Description:
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
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(ThermoFisherScientific#4456740)usingtheNEBNextrRNADepletionKit(Human/Mouse/Rat)andlibrarieswerethenpreparedusingtheNEBNextUltraIIRNAKit.LibrariesweresequencedonanIlluminaNextSeq®500usingpaired-endmode(2x76bp).Salmon0.4.0wasusedforreadmappingandquantificationofallERCCtranscripts.R2valuesforlinearfitareshown.TPM(TranscriptsPerKilobaseMillion)correlationanalysisofthetranscriptsindicatesexcellenttranscriptexpressioncorrelationbetweenthedifferentinputsforUltraIIRNAlibraries(A),includingERCCtranscripts(B).
ViewadditionaldataonlibrarycomplexityKitComponents
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 | |
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