dinucleotideiphonenano

EpigeneticsUNDERSTANDINGHISTONE&DNAMODIFICATIONSNowincludesNEBNextEnzymaticMethyl-seq(EM-seq)beINSPIREDdriveDISCOVERYstayGENUINEOVERVIEW2Forover45years,NewEnglandBiolabshasbeencommittedtounderstandingthemechanismsofrestrictionandmethylationofDNA.
Thisexpertiseinenzymologyhasledtothedevelopmentofasuiteofvalidatedproductsforepigeneticsresearch.
TheseuniquesolutionstostudyDNAandhistonemodificationsaredesignedtoaddresssomeofthechallengesofthecurrentmethods.
NEBNextEnzymaticMethyl-seq(EM-seq)andEpiMarkvalidatedreagentssimplifyepigeneticsresearchandexpandthepotentialforbiomarkerdiscovery.
EpigeneticsisthestudyofheritablechangesinthephenotypeofacellororganismthatarenotencodedintheDNAofthegenome.
ThemolecularbasisofanepigeneticprofilearisesfromcovalentmodificationsoftheproteinandDNAcomponentsofchromatin.
Theepigeneticprofileofacelloftendictatescellmemoryandcellfateand,thusinfluencesmammaliandevelopment.
TheepigeneticcodeishypothesizedtobethecombinedeffectsofhistonemodificationsandDNAmethylationongeneexpression.
Whilethegeneticcodeforanindividualisthesameineverycell,theepigeneticcodeistissue-andcell-specific,andmaychangeovertimeasaresultofaging,diseaseorenvironmentalstimuli(e.
g.
,nutrition,lifestyle,toxinexposure)(1).
Cross-talkbetweenhistonemodifications,DNAmethylationorRNAipathwaysarebeingstudiedinsuchareasascancer,Xchromosomeinactivation,andimprinting.
Epigeneticstableofcontents4–5DNAModifications4DNAMethylationinMammals4MethodsforStudyingDNAMethylationMethylomeAnalysis(5mC&5hmC)6NEBNextEnzymaticMethyl-seq(EM-seq)KitBisulfiteConversion8EpiMarkBisulfiteConversionKit8EpimarkHotStartTaqDNAPolymeraseEnrichmentofMethylatedDNA9EpiMarkMethylatedDNAEnrichmentKit5-Hydroxymethylcytosineand5-methylcytosineIdentificationandQuantification10EpiMark5-hmCand5-mCAnalysisKit11T4Phageb-glucosyltransferaseMethylation-SensitiveRestrictionEnzymesMethylation-DependentRestrictionEnzymes12McrBC13MspJIFamilyofRestrictionEnzymesDNAMethyltransferases15GenomicDNAMethylationusingCpGMethyltransferase(M.
SssI)SamplePreparationforChIP-Seq16NEBNextReagentsRNAMethylation17EpiMarkN6-MethyladenosineEnrichmentKitMethylatedandHypomethylatedDNAChromatinandHistones18EpiMarkNucleosomeAssemblyKitRecombinantHumanHistones19HistoneH1°19HistonesH2A&H2B20HistonesH3&H4HistoneModifications&MethodsforStudyingHistoneModificationsHistoneMethyltransferases22G9aMethyltransferase22HumanPRMT1Methyltransferase22SET7Methyltransferase22SET8Methyltransferase22HumanDNA(cytosine-5)Methyltransferase(DNMT1)6–710–112119–209121817812–1314–151617References1.
Tost,J.
(2010)Mol.
Biotechnol.
44,71-81.
22TOOLS&RESOURCESVisitwww.
epimark.
comtofind:AninteractivetutorialexplainingthephenomenonofepigenticsatthemolecularlevelVideosfromNEBscientistsdiscussingtheconceptofepigeneticsVideosandtutorialsfromNEBscientistsexplainingmethodsfor5hmCand5mCdetectionandquantitationVisitNEBNext.
comtolearn:HowEM-seqcomparestobisulfitesequencingHowEM-seqminimizesDNAdamageandproduceshigh-quality,high-diversitylibrariesWhatmoresensitivedetectionof5mCand5hmCmeansforyourmethylomeanalysisNEBNEXTENZYMATICMETHYL-SEQWORKFLOWFindaninteractivetutorialonepigenetics.
OVERVIEW3ReagentsforEpigeneticStudiesDNAMETHYLATIONANALYSIS(Page4–9)NEBNextEnzymaticMethyl-seqKitEpiMarkBisulfiteConversionKitNEBNextMultiplexOligosforEnzymaticMethyl-seq(UniqueDualIndexPrimerPairs)EpiMarkMethylatedDNAEnrichmentKitNEBNextEnzymaticMethyl-seqConversionModuleEpiMarkHotStartTaqDNAPolymerase5-HYDROXYMETHYLCYTOSINEANALYSIS(Pages10–11)EpiMark5-hmCand5-mCAnalysisKitT4Phageβ-GlucosyltransferaseRNAMETHYLATIONENRICHMENT&ANALYSIS(Page9)EpiMarkN6-MethyladenosineEnrichmentKitDNAMETHYLTRANSFERASES(Pages14–15)HumanDNA(cytosine-5)Methyltransferase(Dnmt1)damMethyltransferaseCpGMethyltransferase(M.
SssI)BamHIMethyltransferasGpCMethyltransferase(M.
CviPI)HhaIMethyltransferaseHpaIIMethyltransferaseTaqIMethyltransferaseMspIMethyltransferaseAluIMethyltransferaseEcoRIMethyltransferaseHaeIIIMethyltransferaseHISTONES(Pages18–21)HistoneH10Human,RecombinantHistoneH3.
3Human,RecombinantHistoneH2AHuman,RecombinantH4Human,RecombinantHistoneH2BHuman,RecombinantEpiMarkNucleosomeAssemblyKitHistoneH3.
1Human,RecombinantHistoneH2A/H2BDimerHistoneH3.
2Human,RecombinantHistoneH3.
1/H4TetramerHISTONE/PROTEINMETHYLTRANSFERASES(Page22)G9aMethyltransferase5-methyl-dCTPPRMT1MethyltransferaseSET8MethyltransferaseSET7MethyltransferaseRESTRICTIONENZYMES(Pages12–13)AbaSIHpaIIDpnILpnPIDpnIIMspIFspEIMspJISAMPLEPREPFORNEXTGENSEQUENCING(Page16)NEBNextMagneticSeparationRackNEBNextModules(seePage23)NEBNextUltraIIDNALibraryPrepKitforIlluminaCONTROLDNAs(Page17)CpGMethylatedJurkatGenomicDNAHeLaGenomicDNA5-Aza-dc–TreatedJurkatGenomicDNACpGMethylatedHeLaGenomicDNANIH3T3MouseGenomicDNAVisitwww.
NEB.
comforthefulllistofreagentsavailableforepigenticstudies.
*seebackcoverfordetailsDOWNLOADTHENEBARAPP*DNAMETHYLATIONDNAcanbemodifiedbymethylationofcytosineandadeninebasesinawidevarietyofprokaryotesandeukaryotes(seeTable2).
Inprokaryotes,DNAmethylationisinvolvedindeterminationofDNA-hostspecificity,virulence,DNArepair,chromosomereplicationandsegregation,cellcycleregulationandgeneexpression.
Inhighereukaryotes,DNAmethylationisinvolvedingeneregulation,chromatinstructure,differentiation,imprint-ing,mammalianXchromosomeinactivation,carcinogenesis,complexdiseasesandaging.
DNAMethylationinMammalsDNAmethylationinmammalsprimarilyoccursonthefifthcarbonofthecytosinebase(5-methylcytosine,5mC,seeTable1)ofCpGdinucleotides,andapproximately70%to80%ofCpGdinucleotidesaremethylatedinsomaticcells.
However,5mCatCpA,CpTandCpCsequenceshavebeenfoundingenomicDNAfrommouseembryonicstemcells,and5mCatCpAsequencesarethoughttoregulateenhancersinmousebrain.
Ofnote,whileDNAmethylationinmammalsprimarilyoccursatCpGdinucleotides,DNAmeth-ylationinplantsmayoccuratCpG,CpHpGandCpHpHsequences,whereHisadenine,cytosine,orthymine.
MethodsforStudyingDNAMethylationStudyoftheDNAmethylationpatternsongenomicDNAhad,untilrecently,takenoneofthreeapproaches–pretreatmentwithsodiumbisulfite,restrictionenzymes,oramethylatedDNA-bindingaffinitymatrix–withsodiumbisulfitetreatmentandso-calledbisulfitesequencingbeingthegoldstandardforanalysisatthesinglebaselevel.
In2019,NEBintroducedagroundbreakingnewmethod,NEBNextEnzymaticMethyl-seq(EM-seq),whichofferedmyriadadvantagesovermethylomeanalysiswithsodiumbisul-fitepretreatment.
ThesetechniquesarecomparedandcontrastedinTable2(nextpage).
BothbisulfitetreatmentandEM-seqcanrevealthemethylationstatusofeverycytosineresidueinthegenome,andtheyarethereforeamenabletomassivelyparallelsequencingmethods.
Methyl-specificdifferentialcleavageofDNArequiresrestrictionenzymes,thatareeithermethylationsensitiveormethylationdependent,tofragmentgenomicDNAforsubsequentanalysis.
Thismethodofferslowerresolutiondataduetotherequirementofarangeofenzymerecognitionsequencesandtheriskforincompletedigestion.
Finally,affinity-basedmethodsusemethylatedDNAbindingproteinsorantibodiestoenrichtheexperimentalDNAsampleformethylatedDNAtobeanalyzedinsubsequentsteps.
Awidevarietyofanalyticalandenzymaticmethodsmaybeemployeddownstreamofmethyl-enrichmentstepstocharacterizegenomicDNA.
Analyticalmethods,includinghigh-performanceliquidchromatography(HPLC)andmatrix-assistedlaserdesorption/ionization-timeofflightmassspectrometry(MALDI-TOFMS),areroutinelyusedtoquantifymodifiednucleobasesincomplexDNA.
