CAJALNeuroscienceTrainingCourseSinglecellprofilingandanalysisinneuroscienceBordeauxSchoolofNeuroscience–June15toJuly3,2020

TheCAJALAdvancedNeuroscienceTrainingProgramme www.cajal-training.org

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During this course, students will get hands-on experience with entire single-celltranscriptomicprojectsfromtissuedissociationtopublishablefigures.Wewillteachtheuseofdifferentkindsofstartingmaterial,threedifferentsequencingtechniques,howtotreattherawsequencingdataandamultitudeofanalyticaltools.Afterattendingthecourse,ourgoal is that the students should be able to go back to their institute and have enoughknowledge and understanding to initiate well-designed single-cell sequencing projects totackleimportantquestionsinNeuroscience.Experimentalprojects(Firstblock)Project1:VisualizationandquantificationofcellularcomplexityoftheCA1regionofthemousebrainInstructor:XiaoyanQian(CartanaAB/StockholmUniversity,SciLifeLab,Sweden)Understanding the function of a tissue requires knowing the spatial organization of itsconstituentcelltypes.Theuseofsingle-cellRNAsequencing(scRNASeq)inbraintissuehasrevealedthegenome-wideexpressionpatternsthatdefinemany,closelyrelatedneuronaltypes. But this method cannot reveal their spatial arrangement nor provide an accuratecountof cells. To revealboth locationandnumberof thedifferent cell types researchershavetoperformtranscriptomicdirectlyintissue.For this project the students will learn the method of probabilistic cell typing by in situsequencing (pciSeq)(Qian et al., Nature Methods, 2019). This is a high through-put andrelatively cheap approach that leverages previous scRNA-seq classification to identify celltypes using multiplexedin situRNA detection. Hippocampal area CA1 is a part of thetrisynaptic circuit that has been central to neuroscience. In this area, in addition to twopyramidalpopulationsatleast23classesofGABAergicneuronshavebeenproposedtodatebasedonelectrophysiologyandconnectivityinrodents.ItwasrecentlyshownthatdistinctscRNAseqprofilesofCA1cellscanbeattributedtothese23functionalclassesinmice.ThestudentswillusepciSeqtoidentifythelocationofandquantifytherelativenumbersofeachoftheseclassesinsectionsofmouseCA1braintissue.Maintechniques:pciSeqmethodAutomatedwide-fieldmicroscopyProject 2: Understanding cellularmaturation during the development of the embryonicnervoussystembywhole-cellRNAseqInstructor:ChristianMayer(MaxPlanckInstituteofNeurobiology,Germany)Oneofthegreatestmysteriesishow,duringdevelopment,anorganascomplicatedasthebraincanarisefromazygote.Single-cellRNAsequencingrevealsRNAabundancewhichisapowerfulindicatorofthestateofindividualcells.Todescribethedevelopmentalprocess,itisnotenoughtoonlyunderstandthefinalcellularcomplexityofthebrainbuthowcelltypesfromdifferent developmental stages are related to each other. Since an RNA-sequencingapproach captures only a static snapshot at a point in time it poses a challenge for theanalysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Tocircumventthisprojectonecaneitherperformlineage-tracingwhichcanbeusedtodirectly

CAJALNeuroscienceTrainingCourseSinglecellprofilingandanalysisinneuroscienceBordeauxSchoolofNeuroscience–June15toJuly3,2020

