Split-Pool Recognition of Interactions by Tag Extension ... · control of a tet-inducible promoter and the Tet transactivator (M2rtTA) from the Rosa26 locus. To induce Xist, doxycycline

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Split-PoolRecognitionofInteractionsbyTagExtension(SPRITE)forDNA:ExperimentalProtocolsSofiaQuinodozsquinodo@lncrna.caltech.eduMitchellGuttmanLaboratoryLastedited:January21,2018ThisdocumentdescribestheexperimentalproceduresforourSPRITE(Split-PoolRecognitionofInteractionsbyTagExtension)methodtobeusedformappinggenome-widehigherorderinteractionsbetweenDNAmolecules.

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TableofContents:1 Materials

1.1 Solutions1.2 Equipment1.3 AdditionalMaterialsandReagents

2 CellCulture3 AdaptorandBarcodeDesign

3.1 DNAPhosphateModified(DPM)Adaptor3.2 OddandEvenTags3.3 TerminalTag3.4 FinalLibraryAmplification3.5 DPMprimersforQCofDPMligation3.6 Adaptorannealingprogram

4 SamplePreparation

4.1 Formaldehyde-DSGCrosslinking4.2 CellLysis4.3 DNAFragmentation

5 SPRITEPreSplit-and-PoolandQCPt.1

5.1 NHSCoupling5.2 PhosphorylationandEndRepair5.3 DPMAdaptorLigation5.4 QC:ChecktoDetermineLigationEfficiencyoftheDPMAdaptor

6 SPRITEandLibraryPreparationPt.2

6.1 SPRITE6.2 LibraryPreparationPt.26.3 EstimatingSequencingDepth

7 SequencingandDataAnalysis

7.1 Tagidentification7.2 Alignment7.3 Filtration7.4 Subsequencepost-processing7.5 QualityControlsofSuccessfulSPRITELibraries

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1 Materials1.1 SolutionsDSGCrosslinkingSolution1XPBS2mMDSGinDMSOScrapingBuffer1xPBSpH7.50.5%BSAStoreat4°CCellLysisBufferA50mMHepespH7.41mMEDTA1mMEGTA140mMNaCl0.25%Triton-X0.5%NP-4010%GlycerolCellLysisBufferB10mMTrispH81.5mMEDTA1.5mMEGTA200mMNaCl10xAnnealingBuffer100mMTris-HClpH7.52MLiCl2mMEDgTA

CellLysisBufferC10mMTrispH81.5mMEDTA1.5mMEGTA100mMNaCl0.1%DOC0.5%NLS10xDNaseBuffer200mMHepespH7.41MNaCl0.5%NP-405mMCaCl225mMMnCl225xDNaseStopSolution250mMEDTA125mMEGTAMyRNKBuffer20mMTrispH7.5100mMNaCl10mMEDTA10mMEGTA0.5%Triton-X0.2%SDS

CouplingBuffer1XPBS0.1%SDSRLT++Buffer1XBufferRLTsuppliedbyQiagen10mMTrispH7.51mMEDTA1mMEGTA0.2%NLS0.1%Triton-X0.1%NP-40M2WashBuffer20mMTrispH7.550mMNaCl0.2%Triton-X0.2%NP-400.2%DOCPBLSD+WashBuffer1XPBS5mMEDTA5mMEGTA5mMDTT(addfresh)0.2%Triton-X0.2%NP-400.2%DOC

Note1:RLT++Buffercontainsguanidinethiocyanatewhichwhenmixedwithbleachproduceshydrogencyanidegasandhydrogenchloridegas.BecarefultoensurethatallliquidRLT++Bufferwasteisdisposedofinitsownwastecontainer.SolidsthathavetouchedRLT++BuffersuchastipsandreservoirsshouldalsobediscardedinaseparatesolidRLT++Buffercontainer.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 AdditionalMaterialsandReagentsBarcodeandadaptorsLow-retentionpipettetipsLow-bind96-wellplateReservoirsPCRStriptubesProteinLo-bind1.5mleppendorftubes15mLFalconconicals50mLFalconconicalsTVPPBSSterileDisuccinimidylglutarate(DSG),50mgbottlefromPierce,broughtto0.5MwiththeadditionofDMSO16%FormaldehydeSolutionAmpulesfromPierce2.5MGlycineProteaseCocktailInhibitorTURBODNasefromThermoFisherScientificProteinaseKfromNewEnglandBiolabsDNACleanandConcentrator-5KitwithCappedColumnsfromZymoResearchGelElectrophoresisSystemPierceNHS-ActivatedMagneticBeads100mMATPfromNewEnglandBiolabsT4PolynucleotideKinasefromNewEnglandBiolabsPolynucleotideKinaseReactionBufferfromNewEnglandBiolabsNEBNextEndRepairEnzymeMixfromNewEnglandBiolabsNEBNextEndRepairReactionBufferfromNewEnglandBiolabsKlenowFragment(3’to5’exo-)FromNewEnglandBiolabsNEBNextdA-TailingReactionBufferFromNewEnglandBiolabsInstantSticky-endLigaseMasterMixfromNewEnglandBiolabsQ5HotStartHigh-Fidelity2XMasterMixfromNewEnglandBiolabsAgencourtAMPureXPMagneticBeadsfromBeckmanCoulter5’DeadenylasefromNewEnglandBiolabsQubitdsDNAHSAssayKitAgilentHighSensitivityDNAKit

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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(M2rtTA)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.The2P_universalprimerand2P_barcodedserveastheRead1andRead2primersforilluminasequencing,respectively.Read1sequencestheDNAmoleculeandtheDPMadaptor.Read2sequencesthemultipletags,ie.uniquebarcode,ligatedtotheDNAmolecules.3.5 DPMprimersforQuality-ControlofDPMligation TheseprimersareusedtoensurethattheDPMadaptorhasbeensuccessfullyligatedtoDNAofthelysate.Ifnolibrariesareobtainedatthisstepafter14-16

