Protocol for ARTseqâ„¢ Ribosome Profiling Kit (Mammalian)

www.epicentre.com Lit.#360•3/2014 1 EPILIT360 Rev. C

ARTseq™ Ribosome Profiling Kit(Mammalian)

Catalog Number: RPHMR12126

2 www.epicentre.com

Quick Protocol for ARTseq™ Ribosome Profiling Kit-Mammalian For experienced users only! The detailed procedure begins at Step 1 on page 6.

Step Procedure Pages

A. Lyse cells and treat with ARTseq nuclease

1. Treat5-50Mcellswithcycloheximide(0.1mg/ml)andwashwithcoldPBS(+cycloheximide).2. Add800µlLysisBufferandscrapecellsinto1.5mltubeonice.Drawupandexpelcellsthrougha

sterilepipette.Incubate10minonicewithgentlemixing.3. Spin@20,000xgfor10min@4°C.Transferclarifiedlysate(~1ml)tofreshtubeonice.4. ObtainA260oflysate,make~100µland200µlaliquotsandkeeponice.5. Remove100µlaliquotandimmediatelyadd10µlof10%SDS.Mixwellandkeeponiceforuseas

‘TotalRNA’sample(seebelowinStepB.3).6. Remove200µlaliquottofreshtubeandadd5UofARTseqnucleaseperA260/mloflysate. Forexample:50A260/mlx0.2mlx5U/A260perml=50UARTseqnuclease.7. [email protected]µlSUPERase∙In™andkeepon

iceorfreezeinliquidnitrogen.

6

B.Purifylysatebysizeexclusionchromatography(SEC)orsucrosecushion ultracentrifugation

1. SizeExclusionChromatography(SEC)a. Equilibratecolumnbygravitywith3bedvolumesofcold1XMammalianPolysomeLysisBuffer.b. [email protected]. Placecolumninfreshcollectiontube,gentlyadd100µlnuclease-treatedsampletotopof

resinandspin2min@600xg.Transferflow-throughtofreshtubeonice.Multiplecolumnspersamplecanbeusedifneededandsamplespooled.

d. Add10µlof10%SDStoeachelutedsampleandextractRNAusingZymoRNAClean&Concentrator™kitseePart2.A.,Step7.B.formodifiedprotocol).EluteRNAwith26µlNuclease-Free Water.

2. SucroseCushionAsucrosecushioncanbeusedasanalternativetotheMicroSpinS-400columnstopurifymonosomes.PleaserefertoAppendix8fordetails.

3. PurifyRNAfrom‘TotalRNA’sample(takeninStepA.5.,above)usingZymoRNAClean&Concentratorkit(>17ntprotocol).EluteRNAwith26µlNuclease-FreeWater.

7

C.Ribo-Zero™depletionofrRNAcontamination frombothTotalandRibosome-ProtectedFragments(RPF)samples

1. FollowRibo-Zeroprotocolexceptomitthe50°Cincubation(Step3.C.3.)andRNApurification.Instead,purifyRibo-ZerotreatedRNAusingZymoRNAClean&Concentratorkit.• ForTotal RNAusethe>200ntprotocol.EluteRNAwith~21µlNuclease-FreeWater.

• ForRPF samples(SeeSection3.3formodifiedprotocol).ElutetheRNAwith~11µl Nuclease-FreeWater.

a. Add10µlDenaturingGelLoadingDyetoRPFsamplesandheatRPFsamplesto95°Cfor5min.

b. PAGEpurify~28-30ntRPFs;usetheRNAControlOligoincludedinthekitsasaguide.

c. ResuspendPAGE-purifiedRPFsin~20µlNuclease-FreeWater.

8

D.FragmentTotalRNAandEnd-RepairbothTotalandRPFsamples

1.Mix: 20.0µl TotalorRPFRNAsample

7.5µl ARTseqPNKBuffer

27.5µl Total

• Keep RPF samples chilled on ice. Do not heat fragment. • HeatfragmentTotalRNAsampleat94°Cfor25minandthenhold@4°C.

• Mix:

44.5µl Nuclease-FreeWater

3.0µl ARTseqPNK

27.5μlSample

75.0µl Total

• Incubate1hour@37°C.

2. PurifyeachsampleusingZymoRNACleanandConcentratorkit.(SeeSection5.5formodifiedprotocol).

• Elutesampleswith10µlNuclease-FreeWater.

9

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Quick Protocol for ARTseq™ Ribosome Profiling Kit-Mammalian continued

Step Procedure Pages

E. 3′AdaptorLigation 1.Mix: 8.0µl RNAsample(orcontrol) 1.0µl ARTseq3′ Adaptor 9.0µl Total

(Note: Be sure to include both CtrlP and CtrlN reactions)• Heatto65°Cfor2minandthenhold@4°C.

2. Tothe9µlfromabove,add: 3.5µl ARTseqLigationBuffer 1.0µl DTT 1.5µl ARTseqLigase 15.0µl Total

• Incubate2hrs@RT(~23°C)3. Add2µlARTseqAREnzymetoeachsample.Incubatefor30min@30°C.

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F. Reverse Transcription

1. PrepareReverseTranscription(RT)MasterMixonice: 4.5µl ARTseqRTReactionMix 1.5µl DTT 6.0µl Nuclease-FreeWater 1.0µl EpiscriptReverseTranscriptase 13.0µl Total

• Add13µlReverseTranscriptionMasterMixtoeach17µlARTseqARenzyme-treatedsamplefromabove.

Incubate30min@50°C.• Add1.0µlARTseqExonucleasetoeachsample.Incubate30min@37°C,15min@80°C,hold

@4°C.• Add1.0µlRNaseMixtoeachsample.Incubate5min@55°C,hold@4°C.• PurifyeachsampleusingZymoRNACleanandConcentratorkit(>17ntprotocol).Elute

sampleswith11µlNuclease-FreeWater. 2. PAGEpurifycDNA.

• ForTotalRNAsamplespurify~80-100ntcDNAsizerange.• ForRPFsamplespurify~70-80ntcDNAsizerange.• PrecipitategelpurifiedcDNAandresuspendin10µlNuclease-FreeWater.

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G.CircularizecDNAwithCircligase

PrepareCircligaseMasterMixonice: 4.0µl ARTseqCircligaseReactionMix 2.0µl ATP 2.0µl MnCl2 2.0µl Circligase 10.0µl Total • Mix10µlcDNAsampleswith10µlCircligaseMasterMix. Incubate2hrs@60°C.Hold@4°C.

