truseq-small-rna-library-prep-guide-15004197-g - 图文(4)

Ligate3'Adapter1

Setuptheligationreactioninasterile,nuclease-free200?μlPCRtubeoniceusingthefollowing:

ReagentRNA3'Adapter(RA3)1?μgTotalRNAinnuclease-freewaterTotalVolumeVolume(μl)15623

Gentlypipettetheentirevolumeupanddown6–8timestomixthoroughly,andthencentrifugebriefly.

Placethetubeonthepreheatedthermalcycler.Closethelidandincubatethetubeat70°Cfor2?minutesandthenimmediatelyplacethetubeonice.

NOTE

ItisimportanttokeeptheRNA3’Adapteroniceafterthe70°Cincubationtopreventsecondarystructureformation.

45

Preheatthethermalcyclerto28°C.

Preparethefollowingmixinaseparate,sterile,nuclease-free200?μlPCRtubeonice.Multiplyeachreagentvolumebythenumberofsamplesbeingprepared.Make10%extrareagentifyouarepreparingmultiplesamples.

ReagentLigationBuffer(HML)RNaseInhibitorT4RNALigase2,DeletionMutantTotalVolumeperSampleVolume(μl)2114NOTE

DonotusethereactionbuffersuppliedwithT4RNALigase2,DeletionMutant.Thisenzymeretainsactivityinligationbuffer.

67

Gentlypipettetheentirevolumeupanddown6–8timestomixthoroughly,andthencentrifugebriefly.

Add4?μlofthemixtothereactiontubefromstep1andgentlypipettetheentirevolumeupanddown6–8timestomixthoroughly.Thetotalvolumeofthereactionis10?μl.

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Part#15004197Rev.G

8Placethetubeonthepreheatedthermalcycler.Closethelidandincubatethetubeat28°Cfor1?hour.

9

Withthereactiontubeonthethermalcycler,add1?μlStopSolution(STP)andgentlypipettetheentirevolumeupanddown6–8timestomixthoroughly.Continuetoincubatethereactiontubeonthethermalcyclerat28°Cfor15?minutesandthenplacethetubeonice.

Ligate5'Adapter1Preheatthethermalcyclerto70°C.

2Add1.1×N?μlRNA5'Adapter(RA5)intoaseparate,nuclease-free200?μlPCRtube,withNequaltothenumberofsamplesbeingprepared.

3

Placethetubeonthepreheatedthermalcycler.Closethelidandincubatethetubeat70°Cfor2?minutesandthenimmediatelyplacethetubeonice.

NOTE

ItisimportanttokeeptheRNA5'Adapteroniceafterthe70°Cincubationtopreventsecondarystructureformation.WhenhandlingtheRNA5'RNAAdapter,pipettefrom1tubeonicetoanothertubeoniceandpipettemixthereactions.

4Preheatthethermalcyclerto28°C.

5

Add1.1×N?μl10mMATPtotheRNA5'Adapteraliquottube,withNequaltothenumberofsamplesbeingprepared.Gentlypipettetheentirevolumeupanddown6–8timestomixthoroughly.

6

Add1.1×N?μlT4RNALigasetotheRNA5'Adapteraliquottube,withNequaltothenumberofsamplesbeingprepared.Gentlypipettetheentirevolumeupanddown6–8timestomixthoroughly.

7

Add3?μlofthemixfromtheRNA5'Adapteraliquottubetothereactionfrom

step?9ofLigate3'Adapter.Gentlypipettetheentirevolumeupanddown6–8timestomixthoroughly.

Thetotalvolumeofthereactionis14?μl.

8

Placethetubeonthepreheatedthermalcycler.Closethelidandincubatethereactiontubeat28°Cfor1?hourandthenplacethetubeonice.

TruSeqSmallRNALibraryPrepGuide

17

LigateAdaptersReverseTranscribeandAmplify

ReversetranscriptionfollowedbyPCRisusedtocreatecDNAconstructsbasedonthesmallRNAligatedwith3'and5'adapters.Thisprocessselectivelyenrichesthose

fragmentsthathaveadaptermoleculesonbothends.PCRisperformedwith2primersthatannealtotheendsoftheadapters.