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cyclesofPCR,westronglyrecommendtonotproceedassubsequentligationoftagsandamplificationoftaggedDNAduringtheSPRITEprotocolwillbeunsuccessful. DPMQCprimerF 5’ TACACGACGCTCTTCCGATCT 3’DPMQCprimerR 5’ TGACTTGTCATGTCTTCCGATCT 3’TheForwardandReverseprimersamplifythetopstrandandbottomstrandoftheDPMadaptor,respectively(section3.1).3.5 AdaptorannealingprogramThefollowingadaptorsareannealedtomakethetagsdouble-strandedadaptorsfordsDNAadaptorligation:

1. DPMadaptors2. Oddadaptors3. Evenadaptors4. TerminalTagadaptors

Mix the top and bottom strandsof each adaptor into a PCR tube or 96-well platewith10xAnnealingBuffer:

Reagents Volume10xAnnealingBuffer 10ulTopAdaptor(200μM) 45ulBottomAdaptor(200μM) 45ul

Total 100ulIncubatewith the following conditions ina thermocycler foradapterannealing todenature any secondary structure within the top and bottom strands of eachadaptor,thenslowlycooltoannealeachstrand:

Temperature(°C) Time(min) Ramp(°C/s) CycleDenaturation 95 02:00 Annealing 85 00:10 -1 60Hold 25 Infinite

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4 SamplePreparationGoal: CrosslinkcellstofixinvivoRNA-DNA-Proteincomplexeswithdisuccinimidylglutarate(DSG)andformaldehydecrosslinkers.LysecellsandfragmentDNAtoappropriatesizesviasonicationandDNase.Optimizationoflysisconditions(amountofsonication,amount/timingofDNase)isacriticalstepinestablishingtheprotocolforthefirsttime.Thelengthofsonicationmightvaryfrom30sectoseveralminutesandDNasetreatmentmightvaryfrom10to20minutes,dependingoncellnumber,ploidy,crosslinkingstrength,andthedesiredDNAfragmentsize.TooptimizeDNasetimingandconditions,remove5µLlysatealiquotsevery2-4minutes,quenchwithEDTAandEGTAonice,andassayDNAsizesforeachtimepointasdescribedintheprotocol.IfanappropriatecombinationofsolubilizationandDNAfragmentsizescannotbeobtainedbyvaryingtheamountofsonicationorDNase,thenreducingthestrengthofthecrosslinkingmaybenecessary.(1)4.1Formaldehyde-DSGCrosslinking

1. Growadherentcellson15-cmplates.Beforecrosslinking,countoneplate.Thisprotocoldetailscrosslinkingmultipleplatesofcellsinonesuspension,butitisimportanttomaintainconsistencyinlysatebatches.Wetypicallystorecellsin10Mpellets.

2. Anhourbeforestarting,warmTVPandwashsolutionat37C.ChillonebottleofPBSat4C,keeponeatroomtemperature.

3. Liftcellsfromplateandwash:Removemediafromplates.Add5mLTVPto

each15cmplateandrockgentlyfor3-4minutes.Afterwards,add25mLwashsolutiontoeachplate.Vigorouslysuspendcellsinthewashsolutionandtransferfromplatetoa50mLconicaltube.Rinsetheplatewithextrawashsolutionandaddtothe50mLconical.Pelletinacentrifugefor3minutesat3300XGatroomtemperature.Washcellsbyresuspendingin4mLroomtemperature1XPBSper10Mcellsandtransfertoa15mLconical.Pelletagain.

4. ResuspendcellsinDSGCrosslinkingSolution,4mLper10Mcells.Rockgentlyatroomtemperaturefor45minutes.NOTE:Uponadditionofcrosslinkerstothepelletitiscriticaltopipetteupanddownrepeatedlytobreakupanyclumpsofcellstoavoidanycell-cellcrosslinking.

5. Pelletcellsfor4minutesat1000XGatroomtemperature.Discard

supernatant.

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6. Washcellswith4mL1xPBSper10Mcells.Pelletasbefore,discardingsupernatant.

7. Resuspendcellpelletin3%formaldehydeinPBS.Rockgentlyatroom

temperaturefor10minutes.NOTE:Uponadditionofcrosslinkerstothepelletitiscriticaltopipetteupanddownrepeatedlytobreakupanyclumpsofcellstoavoidanycell-cellcrosslinking.

8. Add200uLof2.5Mglycinestopsolutionper1mLofcellsuspension.Rock

gentlyatroomtemperaturefor5minutes.

9. Pelletcellsat4Cfor4minutesat1000XGatroomtemperature.Discardformaldehydesupernatantinanappropriatewastecontainer.Fromhere,keepcellsat4C.

10. ResuspendcellpelletincoldScrapingBufferandgentlyrockfor1-2minutes.

11. Pelletcellsat4Cfor4minutesat1000XG.Discardsupernatantin

formaldehydewastecontainer.

12. ResuspendcellpelletincoldScrapingBufferagainandgentlyrockfor1-2minutes.Pelletasbeforeanddiscardsupernatant.

13. Resuspendpelletin1mLofScrapingBufferper10Mcells.

14. Aliquot10McellseachintoMicrocentrifugetubesandpelletat4Cfor5

minutesat2000XG.Removesupernatant.

15. Flashfreezeinliquidnitrogenandstorepelletat-80C.

(1) Engreitz,Jesse“RNAAntisensePurification(RAP):ExperimentalProtocols”4.2CellLysis

1. ChillLysisBuffersA,B,andConice.

2. Ifusinganelectronicchillerforthesonicationchamber,pre-chillto4C.

3. Thaw10Mcellpelletsonice.

4. Add1.4mLofLysisBufferAsupplementedwith1xProteinaseCocktailInhibitor(PIC)toeach10Mcellpelletandresuspend.

5. Incubatemixturesonicefor10minutes.

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6. Pelletcellsat4Cfor9minutesat850XG.

