RealSeq ® -AC miRNA Library Kit for Illumina ® sequencing Cat. No. 500-00012 500-00024 500-00048 20181221_RealSeq ® -AC_CL
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RealSeq®-AC miRNA Library Kit for Illumina® sequencingCat. No. 500-00012 500-00024500-00048
20181221_RealSeq®-AC_CL
Table of Contents
I. Overview . . . . . . . . . . . . . . . . . . . . . . . 5
II. RealSeq®-AC Kit Contents. . . . . . . . . . . . . . 6
III. Warnings and Recommendations . . . . . . . . . . 7
IV. User-supplied Reagents, Consumables, and Laboratory Equipment (not included) . . . . . . 7
V. Input Requirements. . . . . . . . . . . . . . . . . . 8
VI. Experimental Protocol . . . . . . . . . . . . . . . . 9
VII. Appendix A: Thermocycler Programming . . . . . 16
VIII. AppendixB:ExampleLibraryProfile . . . . . . . . 17
IV. Appendix C: Data Analysis . . . . . . . . . . . . 18
V. Appendix D: Reverse Primer Index Sequence . . . 19
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Information in this document is subject to change without notice.
SOMAGENICS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL SOMAGENICS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT SOMAGENICS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES.
NOTICES TO PURCHASER:
LIMITED LICENSE
The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefitofthepurchaser.Norighttoresellthisproductoranyofitscomponentsisconveyedexpressly, by implication, or by estoppel. This product is for internal research purposes onlyandmaynotbetransferredtothirdparties,resold,modifiedforresale,orusedtomanufacture commercial products or to provide a service to third parties without prior written approval of SomaGenics, Inc. For information on obtaining additional rights, please contact: [email protected].
This product is covered by issued and pending patents.
TRADEMARKS
RealSeq® is the Registered Trademark of SomaGenics, Inc. All other brands and names contained herein are the property of their respective owners.
For Research Use Only.
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RealSeq®-ACI. Overview
Step 1. Adapter ligation
Step 2. Adapter blocking
Step 3. Circularization
Step 4. Dimer removal
Step 5. Reverse transcription
Step 6. PCR amplification
Step 7. Size selection
Combine:RNABuffer 1RealSeq® Adapter
Incubate:2 min at 70°C2 min ice
Add:RNase InhibitorBuffer 2Ligase
Incubate:60 min at 25°C
Add:Blocking Agent
Incubate:10 min at 65°CStep down to 37°C
Add:Blocking EnzymeBuffer 3
Incubate:60 min at 37°C20 min at 65°C
Add:RealSeq® EnzymeBuffer 4
Incubate:60 min at 37°C
Add:Dimer Removal Agent
Incubate:10 min at 37°C
Add:RealSeq® Beads
Incubate:10 min at 37°C
Add:RT PrimerdNTPs
Incubate:5 min at 65°C
Add:RT BufferRNase free waterRT EnzymeRNase Inhibitor
Incubate:60 min at 42°C20 min at 65°C
Add:PCR BufferdNTPsFP and RPPCR PolymeraseRNase free water
PCR:30 sec at 94°C5-13 Cycles 15 sec at 94°C 30 sec at 62°C 15 sec at 70°C5 min at 70°C
AMPure XP Beads*
Illumina Sequencing
8 ho
urs
*User supplied
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II. RealSeq®-AC Kit Contents
Core kit box (Store at -20°C) (Box 1 of 2)
Primer box (Store at -20°C) (Box 1 of 2)
Beads (Store at +4°C)
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Tube Component Tube ComponentRB •RNABuffer RTP •RTPrimerA •RealSeq® Adapter dNTP •dNTPsRI •RNaseInhibitor RTB •RTBufferLB •LigationBuffer RTE •RTEnzymeL •Ligase PB •PCRBuffer
BA •BlockingAgent PP •PCRPolymeraseBE •BlockingEnzyme DRA DimerRemovalAgentBB •BlockingBuffer AD AdapterDilutionBuffer
RSE •RealSeq®Enzyme + miRNAControlRSB •RealSeq® Buffer W RNase-FreeWater
Cat. No. Tube Component500-00012 / 24 / 48 FP ForwardPrimer(FP)500-00012 RP1 - 12 ReversePrimers,Index1-12*500-00024 RP1 - 24 ReversePrimers,Index1-24*500-00048 RP1 - 48 ReversePrimers,Index1-48*
*ForsequencesseeAppendixC,page18.
