Description: TheUltraIIRNALibraryPrepKitforIllumina isdesignedfornon-directional(non-strand-specific)RNAlibraryconstruction,anddeliverssignificantlyincreasedsensitivityandspecificityfromyourRNA-seqexperiments,fromever-decreasingamountsofinputRNA.InconjunctionwithribosomalRNA(rRNA)depletionorpoly(A)mRNAenrichment,thekitenablestheproductionofhighqualitylibrariesfrom5ngor10ngofTotalRNA,respectively,upto1µg.
Fordirectional(strand-specific)RNAlibrarypreparation,seetheNEB NextUltraIIDirectionalRNALibraryPrepkitforIllumina.
Features
Getmoreofwhatyouneed,withthehighestlibraryyields GeneratehighqualitylibrariesevenwhenyouhaveonlylimitedamountsofinputRNA: 10ng–1µgTotalRNA(polyAmRNAworkflow) 5ng–1µgTotalRNA(rRNAdepletionworkflow) Minimizebias,withfewerPCRcyclesrequired Increasethecomplexityandtranscriptcoverageofyourlibraries Optimizeyourtimewithstreamlinedworkflows,reducedhands-ontime,andautomationcompatibility Relyonrobustperformance CompatIBL ewithNEBNextpoly(A)mRNAIsolation,rRNADepletionreagentsandmultiplexingadaptorsandprimers Alsoavailablewith optionalSPRIselect®beads. ForextensiveNEBNextUltraIIperformancedata,clickthelinksintheFeaturesaboveanddownloadourtechnicalnoteforpoly(A)mRNAisolationorourtechnicalnoteforrRNAdepletion. LIBRARYYIELDS
Figure1.NEBNextUltraIIRNAproducesthehighestyields,fromarangeofinputamounts Poly(A)-containingmRNAwasisolatedfromHumanUniversalReferenceRNA(Agilent #740000)andlibrarieswerepreparedusingtheNEBNextUltraIIRNAKit(plustheNEBNextPoly(A)mRNAMagneticIsolationModule),andtheIlluminaTruSeqRNASamplePreparationKitv2.InputRNAandnumberofPCRcyclesareindicated.Libraryyieldsfromanaverageofthreereplicatesareshown. Viewadditionaldataonlibraryyields
DUPLICATIONRATES Figure2.NEBNextUltraIIRNAwithNEBNextrRNADepletionresultsinlowerduplicationrates. 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 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 RibosomalRNAwasdepletedfrom1μg,100ngand5ngofHumanUniversalReferenceRNA(Agilent#740000)withrecommendedamountsofERCCRNASpike-InMixI(Thermo FisherScientific#4456740)usingtheNEBNextrRNADepletionKit(Human/Mouse/Rat)andlibrarieswerethenpreparedusingtheNEBNextUltraIIRNAKit.LibrariesweresequencedonanIlluminaNextSeq®500usingpaired-endmode(2x76bp).Salmon0.4.0wasusedforreadmappingandquantificationofallERCCtranscripts.R2 valuesforlinearfitareshown.TPM(TranscriptsPerKilobaseMillion)correlationanalysisofthetranscriptsindicatesexcellenttranscriptexpressioncorrelationbetweenthedifferentinputsforUltraIIRNAlibraries(A),includingERCCtranscripts(B). Viewadditionaldataonlibrarycomplexity
KitComponents Thefollowingreagentsaresuppliedwiththisproduct:
Storeat(°C) Concentration NEBNext® LigationEnhancer -20 NEBNextFirstStrandSynthesisReactionBuffer -20 RandomPrimers -20 NEBNextSecondStrandSynthesisEnzymeMix -20 NEBNextSecondStrandSynthesisReactionBuffer -20 10X NEBNextUltraIIEndPrepEnzymeMix -20 NEBNextUltraIIEndPrepReactionBuffer -20 NEBNext® Ultra™ IILigationMasterMix -20 NEBNext®UltraIIQ5®MasterMix -20 2X NEBNextFirstStrandSynthesisEnzymeMix -20 NEBNextAdaptorDilutionBuffer -20 (0.1X)TEBuffer -20 0.1X Nuclease-freeWater -20