7. Discardthesupernatant,takingcarenottodisturbthepellet.

8. Add1.4mLofLysisBufferBsupplementedwith1XPICtoeach10Mcellpelletandresuspend.

9. Incubatemixturesonicefor10minutes.

10. Pelletcellsat4Cfor9minutesat850XG.

11. Discardthesupernatant,takingcarenottodisturbthepellet.

12. Add550uLofLysisBufferCsupplementedwith1XPICtoeach10Mnuclei

pelletandresuspend.

13. Incubatemixtureonicefor8minutes.

14. Sonicateeachsampleat4-5wattsfor1minute:1pulsefor0.7secondsON,3.3secondsOFF.Duringandaftersonication,keeplysateat4C.ABransonneedle-tipsonicatorkeptat4Cwasusedforthisprotocol.

15. Poolalllysatestogetherandsplitagaininto10Maliquots.Thisensuresthat

allsamplesineachtubeareequallyprocessedandDNAsedinthesubsequentsteps.

16. Flashfreezelysateandstoreat-80C.

4.3DNAFragmentation

1. Thawonetubeoflysateonice.

2. TodeterminetheoptimalamountofDNasetouseforDNAfragmentation,testvaryingDNaseconcentrationson10uLaliquotsoflysate.

StockSolution Volume10XDNaseBuffer 2uLLysate 10uLTurboDNasefromThermoFisher 2/3/4/5/6uLH20 6/5/4/3/2uLTotal 20uL

3. Incubateat37Cfor20minutes.

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4. Add1uLof25XDNaseStopSolutiontoeachsampletoterminatethe

reaction.

5. Reversethecrosslinksineachsample.StockSolution VolumeLysate 21uLMyRNKBuffer 71uLProteinaseK 8uLTotal 100uL

6. Incubateforat65Cforthreehoursattheminimum,optimallyovernight.

7. FollowtheprotocolprovidedintheDNACleanandConcentrator-5Kit,

bindingin6volumesofDNABindingBuffer.Elutein10uLofH20.

8. RuneachDNasesampleonagelwitha100bpDNAladder.AnidealfragmentationsamplewillhavemostDNAaround200bp.Sizeshouldnotgreatlyexceed1kb,orbeoverlyfragmentedwhereDNAis<100bp.RuntracesthathavenolongtailofDNAfragmentsonaDNAHSBioanalyzerofD1000Tapestation.ExampletraceofDNAsizesobtainedforSPRITE.

9. IfnoneoftheseconcentrationsofTURBODNaseledtoidealfragmentation,adjustconcentrationsandrepeattheDNasinguntiloptimalconditionsarefound.

10. DNasethebatchofcrosslinkedlysateattheidentifiedoptimalDNAase

concentration

StockSolution Volume10XDNaseBuffer 110uLLysate 550uL

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TurboDNasefromThermoFisher XuLH20 XuLtoreachfinalvolumeTotal 1100uL

11. Incubateat37Cfor20minutes.

12. Add44uLof25XDNaseStopSolutiontoeachsampletoterminatethe

reaction.

13. FlashfreezeDNaselysateandstoreat-80C.

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5 SPRITEPreSplit-and-PoolandQCPt.1Goal:LysateiscoupledtoPierceNHS-ActivatedMagneticBeadstoallowforeasyDNAlibrarypreparation.DNAoverhangscausedbyfragmentationarerepairedandbluntedbyacombinationofT4PolynucleoideKinase,whichaddsphosphateonto5’ends,andT4DNAPolymerase,whichhas5’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.Todeterminethemicroliteramountoflysate(DNAand/orRNA)tocouplewecalculatethelysatemolaritybyrunninga5%aliquotontheQubitFluorometertodetermineconcentrationofmoleculesperullysateandtheAgilentBioanalyzertodetermineaveragesize.5.1 NHSCouplingNote1:Allwashstepsat4Careperformedinacoldroom.AllwashstepsaboveroomtemperatureareperformedonanEppendorfThermomixer.Ifatemperatureisnotspecified,itisatroomtemperature.Towashbeads,placethetubecontainingthebeadsonamagneticracktocapturethebeads.Waituntilthesolutionisclearandallbeadsarecapturedbeforeremovingtheliquid.Addthewashsolutiontothebeadsandremovethetubefromthemagnet.Gentlypipettewithalow-bindtiptomixthoroughlyuntilallbeadsareinsuspension.IfusinganEppendorfThermomixer,setthethermomixertoshakeat1200RPM.Thenplacethetubebackonthemagnettocapturethebeadsagain.Waituntilthesolutionisclearandallbeadsarecapturedbeforeremovingthewashliquid.NOTE:ThesestepsarecriticaltoavoidlossofbeadsthroughoutprotocolNote2:Theprotocolcanbestoppedatanypointoftheprocess.ToensuretheintegrityoftheDNA,resuspendthebeadsin1mLRLT++andstoreat4Cuntilyouwishtoresume.WashthreetimeswithM2BuffertoremoveallRLTbeforeproceedingwiththeprotocoltopreventdenaturatingenzymesinsubsequentstepsoftheprotocol.Note3:Allstepsinvolvingbeadpipettingshoulduselow-bindpipettetips.

1. GentlyinvertthebottlecontainingthePierceNHS-activatedbeadsinN,N-dimethylacetamide(DMAC)untilthereisauniformsuspension.Beingcareful

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nottointroducewaterintothebottle,transfer2mLofNHSbeadsintoaclean1.7mLtube.Placethetubeonamagneticracktocapturethebeads.

