scriptseq™ v2 rna-seq library preparation kit* - illumina · pdf file2 scriptseq™...

www.epicentre.com Lit.#329•8/2014 1 EPILIT329 Rev. C

* Covered by issued and/or pending patents.

ScriptSeq™ v2 RNA-Seq Library Preparation Kit*

SSV21106 – 6 Reactions

SSV21124 – 24 Reactions

Important! Epicentre’s FailSafe™ PCR Enzyme (available separately) is required for use with this kit.

Epicentre (an Illumina company) warrants that its products will retain full activity for 1 year from the date of receipt by the user if used and stored properly. For full warranty details, see Terms and Conditions available on the Epicentre website at www.epibio.com.

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ScriptSeq™ v2 RNA-Seq Library Preparation Kit

1. Kit Contents and Quality Control

Component Name Tube Label

Volume

Cap Color6-rxn 24-rxn

ScriptSeq v2 cDNA Synthesis Primer cDNA Primer 18µl 55µlGreen

RNAFragmentationSolution FragmentationSolution 10μl 30μl

ScriptSeq v2 cDNA Synthesis PreMix cDNA Synthesis PreMix 25μl 80µl

Red100mMDTT 100mMDTT 100μl 100μl

StarScript AMV Reverse Transcriptase StarScript AMV Reverse Transcriptase 8μl 15µl

ScriptSeqFinishingSolution FinishingSolution 10μl 30µl

ScriptSeqv2TerminalTaggingPreMix TerminalTaggingPreMix 60μl 200µlBlue

DNAPolymerase DNAPolymerase 8μl 15µl

ExonucleaseI Exo I 10µl 30µl

YellowFailSafePCRPreMixE FailSafePCRPreMixE 200μl 650µl

Forward PCR Primer Forward PCR Primer 10μl 30µl

Reverse PCR Primer Reverse PCR Primer 10μl 30µl

Nuclease-FreeWater Nuclease-FreeWater 500μl 500µl Clear

Storage:Storethekitat–15°Cto–25°Cinafreezerwithoutadefrostcycle.

Additional Required Reagents and Equipment:FailSafe™PCREnzymeMix(Epicentre;cat.nos.FSE51100,FSE5101K)

Recommended:WideborepipettipforuseinStep3.C(e.g.,Pure™200Gsteriletip;catalognumber#3531,MolecularBioproducts)

Recommended:2100Bioanalyzer(AgilentTechnologies)

Optional:MinElutePCRPurificationcolumns(Qiagen)

Optional:AgencourtAMPureXPSystem(BeckmanCoulter)andmagneticplate,rack,orstandfor1.5-mltubes

Optional:ScriptSeqIndexPCRPrimers(Epicentre;Cat.Nos.RSBC10948,SSIP1202,SSIP1203,SSIP1204)

Quality Control:

TheScriptSeqv2RNA-SeqLibraryPreparationKitisfunction-testedinacontrolreactionusing5ngofratliverpoly(A)RNA.Atleast 400ngofadi-taggedlibrarymustbeproducedinafinalvolumeof20μlwithapeakbetween150-300bpasassayedusinganAgilentBioanalyzer.

2. PreparationrRNA Removal

TheScriptSeqv2Kitusesarandom-primedcDNAsynthesisreaction.Therefore,thebestsequencingresultsareobtainedusinganRNApreparationdepletedofrRNA.TheScriptSeqv2Kitcanbeusedwithpoly(A)+RNAorwithrRNA-depletedRNA.TomaximizeremovalofrRNA,werecommendusingoneofEpicentre’sRibo-Zero™Kits.

DNA-Free RNATreattheRNAsamplewithDNaseI(forexampleusingBaseline-ZERODNase;EpicentreCat.No.DB0711K,DB0715K)toremovealltracesofDNA.Then,removetheDNaseIpriortoRibo-Zerotreatmentorpoly(A)enrichment.DNAcontaminationwillinterferewithrRNAremovalandisthemaincauseoflossofdirectionalitywhensequencingtheScriptSeqv2library.TheRNAsampleshouldbefreeofsalts(e.g.,Mg2+orguanidiniumsalts)ororganics(e.g.,phenolandethanol).

Automated ScriptSeq Kit ReactionsEpicentredoesnotprovidesupportforanyliquidhandlingroboticplatformorprotocol.Pleasecontactthemanufactureroftheliquidhandlinginstrumentforhardwareandsoftwaresupport.

