SingleCellEncapsulationProtocol,Version2.1
inDropTMSingleCellEncapsulationandReverseTranscriptionProtocol,Version2.1
TableofContents
A. ReagentsneededfortheinDropTMSingleCellReverseTranscriptionProtocol
B. CellCompatibilityC. Step-by-StepProtocolforBarcodingCells
I. InstrumentSetupII. SilanizingthemicrochipIII. HydrogelBeads,CellandRT/LysisBufferPrepsIV. LoadingMaterialsintotheMicrochipV. InitiatingandOptimizingGelBeadEncapsulationVI. cDNASynthesisVII. InstrumentShutdown
SingleCellEncapsulationProtocol,Version2.1 2
A.ReagentsandEquipmentfortheinDropTMSingleCellRTProtocol
WhatisProvidedby1CellBio
• BarcodedHydrogelMicrospheres(BHMs)325μL/kitwillprovide500,000BHMs
• 2xGelConcentrationBuffer• 1.3xRTPremix• WashBuffer• HFE7500• OptiPrep-Density-matchingAgent• Droplet-makingOil• DemulsifyingAgent• 1MMgCl2• MineralOil• Silane• 1mLsyringes• 2Coloredmineraloilsyringeassemblies• Syringeneedles• 3mLsyringe• Collectiontubing• MicrofluidicsChip• ColoredOil• Pipettetipassemblies• BHMloadingassemblies
ReagentsandEquipmentNeededandnotProvidedby1CellBio
• SuperScriptIIIReverseTranscriptase(ThermoFisherCat.No.18080044)• RNaseOUTRecombinantRibonucleaseInhibitor(ThermoFisherCat.No.10777019)• DTT(ThermoFisherCat.No.R0861,alsocomeswithSuperScriptIIIKit)• 1XPhosphateBufferedSaline(VWRScientificCat.No.12001-676)• TrypanBlue(0.4%;ThermoFisher,Cat.No.T10282)• 1.5mLDNALoBindtube(EppendorfAG,Cat.No.022431021)• Thermo-conductivetuberack(VWRScientificCat.No.95045-464)• UVlamp(UVPModelB-100AP,VWRScientificCat.No.36595-020)• Heatblockfor1.5mLtubes(VWRScientificCat.No.12621-084)• Aluminumfoil• Razorbladesorscalpel
Drop
Bead
Cell
Figure1.Collectiontubeofmicrochip,containingallcomponentstocreatebarcodedcDNA
KitStorageConditionsColorCode:
4°C
RoomTemperature
-20°C
SingleCellEncapsulationProtocol,Version2.1 3
• 0.22μmsyringefilterforsilanization(VWR,Cat.No.28145-493)• Centrifuge(EppendorfAG,Cat.No.5424R)• Vortex(VortexGenieorsimilar)• Hemocytometerandcoverslips• Apairoftweezers
B.CellCompatibilityPrior to running the protocol, you should confirm your cell sample meets the followingrequirements:
• The cell suspension has very few, or no, cell doublets or clumps (less than 5%). Cellstrainersmaybeusedtofilteroutcellclumps.
• Thesampleisfreefromcelllysate(aslysatesmaycontainRNA)whichmaycontaminatethesample.
• Cellviabilityin1xPBSishighandremainsstableforatleast30minonice.Ideally,theviabilityofthesamplewillbeabove95%andwillremainabove90%after30minutesonice.
D. Step-by-StepProtocolforBarcodingCellsI.InstrumentSetup
1. Logintothelaptopprovidedby1CellBiO–theloginnameis“inDrop”
2. Clickonthedesktopiconlabeled“Thor”.ThatopensthesoftwarethatcontrolstheinstrumentandimplementstheinDropprocess.
3. Removethehydrogelbeadsyringeassemblyfromitsdesignatedpackageandmountitonthepumpmarked“HydrogelBeads”(Pump0).Note–thesyringeisshippedpre-filledwithhydraulicfluidspecifictotheloadingthehydrogelbeads.Iftheamountoffluidbecomesdepleted,refillthesyringewiththeStorageBuffer.
4. PrimethetubingbyusingThorandinfusingthehydraulicfluidfromthesyringeintothetubing.Onceadropformsatthetipofthetubing,stopthepumpandremovetheexcessfluid.