2. RemovetheDMACandwashbeadswith1mLice-cold1mMHCl.

3. Washbeadswith1mLice-cold1XPBS.

4. Add1mLCouplingBuffertothebeads.Beforemixing,addtheappropriateamountoflysatetothecouplingbuffer.

5. Incubatethelysateandbeadsovernightat4Conamixer.

6. Placebeadsonamagnetandremovea500uLflowthroughaliquottoanother

tube.Thisaliquotcanbeanalyzedtodeterminehowmuchlysatewascoupled.

7. Add500uL1MTrispH7.5(3MethanolaminepH9.0canalsobeused),tothe

beadsandincubateonamixerat4Cforatleast45minutes.ThisensuresthatallNHSbeadswillbequenchedwithproteinfromboundlysateorTris,andwillnotbindenzymesinthefollowingsteps.

8. WashbeadsfourtimesincoldRLT++Bufferat4Cfor3-5minuteseachtime.

9. WashbeadstwiceinPBLSD+WashBufferat50Cfor4-5minuteseachtime.

10. WashbeadsonceatroomtemperatureinPBLSD+buffer.

11. WashbeadsthreetimeswithM2Buffer.

12. Spinthebeadsdownquicklyinamicrocentrifugeandplacebackonthe

magnettoremoveanyremainingliquid.5.2 PhosphorylationandEndRepair

1. Phosphorylatethe5’endsoftheDNAmoleculestoallowsubsequentbarcodeligationbyaddingthefollowingmixturetothebeads:StockSolution VolumeH20 167.5uLT4PolynucleotideKinaseReactionBuffer(10X)

20uL

T4PolynucleotideKinase 10uL100mMATP 2.5uLTotal 200uL

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2. Incubateonathermomixerfor60minutesat37C,1200RPM.

3. WashbeadsthreetimeswithM2Buffer.


magnettoremoveanyremainingliquid.

5. Bluntthe5’and3’endsoftheDNAmoleculestopreventunwantedligationbyaddingthefollowingmixturetothebeads:

StockSolution VolumeH20 212.5uLEndRepairReactionBuffer(10X) 25uLEndRepairEnzymeMix 12.5uLTotal 250uL

6. Incubateonathermomixerfor60minutesat24C,1200RPM.

7. WashoncewithRLT++Buffer.

8. WashthreetimeswithM2Buffer.


magnettoremoveanyremainingliquid.

10. AdddATPtothe3’endsofeachDNAmoleculetoallowforligationoftheDPMadaptorbyaddingthefollowingmixturetothebeads:

StockSolution VolumeH20 215uLdA-TailingReactionBuffer(10X) 25uLKlenowFragment(exo-) 10uLTotal 250uL

11. Incubateonathermomixerfor60minutesat37C,1200RPM.Ifligatingthe

firstadaptorbarcodeonthesameday,setupthereactionduringthisincubation.

12. WashoncewithRLT++Buffer.


14. Spinthebeadsdownquicklyinamicrocentrifugeandplacebackonthemagnettoremoveanyremainingliquid.

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5.3 DPMAdaptorLigationNote:Thereare96adaptorsthataredesignedtoligateontotheDNAmolecules.TheseDPMadaptorsarekeptina96-wellstockplateat45uM.TheligationreactionbetweentheadaptorsandtheDNAoccursina96-wellplate.Thefollowingstepsthatdetailsetuparedesignedforoptimumefficiencyduringtheprocess.Note:AllligationstepsincludeM2buffer,whichcontainsdetergents,topreventbeadsfromaggregationofmultiplebeads,fromstickingtotheplastictipsandtubes,andforevendistributionofthebeadsacrossa96-wellplate.Wehaveverifiedthatthesedetergentsdonotsignificantlyinhibitligationefficiency.

1. Aliquot200uLof2xNEBInstantStickyEndLigaseMasterMix(NOTE:wehavefoundthatotherconcentrationsfrom0.1x-1xfinalmaybeusedbutmighthavedifferencesinligationefficiency)intoeachwellofa12-wellstriptube.Keeponiceuntilreadytouse.

2. CentrifugetheDPMadaptorstockplatebeforeremovingthefoilseal.Aliquot2.4uLfromthestockplateofDPMadaptorstoanewlow-bind96-wellplate.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocesstoavoidcross-contaminationofbarcodes.Useanewpipettetipforeachwell.Aftertransferiscomplete,sealbothplateswithanewfoilseal.

3. CreateadilutedM2Bufferbymixing1100uLofM2Bufferwith792uLofH20.

4. Accountingforbeadvolume,addtheM2+H20mixtothebeadstoachievea

finalvolumeof1700uL.Ensurethatthebeadsareequallysuspendedinthebuffer.

5. Aliquot140uLofthebeadmixintoeachwellofa12-wellstriptube.

6. Centrifugethe96-wellplatecontainingthealiquotedadaptors,andthen

removethefoilseal.

7. Aliquot17.6uLofbeadsintoeachwellofthe96-wellplatethatcontains2.4uLoftheDPMadaptors.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocess.Useanewpipettetipforeachwell.Alsobecarefultoensurethattherearenobeadsremaininginthepipettetip.

8. Carefullyaddanyremainingbeadstoindividualwellsontheplatein1uL

aliquots.

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21

9. Aliquot20uLofInstantStickyEndLigaseMasterMixintoeachwell,mixingbypipettingupanddown10times.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocess.Useanewpipettetipforeachwell.

10. Thefinalreactioncomponentsandvolumesforeachwellshouldbeas

follows:

StockSolution VolumeBeads+M2+H20Mix 17.6uLDPMAdaptor(45uM) 2.4uL2XInstantStickyEndLigationMasterMix 20uLTotal 40uL

11. Sealtheplatewithafoilsealandincubateonathermomixerfor60minutes

at20C,shakingfor15secondsat1600RPMeveryminutetopreventbeadsfromsettlingtothebottomoftheplate.NOTE:ligationtimeiscriticalforhighefficiencyofligationeachround.