11

H.PCRAmplification Combine: 5.0µl CircularcDNAor1:5dilutionofCircularcDNA 16.0µl Nuclease-FreeWater 2.0µl ARTseqForwardPCRPrimer 2.0µl ScriptMiner™IndexIndexedPCRPrimer 25.0µl 2XPhusionHiFiMasterMix 50.0µl Total

• Incubateat98°Cfor30seconds.• Perform9cyclesof94°Cfor15seconds/55°Cfor5seconds/65°Cfor10secondsfollowed

by4°CHold.• Purifysamplesusing1.8xAMPurebeads. Elutepurifiedsamplesfrombeadswith25µlof10mMTrispH8.0.• Visualizesamples:Run2.5µlonan8%NativePAGE,or:Run1µlonAgilentBioanalyzerHigh

SensitivityDNAChip.

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ARTseq™ Ribosome Profiling Kit (Mammalian)

Kit Contents Cat. # Qty. Storage

ARTseq™RibosomeProfilingKit ASLPA1212 1kit –20°C

5XMammalianPolysomeBuffer ASBHMR1212 1bottle –20°C

ScriptMiner™IndexPCRPrimers(1-12) SMIP2124 1kit –20°C

FilterTubes ASFT1212 1pack RT

Additionally Required Materials for Preparation and Isolation of Ribosome Protected Fragments

– Cycloheximide(50mg/mlinAbsoluteEthanol)– SUPERase•In™RNaseInhibitor(LifeTechnologies,Cat.No.AM2696)– Phosphate-BufferedSaline(PBS)– Liquidnitrogen– 100%Isopropylalcohol– Icecold80%Ethanol– NanoDrop®Spectrophotometer

Note: Cycloheximide is available from many vendors. We recommend preparing 15ml 50 mg/ml stock solution using Ethanol as the diluent. Compound is light-sensitive and can be stored up to 1 year at –20°C. Cycloheximide is toxic and proper personal protective equipment should be worn. Please see manufacturer's MSDS for more information.

Additionally Required Materials for Purification of the Ribosome Protected Fragments

– illustra™MicroSpin™S-400HRColumns(GEHealthcare,Cat.No.27-5140-01)– Sucrose

Optional

– 10%NP-40(ThermoScientific,Cat.No.85124)– Sterile22-25gaugeneedle

Additionally Required Materials for ARTseq Library Preparation

– Ribo-Zero™MagneticGoldKit(Human/Mouse/Rat)(Epicentre;CatNo.MRZG12324)– RNAClean&Concentrator™-5kit(ZymoResearch;CatNo.R1015)– RNAClean&Concentrator™-25kit(ZymoResearch;CatNo.R1017)– DenaturingGelLoadingDye(Ambion;Cat.No.8546G)– NativeloadingdyecontainingBromophenolBlue– 20/100Oligoladderat1ng/μl(IDT;Cat.No.51-05-15-02)– 12or15%Polyacrylamide/7-8MUrea/TBEgel– Darkfieldtransilluminator– SYBR®Gold(LifeTechnologies)– 0.5mland1.5mltubes(sterile)– 10%Polyacrylamide/7-8MUrea/TBEgel– 8%NativeTBEPAGEgel– Phusion®Hi-FiPCRMasterMix(NEB;Cat.No.M0531)– AgencourtAMPureXPBeads(Beckman-Coulter;Cat.No.A63880)– 20bpLadder(BayouBiolabs;Cat.No.L-100)– Sterile20gaugeneedle

[email protected]•(800)284-8474 5


Cell Lysis

100 μl 200 μl

Extract RNA

Ribo-Zero™

Heat Fragment

End Repair

3’ Adaptor Ligation

Reverse Transcription

PAGE Puri�cation

Circularization

PCR

Total RNA

Extract RNA

Ribo-Zero™

Purify Monosomes

Nuclease Digestion

RPF PAGE Puri�cation

Ribosome ProtectedFragments (RPFs)

End Repair

3’ Adaptor Ligation


PAGE Puri�cation

Circularization

PCR

Figure 1. Overview of the ARTseq™ workflow.

mRNA

5’ 3’

5’ 3’

5’ 3’

5’ 3’

5’ 3’

5’ 3’

5’ 3’

5’ 3’

5’ 3’ p

Ribosome

Translation

Transcription

End repair (Remove 3’ P)

3’ Adapter Ligation

Ribo-Zero

Ligation + Adaptor removal (AR) enzymes(removes excess 5’ Ap–x primer)

PAGE Purify Ribosomal Protected Fragments

Nuclease digestion and isolate monosomes

Extract and purify RNA

Add Cycloheximide Lyse cells

5’ 3’ p5’ 3’ p

5’ 3’ OH

5’ 5’ Ap X

X

3’ OH

5’

X5’

5’

5’ 3’ OH

5’ 3’ OH

+


(Exo I/RNase)PAGE Purify

dU3’

Circularize

PCR Ampli�cation with index

Sequencing

5’dU3’

dU

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1. Preparation and Isolation of Ribosome Protected Fragments ARTseq™Kitcomponentsusedinthisprocedure:

Kit Contents Volume Cap Color

5XMammalianPolysomeBuffer 50ml

ClearDTT(100mM) 2x1.5ml

10%SDS 0.5ml

10%Triton®X-100 2x1.25ml

RedDNaseI(1U/µl) 100µl

ARTseq™Nuclease(10U/µl) 150µl

Note: Briefly spin tubes to collect reagent at bottom of tubes.

1.A. Mammalian Cell Extract Preparation and Ribosome Footprinting1. Prepare1mlofMammalianLysisBufferforeachsample.Chill the Mammalian Lysis Buffer to 4°C.

Toprepare1mlofMammalianLysisbuffer,combinethefollowing: 200.0µl 5xMammalianPolysomeBuffer 100.0µl 10%TritonX-100 10.0µl 100mMDTT 10.0µl DNaseI(1U/µl) 2.0µl 50mg/mlCycloheximide 10.0µl 10%NP-40(optional)oruseNuclease-FreeWater 668.0µl Nuclease-FreeWater 1.0ml Total2. Growadherentmammaliancellson15cmplatesinliquidmedia.Therequiredcelldensitywilllikelydependonthecelllineand

growthconditions.Acelldensityof5-50millioncells/mlisacceptable(seeStep1.A.6).TreatthecellswithCycloheximidepriortolysis,aspiratethegrowthmediaandsupplementtheplatewithfreshmediacontainingCycloheximide(finalconcentration 0.1mg/ml)andincubatethecellsfor1minute.

3. Aspiratemediaandplacecellsonice.Rinsecellswith10mloficecoldPBSsupplementedwithCycloheximide(finalconcentration0.1mg/ml).