5. Mountthedesignateddropletmakingoilsyringeontothepumpmarked“HydrophobicOil”(Pump1).PrimethetubingbyusingThorandinfusingtheDropletMakingOilfromthesyringe,intothetubing.Onceadropformsatthetipofthetubing,stopthepumpandremovetheexcessfluidwithaKimtechwipe.
SingleCellEncapsulationProtocol,Version2.1 4
6. Removethecoloredoilfilledsyringeassemblymarked“Cells”fromthebagandmounttheassemblyonthecorrespondingpump(Pump2).
7. Placethepipettetipintheholderattachedtomicroscopelightsourcearmwiththetip
pointedupwards.
8. ForPump2,activateInfuseandsetthepumprate=9000uL/hr.Thiswillfillthepipettetipwithmineraloil.Wipeanyexcessoilfromtheendofthepipettetipwhenitfillswithmineraloil.
9. Repeatsteps6-8forthesyringepumpmarked“RT/LysisMix”(Pump3).
10. Movetothenextstepofpreparingthehydrogelbeadsonceallsyringesaremounted
andprimed.
II. Silanizingthemicrochipdevices1. Accessthe1CellBiOwebsite(www.1cell-bio.com)andgototheResourcespage.
2. Downloadandfollowthe“ChipSilanizing”protocoltopreparethemicrochipdevicefor
use.
Critical:Uselowlinttissuetoremoveexcesssilanizingfluid.III. HydrogelBeads,Oil,CellandRT/LysisBufferPreps
A. HydrogelBeadPrep1. Concentratethebarcodedhydrogelmicrospheres(BHMs)ina1.5mLEppendorftubeby
spinningat5000gfor2min.Carefullyexaminethetubetobesureyoucandistinguishthebeadpelletfromthesupernatant.
2. Carefullytransfer300µLoftheconcentratedBHMstoanew1.5mLEppendorftubeandbegintheBHMwashesNote:300µLissufficientforbarcoding20,000-30,000cells.Critical:MinimizetheexposureofBHMstolightpriortoencapsulation.Ideallyallhandlingwilltakeplaceindimlighting.TubesholdingtheBHMsshouldbekeptawayfromdirectlightandcovered,e.g.,wrappedinfoil.
3. WashtheBHMsthreetimesasfollows:a. Add1mLofwashingbuffer.b. Vortexbriefly.c. Centrifugeat1,000gfor1min.
SingleCellEncapsulationProtocol,Version2.1 5
d. Removethesupernatant,dothiscarefullysoasnottodisturbthebeadpellet.e. Inthelastwash,leave~500μLinthetube.
4. Add500µLof 2xGel ConcentrationBuffer, vortex for ~6 seconds, and spin theBHM
suspensionat5,000gfor1min.
5. Usingap1000pipetteremoveasmuchofthesupernatantaspossible.300μLofclose-packedBHMsshouldnowconcentratetoaround150-200μL.
6. Spindownthetubeat5,000gfor1min,anduseap200pipettetiptoremoveanyremainingsupernatant.
B. CellPrep1. Adjust theconcentrationofcells tobe100,000cells/mL,or less, in1XPBS containing
density matching reagent. The cell suspension should have an 18% optiprep to cellsuspension.
Note:Asmuchaspossible,keepthecellsamplecoolonicewhilepreparingforarun.
Note:Wherepossible, preparemore cell phase thanyouanticipate youwill use, ideallyavolumeof500-600μL.Whenthenumberofavailablecellsisverylow,e.g.,atotalnumberof1,000cells,useafinalvolumeofatleast100μL.
C. RT/LysisBufferPrep1. Prepare30μLofRT/LysisMixper1000cellswithanadditional40μL forpriming.For
example,ifyouplantoencapsulateandbarcode10,000cells,prepare340μLofRT/lysismix.
2. Onice,preparetheRT/lysismixbymixing1.3xRTpremixwithMgCl2,DTT,RNaseOUT,
andSuperScriptIII.Here,wespecifytheappropriatevolumefor100μLofRT/LysisMix:Critical: AddRNAseOUTandSuperScriptIIIjustbeforeencapsulationtoreducethetimeonice.Critical:Avoidintroducingairbubblesbypipettinggently.