ThoughHPLCisquantitativeandrepro-ducible,itispoorlysuitedtohigh-throughputapplicationsduetoarequirementforhighinputamounts,althoughrecentworkhasloweredtheminimuminputtonanogramlevels(1).
MALDI-TOFMSisbothquantitativeandamenabletohigherthroughputapplica-tions.
Otherdownstreammethylomeanalysismethodsincludeend-pointPCR,real-timePCR,primerextension,single-strandedconformationalpolymorphismassays,blotting,microarrays,andsequencing.
Selectingamethod(s)willdependonyoursamplesizeandexperimentalgoals(2,seealsowww.
epimark.
com).
DNAModificationsReferences1.
Song,L.
,etal(2005)Anal.
Chem.
,77,504–510.
2.
Laird,P.
W.
(2010)Nat.
Rev.
Genet.
11,191–203.
TOOLS&RESOURCESVisitNEBNext.
comformoreinformationonNEBNextEnzymaticMethyl-seq,anenzyme-basedalternativetobisulfitesequencing4METHYLATEDBASEORGANISMDNAMETHYLATIONSEQUENCEC5-methylcytosineBacteriaVaries(e.
g.
,CCAGG,CCTGG)SomeFungi,SomeInsects,MammalsCpG,CpH*pG,CpH*pHPlantsCpG,CpH*pG,CpH*pHC5-hydroxymethyl-cytosineBacteriophagesVaries(e.
g.
,CCGG,GATC);SomecontainonlymodifiedcytosinesMammalsCpG,CpH*pG,CpH*pHN4-methylcytosineBacteriaVaries(e.
g.
,CTCTTC,CCCGGG)N6-methyladenineBacteria,Bacteriophages,Archaea,Protists,SomeFungi,PlantsVaries(e.
g.
,GATC,GANTC,GAAGAG)Table1:TypesofDNAModifications*=Adenine,Cytosine,orThymineDNAMETHYLATIONTable2:ApproachesforStudyingDNAMethylationMETHODDESCRIPTIONADVANTAGESDISADVANTAGESAPPLICATIONNEBNextEnzymaticMethyl-seq(EM-seq)EM-seqisanewmethodfordetectionof5mCand5hmCatsingle-baseresolution.
Inatwo-stepconversionprocess,TET2andanoxidationenhancerprotectmodifiedcytosinesfromdownstreamdeamination.
TET2enzymaticallyoxidizes5mCand5hmCthroughacascadereactioninto5-carboxycytosine[5-methylcytosine(5mC)→5-hydroxymethylcytosine(5hmC)→5-formylcytosine(5fC)→5-carboxycytosine(5caC)].
Thisprotects5mCand5hmCfromdeamination.
5hmCcanalsobeprotectedfromdeaminationbyglucosylationtoform5ghmcusingtheoxidationenhancer.
Then,APOBECdeaminatescytosinesbutdoesnotaffect5caCand5ghmC.
ComparisonofsequenceinformationbetweenthereferencegenomeandEM-seqDNAcanprovidesingle-nucleotideresolutioninformationaboutcytosinemethylationpatterns.
Superiorsensitivityofdetectionof5mCand5hmCHighmappingefficiencywithuniformGCcoverageGentleenzymaticprocess/minimalDNAdamageMoreCpGdatawithfewersequencingrunsthanWGBSWorkswithdamagedDNA(e.
g.
,FFPE)FasterworkflowthanWGBSResolutionatthenucleotidelevelAutomatedanalysisGives%mCataspecificsiteCannotdistinguishbetween5mCand5hmCIntensivedownstreamanalysis(sameasWGBS)Wholegenome(orsinglelocus)methylationanalysisSodiumBisulfiteConversionTreatmentofdenaturedDNA(i.
e.
,single-strandedDNA)withsodiumbisulfiteleadstodeaminationofunmethylatedcytosineresiduestouracil,leaving5mCintact.
Theuracilsareamplifiedasthymines,and5mCresiduesareamplifiedascytosinesinPCR.
Comparisonofsequenceinformationbetweenthereferencegenomeandbisulfite-treatedDNAcanprovidesingle-nucleotideresolutioninformationaboutcytosinemethylationpatterns.
ResolutionatthenucleotidelevelWorkson5mC-containingDNAAutomatedanalysisGives%mCataspecificsiteRequiresmicrogramsofDNAinput,dependingondownstreamprocessesDNAisoftendamagedMulti-stepprotocolPotentiallyincompleteconversionofDNAIntensivedownstreamanalysisCannotdistinguish5mCand5hmCWholegenomeorasingleDNAlocusmethylationanalysisSequence-SpecificEnzymeDigestionRestrictionenzymesareusedtogenerateDNAfragmentsformethylationanalysis.
Somerestrictionenzymesaremethylation-sensitive(i.
e.
,digestionisimpairedorblockedbymethylatedDNA).
Whenusedinconjunctionwithanisoschizomerthathasthesamerecognitionsite,butismethylationinsensitive,informationaboutmethylationstatuscanbeobtained.
Additionally,theuseofmethylation-dependentrestrictionenzymes(i.
e.
,requiresmethylatedDNAforcleavagetooccur)canbeusedtofragmentDNAforsequencinganalysis.
HighenzymeturnoverWell-studiedEasy-to-useAvailabilityofrecombinantenzymesDeterminationofmethylationstatusislimitedbytheenzymerecognitionsiteOvernightprotocolsLowerthroughputSouthernblotsusingMspI/HpaIIMethylatedDNAImmunoprecipitationFragmentedgenomicDNA(restrictionenzymedigestionorsonication)isdenaturedandimmunoprecipitatedwithantibodiesspecificfor5mC.
TheenrichedDNAfragmentscanbeanalyzedbyPCRforlocus-specificstudiesorbymicroarrays(MeDIP-chip)andmassivelyparallelsequencing(MeDIP-seq)forwholegenomestudies.
RelativelyfastCompatiblewitharray-basedanalysisApplicableforhighthroughputsequencingDependentonantibodyspecificityMayrequiremorethanone5mCforantibodybindingRequiresDNAdenaturationResolutiondependsonthesizeoftheimmunoprecipitatedDNAandformicroarrayexperiments;dependsonprobedesignDatafromrepeatsequencesmaybeoverrepresentedImmunoaffinitycaptureMethylatedDNA-BindingProteinsInsteadofrelyingonantibodiesforDNAenrichment,affinity-basedassaysuseproteinsthatspecificallybindmethylatedorunmethylatedCpGsitesinfragmentedgenomicDNA(restrictionenzymedigestionorsonication).
TheenrichedDNAfragmentscanbeanalyzedbyPCRforlocus-specificstudiesorbymicroarraysandmassivelyparallelsequencingforwholegenomestudies.
Well-studiedDoesnotrequiredenaturationCompatiblewitharray-basedanalysisApplicableforhighthroughputsequencingMayrequirehighDNAinputMayrequirealongprotocolRequiressaltelutionsDoesnotgivesinglebasemethylationresolutiondataCaptureofmethylatedDNA5METHYLOMEANALYSIS(5mC&5hmC)6Themethylomecomprisesthetotalofmethylmarksattachedtothecytosinebaseswithinagenome.
Analyzingthecompletemethylomerequirestoolsthatenablethereliablequantitationofmethylatedcytosines,inmostcasesrequiringtheconversionofmethylatedcytosinesintootherstructuresbeforedeaminationandsequencecomparison.
NEBNextEnzymaticMethyl-seq(EM-seq)KitTheNEBNextEnzymaticMethyl-seqKitprovidesahigh-performanceenzyme-basedalternativetobisulfiteconversionformethylomeanalysisusingIlluminasequencing.
Librariesarepreparedusingaslittleas10nginputDNAandthesuppliedNEBNextUltraIIreagentsandtheoptimizedEM-seqAdaptor.
TET2thenoxidizes5-mCand5-hmC,providingprotectionfromdeaminationbyAPOBECinthenextstep.
Incontrast,unmodi-fiedcytosinesaredeaminatedtouracils.
LibrariesarethenamplifiedusingaNEBNextmastermixformulationofQ5U(amodifiedversionofQ5High-FidelityDNAPolymerase),andsequencedusingIlluminainstrumentation.
TheconsistentlyhighconversionperformanceandminimizedDNAdamagewiththeEM-seqprotocol,incombinationwithhighlyefficientUltraIIlibraryprep,resultinsupe-riordetectionofCpGswithfewersequencingreads.
NEBNextEnzymaticMethyl-seqKitE7120S/LNEBNextEnzymaticMethyl-seqConversionModuleE7125S/LMethylomeAnalysis(5mC&5hmC)ADVANTAGESSuperiorsensitivityofdetectionof5mCand5hmCGreatermappingefficiencyMoreuniformGCcoverageDetectmoreCpGswithfewersequencereadsUniformdinucleotidedistributionLargerlibraryinsertsizesHigh-efficiencylibrarypreparationConversionmodulealsoavailableseparatelyEM-seqandsodiumbisulfiteconversionmethodsSodiumbisulfitemethodConvertedSequencedEM-seqmethodTET2/OxidationEnhancerAPOBECCCGTCGGACCGChmmCCGTCGGACCGCCCGTCGGACCGChmUUGTCGGAUUGChmmTTGTCGGATTGCTTGTCGGATTGCUUGTCGGAUUGCca/gca/gca/gca/gmWe'vebeentestingEM-seqonavarietyofinputs,platforms,andsamples,anditshowsmoreevencoverageacrossCpGislands,thewholegenome,andalsogreaterdetectionofCpGsitesacrossthegenomevs.
WGBS.
–ChristopherMason,WeillCornellMedicalSchoolNewYorkWholegenomebisulfitesequencingistheworkhorsetechniqueinourlaboratoryandwehavetestedrangeofdifferentkits.