12. Afterincubation,centrifugetheplatebeforeremovingthefoilseal.

13. PourRLT++Bufferintoasterileplasticreservoir,andtransfer100uLof

RLT++intoeachwellonthe96-wellplatetostoptheligationreactions.Itisnotnecessarytousenewtipsforeachwell.

14. Poolall96stoppedligationreactionsintoasecondsterileplasticreservoir.

15. Placea15mLconicaltubeonanappropriatelysizedmagneticrackand

transferthepoolintotheconical.Captureallbeadsonthemagnet,disposingallRLT++inanappropriatewastereceptacle.

16. Removethe15mLconicalcontainingthebeadsfromthemagnetand

resuspendbeadsin1mLPBLSD+WashBuffer.Transferthebeadsolutiontoamicrocentrifugetube.

17. WashthreetimeswithPBLSD+WashBufferat50C,1200RPMfor3minutes

eachtime.


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5.4 QualityControl(QC):ChecktoDetermineLigationEfficiencyoftheDPMAdaptor

1. ResuspendthebeadsinMyRNKBuffersothatthefinalbeads+buffervolumeis1mL.Removea5%aliquot(50uL)intoaseparatemicrocentrifugetube.

2. Placetheremaining95%ofbeadsbackonthemagneticrack,removetheMyRNKBuffer,andstorebeadsin1mLofRLT++Buffer.Keepbeadsat4Covernight.

3. RemovetheDNA+DPMadaptormoleculesfromthebeadsandreversethe

crosslinksinthelysate.ProteinaseKwilldegradetheproteincovalentlylinkingthetheproteinsinthecrosslinkedlysatetotheNHSgrouponthebeads,releasingthelysatefromthebeads.ProteinaseKandheatwillfurtherdegradeanyproteininthelysateandremovecrosslinks,leavingnucleicacidinthesample.

StockSolution VolumeSampleonbeadsinMyRNKBuffer 50uLMyRNKBuffer 42uLProteinaseK 8uLTotal 100uL

4. Incubateat65Covernight.(Timecanbereducedto1-4hrsifneeded)5. Placethemicrocentrifugetubeonamagnetandcapturethebeads.Remove

theflowthroughthatcontainstheDNAligatedwithDPMadaptorandplaceinacleanmicrocentrifugetube.

6. Pipette25uLofH20intothetubecontainingthebeads.Vortex,andre-

capturethebeads.Removethe25uLofH20thatnowcontainsanyresidualnucleicacidandaddtothenewsampletube.Discardthebeads.



8. AmplifytheDNAmoleculesthatareligatedtotheadaptors.Theforwardprimershouldprimeoffthe5’endoftheDPMadaptorandthereverseprimershouldprimeoffthe3’endoftheDPMadaptor.Beforeplacingthereactioninthethermocycler,splitthesampleintotwotubeswith50uLineachtube.

StockSolution VolumeSample(cleaned) 10uLDPMQCForwardPrimer(100uM) 2uL

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DPMQCReversePrimer(100uM) 2uLH20 36uLQ5HotStartMasterMix 50uLTotal 100uLPCRProgram:

1. Initialdenaturation:98C-180seconds2. 12-16cycles:

a. 98C-10secondsb. 68C-30secondsc. 72C-60seconds

3. Finalextension:72C-180seconds4. Hold4C

9. CleanthePCRreactionandsizeselectforyourtargetDNAmolecules.Our

DPMadaptorsare30basepairseachandourtargetDNAmoleculesnolessthan100basepairs.AgencourtAMPureXPbeadssizeselectwhilecleaningthePCRreactionofunwantedproducts.

a. Combinethetwo50uLPCRreactionsbackintoonetube.b. Add1.0XAMPureXPbeadstothesampleforatotalvolumeof200uL

andmixthoroughly.c. Incubatefor10minutesatroomtemperature,mixingagainat5

minutes.d. Placethebeadsonanappropriatelysizedmagnettocapturethe

beadsandtheboundDNA.Waitafewminutesuntilallthebeadsarecaptured.

e. Removethesupernatantanddiscard.f. Washbeadstwicewith70%ethanolbypipettingethanolintothetube

whilebeadsarecaptured,movingthetubetotheoppositesideofthemagnetsothatbeadspassthroughtheethanol,andthenremovingtheethanolsolution.

g. Quicklyspindownthebeadsinamicrocentrifuge,re-captureonmagnet,andremoveanyremainingethanol.

h. Air-drybeadswhilethetubeisonthemagnet.i. ElutetheamplifiedDNAfromthebeadsbyresuspendingthebeadsin

12uLofH20.Placethesolutionbackonthemagnettocapturethebeads.RemovetheelutedamplifiedDNAtoacleanmicrocentrifugetube.

10. Runsamplesonagelwitha100bpDNAladdertoconfirmthatDNA

moleculeshaveligatedtotheDPMadaptors.Ifyouhaveenoughsamples,itmaybeworththecosttorunsamplesontheAgilentBioAnalyzertobetterascertainthelibrarydistributionanddetermineaveragepeaksizetocalculatemolarityandmoleculenumber.Beforesamplesarerunonthe

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24

BioAnalyzer,theirconcentrationisdeterminedfromtheQubitFluorometer.Nomorethan5ngofDNAisloadedintoeachwellontheBioAnalyzerchip.FollowinstructionsprovidedwiththeHighSensitivitydsDNAkit.

11. TheDPMamplifiedlibrariesshouldreflectasimilarsizedistribution(~200-400bpaveragesizeafterDPMadaptorligation)thatwasobservedduringtheDNAsedigestionstep.IfaclearlibraryisnotvisibleonDNAHSBioanalyzerorD1000HSafter14-16cycles,itisnotadvisedtoproceed,astheSPRITEsplit-and-poolbarcodingwillbeperformedontoolittlematerial.ExampleDPMlibraryafter12cycleson5%oftheligatedmaterialonaDNAHSBioanalyzer.