[email protected]•(800)284-8474 3


Figure 1. An overview of the procedure for the ScriptSeq™ v2 RNA-Seq Library Preparation Kit.

Fragment RNA

Anneal cDNA Synthesis Primer

Synthesize cDNA

500 pg to 50 ng rRNA-depleted or poly(A)+ RNA

Purify cDNA

Anneal TTO

Synthesize 3'-tagged DNA

Terminal-Tagging Oligo (TTO)3'-end blocked 5' NNNNNX

TaggingSequence

Random Hexamer withTagging Sequence

3' NNNNNNTagging

Sequence

3' 5'

Remove RNA

(AAAA)5'NNNNNN3' NNNNNN3'

5' NNNNNX3' 5'

3' 5'Di-tagged cDNA

(AAAA)5'

Adaptor-tagged RNA-Seq libraryfor directional sequencing

5' 3'3' 5'

Bar Code(optional)

Amplify by PCRPCR Primers

Index/Bar Code (optional)

Amount of RNAThestandardkitreactionuses500pgto50ngofrRNA-depletedRNAorpoly(A)+RNA.Typically,10-15cyclesofPCRaresufficienttogeneratethesequencinglibrary.

RNA from Formalin-Fixed Paraffin-Embedded (FFPE) TissueRNAextractedfromFFPEtissuecanbeusedtoprepareScriptSeqv2libraries.However,thequalityofFFPERNAcanbehighlyvariableduetothetissue-fixationprocedure,ageofthesample,storageconditions,fixationreversalprocess,etc.Therefore,wecannotguaranteesuccesswitheveryFFPERNAsample.

TheuseofFFPERNArequiresproceduralmodificationstotheScriptSeqv2librarypreparationprocedure.BesuretoreadPart3.A,3.D,3.F.2.,andAppendix1beforeproceeding.

StarScript AMV Reverse TranscriptaseTheStarScriptAMVReverseTranscriptaseisisolatedfromAvianMyeloblastosisVirions.Theenzymeiscarefullypurifiedtoensure ≤20-foldAMVgenomecoverageper50Msequencingreads.Thiscorrespondstoapproximately3,020readsina51-basesequencingrunandapproximately1,540readsina100-basesequencingrun.

Sequencing a ScriptSeq RNA-Seq LibraryScriptSeqlibrariesarecompatiblewithsingleread,paired-endandmultiplexsequencingonanyIllumina®sequencer.SeeAppendix2formoredetails.

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Quick Protocol for ScriptSeq™ v2 RNA-Seq Library Preparation KitFor experienced users only! The detailed protocol begins at Step 3.A on the next page.

Step Procedure Pages

Fragment RNA

IfusingRNAfromFFPEsamples,gotoAppendix1(RNAfragmentationisnotrequired).1.Mixthefollowing:xµlNuclease-FreeWateryµlRibo-Zero-treatedRNA(500pgto50ng)1µlRNAFragmentationSolution2µlcDNAPrimer

12µlTotalvolume2.Incubateat85°Cfor5minutesinthermocyclerthenplaceonice.

5

Synthesize cDNA

1.Mixthefollowingperreaction:3.0µlcDNASynthesisPreMix 0.5µl100mMDTT0.5µlStarScriptAMVReverseTranscriptase

4.0µlTotalVolumeofcDNASynthesisMasterMix2.Add4µlofcDNASynthesisMasterMixtoeachreaction.Mixbypipetting.3.Incubateat25°Cfor5minfollowedby42°Cfor20min.4.Coolreactionto37°C.5.Add1.0µlofFinishingSolutiontoeachreaction.Mixbypipetting.6.Incubateat37°Cfor10min.Incubateat95°Cfor3min,coolto25°CandPausethethermocycler.

5

Synthesize3′-TaggedDNA

1.Mixthefollowingperreaction:7.5µlTerminalTaggingPremix0.5µlDNAPolymerase

8.0µlTotalvolumeofTerminalTaggingMasterMix

Solution is viscous!Mixthoroughlybypipettingusingawide-borepipettetip.

2.Add8.0µlofTerminalTaggingMasterMixtoeachreaction.Mixbypipettingusinga wide-borepipettetip.3.Incubatereactionat25°Cfor15minutes.Incubatereactionat95°Cfor3minutes.Coolto4°C. ThecDNAisnowdi-tagged.