NEB'sEM-seqKitprovidesanexcellentalternativethatcausesfarlessdamagetotheDNAandresultsinlargerfragmentswhichmaketheprocessofsequencingmorecosteffective.
Wefoundthatthekitalsoproduceslibrarieswithverylowbiasesinnucleotidecoverageandmethylationestimates.
–DuncanSproul,MRCHumanGeneticsUnitEdinburghWhatusersaresaying:METHYLOMEANALYSIS(5mC&5hmC)7EM-seqidentifiesmoreCpGsthanWGBS,atlowersequencingcoveragedepthwithsuperioruniformityofGCcoverage.
10,50and200ngHumanNA12878genomicDNAwasshearedto300bpusingtheCovarisS2instrumentandusedasinputintoEM-seqandWGBSprotocols.
ForWGBS,NEBNextUltraIIDNAwasusedforlibraryconstruction,followedbytheZymoResearchEZDNAMethylation-GoldKitforbisulfiteconversion.
LibrariesweresequencedonanIlluminaNovaSeq6000(2x100bases).
Readswerealignedtohg38usingbwa-meth0.
2.
2.
A:CoverageofCpGswithEM-seqandWGBSlibrarieswasanalyzedusing324millionpairedendreads,andeachtopandbottomstrandCpGswerecountedindependently,yieldingamaximumof56millionpossibleCpGsites.
EM-seqidentifiesmoreCpGsatlowerdepthofsequencing.
B:GCcoveragewasanalyzedusingPicard2.
17.
2andthedistributionofnormalizedcoverageacrossdifferentGCcontentsofthegenome(0-100%)wasplotted.
EM-seqlibrarieshavesignificantlymoreuniformGCcoverage,andlacktheATover-representationandGCunder-representationtypicalofWGBSlibraries.
00.
511.
522.
533.
50102030405060708090100NormalizedCoverageGCContent(%)EM-seq10ngWGBS10ngEM-seq50ngWGBS50ngEM-seq200ngWGBS200ngA.
B.
EM-seq10ngWGBS10ngEM-seq50ngWGBS50ngEM-seq200ngWGBS200ng01020304050601611162126CpGsCovered(Millions)ObservedCoverageDepthC.
CoverageofCpGswithEM-seqandWGBSlibrarieswasanalyzedusing324millionpairedendreads.
ThenumberofuniqueandcommonCpGsidentifiedbyEM-seqandWGBSat1Xand8Xminimumcoverageforeachinputamountareshown.
EM-seqcoversatleast20%moreCpGsthanWGBSat1Xminimumcoveragethreshold.
ThedifferenceinCpGcoverageincreasestotwo-foldat8Xminimumcoveragethreshold.
EM-seqidentifiesmoreCpGsthanWGBs,atlowersequencingcoveragedepth8Xminimumcoverage0.
7M10.
3M0.
9M1Xminimumcoverage10ng50ng200ng4.
1M12.
5M2.
5M4.
3M2.
8M11M35.
8M17.
9M44.
4M9.
8M0.
2M44.
6M9.
5M0.
2M0.
2MUniqueEM-seqUniqueWGBSCommonWewereveryexcitedbyanopportunitytousethenewEM-seqsystemlaunchednowbyNEB.
Inadditiontoitsattractivefeatures,suchasuser-friendlinessandcleanlinessoftheprocess,forexample,wehaverealizedthatitenablesustodetermineinpreciseandDNAsparingwaythecytosinemethylationstatusevenatlowintegrityDNA.
Ifbisulfiteconversionweretheonlyapproachtoapply,wewoulddefinitelyfailtogeneraterelevantresults.
Thecool,biochemicalapproachtoanalysecytosinemethylationthesystemisutilizing,italsoopensnewavenuestoexplorationsofmethylationatintactlongDNAfragments.
–VladimirBenes,HeadGenomicsCoreFacilityatEMBLHeidelbergC.
BISULFITECONVERSIONBisulfiteConversionEpiMarkBisulfiteConversionKitBisulfiteconversion,involvestheconversionofunmodifiedcytosinestouracil,leavingthemodifiedbases(5mCand5hmC).
TheEpiMarkBisulfiteConversionKitisdesignedforthedetectionofmethylatedcytosine,usingaseriesofalternatingcyclesofthermaldenaturation,followedbyincubationwithsodiumbisulfite.
Thiskitincludesallthereagentsnecessaryforcompletebisulfiteconversion,includingspincolumns.
Amplificationofbisulfite-treatedsamplescanthenbeperformedusingEpiMarkHotStartTaqDNAPolymerase.
EpiMarkBisulfiteConversionKitE3318SEpiMarkHotStartTaqDNAPolymeraseEpiMarkHotStartTaqDNAPolymeraseisamixtureofTaqDNAPolymeraseandatem-peraturesensitive,aptamer-basedinhibitor.
Thisinhibitorbindsreversiblytotheenzyme,inhibitingpolymeraseactivitybelow45°C,butreleasestheenzymeduringnormalPCRcyclingconditions.
ThispermitsPCRreactionstobeassembledatroomtemperatureandeliminatesanactivationstep.
Thisaptamer-basedhotstartactivitycombinedwiththesup-pliedreactionbuffer,thathasbeenoptimizedforamplificationofconvertedDNA,makesEpiMarkHotStartTaqanexcellentchoiceforuseonbisulfite-treatedDNA.
EpiMarkHotStartTaqDNAPolymeraseM0490S/L1μgofgenomicDNAwasbisulfite-treatedusingtheEpimarkBisulfiteConversionKit,and2μlofelutedDNAwasanalyzedbyend-pointPCRusingEpiMarkHotStartTaq.
AmplificationwithprimerpairsforbisulfiteconvertedDNA(lanes1,3,and5),orwithprimerpairsforunconvertedDNA(lanes2,4,and6)wereperformed;lanes2,4,and6shownoamplificationproduct,indicatingcompleteconversion.
EpiMarkKitenablescompleteDNAconversion123456544388731bpOverviewofbisulfiteconversionDenaturationIncubationat95°CfragmentsgenomicDNAFragmentedGenomicDNASamplesConversionIncubationwithsodiumbisulteat65°CandlowpH(5-6)deaminatescytosineresiduesinfragmentedDNADesulphonationIncubationathighpHatroomtemperaturefor15minremovesthesultemoeity,generatinguracilStep1Step2Step3NaHSO3,pH5.
0NaHSO3,pH5.
0+H2O,-NH3OH+NaHSO3UracilCytosine5-Methylcytosine(5mC)5mCand5hmC(notshown)arenotsusceptibletobisulteconversionandremainintactBENEFITSConversionofunmodifiedcytosinestouracilAllreagents,includingpurificationcolumns,areprovided8ENRICHMENTOFMETHYLATEDDNAS&RNASEnrichmentofMethylatedDNAEpiMarkMethylatedDNAEnrichmentKitTheEpiMarkMethylatedDNAEnrichmentKitenablestheenrichmentofdouble-strandedCpGmethylatedDNAbasedonCpGmethylationdensity.
Itutilizesthemethyl-CpGbind-ingdomainofhumanMBD2aproteinasacaptureagent.
TheproteinisfusedtotheFctailofhumanIgG1(MBD2a-Fc),whichiscoupledtoProteinAMagneticBeads(MBD2a-Fc/ProteinABead).
Thisstablecomplexwillselectivelybinddouble-strandedmethylatedCpGcontainingDNA.
Thehighbindingaffinityofthebeadscoupledwithoptimizedreagentsincreasessensitivityandaccuracy.
Thiskitcontainsalltheindividualcomponentsnecessarytoachieveenrichmentinlessthantwohoursusingafourstepprocess:StepI.
FragmentgenomicDNAbysonication,nebulizationorenzymatictreatmenttoanaveragesizeoflessthan1,000bpStepII.
GenerationofbeadmixturebycombiningMBD2a-Fc,ProteinAMagneticBeadsand1XBind/WashReactionBufferStepIII.
CaptureofmethylatedCpGDNAbyincubationwithMBD2a-Fc/ProteinAMagneticBeadmixtureStepIV.
EluteenrichedmethylatedCpGDNAfrombeadsInthefinalstep,enrichedfractionsareelutedinsmallvolumes,simplifyingdownstreamapplications,includingadaptorligationfornextgenerationsequencing.
EpiMarkMethylatedDNAEnrichmentKitE2600SADVANTAGESIncreasedsensitivityEasy-to-useprotocolyieldsenrichedmethylatedDNAinlessthan2hoursAmenabletodownstreamapplications,includingnextgenerationsequencingSuitableforlowlevelsofinputDNA9JustasepigeneticinformationcanbeconveyedviaDNAmodifications,sotoocanitbecon-veyedviaRNAmodifications.
CommonmodificationsofmessengerRNAs(mRNAs)includemethylationofcytosinesandadenosines.
ThemostcommonmRNAmodificationinmam-malsisN6-methyladenosine(m6A),anditisthoughttobeinvolvedinRNAstability,splic-ing,transport,andtolerance(1,2).
EpiMarkN6-MethyladenosineEnrichmentKitTheEpiMarkN6-MethyladenosineEnrichmentKitcanbeusedtoenrichm6Amodi-fiedRNAinimmunoprecipitationprotocolsfordownstreamanalysisbynext-generationRNAsequencingorRT-qPCR.
ThekitcontainsarabbitmonoclonalantibodyspecificforN6-Methyladenosine(m6A).
ThekitalsocontainstwocontrolRNAs,onewithm6Amodification(Gaussialuciferase)andonewithout(Cypridinaluciferase)tomonitorenrich-mentanddepletion.
TheGLucRNAcontrolwastranscribedinthepresenceof20%m6ATPand80%ATP.
Thiskitcanbeusedtoenrichm6AmodifiedRNAinimmuno-precipitationprotocolsfordownstreamanalysisbynext-generationRNAsequencingorRT-qPCR.
