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Methods in Molecular Biology 2584 Raff aele A. Calogero Vladimir Benes Editors Single Cell Transcriptomics Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Single Cell Transcriptomics Methods and Protocols Edited by Raffaele A. Calogero Molecular Biotechnology Center, University of Torino, Torino, Italy Vladimir Benes European Molecular Biology Laboratory, Heidelberg, Germany Editors RaffaeleA.Calogero VladimirBenes MolecularBiotechnologyCenter EuropeanMolecularBiologyLaboratory UniversityofTorino Heidelberg,Germany Torino,Italy ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2755-6 ISBN978-1-0716-2756-3 (eBook) https://doi.org/10.1007/978-1-0716-2756-3 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Thefirstarticleonbonafidesingle-celltranscriptomics(scRNAseq)[1]isnow13yearsold. Undisputedly, this article has paved the way to establishing a flourishing methodology focusingonanalysesoftranscripts’repertoireinindividualcells.Withtheincreasinginterest in this type of analyses and realization of their potential, caveats and limitations of this technology not only in the laboratory but also in analysis of generated data have become apparent too. A “re-purposing” of the MMLV reverse transcriptase originally used for 0 mapping of 5-end [2] termed “SMART” (Switching Mechanism at 5’end of RNA Tran- script)hasstartedthescRNA-seqmethodinearnest[3].Essentially,allfull-lengthaswellas 0 3-end focused single-cell transcriptomic data are generated with the application of this approach. However, fairly soon, it has also turned out that there are very few biological systems withfreelyavailablesinglecellsdirectlyamenabletotheprotocol.Therefore,ithasbeenand stillisnecessarytodevelopprotocolsforfixation,dissociation,andseparationofindividual cells,stepsparticularlycriticalforsuccessofanysingle-celltranscriptomicexperiment.That hasbecomeevenmorerelevantwiththeintroductionofmicrofluidicdroplet-basedsystems [4]. With individual contributions we wish to bring readers’ attention to options available, includingthoseenablingexplorationofplantandbacterialsinglecells.Protocolsincludedin thistomearebynomeansonlypossibilities;everydayweareseeingneweffortstooptimize current methods or provide entirely new protocols, and this trend will undoubtedly continue. Ashappenedwithotherhigh-throughputtechnologies,single-celltranscriptomicsalso require bioinformatics to make the data suitable to further interpretation. Single-cell RNA-seq bioinformatics is today a very active area and provides a wide variety of software toworkwithandinterpretscRNA-seqdata.Thisbookwasdevelopedtoprovidehands-on examples of both basic and advance bioinformatics methodologies. It includes a set of chapters describing, on one side, basic analytical procedures as single-cell data QC, dimensionality reduction, clustering, and cluster-specific features selection. On the other side,thereareasetofchaptersdedicatedtotheanalysisofadvancemethodologiesasRNA velocity,multi-modaldataintegration,andsingle-cellRNAediting. Takentogether,theoverallstructureofthisbookprovidesanup-to-dateinstrumentto facilitateaninformedselectionofsingle-cellwetandbioinformaticsprotocolsbasedonthe researcherexperimentrequirements. Torino,Italy RaffaeleA.Calogero Heidelberg,Germany VladimirBenes References 