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Epigentek/EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina)/P-1055-12/12 reactions

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InputType:DNAResearchArea:DNAMethylation,NextGenSequencingTargetApplication:LibraryConstructionVesselFormat:Columns/Tubes100%Guarantee:6monthsProductOverviewTheEpiNext™Post-BisulfiteDNALibraryPreparationKit isacompletesetofoptimizedreagentstoprepareaDNAlibrary--aftersuccessfulbisulfiteconversion--forvariousIlluminaplatform-basedbisulfitesequencing(bisulfite-seq)assays,suchaswholegenomebisulfitesequencing(WGBS),oxidativebisulfitesequencing(oxBs-seq),reducedrepresentationbisulfitesequencing(RRBS),andotherbisulfite-basednextgenerationsequencingapplications.Theoptimizedprotocolandcomponentsofthekitallowbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrariestobequicklyconstructedusingsub-nanograminputconcentrationsofDNAsincetheDNAisfirstbisulfite-convertedandthenusedforlibrarypreparation. Thekithasthefollowingadvantagesandfeatures:Allowsbisulfite-convertedDNAtobeuseddirectlyforligation,therebyeliminatingthepossibilityofbreakingadapter-ligatedfragments,whichcanoftenoccurincurrentlyusedWGBSandRRBSmethods.Fast5-hourprocedure,frominputstartingmaterialtolibraryamplification.Gel-freesizeselection/purificationsavestimeandpreventshandlingerrors,aswellaslossofvaluablesamples.Highsensitivtyandefficiency--directligationofadaptertobisulfite-convertedDNAfragmentsreduceslossoffragmentsandselectionbias,whichenablespre-bisulfiteinputDNAtobeaslowas1ng. Thekitcanbeusedforbothnon-barcoded(singleplexed)andbarcoded(multiplexed)DNAlibrarypreparation.ComprehensivesetofcomponentstoaccommodateeachstepofDNAlibrarypreparation--ligation,clean-up,sizeselection,andlibraryamplification-- forconvenience,consistency,andreliABIlity.UltraHiFiamplificationenablesachievementofreproducIBLyhighyieldsofDNAlibrarieswithminimalsequencebiasandlowerrorrates.BackgroundInformationSeveralmethods,includingwholegenomebisulfitesequencing(WGBS)andreducedrepresentationbisulfitesequencing(RRBS),arecurrentlyusedforgenome-wideDNAmethlyationanalysis.Thesemethodsconvertunmethylatedcytosinestouracilwhile5-methylcytosinesremainunchangedbythebisulfitetreatment.Thisallowsepigeneticdifferencestobecomegeneticdifferences,whichcanbesubsequentlydetectedviasequencingatthesingle-basedresolutionlevelandonagenome-widescale.However,practicalusewithsuchcurrentmethodsarenotidealasthey(1)needlargeamountsofDNA(>1 µg)asinputmaterial,whichisdifficulttopreparefromlimitedBIOLOGicalsamplessuchastumorbiopsy,earlyembyros,embyronictissues,andcirculatingDNA;(2)requireDNAtofirstbeshearedandthenligatedtoadapters,followedbybisulfiteconversion(post-ligationbisulfiteconversion),whichcausessubstantialamountsofDNAfragmentsontainedintheadapter-DNAfragmentconstructstobebrokenandtherebyformsmono-taggedtemplatesthatbecomeremovedduringlibraryenrichment;and(3)aretime-consuming(2days).The EpiNextPost-BisulfiteDNALibraryPreparationKitisdesignedtoovercometheseweaknesses.Principle&ProcedureWiththiskit,bisulfite-treatedDNA(whichisinsingle-strandedform),isconvertedtodouble-strandedDNAanddirectlyusedforligationwithBisDNA-specificadapterswhicharenecessaryforamplificationandsequencing.ThefragmentsarethensizeselectedandpurifiedwithMQbeads,whichallowsforquickandprecisesizeselectionsofDNA.Size-selectedDNAfragmentsarethenamplifiedwithahigh-fidelityPCRMix,ensuringmaximumyieldsfromminimumamountsofstartingmaterialandprovidingahighlyaccurateamplificationoflibraryDNAwithlowerrorratesandminimalbias.StartingMaterialsInputstartingmaterialmustbebisulfite-treatedDNAgeneratedfromvariousinputDNAamountsof1ngto1 µg.Foroptimalpreparation,theinputDNAamountforthebisulfiteconversionprocessshouldbe100ngto200ngsothatsufficientbisulfite-treatedDNAcanbeyielded.Fig.1.SchematicprocedurefortheEpiNext™Post-BisulfiteDNALibraryPreparationKit.Fig.2.Sizedistributionoflibraryfragmentsasdemonstratedbyapost-bisulfiteDNAlibraryconstructedusingtheEpiNext™ Post-BisulfiteDNALibraryPreparationKitfrom10ngofinputDNA.Fig.3.The EpiNext™ Post-BisulfiteDNALibraryPreparationKitwasusedtopreparealibraryforMethyl-SeqwithanIlluminaHiSeq2500.Readalignmentdataat19kbandbasepairresolutioninchromosome7showsCpGmethylationdifferencesfortreatedandcontrolsamples.Controlsamplesareunmethylated(blue)whiletreatedsamplesaremethylated(red).ProductComponents5XConversionBuffer*ConversionEnzymeMix*ConversionPrimer*10XEndRepairBuffer*EndRepairEnzymeMix*10XdA-TailingBuffer*KlenowFragment(3’-5’exo-)*2XLigationBuffer*T4DNALigase*Adaptors(50µM)*MQBindingBeads*2XHiFiPCRMasterMix*PrimerU(10µM)*PrimerI(10µM)*RNase-freeWater*UserGuide*Spinthesolutiondowntothebottompriortouse.

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