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Methods in Molecular Biology 2611 Georgi K. Marinov William J. Greenleaf Editors Chromatin Accessibility 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. Chromatin Accessibility Methods and Protocols Edited by Georgi K. Marinov and William J. Greenleaf Stanford University, Stanford, CA, USA Editors GeorgiK.Marinov WilliamJ.Greenleaf StanfordUniversity StanfordUniversity Stanford,CA,USA Stanford,CA,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2898-0 ISBN978-1-0716-2899-7 (eBook) https://doi.org/10.1007/978-1-0716-2899-7 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Chapter 6 is licensed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/).Forfurtherdetailsseelicenseinformationinthechapter. 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 The genomic distribution patterns of chromatin accessibility and its dynamics are key featuresoftheregulationofgeneexpressionandmanyotheraspectsofchromatinbiology. The genomes of eukaryotes are usually packaged by nucleosomal particles, which have a generally strong inhibitory effect on transcription and on the occupancy of DNA by regulatory proteins. It is typically active cis-regulatory regions (cREs) in the genome that arecharacterizedbydepletednucleosomaloccupancyandincreasedchromatinaccessibility, which has in turn proven to be a highly useful property enabling the identification of candidate cREs as well as the tracking of their activity across cell types and conditions as accessibleDNAcanbepreferentiallyenzymaticallyorchemicallylabeledinnumerousways. Technological advances in the labeling and readout of accessible DNA have played a major roleindrivingforwardour understandingofchromatinandregulatorybiologyover the last few decades. The last 15 years have seen a particularly dramatic explosion in the varietyandpowerofapproachesforstudyingchromatinaccessibility,drivenbytwosequen- tialtechnologicalrevolutions:first,thedevelopmentofhigh-throughputsequencinginthe mid-2000s,andthentheadventofsingle-cellgenomicsinthe2010s.Thecurrentbookaims to provide a comprehensive resource covering the existing and state-of-the-art tools in the field. We have divided the protocols in the book into several sections, depending on the different aspects of chromatin accessibility that they measure and/or approaches that they take.Inthefirstsection,bulk-cellmethodsforprofilingchromatinaccessibilityandnucleo- somepositioningthatrelyonenzymaticcleavageofaccessibleDNAandproduceinforma- tionaboutrelativeaccessibilityarecovered.Thesecondsectionisdedicatedtomethodsthat usesingle-moleculeandenzymaticapproachestosolvingtheproblemofmappingabsolute occupancy/accessibility. The third section covers the wide array of emerging tools for mappingDNAaccessibilityandnucleosomepositioning insingle cells,aswellasanumber of single-cell multiomics methods that simultaneously measure chromatin accessibility and other features of the cell, such as the transcriptome, the methylome, and protein markers. More recently, imaging-based methods for visualizing accessible chromatin in its nuclear context have emerged; these are