CRISPR droplet sequencing (CROP-seq) protocol (version ...CRISPR droplet sequencing (CROP-seq) Page 3 of 20 3.2 Gibson Assembly Dilute gRNA-encoding ssDNA oligos to 100 µM with EB

CRISPRdropletsequencing(CROP-seq) Page1of20

CRISPRdropletsequencing(CROP-seq)protocol(version2017/01/18)

Authors:PaulDatlinger([email protected]),AndréFRendeiro*,ChristianSchmidl*,ThomasKrausgruber, Peter Traxler, Johanna Klughammer, Linda C Schuster, Amelie Kuchler, Donat Alpar andChristophBock([email protected]).Futureupdatesoftheprotocolwillbesharedviahttp://crop-seq.computational-epigenetics.org.

PartA:Pooled,amplification-freecloningofgRNAlibraries

1.ValidationoftheCROPseq-Guide-PuroplasmidpreparationOrder theCROPseq-Guide-Puroplasmid fromAddgene (86708), performplasmidDNAextraction, andvalidatebyrestrictiondigestionandSangersequencing.

PlasmidvalidationbyrestrictiondigestionRestrictionenzyme Buffer Temperature[°C] Expectedfragments[bp]

SapI CutSmart 37°C 1171,3394,5649SphI NEBBuffer2.1 65°C 1110,3787,5317

PlasmidvalidationbySangersequencing

PrimerID Primersequence[5’>3’] Validateselement(s)86708_p1 GAGGGCCTATTTCCCATGATTCC hU6,BsmBIsite86708_p2 CAAATGCATGCTCTTCAACCTC BsmBIsite,gRNAbackbone,3’LTR86708_p3 GAGACAAATGGCAGTATTCATCCAC EF-1a86708_p4 TAAGTGCAGTAGTCGCCGTG EF-1a,Puro86708_p5 AGTCTTGTAAATGCGGGCCA Puro86708_p6 CACACTGAGTGGGTGGAGAC Puro86708_p7 AGCAACAGATGGAAGGCCTC WPRE86708_p8.1 TTGGGCACTGACAATTCCGT 3’LTR,fillerorgRNA86708_p10 GACGAACCACTGAATTGCCG Psi,Gag86708_p11 GGGAGCTAGAACGATTCGCA RRE86708_p12 TTCCTTGGGTTCTTGGGAGC cPPT86708_p13 CGAGTCAGTGAGCGAGGAAG 5’LTR

2.PreparationofCROPseq-Guide-Purovectorbackbone

2.1Plasmiddigestion

DigestCROPseq-Guide-Puroplasmidandgel-purifythe8,333bpfragmentasindicated.Onceprepared,thebackbonefragmentcanbefrozenat-20°Candre-usedforcloningfurthergRNAlibraries.

Vectordigestionmix Perreaction Mastermixfor#ofdigestionsComponent Volume Amount 4 6 8 10 12CROPseq-Guide-Puro 5µg NEBBuffer3.1 2.5µl 1x 10.0 15.0 20.0 25.0 30.0BsmBI 2µl 20U 8.0 12.0 16.0 20.0 24.0Nuclease-freewater to25µl


Distribute25µlofvectordigestionmixperwellintoatubestrip,incubate:1hourat55°C(digestion),20minat80°C(inactivationoftheenzyme),holdat10°C.2.2UV-freegelextraction<Note>BasedonSNAPUV-FreeGelPurificationKit(Invitrogencat.no.45-0105)

Prepare required number of 0.8% agarose gels with TAE buffer containing 1.8 µg/ml Crystal Violet:Example for one gel: 0.4 g agarose in 50 ml TAE, plus 40 µl of 2 mg/ml Crystal Violet SolutionImportant:useathincombformoreintensebands,TBEisnotcompatiblewiththiskit

Load8µgofDNAladderin25µlofwater,mixedwith5µlof6xCrystalVioletLoadingDyeLoad25µlofeachplasmiddigestion,mixedwith5µlof6xCrystalVioletLoadingDyeRunat80VandobservetheseparationofbandswiththenakedeyeOncethebandsaresufficientlyresolved,stopandisolate8,333bpfragmentwithgel-cuttingtipsTransfergelsliceto1.5mltube,weightoestimatetheagarosevolume(assumingthat1mg~1µl)Add2.5volumesofSodiumIodideSolutionIncubateat42°Cwithoccasionalvortexing,untiltheagaroseiscompletelydissolvedAdd1.5volumesofBindingBuffer,mixandloadontoSNAPPurificationColumnCentrifugeat3,000rcffor30secondsPourtheflowthroughbackontothecolumnandrepeatthecentrifugation,foratotalofthreetimesAfterthelastcentrifugationstep,discardtheflowthroughWashtwotimeswith400µlof1xFinalWashBuffer,discardflowthroughaftersecondcentrifugationCentrifugecolumnfor1minat>10,000rcftoremoveresidualwashbufferTransfer the column to a fresh 1.5ml tube and elutewith 40µl of EB buffer (10,000 rcf for 1min)

Important:incubateforatleast1mintoallowabsorptionintothecolumnTransfertheeluatebacktothecolumn,andeluteasecondtimeasdescribedaboveMeasure1µloftheeluateinaQubitHSassaytodeterminetheconcentrationofdsDNA

<SafeStoppingPoint>Thebackbonefragmentcanbestoredat-20°Candre-used.

<Troubleshooting>Ifrequired,performethanolprecipitationtofurtherconcentratetheDNA:poolmultiple

gel-purificationreactions,add1/10volumeof3Msodiumacetate,pH5.5and2.5volumesof100%ice

coldethanol.PrecipitateDNAfor30minat-80°C,thenspinfor20minat20,000rcfat4°Ctopellet.Wash

pellettwotimeswithroomtemperature70%ethanol,leavetodry(takecarenottoover-drythepellet)

andeluteinasmallervolumeofEBbuffer.