Reagent Amount(μL)
FinalConcentration
1.3xRTpremixMgCl2(1.0M)
761.1
1x11mM
DTT(0.1M) 6.9 6.9mMRNaseOUT(40U/μL) 5.65 2.26U/μLSuperScriptIII(200U/μL) 10.35 20.7U/μLTotal 100
SingleCellEncapsulationProtocol,Version2.1 6
IV. LoadingMaterialsintotheMicrochip
A. BHMLoading1. ComputehowmanyBHMsareneededforthenumberofcellstobeencapsulatedand
fromthatestimatethetimerequiredtoloadtheBHMsintothedeliverytubingbasedontheequationbelow.
2. PrimetubingofBHMpumpby,onthesoftware,increasingtheflowrateto2000uL/hr.Press“Run”andwaituntiladropletoffluidformsatthetipofthetubing.
3. Letthepumprunfor10secondsandpress“Stop”onthesoftware
4. InsertthetipofthetubingintothewashedBHMs.
5. Setthepumpflowrateto500uL/hr.MakesurethepumpisinWithdrawmode.
6. Press“Run”onthesoftware.TheBHMswillnowbeaspiratedintothetubing.Timetheaspirationandclick“Stop”afterthespecifiedtimehaselapsed.
Critical:MakesurethetipofthetubingstaysfullyimmersedintheBHMpellet.IfthetubingtipcomesoutoftheBHMpellet,stopthepumpandre-immersethetubingtipintothepelletandresumetheBHMaspiration.
CalculatetheVolumeofBHMstobeLoadedandLoadingTime
Inputs NumberofcellstobebarcodedNcells Userselected Bead-to-cellratio: 13:1FirstcomputethetotalnumberofBHMs,NBHM,requiredfortheexperiment:
NBHM=13BHM/cellxNcellsSecond,computethevolumeofBHMstobeloaded:
Volume(uL)=NBHM/2,100BHMLastly,computethetime,T,neededtoaspiratethisnumberofBHMs
T(min)=NBHM/(300BHM/s)/60
Table1.ComponentsandspecifiedamountsoftheRT/Lysismixfor100uL
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ExampleNumberofcells= 8,000cellsNumberofBHMs= 104,000BHMBHMvolume= 50ulTimetoaspirate= 6min Note:WithsmallamountsofBHMs,monitorthetubingintheEppendorfcloselytomakesurethetipremainsfullyimmersedinthepelletwhileloading.
B. CellandRT/LysisBufferLoading1. OnceyourcellandRT/Lysismixesareprepared,transfertheEppendorftubestothe
chillerblock.a. Ifyouchoosenottousethechillerblock,transfertheEppendorftubestoasmall
containeroficethatwillfitonthemicroscopestage
2. PlacethechillerblockcontainingtheEppendorftubesontherightsideofthestage.
3. SettheflowratesofPump2(Cells)to2000uL/hrandinfuse,makesuretothereismineraloilallthewaytothebottomofthetip.Wipethetipoffandplaceitinthecellsolution.
4. Repeatstep3usingPump3(RT/LysisBuffer)andplaceyourtipassemblyintoyourRT/Lysissolution.
5. SetPump2and3to‘Withdraw’onthesoftware,andmakesuretheflowrateissetto2000uL/hr.
6. RunPump2andPump3untilthedesiredamountofcellsandbufferarewithdrawnintothetipassembly.
C. ConnectingtotheMicrochip1. SettheflowrateofPump0(HydrogelBeads)to2000uL/hrandinfuseuntilliquidcomes
outofthetip.Stoptheflowandremoveanyexcessliquidfromtheendofthetubing.
2. SettheflowrateofPump1(HydrophobicOil)to2000uL/hrandinfuseuntilliquidcomesoutofthetip.Stoptheflowandremoveanyexcessliquidfromtheendofthetubing.
3. Gentlyinsertthetubingandpipettetipsintothecorrespondinginletsofthemicrochipinthefollowingorder(Figure2&3):
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a. ThepipettetipwithcellsintotheCellinlet.b. ThepipettetipwithRT/lysisBufferintotheBufferinlet.c. ThetubingwithBHMsintheBHMinlet.d. ThetubingwiththehydrophobicoilintheOilinlet.e. Acollectiontubeinthemicrochipoutlettotransfertheemulsiontoan
Eppendorftube.
Critical:DonotpressthetipassembliesallthewayintotheinletofthePDMS,ifpushedtoohard,thetipwillcontacttheglassslideanddisrupttheflow.
