling✔ Note:Allwashstepsat4°Careperformedinacoldroom.AllwashstepsaboveroomtemperatureareperformedonanEppendorfThermomixer.Ifatemperatureisnotspecified,itisatroomtemperature.Towashbeads,placethetubecontainingthebeadsonamagneticracktocapturethebeads.Waituntilthesolutionisclearandallbeadsarecapturedbeforeremovingtheliquid.Addthewashsolutiontothebeadsandremovethetubefromthemagnet.Invertthetubeuntilallbeadsareinsuspension.IfusinganEppendorfThermomixer,setthethermomixertoshakeat1200RPM.Brieflycentrifugethetubetoremovebeadsfromthelid,thenplacethetubebackonthemagnettocapturethebeadsagain.Waituntilthesolutionisclearandallbeadsarecapturedbeforeremovingthewashliquid.Thesestepsarecriticaltoavoidlossofbeadsthroughoutprotocol✔ Note:Theprotocolcanbestoppedatanypointoftheprocess.ToensuretheintegrityoftheDNA,resuspendthebeadsin1mLModifiedRLTBufferandstoreat4°Cuntilyouwishtoresume.WashthreetimeswithSPRITEWashBuffertoremoveallRLTbeforeproceedingwiththeprotocoltopreventenzymedenaturationinsubsequentstepsoftheprotocol.✔ Note:Allstepsinvolvingbeadpipettingshoulduselow-bindpipettetips.

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1. GentlyinvertthebottlecontainingthePierceNHS-activatedbeadsinN,N-

dimethylacetamide(DMAC)untilthereisauniformsuspension.Beingcarefulnottointroducewaterintothebottle,transferXmLofNHSbeadsintoaclean1.7mLlo-bindtube.Placethetubeonamagneticracktocapturethebeads.

2. RemovetheDMACandwashbeadswith1mLice-cold1mMHCl.

3. Washbeadswith1mLice-cold1xPBS.

4. Add1mLCouplingBuffertothebeads.

5. AddXuLofDNasedlysatetothebeads.

6. Incubatethelysateandbeadsovernightat4°Conamixer.

7. Placebeadsonamagnetandremove500uLofflowthrough.OPTIONAL:ThisflowthroughaliquotcanbesavedtodeterminecouplingefficiencyiftheDPMQCfails.

8. Add500uL1MTrispH7.5(3MethanolaminepH9.0canalsobeused)tothe

beadsandincubateonamixerat4°Cforatleast45minutes.ThisensuresthatallNHSbeadswillbequenchedwithproteinfromboundlysateorTris,andwillnotbindenzymesinthefollowingsteps.

9. WashbeadstwiceinModifiedRLTBuffer.

10. WashbeadsthreetimeswithSPRITEWashBuffer.

11. Spinthebeadsdownquicklyinamicrocentrifugeandplacebackonthe

magnettoremoveanyremainingliquid.5.2 PhosphorylationandEndRepair

1. Bluntthe5’and3’endsoftheDNAmoleculestopreventunwantedligationbyaddingthefollowingmixturetothebeads:

StockSolution VolumeH2O 212.5uLEndRepairReactionBuffer(10X) 25uLEndRepairEnzymeMix 12.5uLTotal 250uL

2. Incubateonathermomixerfor60minutesat24°C,1200RPM.

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18

3. WashoncewithModifiedRLTBuffer.

4. WashthreetimeswithSPRITEWashBuffer.

5. Spinthebeadsdownquicklyinamicrocentrifugeandplacebackonthe

magnettoremoveanyremainingliquid.

6. AdddATPtothe3’endsofeachDNAmoleculetoallowforligationoftheDPMadaptorbyaddingthefollowingmixturetothebeads:

StockSolution VolumeH2O 215uLdA-TailingReactionBuffer(10X) 25uLKlenowFragment(exo-) 10uLTotal 250uL

7. Incubateonathermomixerfor60minutesat37°C,1200RPM.Ifligatingthe

DPMadaptorbarcodeonthesameday,setupthereactionduringthisincubation.