Tube ComponentB RealSeq®Beads
III. Warnings and Recommendations• Donotusethekitpasttheexpirationdate.• Donotremoveenzymesfrom-20°Cuntilimmediatelybefore
useandreturnto-20°Cimmediatelyafteruse.• EnsuretheRealSeq® AdapterandmiRNA Controlarealways
onicetominimizedegredation.• Vortexandcentrifugeeachcomponentbeforeuse.• AlwayshavePCRtubesonicewhenhandling.• Do notfreezeRealSeq®BeadsorSPRIselect® Reagent.• Forease,thermocyclerscanbepre-programmedwithallthe
reactionsforacontinuousworkflow.GotoAppendexAforalistoftemperatures.
IV. User-supplied Reagents, Consumables, and Laboratory Equipment (not included)• Sterilenuclease-freePCRtubes
• Sterilenuclease-free1.5mltubes
• MagneticstandforPCRtubes(e.g.Diagenode#B0400001)
• 96-wellaluminumblock
• SPRIselect®Reagent(SomaGenicscatalognumbers510-00012/510-00024/510-00048)*
• 96-100%Ethanol(molecularbiologygrade)
• Bioanalyzer®DNA1000kit(Agilent#5067-1504)orTapeStationD1000DNAkit(Agilent#5067-5582&5037-5583)
• Qubit®Fluorometer(ThermoFisherScientific)andQubit®dsDNAHSAssayKit,100assays(ThermofisherScientific#Q32851)
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*ThisproductcontainsSPRIselectReagentManufacturedbyBeckmanCoulter,Inc.
V. Input Requirements• Thiskitwasoptimizedusing100ngofHumanBrainTotalRNA
(ThermoFisher#AM7962).
• HighqualitytotalRNAwithRNAIntegrityNumber(RIN)>7isrecommendedasinputmaterial.
• UsingpartiallydegradedRNAwillresultinahigherproportionofshortsequencingreads(<15nt)thatcorrespondtodegradedrRNAs,aswellasdecreasedoverallpercentageofmiRNAreads.
• NotallRNAextractionandpurificationkitsisolatesmallRNAs,usersshouldconfirmthatthemethodusedalsoisolatessmallRNAs.
• WhenpreparinglibrariesforthefirsttimewehighlyrecommendusingtheincludedmiRNAcontroltoprepareacontrollibrary.
• Toprepareacontrollibrary,use1µlofthecontrolmiRNAinsteadoftheRNAsample.SeeAppendixB(Figure2)foranexamplelibraryprofilewiththemiRNAcontrol.
Guidelines for different input amounts:
*100ngoftotalRNAisrecommended.HigheramountsofRNAmayenrichforotherclassesofsmallRNAs(i.e.piRNA,snRNA,andsnoRNA)**Foroptimallibraries,prepareanewdilutionforeverystartoftheprotocol.Re-usingthedilutionsmaycausedegredationoftheadapter.
Input Amount RealSeq®-AC Adapter dilution (per library)**
PCR Cycles
1 µg total RNA none 10-13100 ng total RNA* 1µl Tube A + 1µl Tube AD 13-1610 ng total RNA 1µl Tube A + 1µl Tube AD 16-191 ng total RNA 1µl Tube A + 3µl Tube AD 19-22
1 µl miRNA Control 1µl Tube A + 1µl Tube AD 13
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VI. Experimental Protocol1. Adapter Ligation
• Heatthermalcyclerto70°C.• PrepareseparatePCRmicrotubesforeachRNAsample.• RNAsamplescanbeaddeduptoavolumeof3µl.
*RNA Buffer (RB) is very viscous. Pipet slowly. **See Table in Input Requirements (Section V) for Adapter dilutions.• Placeallsampletubesintoathermalcyclerat70°C.• Heatsampletubesfor2minutesat70°Candtransfertoiceforat
leasttwominutes.• Whilethesamplesarestillonice,addthefollowingreagentstothe
sampletube.Mixbypipettingandspindown.
