Description:
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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(linktoE7770ComplexityandTranscriptCoveragedaughterpages)
- Optimizeyourtimewithstreamlinedworkflows,reducedhands-ontime,andautomationcompatibility
- Relyonrobustperformance
- CompatIBLewithNEBNextpoly(A)mRNAIsolation,rRNADepletionreagentsandmultiplexingadaptorsandprimers
AlsoavailablewithoutoptionalSPRIselect?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 | |
| NEBNext?SamplePurificationBeads | 25 | |
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