8. WashoncewithModifiedRLTBuffer.

9. WashthreetimeswithSPRITEWashBuffer.

10. Spinthebeadsdownbrieflyinamicrocentrifugeandplacebackonthemagnettoremoveanyremainingliquid.

5.3 DPMAdaptorLigation✔ Note:Thereare96adaptorsthataredesignedtoligateontotheDNAmolecules.TheseDPMadaptorsarekeptina96-wellstockplateat4.5uM.TheligationreactionbetweentheadaptorsandtheDNAoccursina96-wellplate.Thefollowingstepsthatdetailsetuparedesignedforoptimumefficiencyduringtheprocess.✔ Note:AllligationstepsincludeSPRITEWashBuffer,whichcontainsdetergentstopreventbeadsfromaggregating,stickingtotheplastictipsandtubes,andforevendistributionofthebeadsacrossa96-wellplate.Wehaveverifiedthatthesedetergentsdonotsignificantlyinhibitligationefficiency.

1. CreateadiluteSPRITEWashBufferbymixing1100uLofSPRITEWashBufferwith792uLofH2O.

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2. Accountingforbeadvolume,addthediluteSPRITEWashBuffertothebeadstoachieveafinalvolumeof1.075mL.Ensurethatthebeadsareequallyresuspendedinthebuffer.Distributethebeadsequallyintoa12-wellstriptubebyaliquoting89.6uLofbeadsintoeachwell.

3. MakeLigationMasterMixforfiveroundsofSPRITE(DPM+fourextratags).Splitthemastermixevenlyintoeachwellofa12-wellstriptubebypipetting260uLintoeachwell.Keeponice✔.StockSolution VolumeNEBNextQuickLigationReactionBuffer(5X)

1600uL

InstantSticky-endLigationMasterMix(2X)

1000uL

1,2-Propanediol 600uLTotal 32000uL

✔ Note:LigationMasterMixcanbestoredovernightat-20°C.

4. CentrifugetheDPMadaptorstockplatebeforeremovingthefoilseal.Aliquot2.4uLfromthestockplateofDPMadaptorstoanewlow-bind96-wellplate✔.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocesstoavoidcross-contaminationofbarcodes.Useanewpipettetipforeachwell.Aftertransferiscomplete,sealbothplateswithanewfoilseal.✔ Note:Thisstepcanbedoneinadvanced,inbulk,sothattheseplatesareready-to-use.

5. Centrifugethe96-wellplatecontainingthealiquotedadaptors,andthen

removethefoilseal.

6. Aliquot11.2uLofbeadsintoeachwellofthe96-wellplatethatcontains2.4uLoftheDPMadaptors.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocess.Useanewpipettetipforeachwell.Alsobecarefultoensurethattherearenobeadsremaininginthepipettetip.

7. Carefullyaddanyremainingbeadstoindividualwellsontheplatein1uL

aliquots.

8. Aliquot6.4uLofLigationMasterMixintoeachwell,mixingbypipettingupanddown10times.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocess.Useanewpipettetipforeachwell.

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20

9. Thefinalreactioncomponentsandvolumesforeachwellshouldbeasfollows:

StockSolution VolumeBeads+SPRITEWashBuffer+H2OMix 11.2uLDPMAdaptor(4.5uM) 2.4uLLigationMasterMix 6.4uLTotal 20uL

10. Sealtheplatewithafoilsealandincubateonathermomixerfor60minutesat

20°C,shakingfor30secondsat1600RPMeveryfiveminutestopreventbeadsfromsettlingtothebottomoftheplate✔.✔ Note:Ligationtimeiscriticalforhighefficiencyofligationeachround.Wehavetestedligationat5,15,30,45,and60minutereactiontimesand60minutesligationtimehassignificantlyhigheryieldsovertheothertimes.

11. Afterincubation,centrifugetheplatebeforeremovingthefoilseal.

12. PourModifiedRLTBufferintoasterileplasticreservoir,andtransfer60uLof

ModifiedRLTBufferintoeachwellofthe96-wellplatetostoptheligationreactions.Itisnotnecessarytousenewtipsforeachwell.