3.AssemblyofgRNA-encodingssDNAoligonucleotidesintothevectorbackboneusingGibsonAssembly3.1DesigngRNAsRecommended formanual design: https://benchling.com/, for automateddesign: https://github.com/boutroslab/cld/.OrderasseparatessDNAoligos,with18and35basesofhomologytothehU6promoterandgRNAbackboneforGibsonAssembly.The5’prefixalsoaddsaGasaconstantfirstbaseformoreefficienttranscriptionfromthehU6promoter.Examplesforpositivecontrols(targetingessentialgenes)and negative controls (non-targeting) can be found in the original publication of CROP-seq(SupplementaryTable2).<Example> TGGAAAGGACGAAACACCG(5’prefix,hU6promoterandGasfirstbase)+gRNA+

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC(3’postfix,gRNAbackbone)


3.2GibsonAssembly

Dilute gRNA-encoding ssDNAoligos to 100µMwith EBbuffer. Pool all oligos contained in the library(includingpositiveandnegativecontrols) inequalamounts.Dilute theoligopool1:1000 inEBbuffer,resulting inamolarityof100nM.SetupGibsonassembly reactions foreach libraryand the includedNEBuilderHiFipositivecontrol,asdescribedbelow.

AssemblyofgRNA-encodingssDNAoligosintothevectorbackbonebyGibsonAssembly PerreactionComponent Volume AmountCROPseq-Guide-Purobackbonefragment(8,333bp) 11fmolesLibraryssDNAoligos,pooled(100nM) 2.0µl 200fmolesNEBuilderHiFiDNAAssemblyMasterMix 10µl 1xNuclease-freewater to20µl

<Note>Weusethefollowingonlinetooltocalculatethevolumeofbackbonefragmentrequired:

http://nebiocalculator.neb.com/#!/dsdnaends

Incubateat50°Cfor1hour,thendialyzethereactionagainstwater:

Filla6cmcellculturedishwithHPLC-qualitywater,placemembranefilterontopPipette20µloftheGibsonAssemblyreactionandletfloatontopofthefilterfor30min

<Note>Thisremovessaltsandmakestheelectroporationmoreefficient

About10µloftheGibsonAssemblyreactioncanberecoveredafterthefiltration3.3ElectroporationofLucigenEnduracells

PreparationsPreparemixtureoficeandwaterThawonevialofLucigenEnduraelectrocompetentcellsonice(takesabout20min,sufficientforcloningonegRNAlibrarywithtwoelectroporations)Pre-chilltwo1mmelectroporationcuvettesonicePlacetwoempty2mltubesonicePre-warmLucigenrecoverymediumat37°CPre-warmtwo25x25cmLB-agarplatescontaining100µg/mlcarbenicillinatroomtemperature

ElectroporationAliquot25µlofbacteriaintopre-chilled2mltubes,andkeeponiceAdd10µlofde-saltedGibsonAssemblytothebacteriaandgentlytapthetubetomixTransfertoelectroporationcuvette,taptobringcontentstothebottom,checkforairbubblesPrepareP1000pipettefilledwith1mlofLucigenrecoverymediumElectroporatewiththefollowingsettings:25µF,200Ω,1.5kVImmediatelyafterthepulse,addthepreparedLucigenrecoverymediumGentlypipetupanddowntwotimes,thentransfertoround-bottomtubesTakeanoteofthetimeconstantfortheelectroporation(shouldbe~4.5msec)Incubatefor1hourat37°CforrecoveryPreparea1:100dilution(10µlbacteriaplus990µlSOCmedium),andplate100µlona10cmLB-agardishPlatetheremainingbacteriaontothe25x25cmLB-agarplateIncubatefor20hoursat32°C


3.4Plasmidpreparation

Thenextday,countcoloniesonthe1:1000dilutionplateandcalculatethelibrarycoverageasfollows:(numberofcoloniesx1000)/(numberofgRNAsinlibrary)

Retrievebacteriafromplatesasfollows:Add20mlofpre-warmedLBmediumtotheplate,scrapeoffbacteria,andtransferto50mltubeAddanother30mlofpre-warmedLBmedium,tiltplateandtransferto50mltubeSpinat4,700rcffor15mintocollectabacterialpelletanddiscardsupernatant

ProceedwithplasmidextractionusingtheEndo-FreePlasmidMegakit(Qiagen#12381)accordingtothemanufacturer’sinstructions

4.Next-generationsequencingofgRNAsequencesforlibraryqualitycontrolorpooledscreens

gRNA sequences are amplified from a CROPseq-Guide-Puro plasmid library or from the gDNA oftransducedcells(pooledscreen)inasinglePCRreactionwithspeciallydesignedprimers.ThisstepaddsallsequencesrequiredforcompatibilitywithIlluminasequencers.Inaddition,i7barcodesareprovidedforsamplemultiplexing,andstaggersequencescanincreaselibrarycomplexityforIlluminasequencing.

PrimersforamplificationofgRNAsfornext-generationsequencingPrimerID Primersequence[5’>3’]

CROPseq_libQC_i5_s1 AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTTGTGGAAAGGACGAAACACCG

CROPseq_libQC_i5_s2 AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTATCTTGTGGAAAGGACGAAACACCG

CROPseq_libQC_i5_s3 AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTGATCTTGTGGAAAGGACGAAACACCG

CROPseq_libQC_i5_s4 AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTCGATCTTGTGGAAAGGACGAAACACCG

CROPseq_libQC_i5_s5 AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGATCTTGTGGAAAGGACGAAACACCG

CROPseq_libQC_i5_s6 AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTATCGATCTTGTGGAAAGGACGAAACACCG

CROPseq_libQC_i7:1 CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGTGTCTCAAGATCTAGTTACGCCAAGC

CROPseq_libQC_i7:2 CAAGCAGAAGACGGCATACGAGATTCTCCGGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGTGTCTCAAGATCTAGTTACGCCAAGC

CROPseq_libQC_i7:3 CAAGCAGAAGACGGCATACGAGATAATGAGCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGTGTCTCAAGATCTAGTTACGCCAAGC

CROPseq_libQC_i7:4 CAAGCAGAAGACGGCATACGAGATGGAATCTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGTGTCTCAAGATCTAGTTACGCCAAGC

CROPseq_libQC_i7:5 CAAGCAGAAGACGGCATACGAGATTTCTGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGTGTCTCAAGATCTAGTTACGCCAAGC

CROPseq_libQC_i7:6 CAAGCAGAAGACGGCATACGAGATACGAATTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGTGTCTCAAGATCTAGTTACGCCAAGC Functionalelements5’partofi5ori7Illuminaadapteri7indexforsamplemultiplexing3’partofi5ori7IlluminaadapterstaggersequencetoincreasecomplexityforIlluminasequencingprimerbindingsiteonCROPseq-Guide-Puro