4. AspirateanddiscardthePBSandadd800μlofMammalianLysisBuffertotheplate.Scrapethecellsextensivelyand(optionally)drawupandexpelthroughasterile22-25gaugeneedletolysethecellscompletely.Transferthecelllysatetoafreshtubethathasbeenchilledonice.

5. Incubate10minutesonicewithperiodicinversions.6. Clarifythelysatebycentrifugationfor10minutesat20,000xgat4°C.Transferthesupernatanttoafreshtubethathasbeenchilled

onice.Expecttorecover~1mlclarifiedlysate.7. Preparea1:10dilutionofthecollectedlysateusingNuclease-FreeWater.Useawaterblankanda1:10dilutionofMammalian

LysisBufferasstandard.RecordanA260readingusingaspectrophotometer.CalculatetheA260/mlconcentrationofthelysate(seeequationbelow).ThisnumberwillbeusedtocalculatetheamountofARTseqNucleasetogenerateRibosomeProtectedFragments(RPFs)inStep1.B.1.

(A260 cell lysate – A260MammalianLysisBuffer)x10dilutionfactor=A260/ml.

? Optional Stopping Point (Freeze 100 µl aliquots of the lysate in liquid nitrogen and store at –80°C)8. Foreachcelllysis,createaliquots:100µlvolumealiquot(foruseinStep1.A.9,RibosomeProtectedFragments)and200µlvolume

aliquot(foruseinStep1.B.1,RibosomeProtectedFragments).Keeponice.9. Tothe100µlaliquot,add10µlof10%SDSandmix.Thismaterialwillbeusedas‘TotalRNA’(non-footprintedRNA).Placeonicefor

purificationinStep2.A.7orStep2.B.5.Alternatively,thesamplecanbefrozeninliquidnitrogenandstoredat–80°Cuntilreadyforuse.

1B. Mammalian Ribosome Footprinting with ARTseq Nuclease1. Tothe200μlaliquotofclarifiedlysatefromstep1.A.8,add5UnitsofARTseqNucleaseforeachA260oflysate.

Forexample,50A260/mllysateX0.2mllysateX5Units/A260ARTseqNuclease=50UnitsARTseqNuclease.

Optionally,titratetheARTseqNucleasedigestionasdescribedinAppendix4.

2. Incubatethereactionsatroomtemperaturefor45minuteswithgentlemixing.Freezeanyunusedlysatewithliquidnitrogenandstoreat–80°C.

Note: During incubation, prepare the MicroSpin S-400 columns as described in Step 2A to isolate the nuclease-digested monosomes.

3. Stopthereactionsbyadding15µlSUPERase•InRNaseInhibitorandchillthesamplesoniceforuseinPart2.



2. Purify the Ribosome Protected Fragments RibosomeprotectedfragmentscanbepurifiedbyeitherMicroSpinS-400columns(2.A.)orsucrosecushionultracentrifugation(2.B.).

Note: Do not purify the samples labeled as “Total RNA” with the Microspin S-400 spin columns. Keep these samples frozen for use in Step 2.A.7 or Step 2.B.5.

2.A. Purify monosomes using MicroSpin S-400 columns 1. Foreachsample,prepare3mlof1XMammalianPolysomeBuffer: 600.0μl 5XMammalianPolysomeBuffer 2400.0μlNuclease-FreeWater 3.0 ml Total2. InverttheMicroSpinS-400columnsseveraltimestoresuspendtheresin.Tapoutanybubblesthatmayformintheresinasit

settles. 3. Openthecolumnonbothendsandallowthebuffertodripoutundergravity.Ifthecolumnsarenotdrippingfreely,useaclean

glovedfingertocoverthetopofthecolumn.Pressdowngentlytocreatepressuretoinitiateflow.4. Equilibratetheresinbypassingthrough~3mlof1XMammalianPolysomeBufferundergravityflow.Thisstepmaytakeuptoan

hourforbuffertoflowthroughthecolumn.Donotcentrifuge.Becarefulnottointroducebubblesintotheresin.Thecolumnswilldripmorefreelyoncethe1XMammalianPolysomeBufferreplacestheoriginalbufferinthecolumns.Note: Steps 2.A.2–2.A.4 can be done during the Ribosome Protected Fragment nuclease digestion (Step 1.B.2). Do not allow column to dry. Do not centrifuge the column.

5. Attachacollectiontubeandspinfor4minutesat600xginafixedangletabletopcentrifugeatroomtemperature.Discardflow-through,andtransfercolumnstoa1.5mltube.

6. Immediatelyapply100μlofRibosomeProtectedFragmentnucleasedigestion(fromStep1.B.3).Keepremaining~100μloniceorfreezeinliquidnitrogenandstoreat–80°CincaseneededinPart2.A,Step8.B.Centrifugefor2minutesat600xgand collect the flowthrough.Add10µlof10%SDS.Thismaterialwillbeusedas‘RibosomeProtectedRNA’.

7. PurifybothTotalRNAsamplesandRibosomeProtectedRNAsamples:A. PurifytheTotal RNAsamplesusingtheZymoResearchRNAClean&Concentrator-25kit.Follow the kit protocol for >200 nt

RNA.Elutethesamplesfromthecolumnswith26µlNuclease-FreeWater(expecttorecover~25µl).B. PurifytheRibosome Protected RNAsamplesusingZymoRNAClean&Concentrator-25kitusingamodifiedprotocol

optimizedforsmallfragmentpurification: a) InZymoprotocolStep1,use220μlBindingBuffer(fromZymoKit) b) InZymoprotocolStep2,use495μlAbsoluteEthanol c) Continuewithpurificationaccordingtothemanufacturer’sinstructions d) Elutethesampleswith26µlofNuclease-FreeWater(expecttorecover~25µl)

8. QuantifybyNanoDrop.Youwillneedabout1-5μgofTotalRNAand1-5μgRibosomeProtectedRNAfortheRibo-ZerorRNAremovaltreatmentinStep3.

A. If<1-5μgofTotalRNAhasbeencollected,repeatStep1.A.9withremaininglysatetoachievedesiredamount. B. If<1-5μgofRibosomalProtectedRNAhasbeencollected,repeatStep2withunusedclarifiedlysate(fromStep1.B.3)to

achieve desired amount.9. ContinuetoStep3.A.RemovalofrRNAusingRibo-Zero.

2.B. Purify Monosomes by Sucrose CushionAsucrosecushioncanbeusedasanalternativetotheMicroSpinS-400columnstopurifymonosomes.PleaserefertoAppendix8fordetails.

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Kit components used for ARTseq library preparation.