13. Poolall96stoppedligationreactionsintoasecondsterileplasticreservoir.

14. Placea15mLconicaltubeonanappropriatelysizedmagneticrackand

transfertheligationpoolintotheconical.Captureallbeadsonthemagnet,disposingallModifiedRLTBufferinanappropriatewastereceptacle.

15. Removethe15mLconicalcontainingthebeadsfromthemagnetand

resuspendbeadsin1mLSPRITEWashBuffer.Transferthebeadsolutiontoamicrocentrifugetube.

16. WashthreetimeswithSPRITEWashBuffer.

17. Spinthebeadsdownbrieflyinamicrocentrifugeandplacebackonthe

magnettoremoveanyremainingliquid.5.4 QC:ChecktoDetermineLigationEfficiencyoftheDPMAdaptor

1. ResuspendthebeadsinMyRNKBuffersothatthefinalbeads+buffervolumeis1mL.Removea5%aliquot(50uL)intoaseparate1.7mLmicrocentrifugetube.

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21

2. Placetheremaining95%ofbeadsbackonthemagneticrack,removetheMyRNKBuffer,andstorebeadsin1mLofModifiedRLTBuffer.Keepbeadsat4°Covernight.

3. EluteDNAinthe5%aliquotbyreversalofcrosslinksthroughheatingand

ProteinaseK.

StockSolution VolumeSampleonbeadsinMyRNKBuffer 50uLMyRNKBuffer 42uLProteinaseK 8uLTotal 100uL

4. Incubateat65°Cfortwohoursminimum.5. Placethemicrocentrifugetubeonamagnetandcapturethebeads.Remove

theflowthroughthatcontainstheDNAligatedwithDPMadaptorandplaceinaclean1.7mLmicrocentrifugetube.

6. Pipette25uLofH2Ointothetubecontainingthebeads.Vortex,briefly

centrifuge,andre-capturethebeads.Removethe25uLofH2Othatnowcontainsanyresidualnucleicacidandaddtothenewsampletube.Discardthebeads.

7. CleantheDNAbyfollowingtheprotocolprovidedintheRNACleanand

ConcentratorKit.Elutein40uLofH2O.

8. AmplifytheDNAmoleculesthatareligatedtotheadaptors.Theforwardprimershouldprimeoffthe5’endoftheDPMadaptorandthereverseprimershouldprimeoffthe3’endoftheDPMadaptor.

StockSolution VolumeSample(cleaned) 10uLDPMQCForwardPrimer(100uM) 1uLDPMQCReversePrimer(100uM) 1uLH2O 13uLQ5HotStartMasterMix 25uLTotal 50uLPCRProgram:

1. Initialdenaturation:98°C-120seconds2. 12-16cycles:

a. 98°C-10secondsb. 67°C-30secondsc. 72°C-40seconds

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22

3. Finalextension:72°C-120seconds4. Hold4C

9. CleanthePCRreactionandsizeselectforyourtargetDNAmolecules.Our

DPMadaptorsare30basepairseachandourtargetDNAmoleculesnolessthan100basepairs.AgencourtAMPureXPbeadssizeselectwhilecleaningthePCRreactionofunwantedproducts.

a. Add1.0xvolume(50uL)ofAMPureXPbeadstothesampleforatotalvolumeof100uLandmixthoroughly.

b. Incubatefor10minutesatroomtemperature.c. Placethebeadsonanappropriatelysizedmagnettocapturethebeads

andtheboundDNA.Waitafewminutesuntilallthebeadsarecaptured.

d. Removethesupernatantanddiscard.e. Washbeadstwicewith80%ethanolbypipettingethanolintothetube

whilebeadsarecaptured,movingthetubetotheoppositesideofthemagnetsothatbeadspassthroughtheethanol,andthenremovingtheethanolsolution.

f. Quicklyspindownthebeadsinamicrocentrifuge,re-captureonmagnet,andremoveanyremainingethanol.

g. Air-drybeadswhilethetubeisonthemagnet.h. ElutetheamplifiedDNAfromthebeadsbyresuspendingthebeadsin

12uLofH2O.Placethesolutionbackonthemagnettocapturethebeads.RemovetheelutedamplifiedDNAtoacleanmicrocentrifugetube.