Toretainagoodlibraryrepresentationandreducebias,multiplePCRreactionsshouldbeperformedforeachgRNAplasmidlibraryorgDNApreparation.ThenumberultimatelydependsonthesizeofthegRNAlibrary,butwemostlyusefourparallelPCRreactions.Thesereactionscanusethesamei7barcode,sothattheyaredemultiplexedintothesamefileduringdataprocessing.


qPCRamplificationofgRNAsfromtheplasmidlibraryorgDNAoftransducedcells PerreactionComponent Volume AmountCROPseq-Guide-PuroplasmidlibraryorgDNAfromapooledscreen

10ngor100ng

Q5HotStartHigh-Fidelity2xMasterMix 25µl 1xCROPseq_libQC_i5primer(10µM) 2.5µl 500nMCROPseq_libQC_i7primer(10µM) 2.5µl 500nMOptional:SYBRGreen,100x 0.5µl 1xNuclease-freewater to50µl

ByaddingSYBRGreen,thereactionscanberuninaqPCRmachineandstoppedjustbeforereachingtheplateauphase.Thisensuresthattheoptimalnumberofenrichmentcyclesisused.Whenrunningmultiplesamples,themachinecanbepausedduringthedenaturationphase.Piercethefoiltoremovefinishedreactions,andresumetheamplificationoftheremainingsamples.Incubateasfollows:

98°Cfor30sec(initialdenaturation)40cycles{ 98°Cfor10sec 72°Cfor45sec}stopqPCRreactionsjustbeforetheyreachtheplateauphase

CleanPCRproductswitha2.0xAMPureXPbeadclean-upQualitycontrolonanAgilentBioanalyzerHighSensitivityDNAchipshouldshowasinglebandat282bp

(anexampleisshownonthefollowingpage,intheExpectedresultssection)MeasurethedsDNAconcentrationinaQubitHSassayDilutelibraryto4nMwithEBbuffer+0.1%TweenbasedonsizeandconcentrationmeasurementsSequenceonanIlluminaMiSeqmachine,usinga150-cyclev3flowcellwithareadconfigurationof167

bases(read1)and8bases(i7index).Whilethisisthecheapestoption,NextSeqandHiSeqinstrumentscanbeusedinstead


PartA:Expectedresultsforthepooled,amplification-freecloningofgRNAlibraries

gRNAsamplifiedfromtheplasmidpoolduringlibraryqualitycontrol,orfromgenomicDNAinaclassicalpooledscreen.The282bpPCRproductiscompatiblewithIlluminasequencingwithoutfurtherprocessing.

TypicalrepresentationofthegRNAlibraryintheplasmidpool(left)andwhenamplifiedfromgenomicDNAinapooledscreen(right,day10posttransduction).


PartB:LentivirusproductionandcellcultureforCROP-seqscreens

5.ProductionoflentiviralparticlesPrerequisites:PlasmidpoolofgRNAlibraryclonedasdescribedinpartA,andvalidatedbyNGSPlasmidpreparationsofthe3rdgenerationlentiviralpackagingplasmidspMDLg/pRRE(Addgene12251),pRSV-Rev(Addgene12253),andpMD2.G(Addgene12259)Cellline(s)stablyexpressingCas9aftertransductionwithlentiCas9-Blast(Addgene52962).Thelenti-

virusproductionandtransductionprotocolsbelowcanbeusedtoproducethiscellline<Note>TherequiredscaleofthelentivirusproductiondependsonthesizeofthegRNAlibraryandthe

plannedexperiments.Formostapplications10cmdishes,oreven6-wellplatesshouldsuffice,duetotherelativelyhightitersachievedwiththeCROPseq-Guide-Puroconstruct.Numbersforbothformatsare

providedbelow(color-coded).

<Important>Disposeoflentivirus-containingmaterialsaccordingtoyourinstitute’sbiosafetyguidelines.

Personsperformingexperimentswithlentivirusmustbeproperlytrained. Preparelentiviruspackagingmedium:Opti-MEMI,5%FCS,200µMsodiumpyruvate,noantibioticsSeedHEK293Tcellsat7millioncellsper10cmdishin12mloflentiviruspackagingmedium,orat1.2millioncellsperwellona6-wellplatein2mloflentiviruspackagingmediumThenextmorningproceedwiththetransfectionPreparetransfectionmixturesAandBasindicatedbelow

TransfectionofHEK293Tcellsforlentivirusproduction 10cmdish 6-wellplateTransfectionmix Component Volume Amount Volume Amount

A CROPseq-Guide-PurogRNAlibrary,plasmidpool 10.2µg 1.7µgpMDLg/pRRE 5.4µg 0.9µgpRSV-Rev 5.4µg 0.9µgpMD2.G 5.4µg 0.9µgOpti-MEMI 1.5ml 250µl P3000enhancerreagent 36µl 6µl

B Opti-MEMI 1.5ml 250µl Lipofectamine3000reagent 42µl 7µl

Preparelipid-DNAcomplexesbycombiningtransfectionmixesAandB,andincubatingfor20minatroomtemperatureInthemeantime,remove6ml,or1mlofmediumfromtheHEK293TcellsAdd3mlor500µloflipid-DNAcomplexestotheHEK293Tcells,andgentlyagitateAfter6hofincubationat37°Cand5%CO2exchangemediumfor12mlor2mloffreshlentiviruspackagingmediumCollectlentivirus-containingmediumafter24h,storeat4°C,add12mlor2mloffreshlentiviruspackagingmediumCollectlentivirus-containingmediumafter48h,poolwithsupernatantobtainedthepreviousdayandfilterthrougha0.45µmfiltertoremovecells


Aliquotlentiviruspreparationintocryotubesandfreezeat-80°CProceedwithtitrationofthelentiviruspreparationandpooledscreening6.Optional-Titrationoflentiviruspreparations<Note>Tosavetime,transductionsforpooledscreenscanbeperformedwithvariablevolumesofthe

viruspreparation.Alltransductionconditionsarethenselectedfor2days,andtheconditionwiththe

desiredMOIcanbechosenforthescreen.