Component Name Volume Cap Color

End Repair

ARTseq™PNKBuffer 150µlYellow

ARTseq™PNK 45µl

3′ Adaptor Ligation

ARTseq™RNAControl 20µl

Yellow

ARTseq™3′ Adaptor 15µl

ARTseq™LigationBuffer 49µl

ARTseq™Ligase 22µl

ARTseq™ARenzyme 28µl

DTT(100mM) 2x1.5mlClear

Nuclease-FreeWater 1.5ml


ARTseq™RTReactionMix 65µl

BlueEpiScript™ReverseTranscriptase 15µl

ARTseq™Exonuclease 15µl

ARTseq™RNaseMix 15µl

CircLigase

ARTseq™CLReactionMix 72µl

GreenMnCl2 30µl

CircLigase™ 28µl

ATP 30µl

PCR

ARTseq™ForwardPCRPrimer 75µl Green

ScriptMiner™IndexPCRPrimers 12Indexes Yellow;separatebox

Extras

Glycogen 200µlClear

10%SDS 500µl

Ammonium Acetate 1.2ml Red

FilterTubes 24filtertubes N/A

3. Ribo-Zero Depletion1. Use1-5μgofRibosomeProtectedRNAand1-5µgoftheTotalRNAsamplesthatweregeneratedattheendofPart2.2. FollowtheRibo-Zerokitprocedurewiththefollowingexceptions

A. Omit50°Cincubationstep(Step3.C.3oftheRibo-Zerokitprocedure).B. DonotpurifytherRNAdepletedsamplesasstatedintheRibo-Zeroprocedure.Instead,followthepurificationprocedure

below(Step3.3).3. PurifybothRibo-ZerotreatedTotalRNAsamplesandRibo-ZerotreatedRibosomeProtectedRNAsamples:

A. PurifytheRibo-ZerotreatedTotal RNAsamplesusingtheZymoResearchRNAClean&Concentratorkit.Follow the kit protocol for >200 nt RNA.Elutethesamplesfromthecolumnswith21µlNuclease-FreeWater(expecttorecover ~20µl).Keeponiceorstoreat–20°CuntilreadyforuseinPart5.

B. PurifytheRibo-ZerotreatedRibosome Protected RNAsamplesusingZymoRNAClean&Concentrator-5kit.Adjusteachsamplevolumeto100μlwithNuclease-FreeWater.PurifythereactionsusingamodifiedZymoResearchRNAClean&Concentratorkitprotocol:

a) InZymoprotocolStep1,use200μlBindingBuffer(fromZymoKit) b) InZymoprotocolStep2,use450μlAbsoluteEthanol c) Continuewithpurificationaccordingtothemanufacturer’sinstructions. d) Elutethesampleswith11µlofNuclease-FreeWater(expecttorecover~10µl).QuantifybyNanoDrop.Expecttorecover

~5-20%ofinput. e) ProceedtoPart4.



4. PAGE Purify the Ribosome Protected FragmentsTheRibosomeProtectedFragmentswillbe~28-30ntsinlength.TheARTseqRNAControlthatisprovidedinthekitwillbeusedasasizemarker.TheARTseqRNAControlcontainstwooligosof28ntsand30ntsinlength(seeAppendix1).Do not PAGE purify the Ribo-Zero treated Total RNA samples. Keep these samples on ice or store at –20°C until ready for Part 5.1. PrepareARTseqRNAControl,samples,andladderforPAGEgel.

A. Add5µloftheARTseqRNAControlfromthekittoatube.Add5µlofdenaturinggelloadingdye.B. MixtheRibosomeProtectedRNAsamples(10µl)with10µlofdenaturinggelloadingdye(totalvolume=20µl).C. Topreventcrosscontamination,preparealadderaliquot(4μlIDT20/100ladder,1μlwater,and5μldenaturinggelloading

dye)toloadbetweeneachRibosomeProtectedRNAsampleorARTseqRNAcontrol.2. Denaturethesamplesandladderbyincubatingat95°Cfor5minutes.Placethetubesimmediatelyonice.TheARTseqRNAControl

willbeusedasasizemarker.3. Loadthe10µlaliquotsofeachsampleandladderontoa12%or15%Urea-Polyacrylamidegel.Runthegelat180Vuntilthe

bromophenolbluebandreachesbottomofthegel(~210V*hr;180Vfor70min).Storetheremainingsamplesat–20°C.4. StainthegelwithSYBRGoldandvisualizetheRNAusingaDarkFieldTransilluminator(emitsabluelight).5. Foreachsample,excisethegelslicescorrespondingtothe28ntand30ntcontrolRNAasareference.Cut even if footprints

are not visible.Theladderisoverloadedtomakesizeselectioneasier.However,thistendstooversaturateeyevisionmakingvisualizationoftheribosomeprotectedfragmentschallenging.Note: Also excise the 28 nt and 30 nt ARTseq RNA Control oligo bands. See Appendix 4, Figure 2 for example.

6. Transferthegelslicesto0.5mltubeswithaholepunchedinbottomusingasterile20gaugeneedle,andclosethetubecap.Placethe0.5mltubesinsidea1.5mltubeandcentrifugefor2minutesat~12,000xginamicrocentrifugetodisruptthegelslices.

7. Removeanddiscardthe0.5mltubes.Toeach1.5mlcollectiontube,addtoeachelutedsample: 400.0µl Nuclease-FreeWater 40.0µl 5MAmmoniumAcetate ~2.0µl 10%SDS8. Gentlyrocksamplesatroomtemperaturefor1-2hoursor4°CovernighttoelutetheRNAfromthedisruptedgelslices.9. Transfertheslurryto1.5mlfiltertubesprovided,andcentrifugefor~3minutesat2,300xgtoseparatedisruptedgelpiecesfrom

the eluted RNA solution. 10. Usealargeorifice1mlpipettetiportrimtheendofa1mlpipettetip.Carefullypipettetheaqueoussolutionintonew1.5ml

tubes.Toeachtubeadd2µlGlycogenand700µlof100%Isopropanolandstoreat–20°Cfor>1hour.11. Centrifugetubesat>12,000xgat4°Cfor20minutestopellettheRibosomeProtectedRNA.WashtheRNApelletwithicecold80%

Ethanol and air dry. 12. ResuspendintheappropriateamountofNuclease-FreeWaterasstatedbelow:

A. FortheRibosomeProtectedRNAsamples,resuspendtheRNApelletin20µlofNuclease-FreeWater.ProceedtoPart5orfreezethesamplesat–20°C.

B. FortheARTseqRNAcontrol,resuspendtheRNApelletin8µlofNuclease-FreeWaterforuseinPart6.Storeoniceorat–20°Cuntil ready to use in Part 6.