ModifiedRNAisisolatedfromafragmentedRNAsamplebybindingtotheN6-MethyladenosineantibodyattachedtoProteinGMagneticBeads.
Aftermultiplewashandclean-upsteps,theenrichedRNAiselutedinnuclease-freewaterandisreadyforfur-theranalysis.
EpiMarkN6-MethyladenosineEnrichmentKitE1610SRNAMethylationADVANTAGESCompleteprotocolforenrichmentofm6A-modified–RNAandanalysisbyRT-qPCRincludedRNAcontrols(m6AmodifiedandunmodifiedRNA)enablemonitoringofenrichmentanddepletionAntibodysuppliedinaready-to-usesolutionformReferences1.
Bokar,J.
A.
(2005)Fine-tuningofRNAFunctionsbyModificationandEditing,Springer-Verlag,Berlinpp.
141–178.
2.
Kariko,K.
,Buckstein,M.
,Ni,H.
andWeissman,D.
(2005),Immunity,23,pp.
165–175.
5mCAND5hmCANALYSIS5-Hydroxymethylcytosineand5-methylcytosineIdentificationandQuantificationEpiMark5-hmCand5-mCAnalysisKitTheEpiMark5-hmCand5-mCAnalysisKitcanbeusedtoanalyzeandquantitate5-methylcytosine(5mC)and5-hydroxymethylcytosine(5hmC)withinaspecificlocus.
Thekitdistinguishes5mCfrom5hmCbytheadditionofglucosetothehydroxylgroupof5hmCviaanenzymaticreactionutilizingT4phageβ-glucosyltransferase(T4-BGT).
When5-hmCoccursinthecontextofCCGG,thismodificationconvertsacleavableMspIsitetoanoncleavableone.
EpiMark5-hmCand5-mCAnalysisKitE3317SCCGGGGCChhCCGGGGCCmmmgCCGGGGCCmCCGGGGCCgCCGGGGCCCCGGGGCCCCGGGGCCmmCCGGGGCCggCCGGGGCCCCGGGGCCmmCCGGGGCCgghCCGGGGCCCCGGGGCChhCCGGGGCCmmCCGGGGCCmCCGGGGCCmgCCGGGGCCgCCGGGGCCCCGGGGCCmmCCGGGGCCCCGGGGCChhGlucosylationTreatmentwithT4-BGTandUDP-Glcglucosylatesall5-hmCsites,generating5-ghmC.
IntheabsenceofT4-BGT5hmCremainsintact.
REDigestionMspI(cleaves5mC+5hmCDNA;blockedbyghmC)HpaII(cleavageblockedby5mC,5hmCand5ghmC)indicatescleavagesitePCRAnalysisProductisdetectedwhencleavageisblocked(fragmentsinblueareintactandwillresultinPCRproduct)Step1Step2Step3mixtureof5hmC,5mCandunmethylatedDNAIdentieshydroxymethylatedDNA(5hmC)Controlreaction–NoPCRproductdetectedIdentiestotalmethylation(5mCand5hmC)IdentiestotalamountofDNApresentT4-BGT+UDP-Glc–T4-BGT(UDP-Glconly)1234+MspI+HpaIIUncutcontrol+MspICCGGGGCCmmCCGGGGCChCCGGGGCCmCCGGGGCCmhCCGGGGCChCCGGGGCCIdentiestotalmethylation(5mCand5hmC)IdentiestotalamountofDNApresent(canbeusedasacontroltomeasureanyeectscausedbyT4-BGT)56+HpaIIUncutcontrolT4-BGT:T4Phageβ-glucosyltransferaseUDP-Glc:UDPGlucose5hmC:5-hydroxymethylcytosine(green)5mC:5-methylcytosine(orange)5ghmC:glucosylated(red)5-hydroxymethylcytosineOverviewof5-hmCand5-mCidentificationusingtheEpiMark5-hmCand5-mCAnalysisKitADVANTAGESReproduciblequantitationof5hmCand5mCEasy-to-useprotocolsCompatiblewithexistingtechniquesAmenabletohighthroughput105mCAND5hmCANALYSIST4Phageβ-glucosyltransferaseT4Phageβ-glucosyltransferase(T4-BGT)isalsoavailableasastand-aloneenzymefortheglucosylationof5hmCinDNA.
ThisisthesameenzymeincludedintheEpiMark5-hmCand5-mCAnalysisKit.
T4Phageβ-glucosyltransferaseM0357SAnalysisofthedifferentmethylationstatesinBalb/CmousetissuesamplesusingtheEpiMark5-hmCand5-mCAnalysisKitGlucosylationwithT4-BGTB.
A)EndpointPCRofthe6differentreactionsneededformethylationanalysis.
Theboxedlanesindicatethepresenceof5hmC.
B)Real-timePCRdatawasusedtodetermineamountsof5hmCand5mCpresent.
Theresultsdemonstrateavariationin5hmClevelsinthetissuesourcesindicated.
A.
T4-BGTMspIHpaIIT4-BGTMspIHpaIIT4-BGTMspIHpaIIT4-BGTMspIHpaIIBrainLiverHeartSpleen120100806040200BrainLiverSpleenHeart%5-hmC%5-mC%UnmethylatedCReferences1.
Josse,J.
andKornberg,A.
(1962)J.
Biol.
Chem.
,237,1968-1976.
2.
Tomaschewski,J.
etal.
(1985)NucleicAcidsRes.
,13,7551-7568.
3.
McNicol,L.
A.
etal.
(1973)J.
Mol.
Biol.
,15,76,285-301.
4.
Szwagierczak,A.
etal.
(2010)NucleicAcidsRes.
,inpress.
TreatmentofDNAwithT4-BGTandUDP-Glcglucosylatesall5-hydroxymethylcytosine(5hmC)sites,generatinggluco-sylated5-hydroxymethylcytosine(5ghmC).
C5-hydroxymethylcytosineGlucosylated5-hydroxymethylcytosine+T4-BGT+UDP-GlcAPPLICATIONSGlucosylationof5hmCinDNA(1)Immunodetectionof5hmCinDNA(3)Labelingof5hmCbyincorporationof[3H]-or[14C]-glucoseinto5hmC-containingDNAacceptorafterincubationwith[3H]-or[14C]-UDP-Glc(4)Detectionof5hmCinDNAbyprotectionfromendonucleasecleavage11Learnabout5-hmCdetectioninBalb/Cbraintissue.
RESTRICTIONENZYMESFROMNEBMethylation-SensitiveRestrictionEnzymesMethylation-DependentRestrictionEnzymesSomerestrictionenzymesaremethylation-sensitive(i.
e.
,digestionisimpairedorblockedbymethylatedDNA).
Whenusedinconjunctionwithanisoschizomerthathasthesamerecognitionsitebutismethylationinsensitive,informationaboutmethylationstatuscanbeobtained.
Table4Alistsmethylationsensitiverestrictionenzymesthatcanbeusedinepigeneticstudies.
Somerestrictionenzymesaredependentonmethylationorhydroxymethylationforcleavagetooccur,makingthemparticularlyusefulforDNAmethylationstudies.
McrBCMcrBCisanendonucleasewhichonlycleavesDNAcontainingmethylcytosine(5-methylcytosine,5-hydroxymethylcytosineorN4methylcytosine)ononeorbothstrands(2).
McrBCwillnotactuponunmethylatedDNA(3)andwillnotrecognizeHpaII/MspIsites(CCGG)inwhichtheinternalcytosineismethylated.
McrBCrequiresGTPforcleavage,butinthepresenceofanon-hydrolyzableanalogofGTP,theenzymewillbindtomethylatedDNAspecifically,withoutcleavage(4).
McrBCmakesonecutbetweeneachpairofhalf-sites,cuttingclosetoonehalf-siteortheother,butcleavagepositionsaredistributedoverseveralbasepairsapproximately30basepairsfromthemethylatedbase(5).
Therefore,theenzymedoesnotproducedefinedDNAendsuponcleavage.
Also,whenmultipleMcrBChalf-sitesarepresentinDNA(asisthecasewithcytosine-methylatedgenomicDNA)theflexiblenatureoftherecognitionsequenceresultsinanoverlapofsitesandasmeared,ratherthanasharp,bandingpatternisproduced.
METHYLATIONSENSITIVITYSEQUENCENEB#ISOSCHIZOMERDpnIICleavesdamsites**whichlackadenomethylationandisblockedbycompletedammethylationandprobablybyhemi-methylationR0543MboIDpnIIHpaIIWillnotcleavemethylatedCpGsitesR0171MspIMspINotmethylationsensitiveR0106HpaIITable4A:MethylationSensitiveRestrictionEnzymes**damsites:methylationattheN6positionoftheadenineinthesequenceGATC(GmATC).
APPLICATIONSDifferentiationofmethylationpatternsRESTRICTIONENZYMEDIGESTIONPROTOCOL1.
Addthefollowingcomponentstoasterilemicrocentrifugetube(restrictionenzymeshouldbeaddedlast):component25lreaction50lreactionDNA0.
5g1g10XNEBuffer2.
5l5lNuclease-freewaterto25lto50lRestrictionEnzyme*5units10units2.
Gentlymixthereactionbypipettingupanddownandmicrofugebriefly3.
Incubateattherecommendedtemperaturefor1houror5minutesforTime-Saverqualifiedrestrictionenzymes(seewww.
neb.
com/TimeSaverformoreinformation)4.
TerminatethereactionbyheatinactivationorDNApurificationaccordingtoproductrecommendations*Restrictionenzymescanbedilutedusingtherecommendeddiluentbuffer.
applicationsCpGmethylationstudies(6–10)MethylatedcytosinedetectionMethylatedDNAenrichment(11)125.
.
.
GATC.
.
.
33.
.
.
CTAG.
.
.
55.
.
.
CCGG.
.
.
33.
.
.
GGCC.
.
.
55.
.
.
CCGG.
.
.
33.
.
.
GGCC.
.
.
5RESTRICTIONENZYMESFROMNEBReferences1.
Wang,H.