TheCAJALAdvancedNeuroscienceTrainingProgramme www.cajal-training.org

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inferrelationshipsbetweencellsorcomputationalmethods.Inthisprojectthestudentswillperformsingle-cellRNAsequencingfromthedevelopingbrainattwodifferenttimepoints.They will then use different computational methods, including RNA velocity (a timederivativeofthegeneexpressionstate–whichcanbedirectlyestimatedbydistinguishingbetweenun-splicedandsplicedmRNAsincommonsingle-cellRNAsequencingprotocols),toelucidatethetemporalrelationbetweendifferentstatesofdifferentiation.Maintechniques:EmbryonicbraindissectionanddissociationWholecellsingle-cellRNAsequencingusing10xsystemProject3: GABAergicneuronaldiversityacrossdifferentforebrainstructuresInstructor: Ana Munoz-Manchado (University of Cadiz, Spain / Karolinska Institutet,Sweden)GABAergicinhibitoryneurons,althoughlowinnumber(around20%ofneurons),arecrucialforshapingtheoutputofacircuit.Theyarealsoverydiverseintermsofshape,connectivityandmolecularmake-upwithalargeliteraturedescribingthisheterogeneitydatingbacktoCajal intheendofthe19thcentury.IntheisocortexandhippocampustheGABAergiccellshave been shown to be quite constant in terms of cell types but other telencephalicstructures are lesswell studied. This project aims to describe GABAergic diversity in twodifferent structures allowing for comparison between them and previously describedstructures. Tomaintain a roughly equal coverage of this population (as a comparison toexcitatory cells) to detect the different subtypes it is often necessary to enrich forGABAergiccellspriortosingle-cellsequencing.ThestudentswillthuslearnhowtouseFACSincombinationwithatransgenicmouselinetoenrichforGABAergiccellspriortosingle-cellRNAseq.Maintechniques:AdultbraindissectionanddissociationFACSisolationoffluorescentlylabeledcellsWholecellsingle-cellRNAsequencingusing10xsystemProject4: SinglewholecellsanalysisofanAlzheimer’sdiseasemousemodelInstructor:FatimaMemic(KarolinskaInstitutet,Sweden)Theability to investigate transcriptional changes in individual cells holds greatpromise inthestudyofdisease.Alreadynow,afewpapers,includingoneonAlzheimer’sdiseasehaveappearedwhere this hasbeenused to studydisorders inhumans (Mathys et al.,Nature,2019).Thisapproachopensupnotonlythestudyofdiseasesbutcanalsobeusedasawayto understand which aspects of a disorder are recapitulated by mouse models. For thisproject the students will performwhole-cell single-cell RNA-sequencing of the prefrontalcortexofthe3xTg-ADmousemodelforAlzheimer’sdisease(Oddoetal.,Neuron,2003)toinvestigatesimilaritiesanddifferencestothefindingsfromhumansubjects.Maintechniques:AdultbraindissectionanddissociationWholecellsingle-cellRNAsequencingusing10xsystem

CAJALNeuroscienceTrainingCourseSinglecellprofilingandanalysisinneuroscienceBordeauxSchoolofNeuroscience–June15toJuly3,2020

TheCAJALAdvancedNeuroscienceTrainingProgramme www.cajal-training.org

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Project5:SinglenucleianalysisofanAlzheimer’sdiseasemousemodelInstructor:DannyKitsberg(ELSC,JerusalemUniversity,Israel)Theability to investigate transcriptional changes in individual cells holds greatpromise inthestudyofdisease.Alreadynow,afewpapers,includingoneonAlzheimer’sdiseasehaveappearedwhere this hasbeenused to studydisorders inhumans (Mathys et al.,Nature,2019).Thisapproachopensupnotonlythestudyofdiseasebutcanalsobeusedasawaytounderstandwhichaspectsofadisorderarerecapitulatedbymousemodels.Forstudiesof human subjects, however, we are currently limited to single-nuclei sequencing and arecent studyhave suggested thatusing this techniquemightbias the findings in termsofwhichmRNAsarecaptured(Skeneetal.,NatureGenetics,2018).Inthisprojectthestudentswilllearnhowtoisolatenucleifromfrozen(mouse)tissueofthe3xTg-ADmousemodelforAlzheimer’s disease (Oddo et al., Neuron, 2003), enrich for neuronal populations usingantibody-assisted FACS isolation and use this to compare to data from human and fromwhole-cellsingle-cellRNA-sequencing.Maintechniques:IsolationofnucleifromfrozentissueFACSisolationSingle-nucleiRNAsequencingusing10xsystemProject 6: Single nuclei analysis of GABAergic cells in the dorsal horn in a Chronic painmodelInstructor:DannyKitsberg(ELSC,JerusalemUniversity,Israel)Chronicpathologicalpainisamajorburdenformostsocieties,withhighhuman,socialandeconomic costs. The development of chronic pain is largely thought to result from amalfunction of the spinal neuron network, and more particularly from a dysfunction ofspinal GABAergic neurons located in superficial laminae of the spinal cord. Although thedevelopmentofRNAseqapproacheshasenabledathoroughclassificationofbothexcitatoryandinhibitoryneuronsinthespinalcord,littleisknownaboutthephysiologic,morphologicandtranscriptomicchangesaccompanyingthedevelopmentofchronicpainsymptoms.Forstudiesonhumansubjects,wearecurrentlylimitedtosingle-nucleisequencingandarecentstudy have suggested that using this techniquemight bias the findings in termsofwhichmRNAs are captured (Skene et al., Nature Genetics, 2018). The students will useneuropathic and control animals to investigate transcriptomic changes in FACS sortedneuronalnucleifromspinalsuperficialdorsalhorn.Thiswillgiveinsightstotranscriptomicchangesinbothexcitatoryandinhibitorycells.Maintechniques:IsolationofnucleifromfrozenspinalcordtissueFACSisolationSingle-nucleiRNAsequencingusing10xsystem