ExampleDPMlibraryafter16cycleson5%oftheligatedmaterialon2%agarosegel.

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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

1. Aliquot200uLofInstantStickyEndLigaseMasterMixintoeachwellofa12-wellstriptube.Keeponiceuntilreadytouse.

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26

2. Centrifugethetagstockplatebeforeremovingthefoilseal.Aliquot2.4uLfromthestockplateofbarcodestoanewlow-bind96-wellplate.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocess.Useanewpipettetipforeachwell.Aftertransferiscomplete,sealbothplateswithanewfoilseal.

3. CreateadilutedM2Bufferbymixing1100uLofM2Bufferwith792uLofH20.

4. Accountingforbeadvolume,addtheM2+H20mixtothebeadstoachievea

finalvolumeof1700uL.Ensurethatthebeadsareequallysuspendedinthebuffer.

5. Aliquot140uLofthebeadmixintoeachwellofa12-wellstriptube.

6. Centrifugethe96-wellplatecontainingthealiquotedbarcodes,andthen

removethefoilseal.

7. Aliquot17.6uLofbeadsintoeachwellofthe96-wellplatethatcontains2.4uLofthetags.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocess.Useanewpipettetipforeachwell.Alsobecarefultoensurethattherearenobeadsremaininginthepipettetip.

8. Carefullyaddanyremainingbeadstoindividualwellsontheplatein1uL

aliquots.

9. Aliquot20uLofInstantStickyEndLigaseMasterMixintoeachwell,mixingbypipettingupanddown10times.Becarefultoensurethatthereisnomixingbetweenwellsatanypointoftheprocess.Useanewpipettetipforeachwell.

10. Thefinalreactioncomponentsandvolumesforeachwellshouldbeas

follows:

StockSolution VolumeBeads+M2+H20Mix 17.6uLTag(45uM) 2.4uL2XInstantStickyEndLigationMasterMix

20uL

Total 40uL

11. Sealtheplatewithafoilsealandincubateonathermomixerfor60minutes

at20C,shakingfor15secondsat1600RPMevery5minutes.

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27

12. Afterincubation,centrifugetheplatebeforeremovingthefoilseal.

13. PourRLT++Bufferintoasterileplasticreservoir,andtransfer100uLofRLT++intoeachwellonthe96-wellplatetostoptheligationreactions.Itisnotnecessarytousenewtipsforeachwell.

14. Poolall96stoppedligationreactionsintoasecondsterileplasticreservoir.

15. Placea15mLconicaltubeonanappropriatelysizedmagneticrackand

transferthepoolintotheconical.Captureallbeadsonthemagnet,disposingallRLT++inanappropriatewastereceptacle.

16. Removethe15mLconicalcontainingthebeadsfromthemagnetand

resuspendbeadsin1mLPBLSD+WashBuffer.Transferthebeadsolutiontoamicrocentrifugetube.

17. WashthreetimeswithPBLSD+WashBufferat50C,1200RPMfor3minutes

eachtime.


19. RepeattheprocessstartingatStep1fortheremainingthreeormoreSPRITErounds.

6.2 LibraryPreparationPt.2

1. ResuspendthebeadsinMyRNKBuffersothatthefinalbeads+buffervolumeis1mL.

2. Removefivealiquotsintocleanmicrocentrifugetubes:0.5%,1%,2.5%,5%,and7.5%(5uL,10uL,25uL,50uL,and75uL)andelutethebarcodedDNAfromthebeads.

StockSolution VolumeSampleonbeadsinMyRNKBuffer 5/10/25/50/75uLMyRNKBuffer 87/82/67/42/17uLProteinaseK 8uLTotal 100uL

3. Incubateat65Covernight.

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4. Placethemicrocentrifugetubesonamagnetandcapturethebeads.RemovetheflowthroughthatcontainsthebarcodedDNAandplaceinacleanmicrocentrifugetube.

5. Pipette25uLofH20intothetubecontainingthebeads.Vortex,andre-

capturethebeads.Removethe25uLofH20thatnowcontainsanyresidualnucleicacidandaddtothenewsampletube.Discardthebeads.



7. AmplifythefinalbarcodedDNAthroughPCR.Refertosection3.4fordtailsaboutthefinallibraryamplificationstep.Beforeplacingthereactioninthethermocycler,splitthesampleinintotwotubeswith50uLineachtube.

StockSolution VolumeSample(cleaned) 40uLFirstPrimer(100uM) 2uLSecondPrimer(100uM) 2uLH20 6uLQ5HotStartMasterMix 50uLTotal 100uLPCRProgram:

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

a. 98C-10secondsb. FirstAnnealingTemperature-30secondsc. 72C-90seconds

3. 5cycles:a. 98C-10secondsb. SecondAnnealingTemperature-30secondsc. 72C-90seconds

4. Finalextension:72C-180seconds5. Hold4C

8. CleanthePCRreactionandsizeselectforyourtargetlibraries.Thetotal

lengthofourbarcodeononeamplifiedproductisaround160basepairsandeachtargetDNAmoleculesnolessthan100basepairs.AgencourtAMPureXPbeadsareabletosizeselectwhilecleaningthePCRreactionofunwantedproducts.

a. Combinethetwo50uLPCRreactionsbackintoonetube.b. Add0.7XAMPureXPbeadstothesampleforatotalvolumeof170uL

andmixthoroughly.