6

PurifycDNA ChooseQiagenMinEluteorAgencourtAMPurepurification.Elutein22.5µl. 6

PCRAmplify

IfaddinganoptionalIndex,gotoPart3.EofprotocolandskipthisQuickReferenceProtocol.Ifnot adding an Index: Mixina0.2-mlPCRtube:25µlFailSafePCRPreMixE1µlForwardPCRPrimer1µlReversePCRPrimer22.5µldi-taggedcDNA0.5µlFailSafePCREnzyme(suppliedbytheuser)

50µlTotalvolume PCR cycle conditions:DenaturetheDNAat95°Cfor1minutefollowedbycyclesof:

95°Cfor30seconds55°Cfor30seconds68°Cfor3minutes

Incubateat68°Cfor7minutesafterthefinalcycle

7

PurifyLibrary AMPurepurification.QiagenpurificationonlysuggestedforveryshortFFPERNAsamples(<200nt)andwillresultinadaptor-dimercontamination. 8

QCLibrary QuantifybyQubit™orPicoGreenandvisualizeonAgilentBioAnalyzer 9

3. Kit ProcedureRemoveallcomponentsexceptenzymesandFinishingSolution,allowtothaw,andstoreonice.Centrifugeeachtubebrieflytocollectliquidtothebottomofthetube.ItishighlyrecommendedthatenzymeandFinishingSolutionbestoredinabenchtopcooler(–20˚C)toavoidrepeatedfreeze-thaws.

10-15cyclesusinghighqualityRNA12-15cyclesusingFFPERNA



3.A. Fragment the RNA and Anneal the cDNA Synthesis Primer

Important!1. IfusingseverelyfragmentedRNA,suchasthatobtainedfromFFPEsamples,usetheproceduredescribedin

Appendix 1.

2. TheRNAcanbefragmentedbymethodsotherthanthosedescribedinPart3.A.IffragmentingtheRNAbyothermethods,thefragmentedRNAmustbepurifiedanddissolvedinamaximumof9μlofNuclease-FreeWater.Then,usetheproceduredescribedinAppendix 1toannealthecDNAsynthesisprimerandperformcDNAsynthesis.

RequiredinPart3.A.

Component Name Tube Label Cap Color

cDNA Synthesis Primer cDNA PrimerGreen

RNAFragmentationSolution FragmentationSolution

Nuclease-FreeWater Nuclease-FreeWater Clear

3.A.1. Ina0.2-mlPCRtube,assemblethefollowingreactionmixture.Ifa“notemplate”controlreactionisperformed,substituteNuclease-FreeWaterfortheRNAsample.

Use500pgto50ngofrRNA-depletedorpoly(A)+ RNA per reaction.

x μl Nuclease-FreeWater y μl rRNA-depletedorpoly(A)+ RNA 1 μl FragmentationSolution 2 μl cDNAPrimer 12 μl Totalvolumeperreaction

3.A.2. FragmentRNA:Incubateat85°Cfor5minutesinathermocyclerwithheatedlid.

3.A.3. Stopthefragmentationreactionbyplacingthetubeonice.

3.B. Synthesize cDNARequiredinPart3.B.


ScriptSeq v2 cDNA Synthesis PreMix cDNA Synthesis PreMix

Red100mMDTT 100mMDTT

StarScript AMV Reverse Transcriptase StarScript AMV Reverse Transcriptase

ScriptSeqFinishingSolution FinishingSolution

ThermocyclersettingsforPart3.B: 25°Cfor5minutes(cDNAsynthesis) 42°Cfor20minutes(cDNAsynthesis) 37°CPause/Hold

37°Cfor10minutes(FinishingSolution) 95°Cfor3minutes(InactivateFinishingSolution) 25°CPause/Hold

3.B.1. Onice,preparethecDNASynthesisMasterMix:

Foreachreaction,combineonice:

3.0 μl cDNASynthesisPreMix 0.5 μl 100mMDTT 0.5 μl StarScriptAMVReverseTranscriptase 4.0 μl Totalvolumeperreaction

GentlybutthoroughlymixthecDNASynthesisMasterMixbypipetting.

3.B.2. Add4μlofthecDNASynthesisMasterMixtoeachreactiononicefromPart3.A,Step3,andmixbypipetting.

3.B.3. Incubateat25°Cfor5minutesfollowedby42°Cfor20minutes.

3.B.4. Coolthereactionsto37°CandPause/Holdthethermocycler.

85°C

5:00

37°C 95°C25°C

3:00 Pause10:00

25°C42°C

37°C

20:00 Pause5:00

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3.B.5. Removeonereactionoronestripoftubesatatimefromthethermocycler.Add1.0µlofFinishingSolution,andmixgentlybutthoroughlybypipetting.Returnthereactiontothethermocyclerbeforeproceedingwiththenext.