• Runtheligationreactioninathermalcyclerwiththefollowingpro-file:
• Proceed immediately to next step (Adapter Blocking).
Step Type Temperature TimeHold 25°C 60 minHold 65°C 5 min
Reagent Volume to add•RNA ( 1 µg to 1 ng) up to 3 µl
•RNA Buffer (RB) 3 µl*
•RealSeq® Adapter (A) 1µl**
•RNase Free Water (W) VariableTotal Volume 7 µl
Reagent Volume to add•RNase Inhibitor (RI) 1 µl
•Ligation Buffer (LB) 1 µl
•Ligase (L) 1 µlTotal Volume 10 µl
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2. Adapter Blocking• Thaw,vortexandspin•Blocking Agent (BA).• Add2.5 µlof•Blocking Agent (BA)toeachsampletube.Mix
bypipettingandspindown.• Incubatewiththefollowingprofile:
*Step down from 65°C to 37°C at a rate of 0.1°C per second (approximately 5 mins).
• Addthefollowingreagentstoeachsampletube.Mixbypipettingandspindown.
• RunBlockingreactioninathermalcyclerwiththefollowingprofile:
• Proceed immediately to next step (Circularization).or
*Stopping Point*: Alternativelylibrariescanbestoredovernightat-20°C.Torestart,thawsamplesonicebeforeproceedingtonextstep.
Step Type Temperature TimeHold 65°C 5 min
Step down* 65 to 37°C Approx. 5 min
Reagent Volume to add•Blocking Enzyme (BE) 1.1 µl
•Blocking Buffer (BB) 6.4 µlTotal Volume 20 µl
Step Type Temperature TimeHold 37°C 60 minHold 65°C 20 min
3. Circularization• Performcircularizationbyaddingthefollowingreagentstoeach
sampletube.Mixbypipettingandspindown.
• Placesamplesintoathermalcyclerwiththefollowingprofile:
• Proceed immediately to next step (Dimer Removal).
4. Dimer Removal• WhenCircularizationiscomplete,add1 µlofDimer Removal
Agent (DRA)toeachsampletubeinthethermocycler.Mixbypipetting,andincubateinthethermocyclerwiththefollowingprofile:
• PrepareRealSeq® Beads(Storedat+4°C)
*WARNING*: Do NOT use SPRIselect® Reagent!• Thoroughlyvortexthebeadsforatleast30seconds.• Pipet20µlofthebeadsuspensionintoanewPCRtube.• Placethetubeonthemagneticrackfor1minuteoruntilall
thebeadssettleagainstthesideofthetube.• Removeanddiscardthesupernatant.
• Immediatelyresuspendbeadswithall23µlfromsampletubeandincubatefor10minat37°C.
• Quickyspindownthetubesinamicrocentrifuge,thenplaceonamagneticrackfor1minuteoruntilallbeadssettleagainstthesideofthetube.Transfer20µlofsupernatantintoacleanPCRtube.
• Proceed immediately to next step (Reverse Transcription).
Reagent Volume to add•RealSeq® Enzyme (RSE) 1 µl
•RealSeq® Buffer (RSB) 1 µlTotal Volume 22 µl
Step Type Temperature TimeHold 37°C 60 min
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Step Type Temperature TimeHold 37°C 10 min
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5. Reverse Transcription• Addthefollowingreagentstoeachsampletube.
• Incubatethesamplesat65°Cfor5minutes.Chilloniceforatleasttwominutesandspindown.
• Addthefollowingreagentstoeachsampletube:
• Mixbypipettingandspindown.• Placesamplesintoathermalcyclerwiththefollowingprofile:
• Proceed immediately to next step (PCR Amplification).or
*Stopping Point*: Alternativelylibrariescanbestoredovernightat-20°C.Torestart,thawsamplesonicebeforeproceedingtonextstep.