5. Fragment and End RepairInthisstep,theRibo-ZerotreatedTotalRNAsamplesareheatfragmented.BoththefragmentedTotalRNAsamplesandtheRibosomeProtectedRNAsamplesarethenend-repairedtopreparethemforthe3′AdaptorLigationstep(Part6).RNAend-repairisperformedusingARTseqPNKintheabsenceofATP.

1. AddtoeachoftheTotalRNAandRibosomeProtectedRNAsamples,onice:

20.0µl RNAsample 7.5µl ARTseqPNKBuffer 27.5µl Totalvolume

Hold the Ribosome Protected RNA samples on ice for use in Part 5, Step 3.

2. Heatfragmentonly the Total RNA samplesfor25minutesat94°C.Then,placeoniceorholdat4°C.

3. Toperformendrepair,totheheatfragmentedTotal RNA sample and the Ribosome Protected RNA sampleaddandmixthroughly:

44.5µlNuclease-FreeWater 3.0µl ARTseqPNK 27.5µl SamplefromStep5.1 75.0µl Total

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4. Incubatethesamplesfor1hourat37°C.

5. Adjusteachsamplevolumeto100µlwith25µlNuclease-FreeWater.PurifythereactionsusingamodifiedZymoResearchRNAClean&Concentrator-5kitprotocol:

A. InZymoprotocolStep1,use200µlBindingBuffer(fromZymoKit)

B. InZymoprotocolStep2,use450µlAbsoluteEthanol

C. Continuewithpurificationaccordingtothemanufacturer’sinstructions

D. ElutetheRNAwith10µlofNuclease-FreeWaterandexpecttorecover~8µl

Proceed with the 3′AdaptorLigationprocedureinPart6.

? Optional Stopping Point (Store samples at ≤–20° C)

6. 3′ Adaptor LigationPositive and Negative Controls:

Itisusefultoincludebothpositiveandnegativecontrols.ThepositivecontrolwillalsobeusedindownstreamPAGEGels.

Reactionstoinclude:

CtrlP:PositiveControl=PAGE-purifiedARTseqRNAControlfromPart4,(Step12.B).

CtrlN:NegativeControl=noRNA(mockreaction;use8µlNuclease-FreeWater).

1. Mixonice:

8.0µl RNAsamplesfromPart5(orcontrol) 1.0µl ARTseq3'Adaptor 9.0µl TotalvolumeInathermocyclerwithheatedlid,heatdenaturethesamplesfor2minutesat65°CandthenHoldat4°C.

2. Whilethedenaturingreactionisproceeding,maketheLigationMasterMix.Foreachreaction,prepare:

3.5µl ARTseqLigationBuffer 1.0µl 100mMDTT 1.5µl ARTseqLigase 6.0µl Totalvolume

3. Add6µloftheLigationMasterMixtoeachofthedenaturedRNAsamples.Mixthoroughlybypipettingseveraltimes.Centrifugebrieflytoconsolidatesampleatbottomoftube.Incubateatroomtemperature(23°C)for2hours.

? Optional Stopping Point (Store samples at ≤ –20°C)

4. Add2µlofARTseq™AREnzymetoeachreactionandmixthoroughlybypipetting.Incubateat30°Cfor30minutes.

7. Reverse Transcription1. Foreachreaction,preparetheReverseTranscriptionMasterMixonice:

4.5µl ARTseqRTReactionMix 1.5µl 100mMDTT 6.0µlNuclease-FreeWater 1.0µl EpiScriptReverseTranscriptase 13.0µl Totalvolume

2. Add13µloftheReverseTranscriptionMasterMixtoeachreactionandmixwellbypipetting.

3. Incubatethereactionsfor30minutesat50°Cinathermocyclerwithheatedlid.

4. Add1µlofARTseqExonucleasetoeachreactionandincubatefor:

37°Cfor30minutes

80°Cat15minutes

4°CHold

? Optional Stopping Point (Store samples at ≤–20°C)

5. Add1µlARTseqRNaseMixtoeachreaction.Mixandincubatefor5minutesat55°C.Placethereactionsoniceorholdat4°C.



8. PAGE Purify the cDNAs1. Add18µlofNuclease-FreeWatertoeachreaction.Reactionvolumesarenow50µl.

2. PurifythesamplesusingZymoResearchRNAClean&Concentratorkit.Followmanufacturer’sprotocolfor>17ntRNA.ElutethecDNAwith11µlofNuclease-FreeWaterandexpecttorecover~10µl.

? Optional Stopping Point (Store Samples at ≤–20°C)

3. PreparesamplesandladderforPAGEgel.

A. Add10µlvolumeofdenaturinggelloadingdyetoeachsampleandtheARTseqRNAcontrol.

B. SeparateeachsampleorARTseqRNAcontrolwithanaliquotoftheIDT20/100Oligoladder(4μlladder,1μlwater,and5μldenaturingloadingdye)toavoidcross-contamination.

4. Heatthesamplesto95°Cfor5minutesandthenplaceonice.

5. Load10µlofeachsampleontoasinglelaneofa10%Polyacrylamide/7-8MUrea/TBEgel.Freezetheremaining10µlofsampleat–20°C.

6. Adjustpowersupplyto180VandrungeluntilBromophenolBluedyecompletelymigratesoutofgel(~180V*hr;180Vfor 60min).StaingelwithSYBRgoldandvisualizeunderDarkFieldTransilluminator.(SeeAppendix7formoreinformation).

7. Excisethegelslices.Excise even if material is not visible.Theladderisintentiallyoverloadedtomakesizeselectioneasier.However,thistendstooversaturateeyevisionmakingvisualizationofthecDNAbandsdifficult.

A.FortheRibo-ZerotreatedTotalRNAsamplescut~80-100nt.

B.FortheRibosomeProtectedFragments,CtrlNandCtrlPcontrols,cut~70-80nt.

Note: In the CtrlP sample a distinct cDNA product migrating at 73-75 nt should be clearly visible and excised for use as a positive control. It is also a helpful size marker for comparison to the Ribosome Protected samples. Excise also the same region for the CtrlN sample for use as the negative control. The size ranges are higher than the previous gel purification due to the addition of the 3′ Adaptor and RT primer.

8. Transferthegelslicestoa0.5mltubewithaholepunchedinbottomwitha20gaugeneedleandclosecap.Placethe0.5mltubeinsideofa1.5mltubeandcentrifugefor2minutesat~12,000xginamicrocentrifugetodisruptthegelslice.

9. Removeanddiscardthe0.5mltube.Tothe1.5mltube,add:

400.0µlNuclease-FreeWater 40.0µl 5MAmmoniumAcetate ~2.0μl 10%SDS Shakethesamplesat37°Cfor~1hour(orovernightatroomtemperature)toelutethecDNAfromthedisruptedgelslices.