3.B.6.Incubateat37°Cfor10minutes.

3.B.7. Incubateeachreactionat95°Cfor3minutes.Then,coolthereactionsto25°CandPause/Holdthethermocycler.

PreparetheTerminalTaggingMasterMixasdescribedinPart3.C,Step1.

3.C. Synthesize 3′-Tagged DNARequiredinPart3.C.


ScriptSeqv2Terminal-TaggingPremix TerminalTaggingPreMixBlue

DNAPolymerase DNAPolymerase

Important! The Terminal-Tagging PreMix is a viscous solution. Mix it thoroughly before use.

Recommended: Wide bore pipet tip (e.g., Pure™ 200G sterile tip; catalog number #3531, Molecular Bioproducts) when pipetting the Terminal Tagging PreMix and the Terminal Tagging Master Mix.

ThermocyclersettingsforPart3.C: 25°Cfor15minutes(DNAPolymerase) 95°Cfor3minutes(InactivateDNAPolymerase) 4°CHoldorice

3.C.1. Onice,preparetheTerminalTaggingMasterMix.


7.5 μl TerminalTaggingPremix 0.5 μl DNAPolymerase 8 μl Totalvolumeperreaction

3.C.2. ThoroughlymixtheviscousTerminalTaggingMasterMix.

3.C.3. Removeonereactionorstripoftubesfromthethermocycler(fromPart3.B,Step7)andadd8.0μloftheTerminalTaggingMasterMix.Gentlybutthoroughlymixthereactionbypipetting.Returneachreactiontothethermocyclerbeforeproceedingwith the next.

3.C.4. Incubateeachreactionat25°Cfor15minutes.

3.C.5. Incubateeachreactionat95°Cfor3minutes.Then,coolthereactionsto4°Coniceorinthethermocycler.

3.D. Purify the cDNAThedi-taggedcDNAmustbepurifiedpriortoPCRamplification.WerecommendusingtheMinElutePCRPurificationKit(Qiagen)ortheAgencourtAMPureXPsystem(BeckmanCoulter).If working with FFPE RNA, you must use the MinElute PCR Purification kit.

• IfusingtheMinElutePCRPurificationKit,followthemanufacturer’sdirections.ElutethecDNAusing25μloftheEBBuffer(ElutionBuffer)thatisprovidedintheMinEluteKit.The25-μlvolumetypicallyyieldsafinalvolumeof22.5μl.However,ifnecessary,adjusttheeluateto22.5μlwithEBBuffer.IfusingacolumnpurificationmethodotherthantheMinElutePCRPurificationKit,adjusttheeluatevolumeto22.5μl.

• IfusingtheAMPureXPSystem,thepurificationcanbedoneina96-wellplateorinthemicrofugetubescontainingthedi-taggedcDNAfromPart3.C,Step4.Theproceduredescribedusesa1.8XAMPureXPpurificationscheme.

1. WarmtheAMPureXPbeadstoroomtemperature.Whilethebeadswarm,prepare400µloffresh80%ethanolatroomtemperatureforeachsample.

2. IfperformingtheAMPureXPprocedureusinga96-wellplateformat,transfereachdi-taggedcDNAfromPart3.C,Step4independentlyintoawelloftheplate.Ifusingmicrofugetubes,transfereach70-μlvolumetoaseparate1.5-mltube.

3. Important! Vortex the AMPure XP beads until they are a homogeneous suspension.

4. Add45μlofthebeadstoeachsamplecontainingdi-taggedcDNAfromPart3.C,Step4.

5. Mixthoroughlybygentlypipettingtheentirevolumeofeachwell/tube10times.

6. Incubatethesamplesatroomtemperaturefor15minutes.

7. Placethesamplesinamagneticstandatroomtemperatureforatleast5minutes,untiltheliquidappearsclear.

8. Removeanddiscardthesupernatantfromeachwell/tubeusingapipette.Someliquidmayremainineachwell/tube.Takecarenottodisturbthebeads.

9. Withthesamplesremainingonthemagneticstand,add200μlof80%ethanoltoeachwell/tubewithoutdisturbingthebeads.

25°C 95°C4°C

3:00 Pause15:00



10. Incubatethesamplesatroomtemperatureforatleast30seconds,thenremoveanddiscardallofthesupernatantfromeach.Takecarenottodisturbthebeads.