Reagent Volume to add•RT Primer (RTP) 2 µl
•dNTPs (dNTP) 2 µlTotal Volume 24 µl
Reagent Volume to add• RT Buffer (RTB) 4 µl
• RNase free Water (W) 9 µl
• RT Enzyme (RTE) 2 µl
• Rnase Inhibitor (RI) 1 µlTotal Volume 40 µl
Step Type Temperature TimeHold 42°C 60 minHold 65°C 20 min
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6. PCR Amplification
• PreparePCRreactionmixforeachsample.Mixgentlybyinversionandspindown.
• Add53µlofPCRreactionmixtoeachsample.• Add7 µl of Reverse Primer Index (Primer Box)toeachsample.
Mixbypipettingandspindown.• Runsamplesinathermalcyclerwiththefollowingprofile:
• Proceed immediately to next step (Size Selection).or
*Stopping Point*: Alternativelylibrariescanbestoredovernightat-20°C.Torestart,thawsamplesonicebeforeproceedingtonextstep.
Reagent Volume to add • PCR Buffer (PB) 20 µl
• dNTPs (dNTP) 3 µl
• Forward Primer (FP) 7 µl
• PCR Polymerase (PP) 4 µl
• RNase Free Water (W) 19 µlTotal Volume PCR Master Mix 53 µl
Step Type Temperature TimeHOLD 94°C 30 sec
CYCLE (10-22 cycles)
(See Section V)
94°C 15 sec
62°C 30 sec
70°C 15 secHOLD 70°C 5 min
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7. Size Selection*WARNING*: ForsizeselectionuseSPRIselect®Reagent,DO NOTuseRealSeq®beadsforsizeselection.
• If storing SPRIselect® Reagent at +4°C, take out the SPRIselect®Reagent to the bench top at least 30 minutes before proceeding. This will ensure that the beads warm to room temperature before use.
Size selection with SPRIselect® Reagent• Prepare70%ethanol(500µlpersample).• EnsureSPRIselect®Reagentisatroomtemperature,andresuspend
beforeuse.• VortexandspindowneachPCRreaction.Transfer50µlofsample
tonewPCRtubes.• Add70µlofSPRISelect®Reagenttoeachsample.Mixreagentand
PCRthoroughlybypipettemixing10times.• Letthemixedsamplesincubatefor5minutesatroomtemperature
formaximumrecovery.• Placethesamplesonmagnetuntilallthebeadsseparatefrom
solution(waitforthesolutiontoclearbeforeproceedingtothenextstep).(~3-6minutes)
• Carefullyremovetheclearedsolutionfromthetubeanddiscard.Takecaretonotdisturbthebeadsintheprocess.
• Withoutremovingtubefrommagnet,add200µloffreshlyprepared70%ethanoltoeachsampleandincubatefor30secondsatroomtemperature.Removetheethanolanddiscard.Repeatforatotaloftwowashes.
• Brieflyspinthetubes(~2,000g)tocollecttheremainingliquidatthebottomofeachtube.Placethetubesonthemagneticseparationdevicefor30seconds,thenremoveallremainingliquidwithapipette.
• Letthesampletubesrestopenonthemagnetatroomtemperatureuntilthepelletappearsdryandisnolongershiny.(~3-6minutes)
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• Oncethebeadpellethasdried,removethetubesfrommagnetandadd12.5µlofRNasefreewater(Tube18).Mixthoroughlybypipettingupanddowntoensurecompletebeaddispersion.
• Incubateatroomtemperatureforatleast5minutes.• Placethesamplesonamagnetfor3minutesorlonger,untilthe
solutioniscompletelyclear.• Transfer10µloftheclearsupernatantcontainingpurifiedPCR
productsfromeachtubetoanewtube.Ensurethatnobeadsfollowthelibraryduringthisstep.
• QuantifylibrarywithAgilentBioanalyzer®/TapeStation®andQubit®Fluorometer.