SeedHEK293Tcellsonto24-wellplatesat50,000cellsperwell in500µlofcompleteculturemedium(DMEM,10%FCS,penicillin-streptomycin)Growovernightat37°Cand5%CO2sothatcellsreach30%to50%confluenceExchangeculturemediumfor450µlcompleteculturemedium,supplementedwith8µg/mlpolybreneThawonevialofthelentiviruspreparationfromstorageat-80°CIna96-wellplate,preparea1:5dilutionseriesofthelentiviruspreparationincompleteculturemediumwithpolybrene,rangingover10wells(dilutionfactorsfrom1:5to1:9,765,625)Testtheoriginalstock,andeachdilutioninduplicate,byadding50µlperwelltothe24-wellplatewithHEK293Tcells(thisrequires2x(1+10)=22wells).Theremainingtwowellsserveasuntransducedcontrols.Add50µlcompleteculturemediumcontainingpolybrenetotestfortoxicityofpolybrenetocells(possibleforsomecelltypes).24hposttransductionexchangethemediumfor500µlcompleteculturemediumwithoutpolybrene48hposttransductionstartselectionwithpuromycinandrenewselectivemediumevery2-3days.Thetablebelowprovidescommonantibioticconcentrationsweuseforpuromycinselection,butultimatelytheconcentrationwilldependonthecelllineused.

Examplesofselectionconditionsusedinourlab

Cellline c(Puromycin)[µg/ml]toselectforgRNA

c(Blasticidin)[µg/ml]toselectforCas9

K562 2 30Jurkat 2 25MV4-11 1 20KBM7 0.5 20HEK293T 2.25 22.5

Assoonascellsintheuntransducedcontrolsaredead,removemediumandwashoncewith1xPBSStaincoloniesfor15mininasolutionof1%(w/v)crystalvioletin10%ethanolWashseveralmoretimeswith1xPBS,countcoloniesfortwodilutionfactors,andtworeplicateseachCalculatethevirustiter(transducingunits/ml)as:

(averagenumberofresistantcolonies)x(dilutionfactorofwell)x(1,000µl/50µl)ExampleforatypicalgRNAlibrary: 56and66colonieswerecountedforthewellwithdilutionfactor15,625 ((56+66)/2)x(15,625)x(1,000µl/50µl)=1.9x107TU/ml


7.TransductionofsuspensioncellsforpooledCROP-seqscreensSeed suspension cells in 6-well plates at 5million cells perwell in 2ml of complete culturemedium(dependsoncellline),supplementedwith8µg/mlpolybreneandblasticidin(werecommendtokeepupselectionforlentiCas9-Blastthroughoutthescreen,asitiseasilysilenced;pleaserefertothetableaboveforsuggestionsonblasticidinconcentrationsforvariouscelllines).Option1(virustiterwasmeasured):AddcalculatedvolumeoflentiviruspreparationforthedesiredMOItoonewell,useasecondwellasuntransducedcontrol.Option2(virustiterunknown):Transducefivewellswithdifferentvolumesofthelentiviruspreparation(e.g.250µlor50µlof theoriginalstock,or50µlof1:3,1:6and1:12dilutions).Thesixthwell is leftuntransduced.Immediatelyafteradditionoflentiviralparticles,centrifugetheplatefor45minat1,200rcfand37°C(spinfection),followedbyovernightincubationat37°Cand5%CO2.24hposttransduction,pelletcellsin15mltubes,removesupernatant.TransfercellstoT75cellcultureflasksin30mloffreshcompleteculturemediumsupplementedwithblasticidin(stillonlyselectingforlentiCas9-Blast).48hposttransduction,startselectionforthegRNAlibrarybyaddingpuromycindirectlytotheflask.Renewselectivemediumcontainingblasticidinandpuromycinevery2-3daysforatotalof7-10days toallowforefficientgenomeediting. Ifoption2waschosen,select the flaskwithabout30%survivingcellsafter2daysofpuromycinselection.8.TransductionofadherentcellsforpooledCROP-seqscreens

Seedadherent cells in15 cmdishes at 5million cells perplate in20mlof complete culturemedium(dependsoncellline)supplementedwith8µg/mlpolybreneandblasticidin(werecommendtokeepupselectionforlentiCas9-Blastthroughoutthescreen,asitiseasilysilenced;pleaserefertothetableaboveforsuggestionsonblasticidinconcentrationsforvariouscelllines).Option1(virustiterwasmeasured):AddcalculatedvolumeoflentiviruspreparationforthedesiredMOItooneplate,useasecondplateasuntransducedcontrol.Option2(virustiterunknown):Transducefiveplateswithdifferentvolumesofthelentiviruspreparation(e.g.250µlor50µloftheoriginalstock,or50µlof1:3,1:6and1:12dilutions).Asixthplateisusedastheuntransducedcontrol.Afteradditionoflentiviralparticles,swirltheplategentlytomixandincubateovernightat37°Cand5%CO2.24hourspost transduction,exchangemediumfor freshcompleteculturemediumsupplementedwith blasticidin (selecting for lentiCas9-Blast). At 48 h post transduction, start selection for the gRNAlibrary by addingpuromycin directly to theplate. Renew selectivemediumevery 2-3 days for a totaldurationof7-10daystoallowforefficientediting.Ifoption2waschosen,selecttheplatewithabout30%survivingcellsafter2daysofpuromycinselection.


PartC:Single-cellRNA-seqbasedonDrop-seq

Modifiedfrom:Macoskoetal.2015andadditionalprotocols(http://mccarrolllab.com/dropseq/)

9.Dropletmicrofluidics9.1Preparecells

Collectcellsbycentrifugationat300rcffor5min<Note>Centrifugationspeedandtimemightneedtobeadjustedforothercelltypes

Washoncewith1mlofPBS-0.01%BSA(makefreshonthedayoftherun)Removesupernatantandresuspendin1mlofPBS-0.01%BSAFilterthrougha40µmcellstrainertoobtainasingle-cellsuspensionMeasurecellconcentrationtwiceusingacellcounter

<Note>WeuseaCASYdevice,butanyalternativemethodcanbeused

Diluteto220cellsperµlusingPBS-0.01%BSAinatubethatallowseasyuptakebythesyringesCheckconcentrationofdilutedcellstwice9.2Preparebeads

Mix1.6mlofDrop-seqlysisbuffer(storedat4°C)with80µlofDTT(storedatroomtemperature)DiluteDrop-seqbeadsasfollows

ResuspendDrop-seqbeads(storedat4°C)byverygentleshakingImmediatelytransfertheequivalentof225,000beadstoa1.5mltubeCentrifugefor1minat1,000rcfRemovesupernatantandtakeupbeadsin1.5mloflysisbufferwithDTT(150beadsperµl)Transfertoatubethatallowseasyuptakebythesyringes

9.3Drop-seq

<Generalcomment>Toloadsyringes,cuta200µlpipettipwithoutfilteronbothends(toreduceshearforceandtofacilitateattachmenttothesyringe).Afterloading,invertthesyringe,pullupwhateveris

leftinthetip,discardtip,taporpushtoremoveairbubblesandattachasafe-lockneedleandtubing

usingtweezers(becarefulnottopunctureyourfingers!).Presssyringeuntiltubingisfreeofair

bubbles.

