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NEB//E7770S/96 reactions

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    • Description:

      Ultra II Sample
      ?
      ?

      TheUltraIIRNALibraryPrepKitforIlluminaisdesignedfornon-directional(non-strand-specific)RNAlibraryconstruction,anddeliverssignificantlyincreasedsensitivityandspecificityfromyourRNA-seqexperiments,fromever-decreasingamountsofinputRNA.InconjunctionwithribosomalRNA(rRNA)depletionorpoly(A)mRNAenrichment,thekitenablestheproductionofhighqualitylibrariesfrom5ngor10ngofTotalRNA,respectively,upto1μg.

      Fordirectional(strand-specific)RNAlibrarypreparation,seetheNEBNextUltraIIDirectionalRNALibraryPrepkitforIllumina.

      Features

      • Getmoreofwhatyouneed,withthehighestlibraryyields
      • GeneratehighqualitylibrariesevenwhenyouhaveonlylimitedamountsofinputRNA:
        • 10ng–1μgTotalRNA(polyAmRNAworkflow)
        • 5ng–1μgTotalRNA(rRNAdepletionworkflow)
      • Minimizebias,withfewerPCRcyclesrequired
      • Increasethecomplexityandtranscriptcoverageofyourlibraries
      • Optimizeyourtimewithstreamlinedworkflows,reducedhands-ontime,andautomationcompatibility
      • Relyonrobustperformance
      • CompatIBLewithNEBNextpoly(A)mRNAIsolation,rRNADepletionreagentsandmultiplexingadaptorsandprimers

      Alsoavailablewith?optionalSPRIselect?beads.

      ForextensiveNEBNextUltraIIperformancedata,clickthelinksintheFeaturesaboveanddownloadourtechnicalnoteforpoly(A)mRNAisolationorourtechnicalnoteforrRNAdepletion.


      LIBRARYYIELDS

      Figure1.NEBNextUltraIIRNAproducesthehighestyields,fromarangeofinputamounts
      Yield
      Poly(A)-containingmRNAwasisolatedfromHumanUniversalReferenceRNA(Agilent#740000)andlibrarieswerepreparedusingtheNEBNextUltraIIRNAKit(plustheNEBNextPoly(A)mRNAMagneticIsolationModule),andtheIlluminaTruSeqRNASamplePreparationKitv2.InputRNAandnumberofPCRcyclesareindicated.Libraryyieldsfromanaverageofthreereplicatesareshown.

      Viewadditionaldataonlibraryyields


      DUPLICATIONRATES

      Figure2.NEBNextUltraIIRNAwithNEBNextrRNADepletionresultsinlowerduplicationrates.
      Duplication
      RibosomalRNAwasdepletedfrom1μg,100ngand5ngofHumanUniversalReferenceRNA(Agilent#740000)usingtheNEBNextrRNADepletionKit(Human/Mouse/Rat)orIlluminaRibo-Zero?GoldrRNARemovalKit(Human/Mouse/Rat).LibrarieswerethenpreparedusingtheNEBNextUltraIIRNAKitortheIlluminaTruSeqRNALibraryPrepKitv2,respectively.5nginputwastestedonlywiththeNEBNextkits.LibrariesweresequencedonanIlluminaNextSeq?500usingpaired-endmode(2x76bp).Readsweredownsampledto10millionreadpairsandmappedtothehg19referencegenome.Duplicationrateswerecalculatedasafractionofuniquelymappedreadsusingthe‘ReadDuplication’toolofRSeQCwherereadsmappingtothesamegenomiclocationareregardedasduplicatedreads.

      Viewadditionaldataonlibraryquality


      MAXIMIZINGTRANSCRIPTCOVERAGE

      Figure3.NEBNextUltraIIRNAlibrariesprovideuniformcoverageacrossthegenebodyoftranscripts
      Coverage
      Poly(A)-containingmRNAwasisolatedfromHumanUniversalReferenceRNA(Agilent#740000),andlibrarieswerepreparedusingtheNEBNextUltraIIRNAKit(plustheNEBNextPoly(A)mRNAMagneticIsolationModule),andtheIlluminaTruSeqRNASamplePreparationKitv2.InputRNAamountisindicated.LibrariesweresequencedonanIllumina?NextSeq?500usingpaired-endmode(2x76bp).Thisviewofthe5′to3′coverageofRefSeqtranscriptsrevealsconsistentcoverageforUltraIIRNAlibrariesasinputRNAisdecreasedfrom1μgto10ng.ThechangesapparentinTheTruSeqkitresultsfromlossofcoverageatthe3′endofsometranscripts.

      Viewadditionaldataontranscriptcoverage


      EXCELLENTLIBRARYCOMPLEXITYATLOWINPUTAMOUNTS

      Figure4.LowinputNEBNextUltraIIRNAlibrariesretaincomplexityevenatlowinputamounts
      Complexity
      RibosomalRNAwasdepletedfrom1μg,100ngand5ngofHumanUniversalReferenceRNA(Agilent#740000)withrecommendedamountsofERCCRNASpike-InMixI(ThermoFisherScientific#4456740)usingtheNEBNextrRNADepletionKit(Human/Mouse/Rat)andlibrarieswerethenpreparedusingtheNEBNextUltraIIRNAKit.LibrariesweresequencedonanIlluminaNextSeq?500usingpaired-endmode(2x76bp).Salmon0.4.0wasusedforreadmappingandquantificationofallERCCtranscripts.R2valuesforlinearfitareshown.TPM(TranscriptsPerKilobaseMillion)correlationanalysisofthetranscriptsindicatesexcellenttranscriptexpressioncorrelationbetweenthedifferentinputsforUltraIIRNAlibraries(A),includingERCCtranscripts(B).

      Viewadditionaldataonlibrarycomplexity

      KitComponents

      Thefollowingreagentsaresuppliedwiththisproduct:

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