1. TangF,BarbacioruC,WangY,NordmanE,LeeC,XuNetal(2009)mRNA-Seqwhole-transcrip- tomeanalysisofasinglecell.NatMethods6(5):377–382.https://doi.org/10.1038/nmeth.1315 v vi Preface 2. Chenchik A, Diachenko L, Moqadam F, Tarabykin V, Lukyanov S, Siebert PD (1996) Full-length cDNAcloninganddeterminationofmRNA50and30endsbyamplificationofadaptor-ligatedcDNA. Biotechniques21(3):526–534.https://doi.org/10.2144/96213pf02 3. Picelli S, Bjo¨rklund ÅK, Faridani OR, Sagasser S, Winberg G, Sandberg R (2013) Smart-seq2 for sensitivefull-lengthtranscriptomeprofilinginsinglecells.NatMethods10(11):1096–1098.https:// doi.org/10.1038/nmeth.2639 4. MacoskoEZ,BasuA,SatijaR,NemeshJ,ShekharK,GoldmanM,TiroshI,BialasAR,KamitakiN, Martersteck EM, Trombetta JJ, Weitz DA, Sanes JR, Shalek AK, Regev A, McCarroll SA (2015) Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161(5):1202–1214.https://doi.org/10.1016/j.cell.2015.05.002 Contents Preface ..................................................................... v Contributors................................................................. ix 1 GuidanceonProcessingthe10xGenomicsSingleCellGeneExpression Assay...... ........ ....... ........ ....... ....... ........ ....... ........ 1 KatharinaDanielski 2 BDRhapsody™Single-CellAnalysisSystemWorkflow:FromSample toMultimodalSingle-CellSequencingData......... ........ ....... ........ 29 JannesUlbrich,VadirLopez-Salmeron,andIanGerrard 3 ProfilingTranscriptionalHeterogeneitywithSeq-WellS3:ALow-Cost, Portable,High-FidelityPlatformforMassivelyParallel Single-CellRNA-Seq....... ........ ....... ...... ....... .. .... ... ........ 57 RileyS.Drake,MartinArreolaVillanueva,MikeVilme, DanielaD.Russo,AndrewNavia,J.ChristopherLove, andAlexK.Shalek 4 AMATQ-seq-BasedProtocolforSingle-CellRNA-seqinBacteria.... ........ 105 ChristinaHomberger,Antoine-EmmanuelSaliba, andJörgVogel 5 Full-LengthSingle-CellRNA-SequencingwithFLASH-seq.......... ........ 123 VincentHahautandSimonePicelli 6 PlantSingle-Cell/NucleusRNA-seqWorkflow ...... ........ ....... ........ 165 SandraThibivilliers,AndrewFarmer,SusanSchroeder, andMarcLibault 7 EnsuringQualityCellInputforSingleCellSequencingExperiments byViabilityandSingletEnrichmentUsingCellSorting ...... ....... ..... ... 183 MaltePaulsen 8 TissueRNAIntegrityinVisiumSpatialProtocol(FreshFrozenSamples)...... 191 FedericaAntico,MartaGai,andMaddalenaArigoni 9 Single-CellRNAseqDataQCandPreprocessing .... ........ ....... ........ 205 MartinaOliveroandRaffaeleA.Calogero 10 Single-CellRNAseqComplexityReduction......... ........ ....... ........ 217 FrancescaCorderoandRaffaeleA.Calogero 11 Functional-Feature-BasedDataReductionUsingSparselyConnected Autoencoders ...... ....... ........ ....... ....... ........ .. ..... .. ...... 231 LucaAlessandriandRaffaeleA.Calogero 12 Single-CellRNAseqClustering...... ....... ....... ........ ....... ........ 241 MarcoBeccutiandRaffaeleA.Calogero 13 IdentifyingGeneMarkersAssociatedwithCellSubpopulations....... ........ 251 MariaLuisaRattoandLucaAlessandri vii viii Contents 14 AGuidetoTrajectoryInferenceandRNAVelocity .......... ....... ........ 269 PhilippWeiler,KoenVandenBerge,KellyStreet,andSimoneTiberi 15 IntegrationofscATAC-SeqwithscRNA-SeqData........... ....... ........ 293 IvanBerestandAndreaTangherloni 16 Using“Galaxy-rCASC”:APublicGalaxyInstanceforSingle-Cell RNA-SeqDataAnalysis..... ........ ....... ....... ........ ....... ........ 