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Step Temperature Time
1. Adapter Ligation
70°C 2 minice 2 min
25°C 60 min
65°C 5 min
2. Adapter Blocking
65°C 5 min
Step down 0.1°C/sec --> 37°C
~5 min
37°C 60 min
65°C 20 minOptional Stopping Point -20°C Overnight
3. Circularization 37°C 60 min4. Dimer Removal 37°C ~25-30 min
5. Reverse Transcription
65°C 5 minice 2 min
42°C 60 min
65°C 20 minOptional Stopping Point -20°C Overnight
6. PCR Amplification
Step Temp TimeHOLD 94°C 30 sec
CYCLE(10-22 cycles)
94°C 15 sec
62°C 30 sec
70°C 15 secHOLD 70°C 5 min
Optional Stopping Point -20°C Overnight
IV. Appendix A: Thermocycler Programming
Thermocyclerscanbeprogrammedinadvanceforallreactions.
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VII. Appendix B: Example Library Profiles
Figure 1.ExampleD1000TapeStation®profilefromalibrarywithaninputof100ngBraintotalRNAamplifiedby10cyclesofPCR.miRNAsizedlibrariesareapproximately141bp.
Figure 2.ExampleD1000TapeStation®profilefromalibrarywithaninputof1µlofmiRNAcontrolamplifiedby13cyclesofPCR.miRNAcontrollibrariesareapproximately149bp.
Figure 3.ExampleD1000TapeStation®profilefromalibrarywithaninputof100ngofMCF10Acellsamplifiedby16cyclesofPCR.Thelibrariesareapproximately147-245bp.
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VIII. Appendix C: Data Analysis
• RealSeq®-AClibrariesarecompletelycompatiblewithbioinformaticstoolsdesignedforIllumina’sTruSeqSmallRNAlibraries.
• ThefinalproductofaRealSeq®-AClibrarycontainstheadaptersequenceTGGAATTCTCGGGTGCCAAGG
• Thissequenceneedstobetrimmedfromsequencedreadsbeforemapping.
• Oneofthetoolsthatcanbeusedtoperformtrimmingofadaptersequencesiscutadapt(Martinetal.2011).
• Thefollowingcutadaptcommandwilltrimadaptersequencesandfilterreadswithinsertsshorterthan15nt.
cutadapt-m15-aTGGAATTCTCGGGTGCCAAGGinput.fastq>output.fastq
• Aftertrimmingthealignmentscanbeperformedasnormal.
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IV. Appendix D: Reverse Primer Index Sequence
Tube Sequence Reported*RP1 CGTGAT ATCACGRP2 ACATCG CGATGTRP3 GCCTAA TTAGGCRP4 TGGTCA TGACCARP5 CACTGT ACAGTGRP6 ATTGGC GCCAATRP7 GATCTG CAGATCRP8 TCAAGT ACTTGARP9 CTGATC GATCAG
RP10 AAGCTA TAGCTTRP11 GTAGCC GGCTACRP12 TACAAG CTTGTARP13 TTGACT AGTCAARP14 GGAACT AGTTCCRP15 TGACAT ATGTCARP16 GGACGG CCGTCCRP17 CTCTAC GTAGAGRP18 GCGGAC GTCCGCRP19 TTTCAC GTGAAARP20 GGCCAC GTGGCCRP21 CGAAAC GTTTCGRP22 CGTACG CGTACGRP23 CCACTC GAGTGGRP24 GCTACC GGTAGC
Tube Sequence Reported*RP25 ATCAGT ACTGATRP26 GCTCAT ATGAGCRP27 AGGAAT ATTCCTRP28 CTTTTG CAAAAGRP29 TAGTTG CAACTARP30 CCGGTG CACCGGRP31 ATCGTG CACGATRP32 TGAGTG CACTCARP33 CGCCTG CAGGCGRP34 GCCATG CATGGCRP35 AAAATG CATTTTRP36 TGTTGG CCAACARP37 ATTCCG CGGAATRP38 AGCTAG CTAGCTRP39 GTATAG CTATACRP40 TCTGAG CTCAGARP41 GTCGTC GACGACRP42 CGATTA TAATCGRP43 GCTGTA TACAGCRP44 ATTATA TATAATRP45 GAATGA TCATTCRP46 TCGGGA TCCCGARP47 CTTCGA TCGAAGRP48 TGCCGA TCGGCA
*Note:Reportedarethesequencesreportedbythesequencer.
Pleasevisitsomagenics.comfororderingindexesRP1-RP48.
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