included in the fourth section. The final section features computational methods for the processing and analysis of chromatin accessibility datasets. This book will serve as an extensive and useful reference for researchers studying different facetsofchromatinaccessibilityinawidevarietyofbiologicalcontexts. Stanford,CA,USA GeorgiK.Marinov WilliamJ.Greenleaf v Contents Preface ..................................................................... v Contributors................................................................. ix PART I BULK CLEAVAGE-BASED METHODS 1 Genome-WideMappingofActiveRegulatoryElementsUsing ATAC-seq.......... ....... ........ ....... ....... ........ ....... ........ 3 GeorgiK.Marinov,ZoharShipony,AnshulKundaje, andWilliamJ.Greenleaf 2 MappingNucleosomeLocationUsingFS-Seq....... ........ ....... ........ 21 BarryMilavetz,BrennaHanson,KincaidRowbotham, andJacobHaugen 3 UniversalNicE-Seq:ASimpleandQuickMethodforAccessible ChromatinDetectioninFixedCells......... ....... ........ ....... ...... .. 39 HangGyeongChin,UdayakumarS.Vishnu,ZhiyiSun, V.K.ChaithanyaPonnaluri,GuoqiangZhang,Shuang-yongXu, TouatiBenoukraf,PalomaCejas,GeorgeSpracklin, Pierre-OlivierEste`ve,HenryW.Long,andSriharsaPradhan 4 MeasuringInaccessibleChromatinGenome-WideUsingProtect-seq ......... 53 GeorgeSpracklin,LiyanYang,SriharsaPradhan,andJobDekker 5 DeterminationoftheChromatinOpennessinBacterialGenomes..... ........ 63 MahmoudM.Al-BassamandKarstenZengler 6 ProfilingChromatinAccessibilityonReplicatedDNAwith repli-ATAC-Seq..... ....... ........ ....... ....... ........ ....... ........ 71 KathleenR.Stewart-MorganandAnjaGroth 7 AnalysisofChromatinInteractionandAccessibilitybyTrac-Looping.......... 85 ShuaiLiu,QingsongTang,andKejiZhao PART II METHODS FOR MEASURING THE ABSOLUTE LEVELS OF OCCUPANCY/ACCESSIBILITY 8 Single-MoleculeMappingofChromatinAccessibilityUsing NOMe-seq/dSMF...... ... ........ ....... ....... ........ ....... ........ 101 MichaelaHinks,GeorgiK.Marinov,AnshulKundaje, LacramioaraBintu,andWilliamJ.Greenleaf 9 ORE-Seq:Genome-WideAbsoluteOccupancyMeasurement byRestrictionEnzymeAccessibilities........ ....... ........ ....... ........ 121 ElisaOberbeckmann,MichaelRolandWolff,NilsKrietenstein, MarkHeron,AndreaSchmid,TobiasStraub,UlrichGerland, andPhilippKorber vii viii Contents PART III METHODS FOR PROFILING CHROMATIN ACCESSIBILITY AT THE SINGLE-CELL LEVEL 10 Single-CellJointProfilingofOpenChromatinandTranscriptome byPaired-Seq ...... ....... ........ ....... ....... ........ ....... ........ 155 ChenxuZhu,ZhaoningWang,andBingRen 11 SimultaneousSingle-CellProfilingoftheTranscriptomeand AccessibleChromatinUsingSHARE-seq.... ....... ........ ....... ........ 187 SamuelH.Kim,GeorgiK.Marinov,S.TansuBagdatli,SoonIlHigashino, ZoharShipony,AnshulKundaje,andWilliamJ.Greenleaf 12 SimultaneousMeasurementofDNAMethylationandNucleosome OccupancyinSingleCellsUsingscNOMe-Seq...... ........ ....... .... .... 231 MichaelWasneyandSebastianPott 13 MassivelyParallelProfilingofAccessibleChromatinandProteins withASAP-Seq..... ....... ........ ....... ....... ........ ....... ........ 