311 PietroMandreoli,LucaAlessandri,RaffaeleA.Calogero, MarcoAntonioTangaro,andFedericoZambelli 17 BringingCellSubpopulationDiscoveryonaCloud-HPCUsingrCASC andStreamFlow .... ....... .. ...... ....... ....... ........ ....... ........ 337 SandroG.Contaldo,LucaAlessandri,IacopoColonnelli, MarcoBeccuti,andMarcoAldinucci 18 ProfilingRNAEditinginSingleCells ....... ....... ........ ....... ........ 347 AdrianoFonzino,GrazianoPesole,andErnestoPicardi 19 PracticalConsiderationsforComplexTissueDissociation forSingle-CellTranscriptomics...... ....... ....... ........ ....... ........ 371 StephanieT.Pohl,MariaLlamazares Prada,ElisaEspinet, andRenataJurkowska Index ...................................................................... 389 Contributors MARCOALDINUCCI • DepartmentofComputerScience,UniversityofTurin,Turin,Italy LUCA ALESSANDRI • MolecularBiotechnologyCenter,UniversityofTorino,Turin,Italy; MolecularBiotechnologyCenter,UniversityofTurin,Turin,Italy FEDERICA ANTICO • MolecularBiotechnologyCenter,UniversityofTorino,Torino,Italy MADDALENAARIGONI • MolecularBiotechnologyCenter,UniversityofTorino,Torino,Italy MARCOBECCUTI • DepartmentofComputerScience,UniversityofTorino,Turin,Italy; DepartmentofComputerScience,UniversityofTurin,Turin,Italy IVANBEREST • StructuralandComputationalBiologyUnit,EuropeanMolecularBiology Laboratory,Heidelberg,Germany;DataScience,PharmaResearchandDevelopment, RocheInnovationCenterZurich,Zurich,Switzerland RAFFAELEA.CALOGERO • MolecularBiotechnologyCenter,UniversityofTorino,Torino,Italy IACOPOCOLONNELLI • DepartmentofComputerScience,UniversityofTurin,Turin,Italy SANDROG.CONTALDO • DepartmentofComputerScience,UniversityofTurin,Turin,Italy FRANCESCACORDERO • DepartmentofComputerScience,UniversityofTorino,Turin,Italy KATHARINADANIELSKI • 10xGenomicsB.V,Leiden,TheNetherlands RILEYS.DRAKE • InstituteforMedicalEngineeringandScience(IMES),Massachusetts InstituteofTechnology,Cambridge,MA,USA;DepartmentofChemistry,Massachusetts InstituteofTechnology,Cambridge,MA,USA;KochInstituteforIntegrativeCancer Research,MassachusettsInstituteofTechnology,Cambridge,MA,USA;TheRagon InstituteofMassachusettsGeneralHospital,MassachusettsInstituteofTechnologyand HarvardUniversity,Cambridge,MA,USA;BroadInstituteofMITandHarvard, Cambridge,MA,USA ANDREWFARMER • NationalCenter forGenomeResources,SantaFe,NM,USA ADRIANO FONZINO • DepartmentofBiosciences,BiotechnologyandBiopharmaceutics, UniversityofBari“A.Moro”,Bari,Italy MARTAGAI • MolecularBiotechnologyCenter,UniversityofTorino,Torino,Italy IANGERRARD • BDBiosciencesGmbH,Heidelberg,Germany VINCENTHAHAUT • InstituteofMolecularandClinicalOphthalmologyBasel(IOB),Basel, Switzerland CHRISTINA HOMBERGER • UniversityofWu¨rzburg,Wu¨rzburg,Germany RENATAJURKOWSKA • SchoolofBiosciences,CardiffUniversity,Cardiff,Wales,UK MARCLIBAULT • Center forPlantScienceInnovation,UniversityofNebraska-Lincoln, BeadleCenter,Lincoln,NE,USA;DepartmentofAgronomyandHorticulture,University ofNebraska-Lincoln,BeadleCenter,Lincoln,NE,USA VADIRLOPEZ-SALMERON • BDBiosciencesGmbH,Heidelberg,Germany J.CHRISTOPHERLOVE • KochInstituteforIntegrativeCancerResearch,Massachusetts InstituteofTechnology,Cambridge,MA,USA;TheRagonInstituteofMassachusetts GeneralHospital,MassachusettsInstituteofTechnologyandHarvardUniversity, Cambridge,MA,USA;BroadInstituteofMITandHarvard,Cambridge,MA,USA; DepartmentofChemicalEngineering,MassachusettsInstituteofTechnology,Cambridge, MA,USA ix