Consumables

Item25?mMdNTPMixPCRMix(PML)RNAPCRPrimer(RP1)RNAPCRPrimerIndex(1–48)(RPI1–RPI48)RNARTPrimer(RTP)RNaseInhibitorUltraPureWater5XFirstStrandBuffer100mMDTTHighSensitivityDNAchipNuclease-free200μlPCRtubesSuperScriptIIReverseTranscriptaseQuantity1tube1tube1tube1tubeofeachindexbeingused1tube1tube1tube2?μlpersample1?μlpersample1persample3+1perindexused1?μlpersampleStorage-25°C?to-15°C-25°C?to-15°C-25°C?to-15°C-25°C?to-15°C-25°C?to-15°C-25°C?to-15°C-25°C?to-15°C-25°C?to-15°C-25°C?to-15°C15°Cto30°C15°Cto30°C-25°C?to?-15°CSuppliedByIlluminaIlluminaIlluminaIlluminaIlluminaIlluminaIlluminaUserUserUserUserUserNOTE

TheTruSeqSmallRNALibraryPrepKitscontainupto48differentindexedPCRprimers,eachwithadifferentindex.DuringthePCRstep,RNAPCRPrimerisusedwitheverylibrarywithonly1ofthe48RNAPCRPrimerindexesperlibrary.

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Part#15004197Rev.G

Preparation

}RemovetheIllumina-suppliedconsumables,5XFirstStrandBuffer,100mMDTT,andSuperScriptIIReverseTranscriptasefrom-25°C?to?-15°Cstorageandthawonice.

}Brieflycentrifugethethawedconsumablesat600?×?gfor5?seconds,andthenplacethemonice.

}ReviewtheTruSeqLibraryPrepPoolingGuide(part?#?15042173).}Preheatthethermalcyclerto70°C.

}Choosethethermalcyclerpreheatlidoptionandsetto100°C.

}Labelasterile,nuclease-free,200μlPCRtube12.5?mMdNTPMixwithasmudge-resistantpen.

Dilute25?mMdNTPMix1

Dilutethe25?mMdNTPsbypremixingthefollowingreagentsinasterile,nuclease-free,200?μlPCRtubelabeled12.5?mMdNTPMix.Multiplyeachreagentvolumebythenumberofsamplesbeingprepared.Make10%extrareagentifyouarepreparingmultiplesamples.

ReagentVolume(μl)25?mMdNTPMix0.5UltraPureWater0.5TotalVolumeperSample1.02

Gentlypipettetheentirevolumeupanddown6–8timestomixthoroughly,andthencentrifugebrieflyandthenplaceitonice.

PerformReverseTranscription1

Transfer6μlofeach5'and3'adapter-ligatedRNAtoaseparate,sterile,nuclease-free,200?μlPCRtube.

NOTE

Storetheremaining5'and3'adapter-ligatedRNAat-80°Cforupto7days.

2Add1μlRNARTPrimertoeachtubecontainingadapter-ligatedRNA.

TruSeqSmallRNALibraryPrepGuide

19

ReverseTranscribeandAmplify3456

Gentlypipettetheentirevolumeupanddown6–8timestomixthoroughly,andthencentrifugebriefly.

Placethetubeonthepreheatedthermalcycler.Closethelidandincubatethetubeat70°Cfor2?minutesandthenimmediatelyplacethetubeonice.Preheatthethermalcyclerto50°C.

Preparethefollowingmixinaseparate,sterile,nuclease-free,200?μlPCRtubeplacedonice.Multiplyeachreagentvolumebythenumberofsamplesbeingprepared.Make10%extrareagentifyouarepreparingmultiplesamples.

Reagent5XFirstStrandBuffer12.5?mMdNTPmix100mMDTTRNaseInhibitorSuperScriptIIReverseTranscriptaseTotalVolumeperSampleVolume(μl)20.51115.578

Gentlypipettetheentirevolumeupanddown6–8timestomixthoroughly,andthencentrifugebriefly.

Add5.5?μlofthemixtothereactiontubefromstep4.Gentlypipettetheentirevolumeupanddown6–8timestomixthoroughly,andthencentrifugebriefly.Thetotalvolumeis12.5?μl.

Placethetubeonthepreheatedthermalcycler.Closethelidandincubatethetubeat50°Cfor1?hourandthenplacethetubeonice.

9

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Part#15004197Rev.G

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