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29

c. Incubatefor10minutesatroomtemperature,mixingagainat5minutes.

d. PlacethebeadsonanappropriatelysizedmagnettocapturethebeadsandtheboundDNA.Waitafewminutesuntilallthebeadsarecaptured.

e. Removethesupernatantanddiscard.f. Washbeadstwicewith70%ethanolbypipettingethanolintothetube

whilebeadsarecaptured,movingthetubetotheoppositesideofthemagnetsothatbeadspassthroughtheethanol,andthenremovingtheethanolsolution.

g. Quicklyspindownthebeadsinamicrocentrifuge,re-captureonmagnet,andremoveanyremainingethanol.

h. Air-drybeadswhilethetubeisonthemagnet.i. ElutetheamplifiedDNAfromthebeadsbyresuspendingthebeadsin

100uLofH20.Placethesolutionbackonthemagnettocapturethebeads.RemovetheelutedamplifiedDNAtoacleanmicrocentrifugetube.

j. Repeatthecleanupwith0.7XAMPureXPbeads,elutingfinallyin12uL.

9. DeterminetheconcentrationofeachlibrarywiththeQubitFluorometer.Ourfinallibrariesaregenerallybetween0.5ng/uLand1.5ng/uL.

10. LoadallsamplesontheAgilentBioAnalyzer,followingtheprotocolprovidedwithAgilent’sHighSensitivitydsDNAKit.Finallibrarysizesrangefromaround260basepairsto1000basepairswithpeaksaround400basepairs.

11. UsingtheconcentrationsgatheredfromQubitandtheaveragelibrarysize

gatheredfromtheBioAnalyzer,estimatethenumberofDNAmoleculesineachlibrary.Thesenumbersareusedtodeterminethedepthtosequenceeachlibrary(see6.3).

a. FormulatocalculateLibraryConcentrationnMpost-pcr:i. (Concentrationng/ul)/((649*103*AverageSizebp))*109

b. Formulatocalculatemoleculespost-PCR:i. (10-15*LibconcentrationnM)*(Vol.eluteduL)*(6.022*1023)

c. Formulatocalculatemoleculespre-pcr:i. NOTE:Thishasbeenadjustedtoaccountforlossduring2roundsof0.7xSPRIanddependsonthenumberofcyclesPCRamplified.

ii. (Moleculespost-PCR)/(2(cycles+2))

6.3 Estimatingsequencingdepth

SPRITEinteractionsaredefinedbasedonthesequencesthatsharethesametags.Accordingly,itisessentialtosequenceallofthebarcodedmoleculesinacomplexin

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30

ordertoidentifyinteractionsinasample.Therefore,thenumberofuniquemoleculesthataresequenceddramaticallyaffectsthelikelihoodofidentifyinginteractingmolecules.Toaddressthis,weoptimizedtheloadingdensityofoursequencingsamplebasedonthenumberofuniquemoleculescontainedinthesample.Ourgoalistoloadapproximatelyequimolaruniquemoleculesasthenumberofsequencingreadsgenerated.Specifically,basedonoursimulationsofPoissonsampling,wehavefoundthatsequencingwith~1-3xcoverageofreadsperthenumberofuniquemoleculeswillensurethatmostmoleculesaresampled.ThisfollowsPoissonsamplingwhere1-1/ecofmoleculesaresampledatagivenccoverage.Forexample,3x,2x,and1xcoveragesamplesapproximately95%,86%,and63%ofinteractions,respectively.Inthisstudy,mostlibrariesweresampledwithapproximately1.5-2xcoverage.Todeterminethenumberofuniquemoleculesinoursample,wemeasuretheamountofmaterialpresentonbeadspriortoreversecrosslinkingallinteractions.Todothis,wetakeanaliquotofthesampleandreversecrosslinktoelute(asabove),cleanupDNA,andPCRamplifyfor9-12cycles.WethenmeasurethemolarityusingtheQubitandBioanalyzer(asabove).ThenumberofuniquemoleculespriortoPCRisbackcalculatedfromastandardcurveandadjustedtoaccountforlossduringthecleanup.Specifically,wecalculatethenumberofmoleculespre-pcr(seeabove).ThisisusedtoestimatethenumberofuniquemoleculesinthesamplepriortoPCR.Forexample,ifweestimatethereare70Muniquemoleculespre-pcrinalibrary,wewillsamplethatlibrarywith105-140Mreads.Ifthenumberofmoleculespre-pcrexceedsthenumberofmoleculesthatcanbesampledonasequencer(~300-400M)youmusttakeasmalleraliquotandProKanaliquotwithasmalleramountofmaterialoryouwillnotdetectallinteractionsinasampleandmanymoleculeswillbenon-interacting.Inadditiontooptimizingmolarity,becausethisdilutionresultsinapproximately1%aliquotsofthetotalsamplebeingseparatelyelutedandamplified,thiseffectivelyservesasanotherroundofsplit-poolbarcodingaseachlibraryistaggedwithauniquebarcodedIlluminaprimer.Thisfurtherreducestheprobabilitythatmoleculesindifferentclustersobtainthesamebarcodes.

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7 SequencingandDataAnalysisThe Illumina, Inc. HiSeq v2500 platform was employed for next generationsequencing of the generated librariesusing aTruSeqRapid SBS v1Kit –HS (200cycle)andTruSeqRapidPairedEndClusterKit–HS.AllSPRITEdatainthispaperwasgeneratedusingIlluminapaired-endsequencing.Readsmustbelongenoughtoincorporate all tag information.Most read-pairs in this reportwere (115 bp, 100bp).All code for the SPRITE computational pipeline is found here:https://github.com/GuttmanLab/sprite-pipeline/wiki7.1TagidentificationThisstepisperformedusingcustomin-housesoftware.TheprogramtakesasinputbothFASTQfiles,sortedbynamesothattherecordwithaparticularlinenumberintheread1filecorrespondswiththerecordwiththesamelinenumberintheread2file.Theprogramalsorequiresatextfilecontainingthetagsequenceswithuniqueidentifiers and an identification tolerance -- the number of mismatches toleratedbetweenthetagandthereadwhensearchforthetag.The program first loads the tags from the tag file and stores them in a hashtablekeyed by sequence. Storing these sequences in a hashtable allows rapid (O(1))stringmatching.Additionaltagsaregeneratedaccordingtothegivenidentificationtolerances, and these are also stored. For example, if the tag TTTT has anidentification tolerance of 1, the tag will be inserted into the table, keyed by allsequencesatmostoneHammingdistanceaway:TTTT ATTT TATT TTAT TTTA CTTT TCTT TTCT TTTC GTTT TGTT TTGT TTTG NTTT TNTT TTNT TTTN After storing the tags, the program iterates through the read-pairs by advancingline-by-line through both FASTQ files simultaneously. For a given sequence, the