10. DetermineconcentrationofDNAwiththeDPMadaptorinbyfollowing

directionsprovidedwiththeQubitdsDNAHSAssayKit.

11. DeterminethesizedistributionofDNAwiththeDPMadaptorineachsamplebyfollowingdirectionsprovidedwitheithertheHighSensitivityDNAKitfortheAgilentBioanalyzerortheD1000ScreentapefortheAgilent2200TapeStation.Theaveragesizeshouldberoughlysimilartotheaveragesizeoftheinputlysate(around200-400basepairs).

12. CalculatethenumberofDNAmoleculesinthe5%aliquotbydetermining

molarityfromtheconcentrationandaveragesize✖.

✖ CriticalPoint:The5%aliquotshouldcontain,attheveryminimum,15millionuniqueDNAmoleculesinordertoproceedwithSPRITE.Ifthealiquotcontainslessthanthisnumber,therewillnotbeenoughuniquereadstosequencetheSPRITElibrary.Ifthisisthecase,troubleshootwhatwentwrongbyassayingcouplingefficiencyfromtheflowthroughsavedinStep7oftheNHSCouplingprotocol.Iflysatewassuccessfullycoupled,considerwhethera

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23

mistakewasmadeduringligationoftheDPMadaptororduringoneofthecriticalstepsofcrosslinkingandlysis.

6 SPRITEandLibraryPreparationPt.2

Goal:TheSPRITEmethodprovideseachDNA-DNAcomplexinthesamplelysatewithauniquenucleicacidbarcode.Whenthesecomplexesaredecrosslinked,theindividualDNAmoleculesthatmadeupasinglecomplexretainidenticalbarcodes.TheseDNAlibrariesaresequencedonanIlluminaNext-Generationsequencingplatformandanalyzed.AnyDNAmoleculesfoundtohavethesamebarcodeinteractin-vivo.TheSPRITEmethodworksbysplittingintoa96-wellplateapooledsampleofcrosslinkedlysatewhereDNAmoleculesareligatedtotheDPMadaptor.Eachwellofthe96-wellplatecontainsauniquetag(Odd)towhichtheDNAmoleculesareligated.Theligationreactionsarestopped,pooled,andsplitagainintoanew96-wellplatecontainingdifferent,uniquetagsthanthefirst(Even).Ifnroundsoftagligationareperformed,96nuniquebarcodesaregenerated.Wetypicallyligate5tags,creatingover8billionuniquebarcodes.Afterallbarcodesareligated,thesampleissplitagainintosmallmaliquots(100wellsof1%aliquotsupto10wellsof10%aliquotsaretypicallyuseddependingonthetotalmaterialcoupled)forPCRamplification.ThisfinalsplittingofsampleseffectuallysortstheDNAcomplexesoncemore,sothatthechancethattwodifferentnon-crosslinkedcomplexeswiththesamebarcodeareamplifiedtogetherisnegligible.Thislastdilutionintomwellseffectivelyraisesthenumberofuniquetagstoeachmoleculetom*96n.Forexample,ifthesampleisaliquotedinto1%aliquots,thenover815billionuniquebarcodesaregenerated.ThefirstroundofSPRITEwasalreadycompletedwiththeligationof96uniqueDPMadaptorsthatallowforthesubsequentligationofnewbarcodes.AsdetailedintheAdaptorandBarcodeCreationsection,subsequenttagligationsareperformedinthefollowingorder:

1. ODDTagLigation2. EVENTagLigation3. ODDTagLigation4. TerminalTagLigation

Thefourbarcodeligationslistedaboveareperformedintheexactsamemannerwiththeonlydifferencebeingthetagsequence.Thus,thefollowingsectionwillonlydetailoneroundofSPRITE.6.1 SPRITE

SPRITEProtocol

24

1. RemoveModifiedRLTBufferfromtheremaining95%aliquotandwashthreetimeswithSPRITEWashBuffer.

2. Spinthebeadsdownbrieflyinamicrocentrifugeandplacebackonthe

magnettoremoveanyremainingliquid.

