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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

SingleCellEncapsulationProtocol,Version2.1 7

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):

SingleCellEncapsulationProtocol,Version2.1 8

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

SingleCellEncapsulationProtocol,Version2.1 10

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

SingleCellEncapsulationProtocol,Version2.1 11

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.

SingleCellEncapsulationProtocol,Version2.1 13

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