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program queries the hash table for substrings that correspond to known tagpositions.(Theexactdetailsofthisprocessdependonthebarcodingscheme.)Afterthe identification process for a record is complete, the tags are appended to thenameoftherecord,andthismodifiedrecordisoutputintonewread1andread2FASTQfiles.7.2AlignmentIn our barcoding schemes, only one of the reads in a read pair contains anappreciableamountof genomic sequence.Thesegenomic-readsarealigned to theappropriatereferencewithBowtie2underthedefaultparameters—exceptforthefollowing.Onlyoneof thetwoFASTQfiles isaligned.Wedonotrunapaired-endalignmentdespitehavingpaired-endreads.Before the genomic sequence on the read is an 11-mer DPM tag sequence. Toaccountforthis,werunBowtie2with`--trim511`.After the sequence, there are two possibilities. The readmay extend into the tagsequencesontheotherendofthefragmentifthefragmentistooshort,orthereadmay terminate before the tags if the fragment is long enough. To account for theinclusionoftagsequences,werunBowtie2with`--local`.(ThiswouldalsodealwiththeDPMtagatthestartofthesequence.Wealigntoboththereferencechromosomesandunplacedscaffolds(typicallyendin“random”).WesorttheresultingSAMfileandconvertittoaBAMfile.ThenamesofeachSAMrecordcontaintheidentifiedtags,asthesewerepresentintheinputFASTQfiles.7.3FiltrationTheBAMfileisthenpassedthroughsuccessivefiltrationsteps:i. Remove all alignments with a MAPQ score less than 30. This removes all

unmappedreads.NotethattheMAPQscoredependsonthealignerused;itisnot standardized. If a different aligner is used, this step will need to bereplacedwithadifferentquality-filtrationstep.

ii. RemoveallalignmentsthataligntothereferencewithaHammingscore>2.We only tolerate two mismatches at most between the read and thereference.

iii. Removeallalignmentsthatoverlap(inanyamount)anyregionintherepeat-maskBEDfileprovidedtousbyB.Tabak.Weusedbedtools intersectwiththe`-v`flagset.

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iv. Removeallalignmentsthatoverlap(inanyamount)anyregioninthemaskBEDfilegeneratedbyComputeGenomeMask intheGATKpackage fromtheBroad.Thismaskfilewasgeneratedbyshreddingthereferenceinto35-mersandBLATtingthemagainstthereference.Anynon-uniquelocationthata35-mermapstoismasked.TheoutputofComputeGenomeMaskisnotaBEDfile,but a FASTA filewhere allmasked bases are representedwith 0s, and allunmasked bases are representedwith 1s. Thismask file is converted to aBEDfilewithacustomPythonscript.

7.4Subsequencepost-processingSeetheGuttmanLabGithubpageforthepost-processingscriptstoidentifyinteractionsandQCthelibraryisfoundhere. https://github.com/GuttmanLab/barcoding-post/wiki 7.5QualityControlsofSuccessfulSPRITELibrariesWeusethefollowingmetricstoevaluatewhetherSPRITEtaggingwassuccessfulonthepost-sequencinglibrary:Calculatepercentageofreadswithalltagsligated(get_ligation_efficiency.py)

1. Thepercentageofreadswithalltagsligatedshouldbe>90%eachround.Forexample,ifr=5roundsofSPRITEwereperformed,wecandeterminethisbycalculating the fraction of reads f with all 5 barcodes and calculating theligationefficiencyeachround.Ligationefficiencyperround=f1/r.Thisisanexampleofthedistributionofreadswiththenumberofbarcodesidentified.

2. Percentageof chromatin that is interactingwithother chromatin:Wehave

foundthatover-fragmentationof the lysateviasonication for15-20minuteresults in the majority of SPRITE molecules that are non-interacting.Specifically, the majority of reads after heavy sonication do not sharebarcodeswithothermolecules.Spinningthechromatinafter1-2minuteatahighspeedaftersonicationandonlyperformingSPRITEonthesupernatantalsoresultsinasimilarproblem.

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3. FastQCtoQCqualityofreadsonsequencer.Duetoamonotemplatethatcan

occurbecauseallreadsshareastickyend,weQCwhetheranymonotemplateissueshappentoimpactthequalityofthereadswhenacommonsequenceisreached on themachine.Wehave obtained high quality libraries onHiSeq2500andHiSeq4000andNextSeqloadingat8pM.

4. Calculate sufficient sampling of library to ensure all taggedmolecules aresequenced, which ensures that themajority ofmolecules in a complex areidentifiedonthesequencer.WeusetheprogramPreSeqfromtheSmithLabto ensure that most of the unique molecules (>70%) estimated by theprogram ina libraryare sampled.We typically sampleat adepthof1.5-2xreads per the total number ofuniquemolecules estimated in a library. Forexample,ifweestimatethereare70Muniquemoleculesinalibrary,wewillsamplethatlibrarywith105-140Mreads.Sequencingathigherdepthresultsinmanyduplicateswithoutgainingmanymoremolecules ineachcomplex.Highlyunder-sampling(<40%)howeverresultsinalotofmoleculesthatarenotinteractingwithanyothermoleculeandneedtobesequencedfurther.

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Split-Pool Recognition of Interactions by Tag Extension ... · control of a tet-inducible promoter and the Tet transactivator (M2rtTA) from the Rosa26 locus. To induce Xist, doxycycline