Figure2:Fluidicmicrodevicelayout
Figure3:AfullyconnectedmicrochipV. InitiatingandOptimizingGelBeadEncapsulation
SingleCellEncapsulationProtocol,Version2.1 9
1. Startbyclicking“PrimeChip” inthesoftwarewindow(Figure4).PrimeChipstartsthe
flowoffluidintothemicrochiptopushouttheairbubblesinpreparationforstartinganexperiment.Table2belowdetailstheflowratestouseineachphaseofdeviceoperation.Note:TheseflowratesarepresetintheThorsoftware.
2. OncebeadsareflowingintothebeadinletcloselypackedasshowninFigure5,clickon“RunExperiment”.Thiswilladjusttheflowratestopresetvaluesthatcanbeoverriddenandchangedbytheuser.
Figure4.ImageofThorsoftware
BHM$channel
Cell$channel
RT$Mix$channel
Droplet5making$Oil
Cell$collection$channel
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Figure5.Theimageofclose-packedhydrogelmicrospheresinthebeadchannel.
3. AdjusttheflowrateofBHMstoreach85-95%occupancy.Itmaybenecessarytofinetunetheflowrateinincrementsof5μL/h.
a. Theoccupancyrateismeasuredbytakingavideoofthedropsdownstreamfromthe drop forming region. Counting the drops and hydrogel beads per dropdeterminesthegelbeadencapsulationpercentageasshowninFigure5b.
4. Onceflowratesarestabilizedallowthesystemtorunfor1-2minuntil theemulsions
producedinitiallyaredisplacedfromtheoutlettubing.Table2:MicrodeviceFlowRateConditions
BHMPhase(Pump0)
Droplet-MakingOil(Pump1)
CellPhase(Pump2)
RTPremix(Pump3)
PrimeChip(μL/h) 100 200 200 200
RunExperiment(μL/h) 40 360 250 250
5. Collecttheemulsionintoa1.5mLEppendorftubeplacedinachilledthermos-conductive
rack as seen in Figure 5. Add 200 μL ofmineral oil to a 1.5mL tube and insert thecollectiontubebelowthemineraloil.Critical:Mineraloilisnecessarytopreventdropletcoalescenceduetoevaporation.Critical:Keeptheemulsioncoldbyusingthechillerblock
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6. Monitorthegelbead-cellencapsulationwiththehighspeedcamera.Adjustflowrates,ifnecessary,tomaintainoptimaloperatingconditions.
a. Thereshouldbeatmost1BHM ineachdropletandabout90%ofalldropletsshouldcontainbeads.AvoidhavingtwoBHMsperdroplet.AdjusttheflowrateofBHMsin5μL/hincrementstoensurethattheseconditionsaremaintained.BHMoccupancycanbedetermined fromshortmovies recordedat theoutletof themicrofluidicdeviceorsimplymonitoringthedropletflowusingtheStrobingModeor pausing feature in the software. The Strobing Mode will show the flowconditionsat2fps.
b. Ensurethatthecellencapsulationrateremainsconstantbymonitoringthecellinlet.Toconfirmcellcount,recorda~10seclongmovie,countthenumberofcellsflowingthrough,andcalculatethecellcountpersecond.TheStandardModeinthesoftwarewill record themoviesat150 fps.Whenyouplaya savedmovie,checkthetotalframesofthatmovie.Todeterminethesizeofthatmovie,dividethetotalframesby150fps.
Figure6.LayoutofThorsoftwareidentifyingkeyfeatures
c. Ensure that the droplet size and formation rate remain constant. Confirm the
dropletsize.Dropletvolumeinpicoliters(pL)isgivenby
V=(Fxt)/(cxN)
Flow%rate%controls
Stop%and%run%functions%of%pumps
Pump%Number
Command%Box
Infuse/%Withdraw
Record Play%Frame%by%Frame
Download%picture
Thor%movie%recordings
Image
PlayPause
Stop
Play%in%reverse
Commands
Timer
SingleCellEncapsulationProtocol,Version2.1 12
whereFisthesumflowrates(μL/h)ofallaqueousphases(e.g.hydrogelbeads,cellmix,andRT/lysismix),tisthetime(sec)tocountNdroplets,andcisafactortocorrectforunitdifferences(c=3.6secμL/pL*h).