3. CreateadiluteSPRITEWashBufferbymixing1100uLofSPRITEWashBufferwith792uLofH2O.

4. Accountingforbeadvolume,addthediluteSPRITEWashBuffertothebeadstoachieveafinalvolumeof1.075mL.Ensurethatthebeadsareequallyresuspendedinthebuffer.Distributethebeadsequallyintoa12-wellstriptubebyaliquoting89.6uLofbeadsintoeachwell.

5. Iffrozen,thawthestriptubecontainingtheLigationMasterMixmadeinStep

3oftheDPMAdaptorLigationprotocol.Keeponiceuntilreadytouse.

6. Centrifugethetagstockplatebeforeremovingthefoilseal.Aliquot2.4uLfromthestockplateoftagstoanewlow-bind96-wellplate✔.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocesstoavoidcross-contaminationofbarcodes.Useanewpipettetipforeachwell.Aftertransferiscomplete,sealbothplateswithanewfoilseal.✔ Note:Thisstepcanbedoneinadvanced,inbulk,sothattheseplatesareready-to-use.

7. Centrifugethe96-wellplatecontainingthealiquotedtags,andthenremove

thefoilseal.

8. Aliquot11.2uLofbeadsintoeachwellofthe96-wellplatethatcontains2.4uLofthetags.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocess.Useanewpipettetipforeachwell.Alsobecarefultoensurethattherearenobeadsremaininginthepipettetip.

9. Carefullyaddanyremainingbeadstoindividualwellsontheplatein1uL

aliquots.

10. Aliquot6.4uLofLigationMasterMixintoeachwell,mixingbypipettingupanddown10times.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocess.Useanewpipettetipforeachwell.

11. Thefinalreactioncomponentsandvolumesforeachwellshouldbeas

follows:

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25

StockSolution VolumeBeads+SPRITEWashBuffer+H2OMix 11.2uLTag(4.5uM) 2.4uLLigationMasterMix 6.4uLTotal 20uL

12. Sealtheplatewithafoilsealandincubateonathermomixerfor60minutesat20°C,shakingfor30secondsat1600RPMeveryfiveminutestopreventbeadsfromsettlingtothebottomoftheplate✔.✔ Note:Ligationtimeiscriticalforhighefficiencyofligationeachround.

13. Afterincubation,centrifugetheplatebeforeremovingthefoilseal.

14. PourModifiedRLTBufferintoasterileplasticreservoir,andtransfer60uLof

ModifiedRLTBufferintoeachwellofthe96-wellplatetostoptheligationreactions.Itisnotnecessarytousenewtipsforeachwell.

15. Poolall96stoppedligationreactionsintoasecondsterileplasticreservoir.

16. Placea15mLconicaltubeonanappropriatelysizedmagneticrackand

transfertheligationpoolintotheconical.Captureallbeadsonthemagnet,disposingallModifiedRLTBufferinanappropriatewastereceptacle.

17. Removethe15mLconicalcontainingthebeadsfromthemagnetand

resuspendbeadsin1mLSPRITEWashBuffer.Transferthebeadsolutiontoamicrocentrifugetube.

18. WashthreetimeswithSPRITEWashBuffer.

19. RepeattheprocessstartingatStep6fortheremainingthreeSPRITErounds.

6.2 FinalLibraryPreparation

1. ResuspendthebeadsinMyRNKBuffersothatthefinalbeads+buffervolumeis950uL.

2. Removeeight5%aliquotsintoclean1.7mLmicrocentrifugetubesandelutethebarcodedDNAfromthebeads.Keeptheremaining55%ofthelysateonbeadsinModifiedRLTBufferat4°C.

StockSolution Volume

SPRITEProtocol

26

BeadsinMyRNKBuffer 50uLMyRNKBuffer 42uLProteinaseK 8uLTotal 100uL

3. Incubateat65°Covernight.

4. Placethemicrocentrifugetubesonamagnetandcapturethebeads.Remove

theflowthroughthatcontainsthebarcodedDNAandplaceinacleanmicrocentrifugetube.