Loadoilintoa10mlsyringeLoadcellsintoa3mlsyringePlaceDrop-seqmicrofluidicdeviceonmicroscopestageAttachtubingforoilandcellsandconnecttheoutlettoa50mltube(labelled“waste”)Prepareasecond50mltubeforcollectingdropletsoncetherunisstableLoadbeads

<Criticalstep>Loadingthebeadsisacriticalstep,astheycaneasilysettleorclump.Thiscanclogthe

needleorleadtoanunevenbeadconcentrationthroughouttherun.

Removeplungerfrom3mlsyringe,theninsertmagneticmixingdiscandpushplungerbackSetplungeroftheemptysyringeto1.5ml,attachtothepumpSetupthemagnetto1jumppersecondandkeepswitchedonDetachemptysyringefrompumpUseaP1000togentlymixthebeadsolution(carefulnottoformairbubbles)Loadthesyringewhilegentlyagitatingthebeadsolution;immediatelyinvertthesyringetoavoidcloggingtheneedle(holdsyringewithneedlefacingupwards)Gentlypushthebeadsolutionthroughthetubing,thenconnecttubingforbeadsolutiontothe


microfluidicdeviceFinally,attachthesyringetothepumpwiththeneedlefacingdownwards(themagnetisstillrunningandstartsmixingimmediately)

Setflowrateto1.6ml/hforcellsandbeads,8ml/hfortheoil,andmakesurethepumpissettothespecificsyringetype

Startthepumpsintheordercells>beads>oil<Note>Cellsarestartedfirsttoavoidabackflowoflysisbuffercontainedinthebeadsolution

<Troubleshooting>Ifyouwanttostoptherunforwhateverreason,theorderisbeads>cells>oil

Checkdropletsfromtimetotimeunderthemicroscopebyaddingafewdropsfromtheoutlettubingontoaglassslide.<Note>About5%ofdropletsshouldcontainabead.Cellsarelysedwithinsecondsandarethereforenotvisible.

Startcollectingdropletsina50mltubeifthedropletsizeishomogenousAstherunprogresses,themixingdiskwillstopjumpingbecauseitgetsblockedbytheplunger

(approximatelywhentheplungerreachesthe0.4mlmarking).Atypicalrunlastsfor35-40min,fillingthe50mltubeto7.5–10ml.Makesuretoretrievethemixingdiscfromthebeadsyringeattheend.

Proceedwithdropletbreakageonthesameday.Whenperformingmultiplerunsperday,50mltubescontainingdropletscanbestoredat4°Candfurtherprocessedtogether.

9.4Dropletbreakage

RemoveasmuchoilbelowthedropletlayeraspossibleAdd30mlof6xSSCbufferAdd1mlofperfluoroctanolShakeforcefullybyhand6timestobreakthedropletsBeadcapture

Labelthetopsideofa0.22µmfilterunit,removeplungerfroma20mlsyringe,attachsyringeto0.22µmfilterunit,placeonafresh50mltubeandpourthebead-containingsampleintothesyringeThenplaceplungerbackandfilterasusualDiscard50mltubewithflowthroughandthesyringe,butkeepthefiltercontainingthebeads<Note>Thebeadsareretainedinthe0.22µmfilterunit,duetotheirrelativelylargediameter

WashingbeadsPlacefilterunit(topsideup)onafresh50mltube,pour6xSSCbufferintoareservoir,takeup20mlfromthereservoirwithafreshsyringe,attachtofilteruniton50mltubeandwashthebeadsRepeatonceforatotaloftwowashesKeepthewashfractionandfilter,anddiscardthesyringe<Note>Beadsarestillretainedinthefilterunit,andonlywashedintheprocess

ElutingbeadsInvertthefilterunitandplaceitonafresh50mltubeCuta200µltipwithoutfilteronbothends,itwillserveasanadaptertoconnectthebottomsideofthe0.22µmfiltertoa10mlsyringeThenelutebeadsthreetimeswith10mlof6xSSCbuffer<Note>Beadshavenowbeencollectedinthe50mltubeinatotalvolumeof30ml

ConcentratingbeadsSpin50mltubesforwashandelutionfractionsfor2minat1,250rcfwithbrakespeedsetto50%(oruse‘soft’settingifavailable)<Note>Reducingthebrakespeediscritical,topreventkickingbeadsbackintosolution

Comparepelletsbetweenwashandelutionfraction,discardwashfractionunlessthereisapronouncedpelletaswell


Removesupernatantfromtheelutionfractionuntilonlyabout1mlremainsinthetubeWashbeadpelletoncewith10ml6xSSCbuffer,centrifugingwiththeabovesettingsRemovethesupernatantcarefullywithaP1000pipetteTakeupthepelletin200µlof5xreversetranscription(RT)bufferandtransfertoa1.5mltubeSpin1.5mltubeat1,250for2minwithbrakespeedsetto50%(or‘soft’setting)RemoveasmuchoftheRTbufferaspossiblewithoutdisturbingthebeadsImmediatelyproceedwiththereversetranscription

10.PreparationofcDNAlibrary10.1Reversetranscription

Preparereversetranscriptionmastermixasindicatedinthetablebelow.Thevolumesprovidedalreadycompensatefor10%deadvolume.

Reversetranscriptionmastermix

Perreaction Mastermixfor#ofDrop-seqruns Component Storedat Volume Amount 1 2 3 4 5 6Nuclease-freewater RT 75µl to200µl 82.5 165 247.5 330 412.5 495Maxima5xRTbuffer -20°C 40µl 1x 44 88 132 176 220 26420%FicollPM-400 4°C 40µl 4% 44 88 132 176 220 26410mMdNTPs -20°C 20µl 1mM 22 44 66 88 110 132RecombinantRNaseinhibitor -20°C 5µl 200U 5.5 11 16.5 22 27.5 3350µMtemplateswitchingoligonucleotide -80°C 10µl 2.5µM 11 22 33 44 55 66

MaximaH-RTase -20°C 10µl 2000U 11 22 33 44 55 66Resuspendbeadpellet(oneperrun)fromstep9.4in200µlofreversetranscriptionmastermixTransfertoa96-wellplateandincubateinathermocyclerwithheatedlid

30minat25°C90minat42°CStorageat4°C

<Safestoppingpoint>Thefinishedreversetranscriptionreactioncanbestoredat4°Covernightorleftat4°Cinthethermocycler.