11. Repeatsteps9and10onemoretimeforatotaloftwo80%ethanolwashes.

12. Allowthesamplestoair-dryontheirmagneticstandsfor15minutesatroomtemperature.

13. Add24.5µlofNuclease-FreeWatertoeachwell/tubeandremovefromthemagneticstand.

14. Thoroughlyresuspendthebeadsbygentlypipetting10times.


16. Placethesamplesonthemagneticstandatroomtemperatureforatleast5minutes,untiltheliquidappearsclear.

17. Transfer22.5μloftheclearsupernatant,whichcontainsthedi-taggedcDNA,fromeachwell/tubetoanew0.2-mlPCRtube.

18. PlacethetubesoniceandproceedtoPart3.Eorplaceat–20°Cforlonger-termstorage.

3.E. PCR Amplify the Library and Add an Index (Barcode)ThisstepgeneratesthesecondstrandofcDNA,completestheadditionoftheIlluminaadaptorsequences,incorporatesanIndexorauser-definedbarcode,ifdesired,andamplifiesthelibrarybyPCR.Typically,10-15PCRcyclesareperformed.AtleastonePCRcyclemustbedone.MorePCRcyclescanbeperformedifagreateryieldofthelibraryisneeded.

Adding an Index Read or a user-defined barcode.ThestandardScriptSeqv2reactionusingtheReversePCRPrimerthatisincludedinthekitproducesanonbarcodedlibrary.

• ToaddanIlluminaIndex,replacetheReversePCRPrimerthatisincludedinthiskitwithoneoftheScriptSeqIndexPCRPrimers,availableseparatelyfromEpicentre(seeRelatedProducts).OnlyEpicentre’sScriptSeqIndexPCRPrimersarecompatiblewiththeScriptSeqv2Kitprocedure.CarefullyreadthetheScriptSeqIndexPCRPrimersproductliteraturetoensureproperpoolingofIndexedlibraries.

• Toaddauser-definedbarcode,seeAppendix3.

Choice of PCR enzyme.ThiskitisoptimizedforusewithEpicentre’sFailSafePCREnzyme.WedonotrecommendusingotherPCRenzymes,astheyieldandqualityofthefinallibrarymaybeadverselyaffected.

RequiredinPart3.E.


FailSafePCRPreMixE FailSafePCRPreMixE

YellowForward PCR Primer Forward PCR Primer

Reverse PCR Primer Reverse PCR Primer


Providedbytheuser:FailSafePCREnzyme(Epicentre;cat.nos.FSE51100,FSE5101K)

Important! If you are adding an Index or user-defined barcode to the library, do not use the Reverse PCR Primer that is included in this kit! Instead, use the Index- or barcode-containing oligo as the Reverse PCR Primer in this procedure. Read carefully the ScriptSeq Index PCR Primer product literature to ensure color balancing of the Indexed libraries.

1. Ina0.2-mlPCRtubecombineonice: 22.5 µl ofdi-taggedcDNAfromPart3.D 1 μl ForwardPCRPrimer 1 μl ReversePCRPrimer (orScriptSeqIndexPCRPrimer,oruser-definedbarcodeReversePCRPrimer) 25 μl FailSafePCRPreMixE 0.5 μl FailSafePCREnzyme(1.25U) 50 μl Totalvolumeperreaction

2. PerformPCR

DenaturetheDNAat95°Cfor1minute

Followedby10-15cyclesof:

95°Cfor30seconds

55°Cfor30seconds

68°Cfor3minutes

AftertheappropriatenumberofPCRcycles,incubateat68°Cfor7minutes.

10-15cyclesusinghighqualityRNA12-15cyclesusingFFPERNA

95°C 95°C

55°C

68°C 68°C

:30 :30 3:00 7:001:00

10-15cycles

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SeePart3.GforexamplesofamplifiedScriptSeqRNA-Seqlibraries.DuringthePCR,readPart3.Ftodeterminewhichpost-PCRpurificationprocedureisbestsuitedtoyoursample.AfterthePCRprocedureiscomplete,proceedimmediatelytoPart3.F.

3.F. Purify the RNA-Seq LibraryUsetheAMPureXPsystem(BeckmanCoulter)topurifytheScriptSeqv2kitlibraries,except for libraries prepared from FFPE RNA with an average size <200 nt.TheAMPureXPSystemisbestatremovingthe“primer-dimers”thatcanoccurduringPCR.

Note: Use the MinElute PCR Purification system (Qiagen) only for purifying libraries made from FFPE RNA with an average size <200 nt. Libraries purified using the MinElute columns will be contaminated with primer-dimers.