249 EleniMimitou,PeterSmibert,andCalebA.Lareau 14 ConcomitantSequencingofAccessibleChromatinandMitochondrial GenomesinSingleCellsUsingmtscATAC-Seq...... ........ ....... ........ 269 LeifS.LudwigandCalebA.Lareau PART IV IMAGING METHODS FOR VISUALIZATION OF ACCESSIBLE DNA 15 ATAC-See:ATn5Transposase-MediatedAssayforDetectionof ChromatinAccessibilitywithImaging....... ....... ........ ... .... ..... ... 285 YonglongDang,RamPrakashYadav,andXingqiChen 16 NicE-viewSeq:AnIntegrativeVisualizationandGenomicsMethod toDetectAccessibleChromatininFixedCells....... ........ ....... ........ 293 Pierre-OlivierEste`ve,UdayakumarS.Vishnu, HangGyeongChin,andSriharsaPradhan PART V COMPUTATIONAL ANALYSIS OF CHROMATIN ACCESSIBILITY DATASETS 17 ATAC-seqDataProcessing.......... ....... ....... ........ ....... ........ 305 DanielS.Kim 18 DeepLearningonChromatinAccessibility.......... ........ ....... ... ..... 325 DanielS.Kim Index .......... ........ ....... ........ ....... ....... ........ ....... ........ 335 Contributors MAHMOUDM.AL-BASSAM • DepartmentofPediatrics,UniversityofCalifornia,SanDiego, LaJolla,CA,USA S.TANSU BAGDATLI • DepartmentofGenetics,StanfordUniversity,Stanford,CA,USA TOUATIBENOUKRAF • FacultyofMedicine,CraigL.DobbinGeneticsResearchCentre, MemorialUniversityofNewfoundland,St.John’s,NL,Canada LACRAMIOARABINTU • DepartmentofBioengineering,StanfordUniversity,Stanford,CA, USA PALOMA CEJAS • Center forFunctionalCancerEpigenetics,Dana-FarberCancerInstitute, Boston,MA,USA XINGQI CHEN • DepartmentofImmunology,GeneticsandPathology,UppsalaUniversity, Uppsala,Sweden HANGGYEONGCHIN • NewEnglandBiolabsInc.,Ipswich,MA,USA;GenomeBiology Division,NewEnglandBiolabs,Inc.,Ipswich,MA,USA YONGLONGDANG • DepartmentofImmunology,GeneticsandPathology,Uppsala University,Uppsala,Sweden JOBDEKKER • PrograminSystemsBiology,UniversityofMassachusettsMedicalSchool, Worcester,MA,USA;HowardHughesMedicalInstitute,Boston,MA,USA PIERRE-OLIVIER ESTE`VE • NewEnglandBiolabsInc.,Ipswich,MA,USA;GenomeBiology Division,NewEnglandBiolabs,Inc.,Ipswich,MA,USA ULRICHGERLAND • DepartmentofPhysics,TechnicalUniversityofMunich,Garching, Germany WILLIAMJ.GREENLEAF • DepartmentofGenetics,StanfordUniversity,Stanford,CA,USA; Center forPersonalDynamicRegulomes,StanfordUniversity,Stanford,CA,USA; DepartmentofAppliedPhysics,StanfordUniversity,Stanford,CA,USA;ChanZuckerberg Biohub,SanFrancisco,CA,USA ANJAGROTH • NovoNordiskFoundationCenter forProteinResearch(CPR),Facultyof HealthandMedicalSciences,UniversityofCopenhagen,Copenhagen,Denmark;Biotech ResearchandInnovationCentre(BRIC),FacultyofHealthandMedicalSciences, UniversityofCopenhagen,Copenhagen,Denmark BRENNA HANSON • DepartmentofBiomedicalSciences,SchoolofMedicine,Universityof NorthDakota,GrandForks,ND,USA JACOBHAUGEN • DepartmentofBiomedicalSciences,SchoolofMedicine,UniversityofNorth Dakota,GrandForks,ND,USA MARKHERON • QuantitativeandComputationalBiology,MaxPlanckInstitutefor BiophysicalChemistry,Go¨ttingen,Germany;GeneCenter,FacultyofChemistryand Pharmacy,Ludwig-Maximilians-Universita€tMu¨nchen,Munich,Germany SOONILHIGASHINO • DepartmentofGenetics,StanfordUniversity,Stanford,CA,USA MICHAELAHINKS • DepartmentofGenetics,StanfordUniversity,Stanford,CA,USA DANIELS.KIM • BiomedicalInformaticsProgram,StanfordUniversitySchoolofMedicine, Stanford,CA,USA SAMUELH.KIM • CancerBiologyProgram,SchoolofMedicine,StanfordUniversity, Stanford,CA,USA;MedicalScientistTrainingProgram,StanfordUniversity,Stanford, CA,USA ix x Contributors PHILIPPKORBER • BiomedicalCenter(BMC),DivisionofMolecularBiology,Facultyof Medicine,LMUMunich,Martinsried,Germany NILSKRIETENSTEIN • NovoNordiskFoundationCenter forProteinResearch,Universityof Copenhagen,Copenhagen,Denmark ANSHULKUNDAJE • DepartmentofGenetics,StanfordUniversity,Stanford,CA,USA; DepartmentofComputerScience,StanfordUniversity,Stanford,CA,USA CALEB A.LAREAU • DepartmentsofGeneticsandPathology,StanfordUniversity,Stanford, CA,USA SHUAILIU • LaboratoryofEpigenomeBiology,SystemsBiologyCenter,Divisionof IntramuralResearch,NationalHeart,LungandBloodInstitute,NationalInstitutesof Health,Bethesda,MD,USA HENRYW.LONG • Center forFunctionalCancerEpigenetics,Dana-FarberCancer Institute,Boston,MA,USA LEIF S.LUDWIG • BerlinInstituteofHealthatChariteÚniversita€tsmedizinBerlin,Berlin, Germany;Max-Delbru¨ck-Center forMolecularMedicineintheHelmholtzAssociation, BerlinInstituteforMedicalSystemsBiology,Berlin,Germany GEORGIK.MARINOV • DepartmentofGenetics,StanfordUniversity,Stanford,CA,USA BARRYMILAVETZ • DepartmentofBiomedicalSciences,SchoolofMedicine,Universityof NorthDakota,GrandForks,ND,USA ELENI MIMITOU • Immunai,NewYork,NY,USA ELISAOBERBECKMANN • DepartmentofMolecularBiology,MaxPlanckInstitutefor MultidisciplinarySciences,Go¨ttingen,Germany V.K.CHAITHANYAPONNALURI • NewEnglandBiolabsInc.,Ipswich,MA,USA SEBASTIANPOTT • UniversityofChicago,Chicago,IL,USA SRIHARSAPRADHAN • NewEnglandBiolabsInc.,Ipswich,MA,USA;GenomeBiology Division,NewEnglandBiolabs,Inc.,Ipswich,MA,USA BING REN • LudwigInstituteforCancerResearch,LaJolla,CA,USA;Departmentof CellularandMolecularMedicine,UniversityofCaliforniaSanDiego,SchoolofMedicine, LaJolla,CA,USA;CenterforEpigenomics,InstituteofGenomicMedicine,MooresCancer Center,UniversityofCaliforniaSanDiego,SchoolofMedicine,LaJolla,California,USA KINCAIDROWBOTHAM • DepartmentofBiomedicalSciences,SchoolofMedicine,Universityof NorthDakota,GrandForks,ND,USA ANDREA SCHMID • BiomedicalCenter(BMC),DivisionofMolecularBiology,Facultyof Medicine,LMUMunich,Martinsried,Germany ZOHARSHIPONY • DepartmentofGenetics,StanfordUniversity,Stanford,CA,USA PETERSMIBERT • 10xGenomics,Pleasanton,CA,USA GEORGESPRACKLIN • PrograminSystemsBiology,UniversityofMassachusettsMedicalSchool, Worcester,MA,USA KATHLEENR.STEWART-MORGAN • NovoNordiskFoundationCenter forProteinResearch (CPR),FacultyofHealthandMedicalSciences,UniversityofCopenhagen,Copenhagen, Denmark;BiotechResearchandInnovationCentre(BRIC),FacultyofHealthand MedicalSciences,UniversityofCopenhagen,Copenhagen,Denmark TOBIASSTRAUB • CoreFacilityBioinformatics,BiomedicalCenter(BMC),Facultyof Medicine,LMUMunich,Martinsried,Germany ZHIYISUN • NewEnglandBiolabsInc.,Ipswich,MA,USA QINGSONGTANG • LaboratoryofEpigenomeBiology,SystemsBiologyCenter,Divisionof IntramuralResearch,NationalHeart,LungandBloodInstitute,NationalInstitutesof Health,Bethesda,MD,USA