,etal.
(2011)Nucl.
Acids.
Res.
,39(21)9294-9305.
http://nar.
oxfordjournals.
org/content/39/21/92942.
Sun,Z.
,Terragni,J.
,etal.
(2013)CellReports,3(2)567-576.
http://www.
sciencedirect.
com/science/article/pii/S22111247130000893.
Horton,J.
etal.
(2014)Nucl.
Acids.
Res.
,42(12)7947-7959.
http://nar.
oxfordjournals.
org/content/42/12/7947.
long4.
Zhang,Y.
,etal.
(2010)Nucl.
Acids.
Res.
,38,5527–5534.
5.
Zheng,Y.
,etal.
(2010)Nucl.
Acids.
Res.
,doi;10,1093/nar/gkq3276.
Cohen-Karni,D.
,etal.
(2011)PNAS,108(27)11040-11045.
http://www.
pnas.
org/content/108/27/11040.
long7.
Horton,J.
,etal.
(2012)Nucl.
Acids.
Res.
,40(19)9763-9773.
http://nar.
oxfordjournals.
org/content/40/19/97638.
Horton,J.
,etal.
(2014)Nucl.
Acids.
Res.
,42(19)12092-12101.
9.
Hublarova,P.
etal.
(2009)Int.
J.
Gynecol.
Cancer,19,321–325.
10.
Sutherland,E.
etal.
(1992)J.
Mol.
Biol.
,225,327–334.
11.
Irizarry,R.
A.
etal.
(2008)GenomeRes.
,18,780–790.
12.
Gowher,H.
etal.
(2000)EMBOJ.
,19,6918–6923.
**damsites:methylationattheN6positionoftheadenineinthesequenceGATC(GmATC).
H=AorCorT,notGD=AorGorT,notCTable4B:MethylationDependentRestrictionEnzymesMspJIFamilyofRestrictionEnzymesScientistsatNEBrecentlyidentifiedtheMspJIfamilyofrestrictionenzymes,whicharedependentonmethylationandhydroxymethylationforcleavagetooccur(12).
TheseenzymesexciseDNAfragmentscontainingacentrallylocated5hmCor5mCmodifiedresiduethatcanbeextractedandsequenced.
Duetotheknownpositionofthisepigeneticmodification,bisulfiteconversionisnotrequiredpriortodownstreamanalysis.
ADVANTAGESSpecificitytoepigeneticallyrelevantDNAmodifications(5mCand5hmC)Easy-to-useprotocols(enzymaticdigestionfollowedbygelextraction)LessharshthanbisulfiteconversionSimplifieddataanalysismethylationsensitivitysequenceneb#isoschizomerAbaSI(1,2,3)Recognizes5-glucosylhydroxy-methylcytosine(ghmC)indouble-strandedDNAandcleaves11–13bases3fromthemodifiedCR0665N/ADpnICleavesfully-adenomethylateddam**sites(hemi-adenomethylateddamsites60Xmoreslowly).
CleavageofmammaliangenomicDNAisblockedbyoverlappingCpGmethylation.
R0176DpnIIFspEI(6)CleavesDNAcontaining5-methylcytosineand5-hydroxymethylcytosineR0662N/ALpnPI(6)CleavesDNAcontaining5-methylcytosineand5-hydroxymethylcytosineR0663N/AMcrBCCleavesDNAcontaining5-methylcytosine,5-hydroxymethylcytosineorN4-methylcytosineononeorbothstrands.
5…PumC(N40-3000)PumC…3OptimumspacingisN55-103Pu=AorGCleavagesiteisbetweenthehalf-sitesand~30bpfromoneofthehalf-sitesM0272N/AMspJI(4,5,6,7,8)CleavesDNAcontaining5-methylcytosineand5-hydroxymethylcytosineR0661N/A5.
.
.
CmC(N)12.
.
.
33.
.
.
GG(N)16.
.
.
55.
.
.
CmCDG(N)10.
.
.
33.
.
.
GGHC(N)14.
.
.
55.
.
.
mCNNR(N)9.
.
.
33.
.
.
GNNY(N)13.
.
.
55.
.
.
ghmCN11-13N9-16G.
.
.
33GN9-10N11-13xC.
.
.
5xC=ghmC,hmC,mCorC5.
.
.
GATC.
.
.
33.
.
.
CTAG.
.
.
5CH3CH3GENOMICDNADIGESTION(MspJI)PROTOCOL1.
Setupthefollowingreactioninasterilemicrocentrifugetube(itisimportanttoaddtherecommendedamountofMspJIlast):componentstandardreactionDNA(0.
5to1g)1–5g10XNEBuffer43lBSA1lMspJI0.
5–1l(2to4units)Nuclease-freewaterto30l2.
Incubateat37°Cfor16hours.
13DNAMETHYLTRANSFERASESproductneb#sequencecytosine-c5methyltransferasesHumanDNA(cytosine-5)Methyltransferase(DNMT1)M0230S/LCpGMethyltransferase(M.
SssI)M0226S/LGpCMethyltransferase(M.
CviPI)M0227S/LAluIMethyltransferaseM0220S/LHaeIIIMethyltransferaseM0224S/LHhaIMethyltransferaseM0217S/LHpaIIMethyltransferaseM0214S/LMspIMethyltransferaseM0215S/Lcytosine-n4methyltransferaseBamHIMethyltransferaseM0223S/Ladenine-n6methyltransferasesdamMethyltransferaseM0222S/LEcoRIMethyltransferaseM0211S/LTaqIMethyltransferaseM0219S/LDNAMethyltransferasesNEBoffersaselectionofDNAmethyltransferasesthatcanbeusedtogeneratemethylatedDNAatspecificsitesforgeneexpressionstudies.
OurselectionincludesCpGmethyltrans-ferases,whichisespeciallyusefulforstudyingCpGmethylationeffects.
CH35.
.
.
CG.
.
.
33.
.
.
GC.
.
.
5CH3CH35.
.
.
GC.
.
.
33.
.
.
CG.
.
.
5CH3CH35.
.
.
AGCT.
.
.
33.
.
.
TCGA.
.
.
5CH3CH35.
.
.
GGATCC.
.
.
33.
.
.
CCTAGG.
.
.
5CH3CH35.
.
.
GATC.
.
.
33.
.
.
CTAG.
.
.
5CH3CH35.
.
.
GAATTC.
.
.
33.
.
.
CTTAAG.
.
.
5CH3CH35.
.
.
GGCC.
.
.
33.
.
.
CCGG.
.
.
5CH3CH35.
.
.
GCGC.
.
.
33.
.
.
CGCG.
.
.
5CH3CH35.
.
.
CCGG.
.
.
33.
.
.
GGCC.
.
.
5CH3CH35.
.
.
CG.
.
.
33.
.
.
GC.
.
.
5HumanDNMT1CH35.
.
.
CG.
.
.
33.
.
.
GC.
.
.
5CH3APPLICATIONSBlockingrestrictionenzymecleavageGeneratingpositivecontrolDNAsamplesformethylation-specificPCRorbisulfitesequencingexperimentsStudyingCpGmethylation-dependentgeneexpression[CpGMethyltransferase(M.
SssI),NEB#M0226Probingsequence-specificcontactswithinthemajorgrooveofDNANucleosomefootprintingUniform[3H]-labelingofDNAAlteringthephysicalpropertiesofDNA[e.
g.
,methyl-cytosineslowerthefreeenergyofZ-DNAformation(1),increasethehelicalpitchofDNA(2),alterthekineticsofcruciformextrusion(3)anddecreasereactivitytohydrazine(4)]CH35.
.
.
CCGG.
.
.
33.
.
.
GGCC.
.
.
5CH3CH35.
.
.
TCGA.
.
.
33.
.
.
AGCT.
.
.
5CH314METHYLATIONOFGENOMICDNAGenomicDNAMethylationUsingCpGMethyltransferase(M.
SssI)CpGMethyltransferase(M.
SssI)maybeusefulforstudyingthefunctionofcyto-sinemethylationinhighereukaryotesasitsspecificitymimicsthepatternofmodificationfoundintheirgenomes(1).
Incontrasttothemammalianenzymes(2,3),bothunmethylatedandhemi-methylatedDNAsubstratesaremethylatedwithequalefficiencybythisCpGmethyltransferase(4),makingitamoreusefultoolformodifyingDNA.
CpGMethyltransferasecanbeusedtoblockcleavagebyavarietyofrestric-tionendonucleaseswhoserecognitionsiteseithercontainthesequenceCG,oroverlapthedinucleotide.
ItshouldbenotedthatDNAsmethylatedbytheCpGMethyltransferasearesubjecttoMcrandMrrrestrictioninE.
coli,andthusshouldbetransformedintoMcr-Mrr-E.
colistrains.
ThehighdensityofCpGdinucleotidesinDNAsubstratesshouldbetakenintoaccountwhenmethylatingDNAsinvitro.
Forexample,lambdaDNA(48,502bp)contains3,112CpGsites,andthusa0.
1mgDNA/mlsolutionis19Mwithrespecttomethylacceptorsitesforthemethyltransferase.
Thisissignificantbecausetherecommendedconcentrationofmethyldonor,S-adenosylmethionine(SAM,AdoMet),is160M,an8-foldexcessoveracceptorsites.
ReducingtheDNAconcentration(<0.
02mg/ml)givestwoadvantages.
First,theSAMcon-centrationremainshighenoughtodrivethereaction.
Second,potentialend-productinhibition,arisingfromS-adenosyl-L-homocysteine(SAH,AdoHcy)gen-eratedduringthereaction,islimited.
References1.
Forney,J.
A.
andJack,W.
E.
(1991)NEBTranscript,3(1),5.
2.
Matsuo,K.
etal.
(1994)Nucl.
AcidsRes.
,22,5354–5359.
3.
Doerfler,W.
(1983)Ann.
Rev.
Biochem.
,52,93–124.
4.
Ohmori,H.
etal.
(1978)Nucl.