3.F.1. AMPure XP PurificationThisprocedurewillyieldaScriptSeqlibraryof>200nts(seealsoPart3.G).Thisprocedureusesa1XAMPureXPpurificationscheme.

1. WarmtheAMPureXPbeadstoroomtemperature.Whilethebeadswarm,prepare400µloffresh80%ethanolatroomtemperatureforeachsample.

2. Ifusinga96-wellplateformat,transfereachamplifiedlibraryfromPart3.E,Step2,independentlyintoawelloftheplate.Ifusingmicrofugetubes,transfereach50-μlvolumetoaseparate1.5mltube.

3. Important! Vortex the AMPure XP beads until they are a homogeneous suspension.

4. Add50μlofthebeadstoeachsample.

5. Mixthoroughlybygentlypipettingtheentirevolumeupofeachwell/tube10times.


7. Placethesamplesinamagneticstandatroomtemperatureforatleast5minutes,untiltheliquidappearsclear.

8. Removeanddiscardthesupernatantfromeachwell/tubeusingapipette.Someliquidmayremainineachwell. Takecarenottodisturbthebeads.

9. Withthesampleremainingonthemagneticstand,add200μlof80%ethanoltoeachwell/tubewithoutdisturbingthebeads.

10. Incubatethesamplesatroomtemperatureforatleast30seconds,thenremoveanddiscardallofthesupernatant.Takecarenottodisturbthebeads.

11. Repeatsteps9and10onemoretimeforatotaloftwo80%ethanolwashes.

12. Allowthesamplestoair-dryontheirmagneticstandsfor15minutesatroomtemperature.

13. Add20µlofNuclease-FreeWatertoeachwell/tubeandremovetheplateor1.5-mltubesfromtheirmagneticstand.

14. Thoroughlyresuspendthebeadsbygentlypipetting10times.


16. Placethesamplesonthemagneticstandatroomtemperatureforatleast5minutes,untiltheliquidappearsclear.

17. Transfertheclearsupernatant,whichcontainstheRNA-Seqlibrary,fromeachwell/tubetoanappropriatecollectiontubeforassessmentoflibraryquantityandquality.

3.F.2. MinElute PCR Purification Kit for Highly Fragmented RNA SamplesUsetheMinElutePCRPurificationKittopurifyScriptSeqv2librariesmadefromFFPERNAofaveragesize<200nt.ThisprocedureusestheExonucleaseIenzymeprovidedintheScriptSeqv2Kit.

1. RemoveexcessPCRprimersbyadding1μlofExonucleaseItoeachreactionandincubatethereactionsat37°Cfor15minutes.

2. PurifythelibraryusingtheMinEluteKitproceduredescribedbythemanufacturer.



3.G. Assess Library Quantity and QualityThelibraryshouldbequantifiedbyyourlaboratory’sstandardmethods.Thesizedistributioncanbeassessedusingthe2100Bioanalyzer(Agilent)andaHighSensitivityDNAChip.Fig.3showsrepresentative2100Bioanalyzer(Agilent)profilesofRNA-SeqlibrariesproducedbytheScriptSeqv2Kit.

4. AppendicesAppendix 1: Preparing a Library from Severely Fragmented RNA and FFPE RNAUsethisprocedurewhenpreparinglibrariesfromrRNA-depletedRNA:• Thatishighlyfragmented,suchassometimesobtainedfromFFPEsamples.• ThathasbeenfragmentedusingaproceduredifferentthanthatdescribedinPart3.A.

4.1.A. Anneal the cDNA Synthesis PrimerRequiredinPart4.1.A.


ScriptSeq cDNA Synthesis Primer cDNA Primer Green


1. Ifa“notemplate”controlreactionisperformed,substituteNuclease-FreeWaterfortheRNAsample.

x μl Nuclease-FreeWater y μl rRNA-depletedfragmentedorFFPERNA(500pgto50ng) 2 μl cDNAPrimer 11 μl Totalvolumeperreaction

2. Incubateat65°Cfor5minutesinathermocycler.

3. Stopthereactionbyplacingthetubeonice.

Figure 3. Representative profiles of ScriptSeq™ v2 Kit RNA-Seq libraries. ScriptSeqRNA-Seqlibrarieswerepreparedwiththeindicatedamountofhumanliverpoly(A)+RNA.DuringtheScriptSeqprocedure,thedi-taggedcDNAswerepurified(Part3.D)usingtheMinElute®PCRPurificationKit(Qiagen).InPart3.E,15PCRcycleswereperformedforboththe500pg(Fig.3A)and5ng(Fig.3B)libraries.Figure3Cshowstheprofileofalibrarythathadbeenover-amplified(toomanyPCRcycles)duringStep3.E.If>60%oftheover-amplifiedmaterialisbetween200and1000bp,thelibrarycanbesequenced.ThePCR-amplifiedlibrarieswerepurified(Part3.F)usingtheAMPure®XPprocedure.Purifiedlibrarieswereanalyzedusingthe2100Bioanalyzer(Agilent)withaHighSensitivityDNAChip.