d. ByusingtheflowratesindicatedinTable2,thedropletsizeshould3.0-3.5nLand
canbeadjustedbychangingtheflowrateofthedroplet-makingoil.
e. Toverify,fewdropletscontainmultiplecellseitherbyestimatingthenumberofcellsperdropletusingthecellflowrate,thecelldensity,andthedropletvolume,orcollectasampleofdropletsandestimatetheiroccupancyontheinstrumentusingahemocytometer.
f. Tocalculatethetimeofencapsulationrequiredtocollectthedesirednumberofcells,youcanusethe“Calculator”ontherighttopcornerofthesoftware.Whenyouclickonthecalculatorbutton,anewboxwillopenandaskyoutoenterthenumberofcellsyouwanttocollect,thenumberofcellsyoucounted,thelengthofthemovie(seconds)yousaved.Byenteringthisinformation,theprogramwillcalculatethetimerequiredtocollectthesample.Press“Start”tocommencetheencapsulationcounter.
7. Oncethedesirednumberofcellsisencapsulated,unplugthetubingfromtheoutlet,stop
thepumps,andlettheemulsioninthetubingdrainintothecollectiontubebygravity.
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VI. cDNASynthesisTime:3hours
1. Toreleasethephoto-cleavablebarcodingoligosfromtheBHMs,keepthecollectiontubeonice,exposethecollecteddropletstoUV(6.5J/cm2
at365nm):Usingthespecifiedlampshown in figure 8, fill a Styrofoam container with ice and place the sample tubehorizontallyonice.Placethebulb3to5cmabovethetubeandirradiatefor8min.
2. Heatthetubeto50°Candincubateitfor2hourstoallowcDNAsynthesistooccur(Figure9).
Figure7.Setupforcollectingsample
SingleCellEncapsulationProtocol,Version2.1 14
3. Terminatethereactionbyheatingfor15min@70°C.
4. Removefromheat,andremoveasmuchofthemineraloilandresidualdroplet-makingoilaspossiblewithapipette(Figure10).
Figure8.UVlampontopofsamplesbreakingphotocleavablelinker
SingleCellEncapsulationProtocol,Version2.1 15
5. Ifnecessary,dividetheemulsionintofractionscontainingthedesirednumberofcells.Forexample, if4000cellswerebarcoded,dividetheentirevolumeoftheemulsionintwoequalpartstoget2000-celllibraries.Usingconditionsdescribedinthisprotocol,35μLofemulsiontypicallycontain~1000barcodedcells.
6. Break the emulsion by adding equal volume of demulsifying agent as the samplecollected.Vortexfor5secondsandcentrifugeat1000gfor1min.ThetubeshouldlooklikeFigure11.
Note:Iftheemulsiondoesnotbreakafteraddingthedemulsifyingagent,repeatstep6.
7. ThecDNAcontainedintheaqueousphaseisnowreadytoundergolibrarypreparation.Atthispoint,thetubescanbestoredat-80°Cforatleast3months,orsequencinglibrariescanbepreparedfromthesamplesimmediately.
Figure9.HeatrackfortheRTreaction. Figure10.BeforetheemulsionisbrokenMineraloilhasbeenremovedfromtop
SingleCellEncapsulationProtocol,Version2.1 16
VII. InstrumentShutdown
1. FlushtheblackBHMtubingwiththestoragebufferbyrunningthepumpat2,000uL/hrfor~1min,oruntilallstoragebufferisoutofthesyringe.ItisrecommendedtoflushthetubingintoaLobindEppendorftube.ThetubecanbetreatedasarecoverytubefortheBHMs.Oncecompletewiththeflush,placethesealingcaponthetipofthetubing.
2. Unplugtheoiltubingandinsertthesealingcapintheendofthetubing.Parkthetubeinoneofthetubingholdersonthemicroscope.
3. ForboththeCellandRT/LysisBufferpipettetips,settheflowrateto9,000uL/hrand
click“Withdraw”and“Run”towithdrawthecoloredmineraloilintothetubing.Assoonasthemeniscusofthecoloredmineraloilisvisibleinthetubing,click“Stop”.
4. Removeanddiscardtheusedpipettetips.
5. Inserttheendcapsintoeachtubingandplacethetubinginoneofthetubingholderson
themicroscope.
Figure11.Brokenemulsion,readyforlibraryprep,cDNAcontainedinaqueousphase