AcidsRes.
,5,1479–1485.
5.
Renbaum,P.
etal.
(1990)Nucl.
AcidsRes.
,18,1145–1152.
6.
Murchie,A.
I.
andLilley,D.
M.
(1989)J.
Mol.
Biol.
,205,593–602.
Protocol:1.
Forthestandardreactioninstep2,diluteSAMto1600μMusingthesupplied32mMstock.
(1lSAM,19lNuclease-freewater).
2.
Addthefollowingtoasterilemicrocentrifugetube,intheorderlisted:standardreactionrepresentativelarge-scalereactionNuclease-freewater14μl220μl10XNEBuffer22μl50μlSAM2μlfromstep110μl(32MSAM)GenomicDNA1μl(1μg)200μl(500g/mlλDNA)CpGmethylase(M.
SssI)1μl(4U/μl)20μl(20U/μl)3.
Mixbypipettingupanddownatleastsixtimes.
4.
Incubateforonehourat37°C.
5.
Stopthereactionbyheatingat65°Cfor20minutes.
6.
DNAcanbepurifiedbyphenolextractionfollowedbyethanolprecipita-tionorbyusingacommercialDNApurificationkit.
Forlong-termstorageat-20°C,suspendinTE.
TIPSMgCl2isnotrequiredasacofactor.
InthepresenceofMg2+,methylationbyM.
SssIbecomesdistributiveratherthanproces-siveandalsoexhibitstopoisomeraseactivity(5).
AddingmoreAdoMetafter4hourscanimproveresults,andusingmoreenzymeforlesstimemayimprovemethylation.
Methylationreactions,however,aregreatlyaffectedbyAdoHcy(6),whichisaby-productofthemethylationreactionandbindsmoretightlytomethylasesthandoesAdoMet.
InhibitionbyAdoHcygreatlyreducesthereactionrate.
Theincubationtimecanbeincreasedto4hours.
Overnightincubationsdonotgivesignificantincreasesinmethylation.
ThevolumeofDNAcanbeincreasedto5μl.
WhenusingmorediluteDNA,increasethereactionvolumeto50μl.
UsingtoomuchDNAvolumeinthereactioncancauseinhibitionbychangingthepHorsaltconcentrationofthereaction.
Upto4μgofDNAcanbemethylatedina20μlreaction.
TheSAMconcentrationshouldbeadjustedto640μM.
ConcentratedSssI(NEB#M0226)(1μlof20,000U/ml)shouldbeused.
TheprotocolcanalsobeusedforothertypesofDNA,includingplasmidsandpurifiedPCRproducts.
TOOLS&RESOURCESVisitwww.
neb.
comtofind:AprotocolforlabelinggenomicDNAwith[3H]usingmethyltransferases15ChiP-SeqSAMPLEPREPNEBNextReagentsNEBNextreagentsareaseriesofhighlypurereagentsthatfacilitatelibrarypreparationofDNAorRNAfordownstreamapplications,suchasnextgenerationsequencingandexpressionlibraryconstruction.
Thesereagentsundergostringentqualitycontrolsandfunctionalvalidation,ensuringmaximumyield,convenienceandvalue.
ForsamplepreparationofaChIP-SeqDNAlibrary,NEBofferskits,oligosandmodulesthatsupportstandardorfastworkflows.
Todecidewhichproductstochoose,usetheselectionchartbelow.
SamplePreparationforChIP-SeqADVANTAGESFasthigh-performanceworkflowswithminimalhands-on-timeConvenientformatsincludekitsandmodulesAllreagentsundergostringentqualitycontrols,plussequencingvalidationValuepricingTOOLS&RESOURCESVisitwww.
NEBNext.
comtofind:CompletelistofNEBNextreagentsforsampleprepofDNAorRNAfornextgenerationsequencingProtocols&FAQsOnlinetutorialstohelpwithproductselection,generalhandlingtipsandmoreAccesstoNEBNextSelectorTool,ouronlinetoolforhelpwithselectingtherightNEBNextproductNEBNextcitationsThelatestNEBNextbrochureswhychoosenebnextreagentsforngslibraryprepNEBNextMultiplexOligos–#E7335/#E7500/#E7710/#E7730/#E6609/#E6440/#E6442/#E7535/#E7600/#E7780/#E7140OligosKitNEBNextUltraIIDNALibraryPrepKit–#E7645ModulesNEBNextUltraIIEndRepair/dA-TailingModule–#E7546NEBNextUltraIILigationModule–#E7595NEBNextUltraIIQ5MasterMix–#M0544OligosChIPNEBNextMultiplexOligos–#E7335/#E7500/#E7710/#E7730/#E6609/#E6440/#E6442/#E7535/#E7600/#E7780/#E714016GENOMICDNASMethylatedandHypomethylatedDNAPositiveandnegativecontrolDNAsareespeciallyimportantforstudiesusingsensitivePCR-basedassays.
NEBoffersthreesetsofgenomicDNAthatareuntreatedortreatedwithCpGMethylase(M.
SssI),whichmethylatescytosineresidues(C5)withinthedouble-strandeddinu-cleotidesrecognitionsequence5…CG…3.
Themethylation-positiveDNAsareextensivelytestedforcompletemethylationbyanadditionalmethylgrouptransferassayandmethylation-specificPCR.
ApartiallydemethylatedDNAcontrolhasalsobeencreatedbytreatingJurkatcellswithapotentmethyltransferaseinhibitor(5-Aza-2-deoxycytidine,5-Aza-dc).
Hypomethylationisverifiedusingbisulfiteconversionandsequencingtoanalyzeasectionofintergenic(IGS)repetitiveDNA,whichisnormallyhighlymethylated.
CpGMethylatedJurkatGenomicDNAN4002S5-Aza-dc–TreatedJurkatGenomicDNAN4003SNIH3T3MouseGenomicDNAN4004SHeLaGenomicDNAN4006SCpGMethylatedHeLaGenomicDNAN4007SAPPLICATIONSPCRSNPanalysisSouthernblottingGenomicDNAlibraryconstructionMethylation-specificPCR(MSP)EnzymaticMethyl-seq(EM-seq)BisulfitesequencingMethylation-sensitivesingle-nucleotideprimerextension(ms-SNUPE)Combinedbisulfiterestrictionanalysis(COBRA)BisulfitetreatmentandPCRsingle-strandedconfirmationpolymorphismanalysis(Bisulfite-PCR-SSCP/BiPS)17CHROMATIN&HISTONESChromatinandHistonesIneukaryotes,chromatinisorganizedintonucleosomecoreparticles(NCPs)thatconsistofapproximately147bpofDNAandanoctamercomplexmadeupoftwomoleculesofeachhistone(H2A,H2B,H3andH4).
ThelinkerhistoneH1furthercondenseschroma-tinbybindingtoDNAbetweenthenucleosomecoreparticles(1).
Chromatincanbegen-erallyclassifiedascondensed,transcriptionallysilentheterochromatinorless-condensed,transcriptionallyactiveeuchromatin.
Thedynamicnatureofthechromatinpredictsdif-ferentconformationalformsexistinthenucleusatagiventime.
Furthermore,chromatinstructureisinfluencedbythemodificationofDNAorhistonesthatcompriseitandbyitstranscriptionalstate(2).
Although,mostgenomicDNAisbelievedtobepackedintoheterochromatin(telomeres,pericentricregionsandareasrichinrepetitivesequences),loopingoflargestretchesofchromatinfromachromosometogeneratelocalsecondarystructurepoisedfortranscriptionisobserved(3).
NewEnglandBiolabsoffersaselectionofunmodified,recombinanthumanhistonesthatfunctionassubstratesforhistone-modifyingenzymes.
Sevenhumanhistones,includingthreehistoneH3variants,havebeenindividuallyclonedinE.
coliexpressionvectorsandthenpurifiedfromE.
colicellextracts.
Massspectrometryanalysisdemonstratesthatthesehistonesarefreeofpost-translationalmodifications.
Toaidinstudyingintactnucleosomes,NEBalsoofferstheEpiMarkNucleosomeAssemblyKit.
TheprecisemixingofpreformedrecombinantHumanHistoneH2A/H2BDimerandHistoneH3.
1/H4Tetramergeneratesahumanhistoneoctamer,andinthepresenceofDNA,formsnucleosomes(4,5).
EnzymesthatareunabletomodifyindividualhistoneorDNAmaybeactiveonthesenucleosomecoreparticles,thehistonedimerorthehistonetetramer(6).
References1.
Kornberg,R.
D.
(1977)Annu.
Rev.
Biochem.
46,931–954.
2.
Kim,J.
K.
,Samaranayake,M.
andPradhan,S.
(2009)Cell.
Mol.
LifeSci.
,66,596–612.
3.
Gilbertetal.
,(2004)Cell118,555-566.
4.
Lugeretal.
(1999)MethodsinMol.
Biol.
,119,1–16.
5.
Comb,D.
andMersha,F.
unpublishedobservations.
6.
Li,Yanetal.
(2009)J.
Biol.
Chem.
,284,34283–34295.
EpiMarkNucleosomeAssemblyKitThiskitcontainsthecomponentsnecessarytoformanunmodifiedrecombinanthumannucleosomeusingexperimentalDNAofinterestorthesuppliedcontrolDNA.
Theproto-colrequiresthemixingofalreadyformedandpurifiedrecombinanthumanhistoneH2A/H2BdimerandhistoneH3.
1/H4tetramerinthepresenceofDNAathighsalt,followedbydialysisdowntolowsalttoformnucleosomes.
OnetetramerassociateswithtwodimerstoformthehistoneoctamerontheDNA,generatinganucleosome.
Amethodforassayingnucleosomeformationbygelshiftassayisalsoprovided.
ThesenucleosomesmayserveasbettersubstratesforenzymesthatareinactiveontheDNAoroneofthecorehistonesalone.
Eachdescribedreactioncreatesnucleosomesfrom~50pmolofa208bpDNAandmaybescaleddependingontheexperiment.
