TheEpiNext™5-mC RNABisulfite-SeqEasyKit(Illumina) isacompletesetofoptimizedreagents designedforeasilycarryingoutRNAbisulfiteconversion,followedbya"post-bisulfite"librarypreparationprocessforIlluminaplatform-basedbisulfitesequencing,allinonekit.IntendedapplicationsincludewholetranscriptomeRNAbisulfitesequencingandvariousotherRNAbisulfite-basednextgenerationsequencingtechniquesfor5-methylcytosine(5-mC)basedRNAmethylationanalysis.TheoptimizedprotocolandcomponentsofthekitallowtheRNAtobebisulfiteconvertedandfragmentedsimultaneously,followedbyquicknon-barcoded(singleplexed)and barcoded (multiplexed) libraryconstructionusinglow-nanogramquantitiesofbisulfiteconvertedRNA. - Highsensitivityandefficiency: Byusinganinnovativemethodthatenablesadaptortaggingofbisulfite-convertedRNAtobeintactthroughrandomprobingwithblockingformationofun-taggedorhalf-taggedRNAfragments,bothnon-barcoded(singleplexed)andbarcoded(multiplexed)librarypreparationcanbereliablymadewithhighsensitivityandefficiency,therebyprovidingarobustandreliablemeanstobuild5mCRNA-seqlibrary.TheoptimizedRNAbisulfitemethodandenhancedadaptortaggingeliminateslossoffragmentsandselectionbias,whichenablesinputRNAtobeaslowas5ngwithincreasedefficiencyofcompletelytaggedCDNAlibrary.
- Easyandstreamlinedprocedure:Theentireprocedurecanbefinishedin6hours.Gel-freesizeselection/purificationsavestimeandpreventshandlingerrors,aswellaslossofvaluablesamples.
- Completeconversion:Theinnovativereagentcompositionconvertsunmethylatedcytosineintouracilatalevelgreaterthan99.9%,withnoornegligIBLeinappropriate/errorconversionofmethylcytosinetothymine(<0.1%)whentheindicatedrangeofsampleRNAisused.
- Extremelyconvenient:ThekitcontainsalltherequiredcomponentsforeachstepoftheRNAlibrarypreparationprocess,whichissufficientforbisulfiteconversion,ligation,clean-up,sizeselection,andlibraryamplification,therebyallowingthebisulfiteRNAlibrarypreparationtobestreamlinedforthemostreliableandconsistentresults.
- Minimalbias:UltraHiFiamplificationenablesachievementofreproduciblyhighyieldsofbisulfiteconvertedRNAlibrarieswithminimalsequencebiasandlowerrorrates.
- BroadsamplesuitABIlity:StartingmaterialscanbetotalRNAisolatedfromvarioustissue/cellsamplessuchasfreshandfrozentissue,culturedcellsfromaflaskormicroplate
BackgroundInformation
5-methylcytosine(5-mC)inDNAoccursbythecovalentadditionofamethylgroupatthe5-carbonofthecytosineringbyDNAmethyltransferases.Thisprocesshasbeenwellstudiedandisgenerallyassociatedwithrepressionofgeneexpression.Itwasalsoobservedthatinhumans,5-mCoccursinvariousRNAmoleculesincludingtRNAs,rRNAs,mRNAsandnon-codingRNAs(ncRNAs).Atleast10,2755-mCcandidatesiteswerediscoveredinmRNAsandncRNAs,whichcover10.6%ofthetotalcytosineresiduesinthetranscriptome.5-mCseemstobeenrichedinsomeclassesofncRNA,butrelativelydepletedinmRNAs.However,themajority(83%)oftheircandidatesiteswerefoundinmRNAs.Withinthesetranscripts5-mCappearstobedepletedwithinproteincodingsequences,butenrichedin5’and3’UTRs.Twodifferentmethyltransferases,NSUN2andDnmt2areknowntocatalyze5-mCmodificationineukaryoticRNA.RecentdatastronglysuggestthatRNAcytosinemethylationaffectstheregulationofvariousBIOLOGicalprocessessuchasRNAstabilityandmRNAtranslation.FurThermore,lossof5-mCinvaultRNAscausesaberrantprocessingintoArgonaute-associatedsmallRNAfragmentsthatcanfunctionasmicroRNAs.Thus,impairedprocessingofvaultncRNAmaycontributetotheetiologyofhumandisordersrelatedtoNSun2-defciency. Principle&Procedure
ThiskitincludesallreagentsrequiredforasuccessfulRNAbisulfiteconversionandbisulfiteRNAlibrarypreparationusingbisulfite-convertedRNAgeneratedfromawiderangeofinputRNAamounts(5ngto500ng).Inthispreparation,RNAissimultaneouslybisulfiteconvertedandfragmentedtotheappropriatelengthduringthebisulfiteprocess.Thebisulfite-treatedRNA,whichisinsinglestrandedform,isthensimultaneouslyconvertedtodoublestrandedcDNAandadaptortagged.Thetaggedfragmentsaresizeselectedandpurifiedusing MQbindingbeads,followedbyamplificationwithahigh-fidelityPCR Mix,whichensuresmaximumyieldsfromminimumamountsofstartingmaterialandprovideshighlyaccurateamplificationoflibrarycDNAwithlowerrorratesandminimumbias. StartingMaterials
StartingmaterialscanbetotalRNAisolatedfromvarioustissue/cellsamplessuchasfreshandfrozentissues,culturedcellsfromaflaskormicroplate,microdissectionsamples,andbodyfluidsamples,etc.TheamountofRNAforeachbisulfitereactioncanbe5ngto500ng.Foranoptimalreaction,theinputRNAamountshouldbe100ngto200ng.TheyieldofRNApurifiedafterbisulfiteconversiondependsontheamountofinputRNA,natureofRNA,andsourceofthestartingmaterial. | 
 Fig.1. WorkflowoftheEpiNext™5-mCRNABisulfite-SeqEasyKit(Illumina).
Fig.2. Sizedistributionoflibraryfragments:Post-bisulfitecDNAlibrarywaspreparedfrom50ngofinputmouseRNAusingtheEpiNext™5-mC RNABisulfite-SeqEasyKit(Illumina). |