500pgRNA

Over-amplifiedlibrary

5 ng RNA

A. B.

C.

65°C

5:00

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4.1.B. Synthesize cDNARequiredinPart4.1.B.


RNAFragmentationSolution FragmentationSolution Green

ScriptSeq v2 cDNA Synthesis PreMix cDNA Synthesis PreMix

Red100mMDTT 100mMDTT

StarScript AMV Reverse Transcriptase StarScript AMV Reverse Transcriptase

ScriptSeqFinishingSolution FinishingSolution


ThermocyclersettingsforPart4.1.B:

25°Cfor5minutes(cDNAsynthesis) 42°Cfor20minutes(cDNAsynthesis) 37°CPause/Hold

37°Cfor10minutes(FinishingSolution) 95°Cfor3minutes(InactivateFinishingSolution) 25°CPause/Hold

Note: The RNA Fragmentation Solution is added to supplement Mg2+ in the cDNA synthesis reaction.

1. PreparethecDNASynthesisMasterMix.


1.0 μl FragmentationSolution 3.0 μl cDNASynthesisPremix 0.5 μl 100mMDTT 0.5 μl StarScriptAMVReverseTranscriptase 5 μl Totalvolumeperreaction

GentlybutthoroughlymixthecDNASynthesisMasterMixbypipetting10times.

2. Add5μlofthecDNASynthesisMasterMixtoeachreactiononicefromPart4.1.A, Step3andmixgentlybutthoroughly.

3. Incubateat25°Cfor5minutesfollowedby42°Cfor20minutes.

4. Coolthereactionsto37°Candpausethethermocycler.

5. Removeonereactionorstripoftubesatatimefromthethermocycler,add1.0µlofFinishingSolution,andmixgentlybutthoroughlybypipetting.Returneachreactiontothethermocyclerbeforeproceedingwiththenext.

6. Incubateat37°Cfor10minutes.

7. Incubatethereactionsat95°Cfor3minutes.Then,allowthereactionstocoolto25°CandPause/Holdthethermocycler.

8. ContinuewiththestandardkitprocedurebeginningatPart3.C.

25°C42°C

37°C

20:00 Pause5:00

37°C 95°C25°C

3:00 Pause10:00



Appendix 2: Sequencing the ScriptSeq v2 LibraryScriptSeqRNA-SeqlibrariesarecompatiblewithTruSeq™ClusterKitsandcanbesequencedonanyIllumina®sequencer.

ThesequenceproducedbytheRead1SequencingPrimeristhatofthesensestrandoftheoriginalfragmentedRNAmolecule.TomapScriptSeqlibrarysequencingdatausingTopHat/Cufflinks,usethefr-secondstrand command.

Figure 2. Sequencing a ScriptSeq™ v2 library.

Red = sequenceincorporatedbytheTerminalTaggingprocessandPCRamplification.Blue = sequenceincorporatedduringreversetranscriptionandPCRamplification.Black = sequenceofthecDNA.Rd 1 SP = sequencereadisthatofthesensestrandoftheoriginalfragmentedRNAmolecule.Rd 2 SP = sequencereadisthatoftheantisensestrandoftheoriginalfragmentedRNAmolecule.Index SP = firstnucleotidereadisthatoftheIndexorbarcode.

Appendix 3: Adding a User-Defined Barcode to the LibraryAbarcodeisaddedbytheReversePCRPrimerinPart3.Eoftheprocedure.AReversePCRPrimercontainingauser-definedbarcodesequencemustbesynthesizedbytheuserandisthenusedastheReversePCRPrimerinPart3.Eoftheprocedure.

Theuser-definedReversePCRPrimer(s)must bethefollowingsequence:

5’ CAAGCAGAAGACGGCATACGAGAT desired barcode’s GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 3’ reverse complement

Theprimer(s)shouldbedissolvedtoaconcentrationof10μMinnuclease-freewater.

