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NEB/NEBNext? rRNA Depletion Kit?(Human/Mouse/Rat)/E6310S/24 reactions
  • NEB/NEBNext? rRNA Depletion Kit?(Human/Mouse/Rat)/E6310S/24 reactions

NEB/NEBNext? rRNA Depletion Kit?(Human/Mouse/Rat)/E6310S/24 reactions

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    • Description:











      TheNEBNextrRNADepletionKit(Human/Mouse/Rat)employsanRNaseH-basedmethod(1),(2)todepletebothcytoplasmic(5SrRNA,5.8SrRNA,18SrRNAand28SrRNA)andmitochondrialribosomalRNA(12SrRNAand16SrRNA)fromhuman,mouseandrattotalRNApreparations.ThisproductissuitableforbothintactanddegradedRNA(e.g.FFPERNA).TheresultingrRNA-depletedRNAissuitableforRNA-Seq,random-primedCDNAsynthesis,orotherdownstreamRNAanalysisapplications.

      NowalsoavailablewithoptionalAgencourt?RNAClean?XPbeadsforRNApurification.


      Figure1.EfficientRemovalofrRNAfromIntactandDegradedRNA(FFPE),WhileRetainingCodingandNon-codingTranscripts
      Figure 1. Efficient Removal of rRNA from Intact and Degraded RNA (FFPE), While Retaining Coding and Non-coding Transcripts
      RNA-seqlibrariesweregeneratedfromUniversalHumanReferenceTotalRNA(UHR,Agilent)orBreastCancerFFPERNA(withanarchiveageof1yearand10years).RNA?waseitheruntreatedortreatedwiththeNEBNextpoly(A)mRNAMagneticIsolationModule(NEB#E7490)ortheNEBNextrRNADepletionKit.RNA-seqlibrariesweremadeusingtheNEBNextUltraDirectionalRNALibraryPrepKitforIllumina(NEB#E7420).Readsweremappedtothehg19genomeandreaddistributionsweredeterminedusingPicardRNA-seqMetrics.LibrariesgeneratedfromrRNA-depletedRNAresultincomparablelowrRNAreadstopoly(A)mRNA-enrichedRNA,whilealsoretainingmorenoncodingreads.rRNAdepletionefficiencyisachievedevenwithFFPERNA.


      Figure2.NEBNextrRNADepletionKitWorkflowFigure 2. NEBNext rRNA Depletion Kit Workflow
      Figure3.NEBNextrRNADepletionKitWorkflowTimesFigure 3. NEBNext rRNA Depletion Kit Workflow Times
      Figure4.DepletionEfficiencywithIntactorDegradedRNA
      Figure 4. Depletion Efficiency with Intact or Degraded RNA
      RibosomalRNAwasdepletedfromintactUniversalHumanReferenceTotalRNA(UHR,Agilent)(RIN>9)anddegradedUHRTotalRNA(RIN<3)usingeithertheNEBNextrRNADepletionKit,Ribo-ZeroMagneticGoldKit(Human/Mouse/Rat)(Epicentre)orRibo-ZeroGoldprovidedwithintheTruSeqStrandedTotalRNAKitwithRibo-ZeroGold(Illumina).rRNA-depletedRNAlibrariesweremadeusingeithertheNEBNextUltraDirectionalRNALibraryPrepKitforIllumina(NEB#E7420.Greenandbluebars)ortheTruSeqStrandedTotalRNAKitwithRibo-ZeroGold(Illumina,redbars).TotalrRNA-alignedreadsweredeterminedusingBowtie2.0(local,sensitive).NEBNextrRNA-depletedlibrariescontainaminimalpercentageofrRNAreadsregardlessofthequalityoftheRNA.
      Figure5.DepletionEfficiencywithFFPERNAFigure 5. Depletion Efficiency with FFPE RNA

      0.1μg,0.5μgor1μgofbreastcancerFFPERNAsamples(witharchiveagesofoneyearand10years)weredepletedofrRNAusingeithertheNEBNextrRNADepletionKitortheTruSeq?StrandedTotalRNAKitwithRibo-ZeroGold(Illumina#RS-122-2301).rRNA-depletedRNAlibrariesweremadeusingeithertheNEBNextUltraDirectionalRNALibraryPrepKitforIllumina(NEB#E7420)(greenbars)ortheTruSeqStrandedTotalRNAKitwithRibo-ZeroGold(bluebars).TotalrRNA-alignedreadsweredeterminedusingBowtie2.0(local,sensitive).NEBNextrRNA-depletedFFPElibrariesresultinaminimalpercentageofrRNAreads,regardlessofthearchiveageoftheFFPERNA.
      Figure6.rRNADepletionEfficiencyonMouseandRatRNAFigure 6. rRNA Depletion Efficiency on Mouse and Rat RNA

      RibosomalRNAwasdepletedfrommouseandratkidneytotalRNA(twotechnicalreplicates)usingtheNEBNextrRNADepletionKit.RNA-seqlibrariesweremadefromnon-depletedTotalRNAorrRNA-depletedRNA,usingtheNEBNextUltraDirectionalRNALibraryPrepKitforIllumina(NEB#E7420).TotalrRNA-alignedreadsweredeterminedusingBowtie2.0(local,sensitive).NEBNextrRNA-depletedmouseandratlibrariescontainaminimalpercentageofreadsmappingtorRNA.
      Figure7.TranscriptExpressionCorrelationwithNon-depletedLibrariesFigure 7. Transcript Expression Correlation with Non-depleted Libraries

      LibrariesweremadefromUHRRNA(Agilent)andBreastCancerFFPERNA(witharchiveageofoneyearand10years),bothnon-depletedanddepletedrRNAusingtheNEBNextrRNADepletionKit.AlllibrariesweremadeusingtheNEBNextUltraDirectionalRNALibraryPrepKitforIllumina(NEB#E7420).TopHat2andCufflinkswereusedforreadmappingandtranscriptassemblyandquantification.FPKM(FragmentsPerKilobaseoftranscriptperMillionmappedreads)correlationanalysisindicatesverygoodtranscriptexpressioncorrelation(R>0.93)betweenDepletedandNon-Depletedlibraries.NEBNextrRNAdepletiondoesnotaffecttranscriptexpressionlevels.


      ReagentsSupplied

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      RNaseHReactionBuffer-2010X
      NEBNextrRNADepletionSolution-20
      NEBNextProbeHybridizationBuffer-20
      DNaseI(RNase-free)-20
      DNaseIReactionBuffer-2010X
      Nuclease-freeWater-20
      NEBNextRNaseH-20
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