5. Pipette25uLofH2Ointothetubecontainingthebeads.Vortex,andre-

capturethebeads.Removethe25uLofH2Othatnowcontainsanyresidualnucleicacidandaddtothenewsampletube.Discardthebeads.

6. FollowtheprotocolprovidedintheRNACleanandConcentratorKit.Elutein

20uLofH2O.

7. AmplifythebarcodedDNA.Refertosection3.4fordetailsaboutthefinallibraryamplificationstep.

StockSolution VolumeBarcodedDNA(cleaned) 20uLFirstPrimer(100uM) 1uLSecondPrimer(100uM) 1uLH2O 3uLQ5HotStartMasterMix 25uLTotal 50uLPCRProgram:

1. Initialdenaturation:98°C-180seconds2. 4cycles:

a. 98°C-10secondsb. 68°C-30secondsc. 72°C-40seconds

3. 7cycles:a. 98°C-10secondsb. 70°C-30secondsc. 72°C-40seconds

4. Finalextension:72°C-180seconds5. Hold4°C

8. CleanthePCRreactionandsizeselectforyourtargetlibraries.Thetotal

lengthofourbarcodeononeamplifiedproductisaround160basepairsandeachtargetDNAmoleculesnolessthan100basepairs.AgencourtAMPureXP

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27

beadsareabletosizeselectwhilecleaningthePCRreactionofunwantedproducts.

a. Add0.7xvolume(35uL)AMPureXPbeadstothesampleforatotalvolumeof85uLandmixthoroughly.

b. Incubatefor10minutesatroomtemperature.c. Placethebeadsonanappropriatelysizedmagnettocapturethebeads

andtheboundDNA.Waitafewminutesuntilallthebeadsarecaptured.

d. Removethesupernatantanddiscard.e. Washbeadstwicewith80%ethanolbypipettingethanolintothetube

whilebeadsarecaptured,movingthetubetotheoppositesideofthemagnetsothatbeadspassthroughtheethanol,andthenremovingtheethanolsolution.

f. Quicklyspindownthebeadsinamicrocentrifuge,re-captureonmagnet,andremoveanyremainingethanol.

g. Air-drybeadswhilethetubeisonthemagnet.h. ElutetheamplifiedDNAfromthebeadsbyresuspendingthebeadsin

50uLofH2O.i. Repeatthesize-selectcleanupwith0.7xAMPureXPbeads(add

directlytotheelutedDNA/beadmix),elutingfinallyin12uLH2O✔.✔ Note:Toensurealllibrarymaterialiselutedfrombeads,elutetwicewith6uLH2O.Mostofthematerialwillberemovedinthefirstelution,andanyremainingmaterialwillberemovedinthesecond.

9. DetermineconcentrationofthebarcodedlibrariesbyfollowingdirectionsprovidedwiththeQubitdsDNAHSAssayKit.Finallibrariesaregenerallybetween0.3ng/uLand1.5ng/uL.

10. DeterminethesizedistributionandaveragesizeofthebarcodedlibrariesbyfollowingdirectionsprovidedwiththeHighSensitivityDNAKitfortheAgilentBioanalyzer.Averagesizesaregenerallyaround350-450basepairs.

11. CalculatethenumberofDNAmoleculesineachbarcodedlibraryby

determiningmolarityfromtheconcentrationandaveragesize.Wetypicallypooltogether300millionuniqueDNAmoleculesforsequencing.

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28

7 SequencingandDataAnalysisThe Illumina, Inc. HiSeq v2500 platform was employed for next generationsequencingofthegeneratedlibrariesusingaTruSeqRapidSBSv1Kit–HS(200cycle)andTruSeqRapidPairedEndClusterKit–HS.