10. Transfertheslurrytoanew1.5mlfiltertubeandcentrifugefor~3minutesat2,300xgtoseparatetheshreddedgelpiecesfromtheelutedcDNAsolution.

11. Usealargeorifice1mlpipettetiportrimtheendofa1mlpipettetip.Carefullypipettetheaqueoussolutiontonew1.5mltubes.Toeachsample,add2µlGlycogenand700µl100%IsopropanolAlcohol.Mixandstoreat–20°Cfor>1hour.

12. Centrifugeat>12,000xg@4°Cfor15minutestopelletthecDNA.Washthepelletwithicecold80%Ethanolandairdry.ResuspendthecDNApelletin10µlofNuclease-FreeWater.

13. ProceedtoPart9orfreezethesamplesat–20°C.

9. Circularize the cDNA1. ForeachsampleprepareCircLigaseMasterMixonice:

4.0µl ARTseqCircLigaseReactionMix 2.0µl ATP 2.0µlMnCl2 2.0µl CircLigase 10.0µl Totalvolumeperreaction

2. Add10µloftheCircLigaseMasterMixtoeachreaction.Mixthetubescontentsbygentlemixingandbrieflycentrifuge.Incubatethereactionsat60°Cfor2hours,thenplaceoniceorholdat4°C.

10. PCR Amplification ToomuchtemplateortoomanyPCRcyclescanresultin“overamplification”–theappearanceofhigher-than-expectedmolecularweightbands,smearedPCRproducts,andadapterdimer-derivedproducts.ExamplesofoveramplifiedsamplescanbeseeninAppendix5.

Formostsamples,use1-5μlofthecircularizedcDNAfromPart9.NinePCRcycleswilltypicallyyieldsufficientamountsofthecorrectPCR product.



AgoodstartingstrategyistosetuptwoPCRreactionsforeachsample.ForonePCRreaction,use5μlof1:5dilution(withwater)ofthecircularizedcDNAastemplate.ForthesecondPCR,use5μlofundilutedcircularizedcDNA.

TheremainingcircularizedcDNA(~14μl)canberetainedandstoredat–20°Cincasethetemplateinputand/orPCRcyclenumberneedtobeoptimized.

Note:

A) ChooseoneoftheScriptMinerIndexPCRPrimers(provided)foruseastheReversePCRPrimerinthePCRreaction.

B) Ifpoolinglibrariesforsequencinglater,besuretochecktheindicesforcolorbalanceduringsequencing.SeeendofAppendix2forrecommendationsonindicescombinationsforsequencing.

C) ThePCRreactionisoptimizedforusewiththe2XPhusionMasterMix(NEB;cat.no.M0531)

1. PreparethePCRMasterMix.Foreachsample,combineonice:

16.0µlNuclease-FreeWater 2.0µl ARTseqForwardPCRPrimer 2.0µl ScriptMinerIndexPCRprimerofchoice 25.0µl 2XPhusionMasterMix 45.0µl Totalvolumeperreaction

2. Add45µlofthePCRMasterMixtoeachofthe5µlsamplesofboth1:5dilutedandundilutedcDNA.Mixthetubesthoroughly.

3. PlacealltubesinPCRthermocyclerandrunthefollowingprogram:

98°Cfor30seconds

94°Cfor15seconds

55°Cfor5seconds 9cycles

65°Cfor10seconds

4°CHold

4. PurifythePCRproductsusing90μl(1.8X)ofAgencourt®AMPure®XPbeads.Purifyaccordingtothemanufacturer’sinstructions.Elutethelibrarieswith25µlofNuclease-FreeWateror10mMTris-HClpH8.0.

5. Remove2.5μlaliquotsfromeachPCRreactiontoanewtubeatroomtemperature.Add1.5μlwaterand1μlof6xNativeGelLoadingDyecontainingBromophenolBlue

6. Load5µlofPCRreactionaliquotsfromStep10.5and20bpladderonan8%NativePAGE-1xTBE.Runthegelat200Vuntilbromophenolbluereachesbottomofgel(~83V*hr;200Vfor25min).StaingelwithSYBRGoldandvisualizeunderUVlightorDarkFieldTransilluminator.

7. TheexpectedsizeofthePCR-amplifiedlibraryis140-160bp.SelectthetubesthatcontainthecorrectPCRproductforpurificationandsequencing.

Note: If excess adaptor-only product (~113 bp) is observed, PAGE-purify the desired product using an 8% native polyacrylamide gel (see Appendix 6).

Optional:Ifnecessaryordesired,preparemorePCRamplifiedproductsusingthe14µlofcDNAretainedfromabove.IncreaseordecreasetheamountofinputcDNAand/orthenumberofPCRcyclestoobtainoptimalyieldofthedesired140–160bpamplicon(s).SeeAppendix5forexample.

8. ThesizedistributionofthelibrarycanalternativelybecharacterizedusingtheAgilentHighSensitivityDNAAssay.SeeAppendix6formoredetails.

9. Proceedtosequencing.SeeAppendix3forsequencinginformation.

11. Appendices

Appendix 1ARTseq Sequences:

Control RNA oligos (two):

5′NNGUACACGGAGUCGACCCGCAACGCNN3′(28nts)

5′NNGUACACGGAGUCAAGACCCGCAACGCNN3′(30nts)

3′ Adaptor:

5′AGATCGGAAGAGCACACGTCT3′

Forward PCR Primer:

5′AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACG3′



Appendix 2ThesequencesoftheScriptMinerIndexPCRPrimersfunctionastheReversePCRprimersintheARTSeqprocedure.Theindexsequenceforeachprimerisunderlinedandindexbaseshavebeencoloredtoassistinbalancingoflasercolorsduringsequencingindexreads.Seebelowforforrecommendedindexcombinations.

Index 1 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 2 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 3 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 4 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 5 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 6 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 7 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 8 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 9 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 10 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 11 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Index 12 PCR Primer

5'CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3'

Recommended Index Combinations for Sequencing Library Pools

Duplex(twoindices) Index06andIndex12 (Perfectbasecolorbalance)

Triplex(threeindices) Index04andIndex08 Addone:Index01,02,05,06,09,or10



Four or more indices Index06andIndex12 Addanyotherindexes

Appendix 3

Sequencing an ARTseq Ribosome Protected Fragment.Purified,Adaptor-taggedARTseqlibrariesarecompatiblewithIllumina®TruSeq™ClusterKitsandcanbesequencedusingtheIlluminasequencingplatforms.WiththeARTseqlibraryprepkit,thesequencegeneratedbyRead1isthatofthesensestrandoftheoriginalRNA.RecommendedsequencingreadlengthforARTseqlibrariesis51cycles(Read1onlyorRead1plusIndexRead);BioAnalyzeranalysisofpreparedlibrariesshowsapproximately150bpfragments(seeAppendix6).Actualinsertsizeisapproximately30nt(SeeAppendix6).

