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    NEB//E7760S/96 reactions
    • NEB//E7760S/96 reactions

    NEB//E7760S/96 reactions

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    貨號: E7760S-96reactions
    品牌: NEB
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      • Description:

        Ultra II Sample
        ?
        ?
        TheUltraIIDirectionalRNALibraryPrepKitforIlluminadeliverssignificantlyincreasedsensitivityandspecificityfromyourRNA-seqexperiments,fromever-decreasingamountsofinputRNA.InconjunctionwithribosomalRNA(rRNA)depletionorpoly(A)enrichment,thekitenablestheproductionofhighqualitylibrariesfrom5ngor10ngofTotalRNA,respectively,upto1μg.

        Strand-specific/directionalmethodsforsequencingRNAprovideinformationontheDNAstrandfromwhichtheRNAstrandwastranscribed.Thisisusefulformanyreasonsincluding:Identificationofantisensetranscripts,determinationofthetranscribedstrandofnoncodingRNA,andmeasurementofexpressionlevelsofcodingornoncodingoverlappingtranscripts.Overall,theABIlitytodeterminetheoriginatingstrandcansubstantiallyenhancethevalueofaRNA-seqexperiment.

        TheNEBNextUltraIIDirectionalRNALibraryPrepKitderivesitsdirectionalityfromthe“dUTP”methodforstrand-specificity,withprovensuperiorityforthisapplication.

        SeewhatcustomersaresayingaboutNEBNextUltraIIDirectionalRNA.

        Features
        • Getmoreofwhatyouneed,withthehighestlibraryyields
        • GeneratehighqualitylibrariesevenwhenyouhaveonlylimitedamountsofinputRNA:
          • 10ng–1μgTotalRNA(polyAmRNAworkflow)
          • 5ng–1μgTotalRNA(rRNAdepletionworkflow)
        • Minimizebias,withfewerPCRcyclesrequired
        • Increasethecomplexityandtranscriptcoverageofyourlibraries
        • Optimizeyourtimewithstreamlinedworkflows,reducedhands-ontime,andautomationcompatibility
        • Relyonrobustperformance,evenwithlowqualityRNA,includingFFPE
        • CompatIBLewithNEBNextpoly(A)mRNAIsolation,rRNADepletionreagentsandmultiplexingadaptorsandprimers.
        AlsoavailablewithoptionalSPRIselect?beadsforclean-upandsize-selectionsteps.

        Pleasenotethatadaptors,primers,rRNAdepletionreagentsandpoly(A)mRNAisolationreagentsarenotincludedinthekitandareavailableseparately.

        ForextensiveNEBNextUltraIIperformancedata,clickthelinksintheFeaturesaboveanddownloadourtechnicalnoteforpoly(A)mRNAisolationorourtechnicalnoteforrRNAdepletion.

        LIBRARYYIELDS

        Figure1.NEBNextUltraIIDirectionalRNAproducesthehighestyields,fromarangeofinputamounts
        Yield
        Poly(A)-containingmRNAwasisolatedfromHumanUniversalReferenceRNA(Agilent#740000),andlibrariesweremadeusingtheNEBNextUltraIIDirectionalRNAKit(plustheNEBNextPoly(A)mRNAMagneticIsolationModule),KapaStrandedmRNA-SeqKit,KapamRNAHyperPrepKitandIlluminaTruSeqStrandedmRNAKit.TheinputRNAamountandnumberofPCRcyclesareindicated.Libraryyieldsfromanaverageofthreereplicatesareshown.

        Viewadditionaldataonlibraryyields.



        GCCONTENTDISTRIBUTION

        Figure2.NEBNextUltraIIDirectionalRNAlibrariesprovideuniformGCcontentdistribution,atabroadrangeofinputamounts
        CG Plot
        Poly(A)-containingmRNAwasisolatedfromHumanUniversalReferenceRNA(Agilent#740000),andlibrariesweremadeusingtheNEBNextUltraIIDirectionalRNAKit(plustheNEBNextPoly(A)mRNAMagneticIsolationModule),IlluminaTruSeqStrandedmRNAKit,KapaStrandedmRNA-SeqKitandKapamRNAHyperPrepKit.LibrariesweresequencedonanIlluminaNextSeq?500usingpaired-endmode(2x76bp).Readsweremappedtothehg19referencegenome.GCcontentdistributionforeachlibrarywascalculatedusingmappedreads.UltraIIDirectionalRNAlibrarieshaduniformGCcontentdistributionacrossarangeofinputamounts,whereasforotherkitstheGCcontentdistributionchangedwithdifferentinputamounts,indicatingtheintroductionofinput-dependentsequencebias.

        Viewadditionaldataonlibraryquality.



        MAXIMIZINGTRANSCRIPTCOVERAGE

        Figure3.NEBNextUltraIIDirectionalRNAlibrariesprovideuniformcoverageacrossthegenebodyoftranscripts
        coverage
        Poly(A)-containingmRNAwasisolatedfromHumanUniversalReferenceRNA(Agilent#740000),andlibrariesweremadeusingtheNEBNextUltraIIDirectionalRNAKit(plustheNEBNextPoly(A)mRNAMagneticIsolationModule),IlluminaTruSeqStrandedmRNAKit,KapaStrandedmRNA-SeqKitandKapamRNAHyperPrepKit.LibrariesweresequencedonanIlluminaNextSeq?500usingpaired-endmode(2x76bp).Thisviewofthe5′to3′coverageofRefSeqtranscriptsrevealsconsistentcoverageforUltraIIDirectionalRNAlibrariesasinputRNAisdecreasedfrom1μgto10ng.Thechangesapparentinotherkitsresultfromlossofcoverageatthe3′endofsometranscripts.