EpiMarkNucleosomeAssemblyKitE5350SHighlyPurifiedHistonesfromNEBExperiencethepurityofHistonesfromNEB.
SDS-PAGEanalysisofthehistonesavailablefromNEB.
1.
HistoneH1°(NEB#M2501)1g2.
HistoneH2A(NEB#M2502)1g3.
HistoneH2B(NEB#M2505)1g4.
HistoneH3.
1(NEB#M2503)1g5.
HistoneH3.
2(NEB#M2506)1g6.
HistoneH3.
3(NEB#M2507)1g7.
HistoneH4(NEB#M2504)1g8.
HistoneH2A/H2BDimer(NEB#M2508)2g9.
HistoneH3.
1/H4Tetramer(NEB#M2509)2g10.
NEBProteinLadder(NEB#P7703)APPLICATIONSPurificationandcharacterizationofenzymesthatmodifyhistoneproteinsFormationofunmodifiednucleosomecoreparticles,whichmaybemodifiedbyenzymesthatareinactiveonindividualhistonesorDNATOOLS&RESOURCESVisitwww.
epimark.
comformoreinformationonhistonemodificationskDa8060504030252015101234567891018HISTONESRecombinantHumanHistonesHistoneH1HistoneH1actsonthelinkerregionofpolynucleosomeDNAtocondensethechromatinintostructuresof~30nm(1)andisnotnecessaryforoctamerornucleosomecoreparticleformation.
EightdifferenthistoneH1proteinshavebeenidentifiedinthehumangenome(2).
HistoneH1°isanonreplication-dependenthistonethatishighlyexpressedincellsthathaveterminallydifferentiated(3).
RecombinanthumanhistoneH1fromNEBisexpressedinE.
coliusingtheH1F0orH1FVgene(Genbankaccessionnumber:X03473).
HistoneH1Human,RecombinantM2501SHistonesH2A&H2BHistoneH2AinteractswithhistoneH2BtoformtheH2A/H2Bheterodimer.
TwoH2A/H2BheterodimersinteractwithanH3/H4tetramertoformthehistoneoctamer(1,4).
HistonesH2AandH2Baremodifiedbyvariousenzymesandhavebeenshowntobeimportantingenetranscription(5).
RecombinanthumanhistonesH2AandH2BareexpressedinE.
coliusingtheHIST3H2Agene(Genbankaccessionnumber:AY131974)andtheHIST2H2BEorH2BFQgene(Genbankaccessionnumber:AY131979),respectively.
NEBalsooffersthepreformedhistoneH2A/H2Bdimer.
Thisisgeneratedbyrefoldingthedenatured,purifiedsubunitsH2AandH2B,followedbygelfiltration.
HistoneH2AHuman,RecombinantM2502SHistoneH2BHuman,RecombinantM2505SHistoneH2A/H2BDimerHuman,RecombinantM2508SReferences1.
vanHolde,K.
E.
(1989)Chromatin,1–497.
2.
Gongidi,P.
,etal.
(2002)Genomics,80,487–497.
3.
Pehrson,J.
R.
andCole,R.
D.
(1982)Biochem,21,456–460.
4.
Kornberg,R.
D.
(1977)Annu.
Rev.
Biochem.
,46,931-954.
5.
Wyrick,J.
J.
andParra,M.
A.
(2009)BiochimBiophysActa,1789,37–44.
MassSpectroscopyAnalysisofHistoneH1°Human,RecombinantMassSpectroscopyAnalysisofHistoneH2AHuman,Recombinant13,788.
5010,00012,00014,00016,00018,000Mass(Da)Intensity,Counts(x106)1.
02.
03.
04.
00.
0MassSpectroscopyAnalysisofHistoneH2BHuman,Recombinant17,00019,00021,00023,00025,000DeconvolutedMass(Da)20,731.
8520,862.
87Intensity,Counts(x105)0243176513,990.
2714,032.
720102030405060708090100%Intensity10,00012,00014,00016,00018,000Mass,Da19HISTONESHistonesH3&H4HistoneH3interactswithhistoneH4toformtheH3/H4tetramer.
TwoH2A/H2Bhet-erodimersinteractwithanH3/H4tetramertoformthehistoneoctamer(1,2).
HistoneH3.
1,anH3variantthathasthusfaronlybeenfoundinmammals,isreplica-tion-dependentandisassociatedwithgeneactivationandgenesilencing(3).
HistoneH3.
2,anH3variantthatisfoundinalleukaryotes,exceptbuddingyeast,isreplication-dependentandisassociatedwithgenesilencing(4).
HistoneH3.
3,anH3variantthatisfoundinalleukaryotesfromyeasttohuman,isreplicationandcellcyclephase-inde-pendentandisthemostcommonH3innon-dividingcells(5).
Ithasbeenshowntobeenrichedincovalentmodificationsassociatedwithgeneactivation(4,6).
RecombinanthumanhistonesH3.
1,H3.
2andH3.
3aresynthesizedinE.
coliusingtheHIST1H3AorH3FAgene(Genbankaccessionnumber:AF531274),HIST2H3AorHIST2H3Cgene(Genbankaccessionnumber:BC130637)andH3F3AorH3F3Bgene(Genbankaccessionnumber:AK311905),respectively.
Recombinanthumanhis-toneH4issynthesizedinE.
coliusingtheHIST2H4gene(Genbankaccessionnumber:AF525682).
NEBalsoofferspreformedrecombinanthistoneH3.
1/H4tetramer.
Thisisgeneratedbyrefoldingthedenatured,purifiedsubunitsH3.
1andH4,followedbygelfiltration.
HistoneH3.
1Human,RecombinantM2503SHistoneH3.
2Human,RecombinantM2506SHistoneH3.
3Human,RecombinantM2507SHistoneH4Human,RecombinantM2504SHistoneH3.
1/H4TetramerHuman,RecombinantM2509SReferences1.
Kornberg,R.
D.
(1977)Annu.
Rev.
Biochem.
,46,931–954.
2.
vanHolde,K.
E.
(1989)Chromatin,1–497.
3.
Gill,S.
C.
andvonHippel,P.
H.
(1989)Anal.
Biochem.
,182,319–326.
4.
Hake,S.
B.
etal.
(2006)J.
Biol.
Chem.
,281,559–568.
5.
Gabriellietal.
(1984)Mol.
Cell.
Biochem.
,65,57–66.
6.
Henikoff,S.
etal.
(2004)PNAS,101,1525–1530.
MassSpectroscopyAnalysisofHistoneH3Human,RecombinantMassSpectroscopyAnalysisofHistoneH4Human,RecombinantMass,Da6,0008,00010,00012,00014,00016,0000102030%Intensity10090807060504011,235.
740102030405060708090100%IntensityMass,Da15258.
0615,00010,00013,00017,00019,0000102030405060708090100%Intensity15196.
9615239.
38Mass,Da12,00014,00016,00018,00020,000H3.
1H3.
2H3.
3%Intensity10090807060504030201015,273.
2010,00012,00014,00016,00018,000Mass,Da020,00020HISTONEMODIFICATIONSHistoneModificationsThecorehistonesconsistofaglobularC-terminaldomainandanunstructuredN-terminaltail.
Althoughavarietyofmodificationsoccurthroughoutthehistoneprotein(seeTable1),theyoccurprimarilyontheN-terminaltail(1-5).
Throughtheirpotentialcombinatorialmodificationonagivenhistoneanditsreversibility,thesemodificationsdynamicallyrestrictorrecruitnumerousotherproteinsorproteincomplexesontochromatin(5).
Thestudyoftheirrolesingeneregulation(6),cellularstressevents(6),agingandDNArepair(7)isrevealingthemultiplefunctionsofhistonemodificationsindeterminingthefateofacell.
Additionalvariabilityisincorporatedintothesystembyhistonevariants.
ActingindividuallyorinconjunctionwithDNAmodification,histonemodificationsandhistonevariantsarethoughttoestablishanepigeneticcodeorepigeneticsignatureforgeneregulation(5).
AMINOACIDMODIFICATIONLysineMethylation,Acetylation,Ubiquitination,Sumoylation,ADP-RibosylationArginineMethylationSerinePhosphorylationThreoninePhosphorylationTable1:TypesofHistoneModificationsOneofthemostwidelyusedmethodsforstudyinghistonemodificationsinvivoischromatinimmunoprecipitation(ChIP).
Inbrief,proteinandDNAaregenerallycross-linkedbyformaldehydetreatment.
Afterthechromatinisfragmentedbysonication,antibodiesspecificforahistonemodificationorchromatinbindingproteinareusedtoimmunoprecipitatetheDNA.
ThehistonesfromNEBcanbeusedascarrierchromatininCChIP(CarrierChromatinImmunoPrecipitation)assays(8).
Forlarge-scaleanalyses,theisolatedDNAcanbeanalyzedonamicroarray(ChIP-chip)orbysequencing(ChIP-seq,seepage20).
ThelimitationsoftraditionalChIP(e.
g.
,qualityoftheantibody,biasfromfixationandfragmentation,andinterferencefromotherhistone-bindingproteins)arepartiallyaddressedbyalternativemethods,suchasN-ChIP(Native-ChIP),biotin-tagaffinitypurification,andDamID(reviewedin9).
MethodsforStudyingHistoneModificationsReferences1.
Kouzarides,T.
(2007)Cell128,693–705.
2.
Santos-Rosa,H.
andCaldas,C.
(2005)Eur.
J.
Cancer41,2381–2402.
3.
Peterson,C.
L.
andLaniel,M.
A.
(2004)Curr.
Biol.
14,R546–R551.
4.
Bhaumik,S.
R.
,etal.
(2007)Nat.
Struct.
Mol.
Biol.
14,1008–1016.
5.
Kim,J.
K.
,Samaranayake,M.
andPradhan,S.
(2009)Cell.
Mol.
LifeSci.
,66,596–612.
6.
Huang,J.
etal.