10.2ExonucleaseItreatment

Wash1(centrifuge96-wellplatefor2minat1,250rcfwithbrakespeedsetto50%throughout)Washbeadsoncewith200µlofTE-SDSWashbeadstwicewith200µlofTE-TWWashbeadsoncewith200µlof10mMTris,pH8.0

PrepareexonucleaseImastermixasindicatedinthetablebelow.Theindicatedvolumesalreadyaccountfor10%deadvolume


ExonucleaseImastermix /reaction Mastermixfor#ofDrop-seq

runs

Component Storedat Volume Amount 1 2 3 4 5 6Nuclease-freewater RT 170µl to200µl 187 374 561 748 935 112210xExonucleaseIbuffer -20°C 20µl 1x 22 44 66 88 110 132ExonucleaseI -20°C 10µl 200U 11 22 33 44 55 66

Removesupernatantcarefully,andresuspendthebeadpelletin200µlofexonucleaseImastermixIncubatefor45minat37°CWash2(centrifuge96-wellplatefor2minat1,250rcfwithbrakespeedsetto50%throughout)

Washbeadsoncewith200µlofTE-SDSWashbeadstwicewith200µlofTE-TW

<Safestoppingpoint>SamplecanbestoredinTE-WEat4°C10.3SMARTPCRtoenrichcDNAlibrary

Pelletbeadsbycentrifugingfor2minat1,250rcfwiththebrakespeedsetto50%Removesupernatantandresuspendbeadpelletin400µlofnuclease-freewaterEstimatebeadconcentration

<Note>Drop-seqbeadshaveaveryhighsedimentationrate,makesuretomixthoroughly

immediatelybeforepipettingbead-containingsolution.PreparePCRtubestrip,containing7.5µlof6xgelloadingdyeperwell(onewellperbeadsample)<Note>ThegelloadingdyehelpstoevenlydistributebeadsinthecountingchamberlateronMixbeadsolutionbypipettingupanddownwithaP200Quicklyremove37.5µlofbeadsuspension,mixwiththe6xgelloadingdyeinthetubestripandload20µlintoeachofthetwochambersofadisposableIncytoC-ChipwithFuchs-RosenthalgridCount>4squaresforbothchambersandcalculatetheaveragenumberofbeadspersquare

Calculatetotalamountofbeadspertubeas[(Averagenumberofbeadspersquare)x5,000x1.2]*0.3625=xbeadsinoriginaltubewhere5,000isthechambervolumefactor,1.2thedilutionfactor(gelloadingdye)andmultiplicationby0.3625convertstothetotalamountofbeadsleftintheoriginaltube(nowcontaining362.5µl)

Dilutebeadsolutionto220beadsperµlasfollowsPelletbeadsbycentrifugingfor2minat1,250rcfwiththebrakespeedsetto50%Removesupernatantandresuspendin(x/220)=yµlofnuclease-freewater,wherexisthetotalnumberofbeadsinthepellet,ascalculatedabove

CalculatenumberofPCRreactionsasy/(20+1)=nPCRreactionswhereyisthevolumeusedfordilutingbeads,20isthevolumefor4,400beadsusedinasinglePCRreaction,and+1accountsfor5%deadvolumeduringpipetting

PrepareSMARTPCRmastermixasindicatedinthetablebelow

SMARTPCRmastermix /reactionComponent Storedat Volume AmountNuclease-freewater RT 4.6µl to50µl100µMSMARTPCRprimer -20°C 0.4µl 0.8µM2xKapaHiFiHotStartReadyMix -20°C 25µl 1x


Aliquot20µlofbeadsinto96-wellplates<Note>Thiscorrespondsto4,400beadsorapproximately220cellsperPCR

Add30µlofSMARTPCRmastermixperwellIncubatePCRasfollows

95°Cfor3min4x { 98°Cfor20sec, 65°Cfor45sec, 72°Cfor3min }10x { 98°Cfor20sec,

67°Cfor20sec, 72°Cfor3min }

72°Cfor5minStorageat4°C

<Note>Dependingonthecelltype,thenumberofPCRcyclesmightneedoptimization.Thesecycle

numbershaveworkedwelloncelllines.

10.4Purification,qualitycontrolandpoolingofSMART-PCRenrichedcDNA

PurificationofSMARTPCRproducts<Note>Performwithmultichannelpipettesin96-wellformatAdd30µlofroomtemperatureAMPureXPbeadstoeachPCRreaction(0.6xbeadtosampleratio)Incubatefor10minatroomtemperaturePlaceonmagnetfor5minWashtwicewith80%ethanol(5secperwash)Leavetodryfor5minElutein10µlnuclease-freewater

Run1µlofrandomlyselectedwellsonaBioanalyzerHighSensitivityChipaccordingtothemanufacturer’sinstructions.ThisshouldconfirmhighqualityofthecDNA(peakmaximum>1000bpwithoutbandsthatwouldindicatedegradation),andloworundetectableamountsofprimerdimerformation(peakat150bp).

<Troubleshooting>Ifapronouncedprimerdimerpeakispresentat150bp,repeatthe0.6xAMPureXP

beadclean-uponemoretime.Usually,thisremovesthepeakentirely.

MeasurethedsDNAconcentrationforallwellsusingaQubitHSDNAassayBasedonthesemeasurements,poolallSMARTPCRsforaparticularDrop-seqruninequalamountsMeasurethedsDNAconcentrationofeachpoolusingaQubitHSDNAassay,thisconcentrationis

neededfortheNexteraXTlibrarypreparation.<Safestoppingpoint>EnrichedcDNApoolscanbekeptat4°C,orstoredlong-termat-20°C

11.NexteraXTlibrarypreparation11.1Tagmentation

InaPCRtubestrip,prepare1ngofpooledcDNAperDrop-seqruninatotalvolumeof5µlofnuclease-freewater

Preheatathermocyclerto55°CTransferNexteraXTNeutralizationBufferfromstorageat4°CtoroomtemperaturePrepareNexteraXTtagmentationmastermixasindicatedinthetable,thevolumesarealreadyaccounting

for10%deadvolume


NexteraXTtagmentationmastermix Mastermixfor#ofDrop-seqrunsComponent Storedat Volume 1 2 3 4 5 6NexteraTDbuffer -20°C 10µl 11 22 33 44 55 55Amplicontagmentenzyme -20°C 5µl 5.5 11 16.5 22 27.5 27.5

Add15µloftagmentationmastermixperwell,andpipetteupanddowntomixIncubateat55°Cfor5minImmediatelyadd5µlofNeutralizationBuffertostopthereactionIncubateatroomtemperaturefor5min11.2NexteraXTPCRenrichment

PrepareNexteraXTPCRmastermixasindicatedinthetablebelow.Thevolumesprovidedalreadyaccountfor10%deadvolumelostduringpipetting.