SequencingRead1requireseitherHP6orHP10primer;HP6isincludedintheTruSeqPEClusterKits(e.g.PE-401-3001).HP10istheRead1primerincludedintheMiSeq™ReagentKits(MS-102-2001,MS-102-2002,MS-102-2003,MS-102-3001,andMS-102-3003).

TheIndexReadrequires7cycles(6basebarcodes)andrequiresHP12primerwhichisincludedinTruSeqDualIndexSequencingPrimerBox(FC-121-1003,PE-121-1003).SeeAppendix2forpoolingsuggestions.

Note: To use IEM (Illumina's Experiment Manager) select TruSeq LT to access appropriate barcodes.



ARTseqlibrarieshavesufficientcomplexityforsingle-sample-per-lanesequencing;multiplexingisoptionalandthereforegenerousPhiXspike-ins(topreventregistrationissues)isnotneeded.PhiXmaybeaddedtomonitorerrorratesduringsequencing,ifdesired.

Theminimumrecommendednumberofreadsforeachsamplelibraryis40million.Forguidanceonanalyzingsequencingdata,pleaserefertotheARTseq™BioinformaticsUserGuide.

Appendix 4.

Optimizing nuclease digestion to generate the Ribosome Protected Fragments. RibosomeProtectedFragments(RPF)canbecontaminatedwithpartiallydegradedtRNAfragments.Therefore,itmaybedesirabletofirstoptimizetheARTseq™NucleasedigestionstepinordertomaximizeRPFrecoveryandminimizecontaminations.TheARTseqNucleaseisprovidedataconcentrationof10U/µl.

1. SetupatitrationseriesofARTseqNucleaseusingindividualaliquotsofcelllysate.Forexample,tofourseparate200µlaliquotsofthecelllysate,add0.3µl(3Units),1µl(10Units),3µl(30Units)and6µl(60Units)oftheARTseqNucleaserespectively. Forexample,50A260/mllysateX0.2mllysateX5Units/A260ARTseqNuclease=50UnitsARTseqNuclease.

2. Incubatethereactionsatroomtemperaturefor45–60minutes.

3. Stopthereactionsbyadding15µlofSUPERase•InRNaseInhibitorandchillthesamplesonice.

4. Purify100µlofeachreactionusingtheMicroSpinS-400columnsandextractRNAasdescribedinPart2.1through2.8ofthestandardprocedure.Freezetheremainingsampleinliquidnitrogenandstoreat–80°C.

5. PrepareARTseqRNAControl,RibosomeProtectedFragmentsamples,andladderforPAGEgel.

A. Add1µloftheARTseqRNAControlfromthekittoa0.5mltube.Add9µlofdenaturinggelloadingdye.

B. ForeachoftheresuspendedRibosomeProtectedFragment(RPF)samplesfromAppendix4.4,mix250ngwithdenaturinggelloadingdyeupto10μlfinalvolume.

C. Preparealadderaliquot(2μlIDT20/100ladder,3μlwater,and5μldenaturinggelloadingdye).

6. Heatallsamplesto95°Cfor5minutes.Placethetubesimmediatelyonice.TheARTseqRNAControl(28ntsand30nts)oligowillbeusedforsizemarker.

7. Load10µlaliquotsofeachsampleontoa12or15%Urea-Polyacrylamidegel.Runthegelat180Vuntilbromophenolbluebandreachesbottomofthegel(~210V*hr;180Vfor70min).StainthegelwithSYBRGold.VisualizegelwithUVlightorDarkFieldTransilluminator.

8. ContinueRibo-ZerorRNAremoval(Part3.A)withthetitratedsamplefromAppendix4.4thathasadistinctRPFbandsimilartothebracketedregionofthe60UnitsampleshowninFigure2.

Figure 2. Denaturing PAGE analysis of the effect of increasing amounts of ARTseq Nuclease on Ribosome Protected Fragment recovery. 60UnitsofARTseqNucleasewasoptimaltoobserveRNase-resistantmaterialcorrespondingtothesizeofRPFs(bracket).



Appendix 5.

Effect of over-amplifying Ribosome Protected cDNA.Over-amplificationoftheRibosomeProtectedFragmentsinPart10(PCRAmplification)canleadtotheappearanceofhigher-than-expectedamplicons,smearedampliconsandadapter-dimerproducts.Intheexampleshown,9cyclesofPCRyieldedtheoptimalamountoftheexpectedPCRproduct.

Figure 3. Effect of PCR cycle number on product size. “+”librarieswerepreparedfromthe28ntARTseqControlRNA;“-“negativecontrol(noRNA)library(bandappearingat~120bpin12and15cyclesisadaptordimer);S1andS2;Ribosomeprotectedsamples.

Appendix 6.

BioAnalyzer profiles of ARTseq libraries and optional gel purification.

Figure 4. BioAnalyzer profiles of ARTseq libraries. ARTseqlibrariesmadefromtwoRibosomeProtectedmRNAsamples,amplifiedforninecyclesofPCRandpurifiedusingAMPurebeads.SampleswereanalyzedbyAgilentHighSensitivityDNAAssay.A)A‘good’sample.The~140-160bppeakistheexpectedsizerangeandnofurtherpurificationneeded.B)Asamplecontaininganexcessiveamountofadaptordimeramplifiedproduct(~120bp)comparedtothedesiredproduct(~140-160bp).ThissampleshouldbepurifiedfurtherusingthePAGEpurificationprocedure.

A

B



PCR PAGE Purification1. Preparesamples,positivecontrol,andladdersforPAGEgel.

A. Foreach~25µlPCRsampletobepurifiedandtheamplifiedpositivecontrol,add6µlofNativeGelLoadingDyecontainingBromophenolBlue.

B. Separatesampleswitha5µl20bpladderaliquotoranaliquotofdiluted1µlNativeGelLoadingDyecontainingBromophenolBlue,5µlwater.

2. Load15µlperlane(samplessplitacrosstwolanes)ofa6or8%NativePAGE-1xTBEgelusingaladderorNativeGelLoadingDyecontainingBromophenolBluealiquotbetweensamplestoavoidcrosscontamination.

3. Adjustto200VandrungeluntilBromophenolBluedyereachesthebottomofthegel(~83V*hr;200Vfor25min).StaingelwithSYBRgoldandvisualizeunderDarkFieldTransilluminator.

