Description:
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
Poly(A)-containingmRNAwasisolatedfromHumanUniversalReferenceRNA(Agilent#740000),andlibrariesweremadeusingtheNEBNextUltraIIDirectionalRNAKit(plustheNEBNextPoly(A)mRNAMagneticIsolationModule),KapaStrandedmRNA-SeqKit,KapamRNAHyperPrepKitandIlluminaTruSeqStrandedmRNAKit.TheinputRNAamountandnumberofPCRcyclesareindicated.Libraryyieldsfromanaverageofthreereplicatesareshown.
Viewadditionaldataonlibraryyields.
GCCONTENTDISTRIBUTION
Figure2.NEBNextUltraIIDirectionalRNAlibrariesprovideuniformGCcontentdistribution,atabroadrangeofinputamounts
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
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
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
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
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 | -20 | 10X |
| NEBNextUltraIIEndPrepEnzymeMix | -20 | |
| NEBNextUltraIIEndPrepReactionBuffer | -20 | |
| NEBNext®Ultra™IILigationMasterMix | -20 | |
| NEBNext®LigationEnhancer | -20 | |
| NEBNextUSER®Enzyme | -20 | |
| NEBNext®UltraIIQ5®MasterMix | -20 | 2X |
| NEBNextAdaptorDilutionBuffer | -20 | |
| NEBNext®SamplePurificationBeads | 25 | |
| NEBNextFirstStrandSynthesisReactionBuffer | -20 | |
| RandomPrimers | -20 | |
| NEBNextSecondStrandSynthesisEnzymeMix | -20 | |
| (0.1X)TEBuffer | -20 | 0.1X |
| Nuclease-freeWater | -20 | |
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