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NEB//E7765S/96 reactions

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    • Description:

      Ultra II Sample
      ?
      ?

      UltraIIDirectionalRNALibraryPrepwithSamplePurificationBeadsdeliverssignificantlyincreasedsensitivityandspecificityfromyourRNA-seqexperiments,fromever-decreasingamountsofinputRNA.InconjunctionwithribosomalRNA(rRNA)depletionorpoly(A)enrichment,thekitenablestheproductionofhighqualitylibrariesfrom5ngor10ngofTotalRNA,respectively,upto1μg.

      ThiskitcontainsNEBNextSamplePurificationBeads(SPRIselect?beadsfromBeckmanCoulter)forsizeselectionandenzymereactioncleanup.

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

      TheNEBNextUltraIIDirectionalRNALibraryPrepKitderivesitsdirectionalityfromthe“dUTP”methodforstrand-specificity,withprovensuperiorityforthisapplication.


      Features

      • Getmoreofwhatyouneed,withthehighestlibraryyields
      • GeneratehighqualitylibrariesevenwhenyouhaveonlylimitedamountsofinputRNA:
        • 10ng–1μgTotalRNA(polyAmRNAworkflow)
        • 5ng–1μgTotalRNA(rRNAdepletionworkflow)
      • Minimizebias,withfewerPCRcyclesrequired
      • Increasethecomplexityandtranscriptcoverageofyourlibraries
      • Optimizeyourtimewithstreamlinedworkflows,reducedhands-ontime,andautomationcompatibility
      • Relyonrobustperformance,evenwithlowqualityRNA,includingFFPE
      • EnjoytheflexibilityandreliabilityofthegoldstandardSPRIselectsizeselectionandclean-upbeads,suppliedinjusttheamountsyouneed
      AlsoavailablewithoutSPRIselect?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
      NEBNextFirstStrandSynthesisEnzymeMix-20
      NEBNextStrandSpecificityReagent-20
      NEBNextSecondStrandSynthesisReactionBufferwithdUTPMix-2010X
      NEBNextUltraIIEndPrepEnzymeMix-20
      NEBNextUltraIIEndPrepReactionBuffer-20
      NEBNext?Ultra?IILigationMasterMix-20
      NEBNext?LigationEnhancer-20
      NEBNextUSER?Enzyme-20
      NEBNext?UltraIIQ5?MasterMix-202X
      NEBNextAdaptorDilutionBuffer-20
      NEBNext?SamplePurificationBeads25
      NEBNextFirstStrandSynthesisReactionBuffer-20
      RandomPrimers-20
      NEBNextSecondStrandSynthesisEnzymeMix-20
      (0.1X)TEBuffer-200.1X
      Nuclease-freeWater-20
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