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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