CAJALNeuroscienceTrainingCourseSinglecellprofilingandanalysisinneuroscienceBordeauxSchoolofNeuroscience–June15toJuly3,2020

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Project7:SinglewholecellsanalysisofGABAergiccellsinthedorsalhorninaChronicpainmodelInstructor:MartinHäring(UniversityofVienna,Austria)Chronicpathologicalpainisamajorburdenformostsocieties,withhighhuman,socialandeconomic costs. The development of chronic pain is largely thought to result from amalfunction of the spinal neuron network, and more particularly from a dysfunction ofspinal GABAergic neurons located in superficial laminae of the spinal cord. Although thedevelopmentofRNAseqapproacheshasenabledathoroughclassificationofbothexcitatoryandinhibitoryneuronsinthespinalcord,littleisknownaboutthephysiologic,morphologicandtranscriptomicchangesaccompanyingthedevelopmentofchronicpainsymptoms.ThestudentswilluseneuropathicandcontrolanimalstoinvestigatetranscriptomicchangesinFACS sortedGABAergicneurons fromspinal superficialdorsalhorn.Relevanceof thesechanges with respect to previously characterized alterations of morphological andelectrophysiologicalpropertieswillbeinvestigated.Maintechniques:Spinalcord/dorsalhorndissectionFACSisolationWholecellsingle-cellRNAsequencingusing10xsystemProject8:Largescalesingle-cellRNA-sequencingofbraintissueusingSPLiT-SeqInstructor:SongCheng(UniversityofSanDiego,US)The students will learn how to apply SPLiT-Seq (SplitPool Ligation-based Transcriptomesequencing)which isa techniquebywhichonecanperform large-scalesinglecell (nuclei)sequencing without the need for advanced equipment. We will be open for student’ssuggestionwithregardstowhichbrainareatoinvestigate.See this webpage for methodological explanation and more information:https://sites.google.com/uw.edu/splitseq.Maintechniques:SPLiT-seqComputationalprojects(Secondblock)In the second, computational part, all students will learn state-of-the-art approaches forcomputational analysis and interpretationof single-cell RNA-seqdata. The instructorswillexplain the statistical and computational underpinnings of different methods, and thenproceed with practical examples and walkthroughs. In addition to going through thecommonexercises,each individualgroupwill thenapply therelevantapproachestocarryouttheanalysisofthedatatheyhavegeneratedinthefirstpartofthecourse.The students will learn how to carry out the initial alignment and demultiplexing steps,calculate and interpret various quality controlmetrics. Installation and setup of differentanalysisframeworks,includingScanpy,Seurat,scVIandConoswillbecovered.Thestudentswill examinedifferent normalization anddimensionality reduction techniques,monitoringfor common technical sideeffects,exploredifferentoptions for clustering subpopulations

CAJALNeuroscienceTrainingCourseSinglecellprofilingandanalysisinneuroscienceBordeauxSchoolofNeuroscience–June15toJuly3,2020

TheCAJALAdvancedNeuroscienceTrainingProgramme www.cajal-training.org

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andtestingfordifferentialexpression.Thecoursewillcoverintegrationandcomparisonofmultiple datasets, techniques for transferring and automating cell annotations. For theanalysis of dynamical processes, studentswill use trajectory fitting techniques aswell asRNA velocity estimations. Many of these techniques will be directly applicable to theexperimentaldatathatwillbegeneratedinthefirstpartofthecourse,whichwillbedoneunderthesupervisionoftheinstructors.Instructors:ViktorPetukhov(UniversityofCopenhagen,Denmark)MalteLücken(ICB,HelmholtzZentrumMünchen,Germany)GioeleLaManno(SchoolofLifeSciences,EPFL,Lausanne,Switzerland)RomainLopez(EECS,UCBerkeley)MarkusHilscher(SciLifeLabs,Stockholm,Sweden)LisaBast(KarolinskaInstitutet,Sweden)