Important! The user-defined barcode sequence of the of the custom synthesized Reverse PCR Primer should be the reverse complement of the sequence read. For example, using the Illumina Multiplexing Index Read Sequencing Primer, the user-defined barcode sequence:

5’…ACGTAC… 3’ willbereadas: 5’…GTACGT…3’

PleasecontactTechnicalSupportifyouhavequestionsaboutaddinguser-definedbarcodesorsynthesizingcustomreversePCRprimers.

➞

➞ ➞

5’ AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT3’ TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA

5’ ACACTCTTTCCCTACACGACGCTCTTCCGATCT

AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC(Barcode)ATCTCGTATGCCGTCTTCTGCTTG 3’TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG(Barcode)TAGAGCATACGGCAGAAGACGAAC 5’

TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG 5’

5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCAC

---cDNA (sense orientation)----------cDNA (antisense orientation)---

Rd 1 SP

Rd 2 SP

Index SP



Appendix 4. Troubleshooting and FAQs

Observation/Question Recommendation

Library Preparation

DoesPCRhavetobeperformed? Yes.PCRcompletestheadditionoftheIlluminaadaptorsequences.AtleastonecycleofPCRmustbeperformed.

ThereisnotenoughoftheTerminalTaggingOligo(TTO)Solution

TheTerminalTaggingSolutionisveryviscous.Pleasespindownthetubeinamicrocentrifugeatmaximumspeedfor30seconds.Makesureyourpipettesarecalibrated.Pipettethesolutionslowly.BesurethatTTOsolutiondoesnotadheretotheoutsideofthepipettetipwhenitiswithdrawnfromthesolution.Ifpossible,useawide-borepipettetipwhenpipettingtheTTOsolution.

Thereisabi-modalpeakinbioanalyzertraceofmyScriptSeqlibrary

ThelibrarywasoveramplifiedduringthePCR.Re-createthelibrarieswithfewercycles.AlternativelyadoubleSPRIcleanupcanremovelargefragments.SeealsoFigure4CinStep3.G.

Sequencing

WhichTruSeqClusterkitsarecompatiblewithScriptSeqLibraries?

TruSeqClusterkitsthatusetheHP8andHP10sequencingprimersarecompatiblewithSctiptSeqlibraries.

HowdoIchoosewhichIndexestousewhenmakingIndexedScriptSeqlibraries?

PleaseseetheScriptSeqIndexPCRPrimersprotocolformoreinformtation.

Onthesequencingsamplesheet,whatkitsettingshouldIusetosequencetheScriptSeqlibraries?

Tru-SeqLT

CanIusetheTruSeqLTIndexadaptorswhenmakinganIndexedScriptSeqlibrary?

No.OnlytheScriptSeqIndexPCRPrimerscanbeusedtoaddanIndextoaScriptSeqlibrary.Althoughthe6-nucleotideIndexsequenceisthesameforeachScriptSeqIndexPrimerandthecorrespondingTruSeqIndexAdapter,theflankingsequencesaredifferent.Therefore,theScriptSeqIndexPrimersandTruSeqIndexAdapterscannotbeusedinterchangably.

Data Anlaysis

Whendoingdataanalysis,whichstranddoImaptoinTopHat/Cufflinks?

Pleaseusethe fr-secondstrand commandwhenperformingScriptSeqlibraryanalysis.

AreScriptSeqlibrariesstranded? Yes.ThesequencegeneratedbytheRead1SequencingPrimercorrespondstothesensestrandoftheoriginalfragmentedRNAmolecule.SeeAppendix2.

5. Additional RNA-Seq Sample Prep Products

Ribo-Zero™ rRNA Removal Kits and Globin-Zero™ Gold Kit for globin mRNA/rRNA removal are available separately for many sample types.

ScriptSeq™ Complete Kits, combining a Ribo-Zero™ rRNA removal or Globin-Zero™ globin mRNA/rRNA removal module and a ScriptSeq™ v2 RNA-Seq Library Preparation Kit module, are available for many sample types.

ScriptSeq™ Index PCR Primers for adding an Index to the ScriptSeq libraries.

FailSafe, Ribo-Zero, ScriptSeq, and StarScript are trademarks of Epicentre, Madison, Wisconsin.

Illumina is a registered trademark and TruSeq is a trademark of Illumina Inc., San Diego, California.

AMPure is a registered trademark of Beckman Coulter, Inc., Danvers, Massachusetts.

MinElute is a registered trademark of Qiagen Inc., Valencia, California.

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