NexteraXTPCRmastermix /reactionMastermixfor#ofDrop-seqrunsComponent Storedat Volume 1 2 3 4 5 6

Nuclease-freewater RT 8µl 8.8 17.6 26.4 35.2 44 44NexteraPCRmix -20°C 15µl 16.5 33 49.5 66 82.5 82.510µMNew-P5-SMARTPCRhybridoligo -20°C 1µl 1.1 2.2 3.3 4.4 5.5 5.5

10µMNexteraN70Xoligo -20°C 1µl 1.1 2.2 3.3 4.4 5.5 5.5Add25µlofNexteraXTPCRmastermixtothetagmentationreaction,bringingthevolumeto50µlIncubatePCR

95°Cfor30sec10x { 95°Cfor10sec, 55°Cfor30sec, 72°Cfor30sec }72°Cfor5minStorageat4°C

11.3Purificationofthefinallibrary

Clean-up1(performwithmultichannelpipettesinthePCRtubestrip)Add30µlofroomtemperatureAMPureXPbeadstoeachPCRreaction(0.6xbeadtosampleratio)Incubatefor10minatroomtemperaturePlaceonmagnetfor5minWashtwicewith80%ethanol(5secperwash)Leavetodryfor5minElutein32µlofnuclease-freewater,thentransfer30µltoafreshPCRtubestrip

Clean-up2<Note>Bothclean-upstepsarerequiredtoremovePCRprimersthatmightinterferewithsequencing

Add30µlofroomtemperatureAMPureXPbeadstoeachPCRreaction(1.0xbeadtosampleratio)Incubatefor10minatroomtemperaturePlaceonmagnetfor5min


Washtwicewith80%ethanol(5secperwash)Leavetodryfor5minElutein10µlofnuclease-freewater,thentransferto1.5mltubesforlong-termstorageat-20°C

12.Libraryqualitycontrolandnext-generationsequencing12.1QualitycontrolanddilutionoftheCROP-seqlibrary

Run1µlofthefinallibraryonaBioanalyzerHighSensitivityDNAchipaccordingtothemanufacturer’sinstructions.<Note>ToincreasethelikelihoodthatfragmentscontainthegRNAsequence,CROP-sequsesa

slightlylargerlibraryfragmentsize.ThisisachievedbyaddingmorecDNAasinputfortheNextera

XTtagmentationreaction.Sometimes,thefinallibrarymightappearabitskewedtowardslarger

fragments,whichdoesnothaveanegativeimpactonthesequencingresults.EstimatetheaveragesizeofthelibraryfromtheBioanalyzerprofile.

<Note>Forsymmetricallibrariesthepeakmaximumcanbeused;forskewedlibrariestheaverage

fragmentsizeisestimatedbasedonthefragmentdistribution.Weusuallyuse575bp.

MeasuretheconcentrationofdsDNAinaQubitDNAHSassay,accordingtothestandardprotocolCalculatethemolarityofyourlibraryas

c(dsDNA)[ng/ul]/(averagesize[bp]x660g/mol)x1,000,000=molarityoflibrary[nM]where660g/molisthemolecularweightofaDNAbasepairand1,000,000isaconversionfactor

DiluteyourlibrarywithEB-0.01%Tweentoreachamolarityof4nM

12.2Next-generationsequencingonIlluminaHiSeq3000/4000instruments

Drop-seqCustomRead1PrimerisspikedintotheHP10primersolution,locatedincolumn11ofthecBotReagentPlateat1μM final concentration.High sequencecomplexityneeded foroptimalbasecallingperformance is achieved by adding 20-30% PhiX as spike-in. Cluster generation and Read 1 primerhybridizationshouldbecompletedusingtheIlluminacBotprotocol‘HiSeq_3000_4000_HD_Exclusion_Amp_v1.0’.12.3Next-generationsequencingonIlluminaNextSeq500instruments

LibrariesaresequencedonanIlluminaNextSeq500instrumentusingthe75cycleHighOutputv2kit.Load1.8pMlibrarywithoutPhiXspike-inandprovideDrop-seqCustomRead1Primerat0.3μMinposition7ofthereagentcartridge.Setthereadconfigurationto20bases(CustomRead1),8bases(Index1)and64bases(Read2).

<Note>WhensettinguptherunintheIlluminaBasespace,thereadlengthofonly20basesfor

CustomRead1resultsinawarning.Weignorethiswarningandproceedwiththerun,becauseany

additionalbaseswouldjustreadfromtheA-tail,thusreducingsequencingquality.However,when

sequencingwiththissetup,thedatahastobedemultiplexedmanually.


PartC:Expectedresultsforsingle-cellRNA-seqbyDrop-seq

TypicalBioanalyzerprofilesforSMART-PCRenrichedcDNAandfinalNexteraXTlibraries

MeasuredcelldoubletrateforthedescribedDrop-seqsetup,measuredinaspeciesmixingexperimentonHEK293T(human)and3T3(mouse)celllines.Themixingratiowas1:1.