(2006)Nature,444,629–632.
7.
Pahlich,S.
,Zakaryan,R.
P.
andGehring,H.
(2006)Biochim.
Biophys.
Acta.
,1764,1890–1903.
8.
O'Neill,L.
P.
,etal.
(2006)Nat.
Genet.
,38,835–841.
9.
Bernstein,B.
E.
,etal.
(2007)Cell128,669-681.
21PROTEINMETHYLTRANSFERASESHistoneMethyltransferasesLysineorarginineresiduesinhistonesundergoenzymaticmethylationviatheattachmentofone,twoorthreemethylgroups.
Thetimingoftheappearanceofthesemodificationsisoftendynamicandwilldependonthesignalingconditionofthecell.
Histonemodifi-cationsparticipateintranscription,repair,replicationandchromatincondensation.
NEBoffersaselectionofproteinmethyltransferasesspecificforhistoneH3.
1,H3.
2,H3.
3andhistoneH4.
G9aMethyltransferaseG9amethyltransferasemethylateslysine9(Lys9)ofhistoneH3(1-3).
Methylationoccursattheεaminogroupoflysineresidues.
MethylationofhistoneH3Lys9isahallmarkofsilentchromatinandisgloballydistributedthroughouttheheterochromaticregions,suchascentromeresandtelomeres(4,5).
TheG9aenzymefromNEBisexpressedfrommouseG9acDNA(1,2).
HumanPRMT1MethyltransferasePRMT1isamajorproteinargininemethyltransferase(6).
Itspecificallymethylatesargi-nine3(Arg3)ofhistoneH4.
Furthermore,methylationofhistoneH4atArg3facilitatestranscriptionalactivationbynuclearhormonereceptors(7).
Inaddition,theorderedcoop-erativefunctionsofPRMT1,p300andCARM1intranscriptionalactivationbyp53isobservedontheGADD45genefollowingectopicp53expressionand/orUVirradiation(8).
ThePRMT1enzymefromNEBisexpressedfromratPMRT1cDNA.
SET7MethyltransferaseSET7Methyltransferasemethylateslysine4(Lys4)ofhistoneH3(9).
Methylationoccursattheεaminogroupoflysineresidues(10,11).
Di-andtri-methylationofhistoneH3Lys4isahallmarkoftranscriptionallyactivechromatinandisgloballydistributed(12,13).
TheSET7enzymefromNEBisexpressedfromhumanSET7cDNA.
SET8MethyltransferaseSET8(PR-Set7)Methyltransferasemono-methylateslysine20ofhistoneH4(H4-K20)attheεaminogroupoflysineresidues.
SET8-mediatedhistoneH4methylationisimplicatedingenomereplicationandstability;andplaysanimportantroleinthenodalpathwaysofembryodevelopment.
HumanDNA(cytosine-5)Methyltransferase(DNMT1)DNMT1methylatescytosineresiduesinhemimethylatedDNAat5…CG…3sites(14,15).
MammalianDNAmethylationaffordedbyDNMT1isinvolvedincarcinogenesis,embryonicdevelopmentandseveralotherbiologicalfunctions(16-18).
G9aMethyltransferaseM0235SHumanPRMT1MethyltransferaseM0221SSET7MethyltransferaseM0233SSET8MethyltransferaseM0428SHumanDNA(cytosine-5)Methyltransferase(DNMT1)M0230S/LReferences1.
Tachibana,M.
etal.
(2001)J.
Biol.
Chem.
,276,25309–25317.
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Patnaik,Detal.
(2004)J.
Biol.
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,279,53248–53258.
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Esteve,P.
O.
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(2005)Nucl.
AcidsRes.
,3211–3223.
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Strahl,B.
D.
andAllis,C.
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(2000)Nature,403,41–45.
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Noma,K.
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(2001)Science,293,1150–1155.
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Tang,J.
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(2000)J.
Biol.
Chem.
,275,7723–7730.
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Wang,H.
etal.
(2001)Science,293,853–857.
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An,W.
etal.
(2004)Cell,117,735–748.
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Wang,H.
etal.
(2001)MolCell,6,1207–1217.
10.
Xiao,B.
etal.
(2003)Nature,421,652–656.
11.
Wilson,J.
R.
etal.
(2002)Cell,111,105–115.
12.
Schneider,R.
etal.
(2004)Nat.
CellBiol.
,6,73–77.
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Santos-Rosa,H.
etal.
(2002)Nature,419,407–411.
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Pradhan,S.
etal.
(1999)J.
Biol.
Chem.
,274,33002–33010.
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Bacolla,A.
etal.
(1999)J.
Biol.
Chem.
,274,33011–33019.
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Schmutte,C.
etal.
(1998)Biol.
Chem.
,379,377–388.
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Laird,P.
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(1995)Cell,81,197–205.
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Li,E.
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(1992)Cell,12,915–926.
APPLICATIONSPurificationandcharacterizationofenzymesthatmodifyhistoneproteinsOctamermodificationstudiesCarrierChromatinImmunoprecipitation(CChIP)22ORDERINGINFORMATIONPRODUCTNEB#SIZEMETHYLOMEANALYSISNEBNextEnzymaticMethyl-seq(EM-seq)KitE712024/96reactionsNEBNextEnzymaticMethyl-seqModuleE712524/96reactionsNEBNextMultiplexOligosforEnzymaticMethyl-seq(UniqueDualIndexPrimerPairs)E7140S/L24/96reactionsEpiMark5-hmCand5-mCAnalysisKitE3317S20reactionsEpiMarkBisulfiteConversionKitE3318S48reactionsEpiMarkHotStartTaqDNAPolymeraseM0490S/L100/500reactionsEpiMarkN6-MethyladenosineEnrichmentKitE1610S20reactionsEpiMarkMethylatedDNAEnrichmentKitE2600S25reactionsAbaSIR0665S1,000unitsDpnIR0176S/L1,000/5,000unitsDpnIIR0543S/T/L/M1,000/1,000/5,000/5,000unitsFspEIR0662S/L200/1,000unitsHpaIIR0171S/M/L2,000/10,000/10,000unitsLpnPIR0663S/L200/1,000unitsMspIR0106S/T/M/L5,000/5,000/25,000/25,000unitsMspJIR0661S/L200/1,000units5-Methyl-dCTPN0356S1μmolMcrBCM0272S/L500/2,500unitsMETHYLTRANSFERASES&ANTIBODIESG9aMethyltransferaseM0235S100unitsPRMT1MethyltransferaseM0221S50unitsSET7MethyltransferaseM0233S100unitsSET8MethyltransferaseM0428S100unitsHumanDNA(cytosine-5)Methyltransferase(Dnmt1)M0230S/L50/250unitsCpGMethyltransferase(M.
SssI)M0226S/M/L100/500/500unitsGpCMethyltransferase(M.
CviPI)M0227S/L200/1,000unitsHpaIIMethyltransferaseM0214S/L100/500unitsMspIMethyltransferaseM0215S/L100/500unitsEcoRIMethyltransferaseM0211S/L10,000/50,000unitsdamMethyltransferaseM0222S/L500/2,500unitsBamHIMethyltransferaseM0223S/L100/500unitsHhaIMethyltransferaseM0217S/L1,000/5,000unitsTaqIMethyltransferaseM0219S/L1,000/5,000unitsAluIMethyltransferaseM0220S/L100/500unitsHaeIIIMethyltransferaseM0224S/L500/2,500unitsSAMPLEPREPFORChIP-SEQNEBNextUltraIIDNALibraryPrepKitforIlluminaE7645S/L24/96reactionsNEBNextUltraIIDNALibraryPrepwithSamplePurificationBeadsE7103S/L24/96reactionsNEBNextMultiplexOligosforIllumina(IndexPrimersSet1)E7335S/L24/96reactionsNEBNextMultiplexOligosforIllumina(IndexPrimersSet2)E7500S/L24/96reactionsNEBNextMultiplexOligosforIllumina(IndexPrimersSet3)E7710S/L24/96reactionsNEBNextMultiplexOligosforIllumina(IndexPrimersSet4)E7730S/L24/96reactionsPRODUCTNEB#SIZENEBNextMultiplexOligosforIllumina(96IndexPrimers)E6609S/L96/384reactionsNEBNextMultiplexOligosforIllumina(96UniqueDualIndexPrimerPairs)E6440S/L96/384reactionsNEBNextMultiplexOligosforIllumina(96UniqueDualIndexPrimerPairsSet2)E6442S/L96/384reactionsNEBNextMultiplexOligosforIllumina(MethylatedAdaptor,IndexPrimersSet1)E7535S/L24/96reactionsNEBNextMultiplexOligosforIllumina(DualIndexPrimersSet1)E7600S96reactionsNEBNextMultiplexOligosforIllumina(DualIndexPrimersSet2)E7780S96reactionsNEBNextUltraIIEndRepair/dA-TailingModuleE7546S/L24/96reactionsNEBNextUltraIILigationModuleE7595S/L24/96reactionsNEBNextUltraIIQ5MasterMixM0544S/L50/250reactionsCONTROLDNACpGMethylatedJurkatGenomicDNAN4002S15g5-Aza-dcTreatedJurkatGenomicDNAN4003S15gNIH3T3MouseGenomicDNAN4004S15gHeLaGenomicDNAN4006S15gCpGMethylatedHeLaGenomicDNAN4007S15gHISTONESEpiMarkNucleosomeAssemblyKitE5350S20reactionsH10Human,RecombinantM2501S100gH2AHuman,RecombinantM2502S100gH2BHuman,RecombinantM2505S100gH3.
1Human,RecombinantM2503S100gH3.
2Human,RecombinantM2506S100gH3.
3Human,RecombinantM2507S100gH4Human,RecombinantM2504S100gHistoneH3.
1/H4TetramerHuman,RecombinantM2509S1nmolHistoneH2A/H2BDimerHuman,RecombinantM2508S2nmolNucleosomeControlDNAN1202S0.
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