truseq-small-rna-library-prep-guide-15004197-g - 图文(2)

Part?#15004197RevisionBDateFebruary201115004197ANovember2010DescriptionofChange?Correctedindextable?CorrectedRNAPCRPrimer(RP1),part#15013198?AddedTrackingToolssection?UpdatedfigureforSmallRNALibraryfromTotalRNASamples?Changed\to\inDNAchiptracefigures?Addedinstructionsonhowtouse0.5mltubeasanalternativetogelbreakertubes?Convertedcentrifugerpmto×?gInitialReleaseIntroduction

TheIllumina?TruSeq?SmallRNALibraryPrepprotocolisusedtopreparevariousRNAspecies.TheprotocoltakesadvantageofthenaturalstructurecommontomostknownmicroRNAmolecules.MostmaturemiRNAshavea5'-phosphateanda

3'-hydroxylgroupasaresultofthecellularpathwayusedtocreatethem.Becauseofthis,theIlluminaadaptersinthiskitaredirectlyligatedtomiRNAs.

Thisguideexplainshowtopreparelibrariesforsubsequentclustergeneration,usingtotalRNAorpurifiedsmallRNAasinput.Theprotocoldescribesthestepsforadapterligation,reversetranscription,PCRamplification,andpooledgelpurificationtogeneratealibraryproduct.

Figure1FragmentsafterTruSeqSmallRNALibraryPrepKit

TheRNA3'adapterismodifiedtotargetmicroRNAsandothersmallRNAsthathavea3'hydroxylgroupresultingfromenzymaticcleavagebyDicerorotherRNAprocessingenzymes.TheadaptersareligatedtoeachendoftheRNAmoleculeandan?RTreaction

TruSeqSmallRNALibraryPrepGuide

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IntroductionisusedtocreatesinglestrandedcDNA.ThecDNAisthenPCRamplifiedusinga

commonprimerandaprimercontaining1of48indexsequences.TheintroductionoftheindexsequenceatthePCRstepseparatestheindexesfromtheRNAligationreaction.Thisdesignallowsfortheindexestobereadusingasecondreadandsignificantlyreducesbiascomparedtodesignsthatincludetheindexwithinthefirstread.

TheTruSeqSmallRNALibraryPrepKitallowsfortheuseof48differentindextagsformultiplexingandanalysisofdirectionalandsmallRNAsamples.Formoreinformation,seeIndexSequencesonpage43.Illuminamultiplexedsequencinguses6-baseindexestodistinguishdifferentsamplesfromeachotherinasinglelaneofaflowcell.

Thekitsareconfiguredfor24reactionswith12differentindexesperkit.The48indexesaredividedinto4kits:

Table1TruSeqSmallRNALibraryPrepKitsCatalog#ContainsaCoreSolutionsBoxIndexesandthefollowingIndicesBox1–12RS-200-0012A13–24RS-200-0024B25–36RS-200-0036C37–48RS-200-0048D8

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AdditionalResources

ThefollowingdocumentationisavailablefordownloadfromtheIlluminawebsite.

ResourceDescriptionTruSeqSmallRNAExperiencedProvidesprotocolinstructions,butwithlessdetailthanUserCard(part#15012191)whatisprovidedinthisguide.NeworlessexperiencedusersareadvisedtofollowthisguideandnottheExperiencedUserCard(EUC).TruSeqLibraryPrepPoolingProvidesTruSeqpoolingguidelinesforpreparinglibrariesGuide(part?#?15042173)forIlluminasequencingsystemsthatrequirebalancedindexcombinations.Reviewthisguidebeforebeginninglibrarypreparation.IlluminaExperimentManagerProvideinformationaboutcreatingandeditingappropriateGuide(part?#?15031335)andIEMsamplesheetsforIlluminasequencingsystemsandanalysisTruSeqSmallRNAQuicksoftwareandrecordparametersforyoursampleplate.ReferenceCard(part?#?15037153)BaseSpaceUserGuide(part#ProvidesinformationabouttheBaseSpacesequencingdata15044182)analysistoolthatalsoenablesyoutoorganizesamples,libraries,pools,andsequencingrunsinasingleenvironment.VisittheTruSeqSmallRNALibraryPrepKitsupportpageontheIlluminawebsiteforaccesstorequirementsandcompatibility,additionaldocumentation,softwaredownloads,onlinetraining,frequentlyaskedquestions,andbestpractices.

TruSeqSmallRNALibraryPrepGuide

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AdditionalResourcesRNAInputRecommendations

ItisimportanttofollowtheTruSeqSmallRNALibraryPrepKitinputrecommendations.

TotalRNAInput}Optimization

?TheTruSeqSmallRNALibraryPrepKitprotocolisoptimizedfor1?μgoftotal

RNAin5?μlnucleasefreewater,asquantifiedwithafluorometricmethod.?Loweramountsmightresultininefficientligationandlowyield.}Testing

?TheTruSeqSmallRNALibraryPrepKitprotocolhasbeentestedusing1?μgof

high-qualityuniversalhumanreferencetotalRNAasinput.

?SmallRNApopulationscanvarysignificantlybetweendifferenttissuetypes

andspecies.

?UseofRNAfromotherspecies,tissues,orqualitiesmightrequirefurther

optimizationregardingtheinitialinputamountandselectionofdesiredbandsduringthefinalgelexcision.

?ThetypesandcoverageofsmallRNAssequencedvarydependingonwhich

bandsareselectedduringgelexcision.

}ItisimportanttoknowthequalityoftheRNAstartingmaterial.

?UseofdegradedRNAcanresultinlowyield,changesinobservedexpression

patterns,orfailureoftheprotocol.

?IlluminarecommendsthatyouchecktotalRNAintegrityfollowingisolation,

usinganAgilentTechnologies2100Bioanalyzer,forhuman(ormammalian)sampleswithanRNAIntegrityNumber(RIN)value≥8.AlthoughthisdoesnotdirectlymeasuresmallRNA,anRNAsamplewithdegradedmRNAmostlikelyhasdegradedsmallRNAaswell.

?RNAthathasDNAcontaminationresultsinanunderestimationoftheamount

ofRNAused.

?IlluminarecommendsincludingaDNasestepwiththeRNAisolationmethod.

However,contaminantDNAisremovedduringsmallRNApurification.

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Part#15004197Rev.G

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