Single Cell Transcriptomics: Methods and Protocols

394 Pages·2022·10.482 MB·English

by  Raffaele A. Calogero, Vladimir Benes

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
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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
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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,
andJ¨orgVogel
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