7.1ComputationalpipelineThesequencingdataisoutputastwoFASTQfiles:onefileforreadone(forwardstrand)thatcontains informationabout thegenomicsequence,andone file forread two (reverse strand) that comprises information about the attachedbarcodes. In addition to the sequence information, in this case as well as ingeneral,theFASTQfilesalsocontaininformationaboutthesequencingqualityandtheflowcellpositionforeachread,aswellasauniquenameforeachread.Thisinformationisretainedthroughoutthefollowingprocess.First,thetagsequenceswithinthereadsareidentified.Theprocessstartswithahashtablecontainingallbarcodesusedforthespecificexperiment.Thishashtablemapsnucleotidesequencetobarcodename,allowinguptotwomismatchesfortheEVENandODDtagsequences,butrequiringnomismatchesfortheDPMandtheYshapeadapter.Theenginequeriesthehashtableforsubsequencesofthereads,firstlookingatthebeginningof readone for aDPMsequence, then “walkingdown” read twolookingsequentiallyfortheYshapeadapter,anODDbarcode,anEVENbarcode,andafinalODDbarcode.TheoutputofthisprocessistwoFASTQfilesidenticaltotheinputfiles,butwitheachreadnameadjustedtoadditionallycontainthefiveidentified sequences. (If fewer than five sequences are found, the read namecontainsallof the foundsequences,aswellasa“NOT_FOUND”placeholder foreachbarcodenotfound.)Thefirstelevenbases,whichshouldbetheDPMsequence,aretrimmedfromtheread-onesequences.TheremaindershouldbewhollygenomicDNA,andthis isalignedbyBowtie2(localalignment,defaultscoring,-D15-R2),firsttothemousemm9assembly(nucleotidesequencesofreferencechromosomesandscaffolds),then separately to the human hg19 assembly. Local alignment is chosen overglobalalignmentincasethesequence“readsthrough”thegenomicdataintothebarcodesontheoppositeside.Somesequencesmayaligntobothmm9andhg19,sothetworesultingBAMfilesarecomparedagainsteachother,andanyreadssequences,whichaligntobothassemblies,areremoved.Finally,theBAMfilesarefilteredtokeeponlythosereadsinwhichallbarcodesweresuccessfullyidentified.Ultimately,eachBAMrecordcontainsthefollowinginformation:

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1. Readname2. Locationwithintheflowcell(lane,swath,tile)3. Sequencingquality4. Alignmentcoordinates(chromosomeandnucleotideposition)5. Thesequenceofidentifiedbarcodes6. Thesample identifier (i.emolecule-to-bead ratio tracked indirectly, from

theDPM)7. Alignmentscore(howwellthereadaligned)8. Theidentificationofmismatches/indels

7.2DNAcontactmapsIncaseofdataoriginatingfromDNA-DNAbarcodingwithapooledsample,eachconditionwasspecificallylabeledwithasubsetofDPMadapterbarcodes.Tode-convolutethedifferentindividualsamplesascriptisrunthatidentifiestheDPMadapterbarcodesintheBAMfilesresultingfromthecomputationalpipelineandgeneratesafileforeachDPMadapterbarcodesubset.Thisstepisnotnecessaryin case of RNA-DNA barcoding. Next, all reads within an individual samplepossessing the same barcodes are grouped together and contact frequenciesbetweenbinsofagivensize(forinstance1Mb)arecalculated.Sincethenumberof contacts that eachbinparticipates in isnotuniform,wemust take this intoaccount in order to accurately compare contact frequencies between differentpairsofbins.Todothis,wenormalizethecontactfrequenciesbycalculatingtheexpectednumberofcontactsforeachpairofbinsbasedonauniformdistributionofcontactsandthendividingtheobservednumberofcontactsbytheexpectednumber of contacts. This information is then translated into amatrix (contactfrequencymap)thatcanploteithertheobserved/expectedratiooraZ-scoreofthisratio.Theplotcanbemadeforsinglechromosomeand/oragenome-widecontactfrequencymap.IterativecorrectionandeigenvectordecompositionofHi-CdatacanalsobeperformedtonormalizeDNAcontactsasdescribedbyImakaevetal.NatureMethods(2012).Thesignaltonoiselevelismeasuredbycalculatingthepercentageofobservedover expected contacts containing reads from both specimens (human andmouse)wherebyexpectedisthepercentofhuman-mousecontactsatrandom.