        Viewadditionaldataontranscriptcoverage.



        SUPERIORLIBRARYCOMPLEXITYATLOWINPUTAMOUNTS

        Figure4.LowinputNEBNextUltraIIDirectionalRNAlibrariesretainsuperiorcomplexity
        Transcription
        Poly(A)-containingmRNAwasisolatedfromHumanUniversalReferenceRNA(Agilent#740000),andlibrariesweremadeusingtheNEBNextUltraIIDirectionalRNAKit(plustheNEBNextPoly(A)mRNAMagneticIsolationModule),IlluminaTruSeqStrandedmRNAKit,KapaStrandedmRNA-SeqKitandKapamRNAHyperPrepKit.LibrariesweresequencedonanIlluminaNextSeq?500usingpaired-endmode(2x76bp).Salmon0.4.0wasusedforreadmappingandquantificationofallGENCODEv25transcripts.TPM=TranscriptsPerKilobaseMillion.R2valuesforthelinearfitareshown.CorrelationanalysisofthetranscriptsindicatessuperiortranscriptexpressioncorrelationbetweenthedifferentinputsforUltraIIDirectionalRNAlibraries.
        Viewadditionaldataonlibrarycomplexity.


        SUPERIORPERFORMANCEWITHFFPERNA

        Figure5.NEBNextUltraIIDirectionalRNAwithNEBNextrRNADepletionresultsinthelowestremainingribosomalRNAlevelswithFFPEsamples
        FFPE low
        RibosomalRNAwasdepletedfromhumanadultnormallivertissueFFPETotalRNA(Biochain#R2234149.RIN2.5)andlibrariesweremadeusingNEBNextUltraIIDirectionalRNAKit(plustheNEBNextrRNADepletionKit(Human/Mouse/Rat)),KapaStrandedRNA-SeqKitwithRiboErase,KapaHyperPrepKitwithRiboErase,andIlluminaTruSeqStrandedTotalRNALibraryPrepKitwithRibo-Zero?Gold.LibrariesweresequencedonanIlluminaNextSeq?500usingpaired-endmode(2x76bp).ReadpairswereassessedtoberRNAiftheycontain6ormore32basematchesto18S,28S,5S,5.8S,16Sor12ShumanrRNAsequences(mirabait4.9).PercentrRNAremainingwascalculatedbydividingrRNAreadsbythetotalnumberofreadspassinginstrumentqualityfiltering.AveragepercentrRNAremainingisshownforthreereplicates.TheNEBNextrRNADepletionUltraIIDirectionalRNAworkflowisthemostefficientinremovingrRNAfromtotalFFPERNA.


        Figure6.UniformityofCoverageacrosstheAP000769.1-201transcript
        FFPR ERRC
        RibosomalRNAwasdepletedfromhumanadultnormallivertissueFFPETotalRNA(Biochain#R2234149.RIN2.5),andlibrariesweremadeusingNEBNextUltraIIDirectionalRNAKit(plustheNEBNextrRNADepletionKit(Human/Mouse/Rat)),IlluminaTruSeqStrandedTotalRNALibraryPrepKitwithRibo-Zero?Gold,KapaStrandedRNA-SeqKitwithRiboEraseandKapaHyperPrepKitwithRiboErase.LibrariesweresequencedonanIlluminaNextSeq?500usingpaired-endmode(2x76bp).Coverageacrossthelengthofthisindividualtranscript(ENST00000625158.1;AP000769.1-201)wasassessedbymappingreadsdirectlytotheGENCODEv25transcriptsandexamining100binsalongthetranscriptlength.NEBNextUltraIIDirectionalRNAlibrariesprovidedcoverageacrosstheentirelengthofthetranscriptevenasinputwasdecreasedfrom100ngto10ng.

        ViewadditionaldataonFFPERNAsamples.

        KitComponents

        Thefollowingreagentsaresuppliedwiththisproduct:

        Storeat(°C)Concentration
        NEBNext?LigationEnhancer-20
        NEBNextFirstStrandSynthesisReactionBuffer-20
        RandomPrimers-20
        NEBNextSecondStrandSynthesisEnzymeMix-20
        NEBNextSecondStrandSynthesisReactionBufferwithdUTPMix-2010X
        NEBNextUSER?Enzyme-20
        NEBNextUltraIIEndPrepEnzymeMix-20
        NEBNextUltraIIEndPrepReactionBuffer-20
        NEBNext?Ultra?IILigationMasterMix-20
        NEBNext?UltraIIQ5?MasterMix-202X
        NEBNextFirstStrandSynthesisEnzymeMix-20
        NEBNextAdaptorDilutionBuffer-20
        (0.1X)TEBuffer-200.1X
        Nuclease-freeWater-20
        NEBNextStrandSpecificityReagent-20
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