TablesummarizingsequencingresultsforatypicalNextSeq500run


Appendix:Materials

ReagentsrequiredforPartA(Pooled,amplification-freecloningofgRNAlibraries)

Reagent(alphabeticalorder) Supplier Cat.No. CommentonuseAbsoluteethanol Merck 64-17-5

Agar,powder Sigma A5054-250G

Agarose Sigma A9539-100G

AgilentBioanalyzerHighSensitivityDNAchip Agilent 5067-4626

AMPureXPbeads BeckmanCoulter A63880

Bioassaydish,245x245x25mm Sigma D4803-1CSUsedforgrowingbacteria

electroporatedwithgRNAlibraries

BsmBIrestrictionenzyme NEB R0580S

Carbenicillin,5g Roth 6344.2

CROPseq-Guide-Puro Addgene 86708

EBbuffer Qiagen 1014609

Electroporationcuvettes,1mm BioRad 1652089

Endo-FreePlasmidMegakit Qiagen 12381Forpreparingplasmidcontaining

entiregRNAlibraries

Enduraelectrocompetentcells Lucigen 60242-2

Gel-cuttingtips Biostep 99-90-306

GeneRuler1kbDNALadder ThermoFisherScientific SM0311

LB(Miller),powder Sigma L3522-1KG

Membranefilter Merck VMWP04700FordesaltingGibsonassembly

reactions

NEBuilderHiFiDNAassemblymastermix NEB E2621S

PlasmidPlusMidikit,100preparations Qiagen 12945ForCROPseq-Guide-Puroand

lentiviruspackagingplasmids

Q5HotStartHigh-Fidelity2xMasterMix NEB M0494L

QubitdsDNAHSAssaykit ThermoFisherScientific Q32854

Round-bottomtubes Falcon 352051

SapIrestrictionenzyme NEB R0569S

SNAPUV-FreeGelPurificationkit Invitrogen 45-0105

SOCmedium NEB B9020S

Sodiumacetate,3M,pH5.5 Ambion AM9740

SphIrestrictionenzyme NEB R0182S

SYBRGreen;10,000xconcentrateinDMSO ThermoFisherScientific S7563

TAEbuffer,50x Invitrogen 24710-030

Tween Sigma P7949-500ML


ReagentsrequiredforPartB(LentivirusproductionandcellcultureforCROP-seqscreens)

Reagent(alphabeticalorder) Supplier Cat.No. CommentonuseBlasticidin Invivogen ant-bl-5

Crystalviolet Sigma 61135-25GTostainresistantcoloniesfortiterestimation

DMEM Gibco 10569010DPBS,1x Gibco 14190-094FCS Sigma N/AHEK293TcelllineLipofectamine3000transfectionreagents Invitrogen L3000015Opti-MEMI Gibco 51985-034Penicillin-streptomycin;5,000U/ml ThermoFisherScientific 15070063

pMD2.G Addgene 122593rdgenerationlentiviralpackagingsystem

pMDLg/pRRE Addgene 122513rdgenerationlentiviralpackagingsystem

Polybrene Sigma H9268-5GToenhancecontactbetweenvirusandcells

pRSV-Rev Addgene 122533rdgenerationlentiviralpackagingsystem

Puromycin ThermoFisherScientific A1113803Sodiumpyruvate Gibco 11360-070

Syringefilter,0.45µm VWR 514-8021Forremovingcellsfromlentiviruspreparations


ReagentsrequiredforPartC(Single-cellRNA-seqbasedonDrop-seq)

Modifiedfrom:Macoskoetal.2015andadditionalprotocols(http://mccarrolllab.com/dropseq/)

Reagent(alphabeticalorder) Supplier Cat.No. CommentonuseBarcodedBeadSeqB ChemGenes MACOSKO-2011-10 Drop-seqbeadsBovineserumalbumin(BSA) Sigma A7030-50GC-ChipDisposableHemacytometers,Incyto,Pkg.of50 FisherScientific 22-600-102 ForestimatingbeadnumberCellstrainer,100µm(forbeads) VWR 21008-950Cellstrainer,40µm(forcells) VWR 21008-949dNTPsolution,10mM ThermoFisherScientific R0192DropletGenerationOilforEvaGreen,QX200 BioRad 1864006 Drop-seqoilDTT Sigma 646563-10x.5ML AddondayofrunEDTA,0.5M,pH8.0 ThermoFisherScientific AM92606ExonucleaseI NEB M0293LFicollPM-400 Sigma F5415-50MLKAPAHiFiHotStartReadyMix KAPA KK2602MaximaHMinusReverseTranscriptase,4x50ul ThermoFisherScientific EP0753Microfluidicdevices FlowJem seeordersheetfordetailsN-Lauroylsarcosinesodiumsaltsolution,20% Sigma L7414NeedleBDMicrolance3(brown) BD 300300NeedleNeoject25G0.5x16mm Hartenstein EU23NexteraXTkitfor24samples Illumina FC-131-1024Perfluoroctanol(1H,1H,2H,2H-Perfluoro-1-octanol) Sigma 370533-25G FordropletbreakagePolyethylenetubing,ID0.38mm Scicominc BB31695-PE/2RecombinantRnaseinhibitor Takara 2313ASSCbuffer,6x Promega V4261 FordropletbreakageSyringeBDDiscarditII10ml BD 309110SyringeBDDiscarditII20ml BD 300296SyringeBDLuer-Lok1ml BD 309628SyringeBDLuer-Lok3ml BD 309657Syringefilterunit,Millex-GV,0.22µm,PVDF MerckMillipore SLGV033RSTris,2M,pH7.5 Sigma T2944-100MLTween-20 Sigma P7949-500ML

Oligonucleotide(orderofuse) Supplier Sequence Comment

Templateswitchingoligonucleotide(TSO) Exiqon AAGCAGTGGTATCAACGCAGAGTGAATrGrGrGr…indicatesRNAbases,storeat-80°Cafterresuspending

SMARTPCRprimer Sigma AAGCAGTGGTATCAACGCAGAGT

New-P5-SMARTPCRhybridoligo SigmaAATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT*A*C

CustomRead1primer Sigma GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACCustomsequencingprimerforRead1onIlluminamachines

Solution(orderofuse) RecipeDrop-seqlysisbuffer For40ml(it'spossibletostorelargestockswithoutDTTat4°C):

20mlNuclease-freewater12ml20%FicollPM-400400ul20%N-Lauroylsarcosinesodiumsaltsolution(Sarcosyl),<Caution>toxic1.6ml0.5MEDTA4ml2MTrispH7.550ulpermlLysisbuffer1MDTT→addthisonthedayoftheexperiment

TE-SDS 10mMTris,pH8.01mMEDTA0.5%SDS

TE-TW 10mMTris,pH8.01mMEDTA0.01%Tween

Documents

CRISPR droplet sequencing (CROP-seq) protocol (version ...CRISPR droplet sequencing (CROP-seq) Page 3 of 20 3.2 Gibson Assembly Dilute gRNA-encoding ssDNA oligos to 100 µM with EB