Note: Chill SYBR gold solution and stain gel at 4°C to avoid diffusion of signal in gel.

4. Excisetheuppermostband(~140-160bp)beingcarefultoavoidtheadaptordimer(~113bp).

5. Transferthegelslicestoa0.5mltubewithholepunchedinbottomwitha20gaugeneedleandclosecap.Placethe0.5mltubeinsideofa1.5mltubeandcentrifugefor2minutesat~12,000xginatabletopmicrofugetodisruptgelslice.

6. Removeanddiscardthe0.5mltubes.Tothe1.5mltubes,add:

400.0µlNuclease-FreeWater 40.0µl 5MAmmoniumAcetate ~2.0μl 10%SDS Gentlyrockthesamplesat37°Cfor~1hour(orovernightatroomtemperature)toelutethePCRproductsfromthedisruptedgel

slices.

7. Transfertheslurrytonew1.5mlfiltertubesandcentrifugefor~3minutesat2,000xgtoseparatethedisruptedgelpiecesfromthe eluted PCR product solution.

8. Usealargeorifice1mlpipettetiportrimtheendofa1mlpipettetip.Carefullypipettetheaqueoussolutiontonew1.5mltubes.Toeach,add2µlGlycogenand700µl100%IsopropanolAlcohol.Mixeachtubeandstoreat–20°Cfor>1hour.

Note: It has not been observed that the addition of Glycogen has interfered with downstream applications. However, some prefer to use GlycoBlue™ with a dye added to better visualize the pellet. This is not advised for this step since the dyes can interfere with the quantification of nucleic acid concentration.

9. Centrifugethetubesat>12,000xgat4°Cfor15minutestopelletthePCRproducts.Washthepelletwithcold80%Ethanolandairdry.

10. Resuspendthepelletin15µlofNuclease-FreeWateror10mMTris-HClpH8.0.Pooltubesofsamesampletogether(samplevolume=30μl).

11. Check1μlofeach.SampleontheBioAnalyzerusingtheAgilentHighSensitivityDNAAssay.(seeFigure4).



Appendix 7. Positive and Negative Controls.Note: CtrlP and Sample cDNA at 70-100 nt may/may not be visible on purification gel. Continue to excise gel regions within this range. The ladder is overloaded to make size selection easier but tends to make visualization of the cDNA bands difficult.

Figure 5. PAGE purification of cDNA. ThecDNAreactionsfromtheCtrlN(lanes2&3)andCtrlP(lanes5&6)samplesfromPart8.6oftheprotocol.Topurify,excisegelregionscorrespondingtosizes~70-80ntforRPFsamplesandtheCtrlNandCtrlPsamples(seewhitedottedlines)andextractthecDNAasdescribedintheprotocol.73and75ntcDNAproductsshouldbeobservedintheCtrlPlane(s)correspondingtothe28ntand30ntcontrolRNAoligos.ForRibo-ZerotreatedTotalRNAsamples,excisethesizerange~80-100ntandextractthecDNAasdescribedintheprotocol.

Appendix 8. Purify Monosomes by Sucrose Cushion

1. Foreachsample,prepare1mlofSucroseCushionBufferasfollows:

200.0μl 5XMammalianPolysomeBuffer 0.5g Sucrose 5.0μl SUPERase•In(20U/μl) 140.0μl MgCl2(25mM) 5.0μl DTT(100mM) 2.0μl Cycloheximide(50mg/ml) Bringsolutionupto1mlwithNuclease-FreeWater.

2. Carefullylayer200μloftheARTseqNuclease/SUPERase•Intreatedlysateover1mloficecoldSucroseCushionBufferinanappropriateultracentrifuge-compatibletube.

3. Pellettheribosomesbycentrifugationat4°Cfor4hoursat70,000rpminaTLA-110rotor.

4. RemovethesupernatantandresuspendtheRNApelletsin100μlNuclease-FreeWateror10mMTris-HClpH7.0.Add10μlof10%SDS.Thismaterialwillbeusedas"RibosomeProtectedRNA."Storesamplesat–80°Corproceedtonextstep.

5. PurifybothTotalRNAsamplesandRibosomeProtectedRNAsamples:

A. PurifytheTotal RNAsamplesusingtheZymoResearchRNAClean&Concentratorkit.Follow the kit protocol for >200 nt RNA.Elutethesamplesfromthecolumnswith26µlNuclease-FreeWater(expecttorecover~25µl).

B. PurifytheRibosome Protected RNAsamplesusingZymoRNAClean&Concentrator-25kitusingamodifiedprotocoloptimizedforsmallfragmentpurification:

a) InZymoprotocolStep1,use220μlBindingBuffer(fromZymoKit)

b) InZymoprotocolStep2,use495μlAbsoluteEthanol

c) Continuewithpurificationaccordingtothemanufacturer’sinstructions

d) Elutethesampleswith26µlofNuclease-FreeWater(expecttorecover~25µl)

6. QuantifybyNanoDrop.Youwillneedabout1-5μgofTotalRNAand1-5μgRibosomeProtectedRNAfortheRibo-ZerorRNAremovaltreatmentinStep3.

A. If<1-5μgofTotalRNAhasbeencollected,repeatstep1.A.9withunusedclarifiedlysatetoachievedesiredamount.

B. If<1-5μgofRibosomalProtectedRNAhasbeencollected,repeatstep1.Band2.Bwithunusedclarifiedlysatetoachievedesired amount.

7. ContinuetoStep3.A.RemovalofrRNAusingRibo-Zero.

Reference:Ingolia,N.T.Genome-WideTranslationalProfilingbyRibosomeFootprinting.MethodsinEnzymology470:119-142(2010).



ARTseq and Ribo-Zero are trademarks of Epicentre, Madison, Wisconsin.

Agencourt and AMPure are registered trademarks of Beckman Coulter, Inc., Brea, California.

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

NanoDrop is a registered trademark of NanoDrop Technologies, Inc., Wilmington, Delaware.

Phusion is a registered trademark of NEB, Ipswich, MA.

SYBR is a registered trademark of Molecular Probes, Inc., Eugene, Oregon.

Clean & Concentrator is a trademark of Zymo Research Corp., Irvine, CA.

Illustra and MicroSpin are trademarks of GE Healthcare, Fairfield, CT.

GlycoBlue is a trademark of Life Technologies, Carlsbad, CA.

SUPERase∙In is a trademark of Life Technologies, Carlsbad, CA.

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Protocol for ARTseqâ„¢ Ribosome Profiling Kit (Mammalian)

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Transcript of Protocol for ARTseqâ„¢ Ribosome Profiling Kit (Mammalian)