Polycomb Group Proteins: Methods and Protocols

Preface
References
Contents
Contributors
Chapter 1: Analyzing the Genome-Wide Distribution of Histone Marks by CUT&Tag in Drosophila Embryos
1 Introduction
2 Material and Reagents
2.1 Embryo Collection
2.2 Protein-DNA Cross-Link
2.3 General Chemicals and Solutions
2.4 Nuclear Isolation, Binding to Concanavalin Beads and Primary Antibody Binding
2.5 Secondary Antibody Binding and Tagmentation
2.6 DNA Purification
2.7 Library Preparation
2.8 Antibodies
2.9 Primers
3 Methods
3.1 Embryo Collection
3.2 Embryo Collection for PFA Fixation (3 h)
3.3 Fixation (0.5-1 h)
3.4 Alternative: Native Collection of Embryos (0.5-1 h)
3.5 Nuclei Isolation (0.5 h)
3.6 Preparation of Concanavalin A Beads and Binding of the Nuclei (0.5 h)
3.7 Binding of the Primary Antibody (2.5 h - O/N)
3.8 Binding of the Secondary Antibody (1.5 h)
3.9 Binding of the Protein A-Tn5 (1.5 h)
3.10 Chromatin Digestion, Clean- Up, and Lambda-DNA Spike-In (1.5 h)
3.11 Library PCR and Indexing (1.5 h)
3.12 Clean-up of the Libraries and Quality Control (1.5 h)
3.13 Bioinformatic Analysis of CUT&Tag Data
4 Notes
References
Chapter 2: Chromatin Preparation and Chromatin Immunoprecipitation from Drosophila Heads
1 Introduction
2 Materials
2.1 Material for Heads Isolation (Fig. 2a-c)
2.2 Material for Chromatin Preparation and Immunoprecipitation
2.3 Buffers
3 Methods
3.1 Head Isolation from Adult Flies
3.1.1 Fly Collection
3.1.2 Head Removal
3.2 Chromatin Preparation
3.2.1 Homogenization
3.2.2 Formaldehyde Cross-Linking
3.2.3 Nuclei Isolation
3.2.4 Chromatin Shearing
3.2.5 DNA Fragment Size Check
3.3 Chromatin Immunoprecipitation
3.3.1 Binding Antibodies to Magnetic Beads
3.3.2 Immunoprecipitation and Washes
3.3.3 Elution and Cross-Link Reversal
3.3.4 DNA Purification and Recovery
3.3.5 Quality Controls
3.4 Library Preparation and Next-Generation Sequencing
3.4.1 Library Preparation for Illumina Sequencing
3.4.2 Sequencing
4 Notes
References
Untitled
Chapter 3: Detecting Cell Compartment-Specific PRC2-RNA Interactions via UV-RIP
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffers
2.3 Equipment
2.4 Consumables
3 Methods
3.1 Cells Seeding and UV Cross-Linking
3.2 Cell Fractionation and Sonication
3.3 Immunoprecipitation and RNA Isolation
4 Notes
References
Chapter 4: Chromosome Conformation Capture Followed by Genome-Wide Sequencing (Hi-C) in Drosophila Embryos
1 Introduction
2 Material and Reagents
2.1 Embryo Collection
2.2 Protein-DNA Cross-Link
2.3 Nuclear Isolation
2.4 Chromatin Digestion
2.5 Biotinylation of the Chromatin
2.6 Ligation of the Chromatin
2.7 Chromatin Digestion
2.8 DNA Purification
2.9 Biotin Removal
2.10 DNA Shearing
2.11 Biotin Pull Down
2.12 Library Preparation
3 Methods
3.1 Embryo Collection (3 h)
3.2 Fixation (0.5-1 h)
3.3 Cell Lysis and Nuclei Isolation (0.5 h)
3.4 Chromatin Digestion (3.5 h)
3.5 Biotinylation of the Chromatin (1.5 h)
3.6 Ligation of the Chromatin (4.5 h)
3.7 Chromatin Digestion and Reverse Cross-Linking (0.5 h and Overnight)
3.8 DNA Clean-up (1 h)
3.9 Biotin Removal (1 h)
3.10 DNA Shearing (0.5 h)
3.11 Biotin Pull Down (1 h)
3.12 Library Preparation (3 h)
3.13 DNA Fragment Size Control and Deep-Sequencing Analysis
4 Data Analysis
4.1 Hi-C Matrices Building
4.2 TAD and Compartment Calling
5 Notes
References
Chapter 5: Physics-Based Polymer Models to Probe Chromosome Structure in Single Molecules
1 Introduction
2 Materials
3 Methods
3.1 The SBS Polymer Model of Chromosome Folding
3.2 Validation of the Model Against Independent Super-resolution Single-Cell Imaging Experiments
3.3 Single-Polymer Structures Are Used to Benchmark in Silico Hi-C, GAM, and SPRITE Methods
4 Notes
References
Chapter 6: Co-Immunoprecipitation (Co-Ip) in Mammalian Cells
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffers
2.3 Equipments
3 Methods
3.1 Lysis and Denaturation of Cellular Extracts
3.2 Preclearing of the Antibody with the Beads
3.3 Incubation of the Lysate with the Resin/Coupled Antibody
3.4 Washes and Elution of Immune-Precipitated Sample
4 Notes
References
Chapter 7: Site-Directed Mutagenesis Protocol to Determine the Role of Amino Acid Residues in Polycomb Group (PcG) Protein Fun...
1 Introduction
2 Materials
2.1 General Reagents
2.2 PCR Reagents
2.3 DpnI Treatment Reagents
2.4 Transformation Reagents
2.5 Colony PCR Reagents (Optional)
2.6 Mutant Purification Reagents
2.7 Equipment
3 Methods
3.1 Design of Primers
3.2 PCR Amplification
3.3 Dpn I Digestion
3.4 DNA Transformation
3.5 Colony PCR (Optional)
3.6 Mutant Purification and Confirmation
4 Notes
References
Chapter 8: Isolation of Chromatin Proteins by Genome Capture
1 Introduction
2 Materials
2.1 Media Components and Reagents
2.1.1 Solutions
2.1.2 Buffers
3 Methods
3.1 EdU Labeling, Cell Fixation, and Permeabilization
3.2 Biotinylation and Chromatin Fragmentation
3.3 Pre-block of Beads
3.4 Genome Capture and Protein Elution
4 Notes
References
Chapter 9: Dynamic Interactome of PRC2-EZH1 Complex Using Tandem-Affinity Purification and Quantitative Mass Spectrometry
1 Introduction
2 Materials
2.1 Cells and Media
2.2 Buffer Components and Reagents
2.2.1 Cell Lysis, Sonication and Immunoprecipitation (IP)
2.2.2 IPs and Peptide Elution
2.2.3 Silver Staining and Immunoblot Assay
2.2.4 Filter Aided Sample Preparation (FASP) and Desalting Using Zip-Tip
2.2.5 Mass Spectrometry
2.2.6 Antibodies
2.3 Equipment
2.4 Disposables
2.5 Solutions
2.5.1 C2C12 Growth Medium (GM)
2.5.2 C2C12 Differentiation Medium (DM)
2.5.3 Solutions
3 Methods
3.1 Culture of C2C12 Cells
3.2 Differentiation of C2C12 Myoblast to Myotube
3.3 Nuclear Extracts Preparation
3.4 Tandem Affinity Purification
3.5 Silver Staining and WB
3.6 Solution Digestion (FASP)
3.7 Desalting with Zip-Tip
3.8 LC-MS Data Acquisition Using DIA Mode
3.9 DIA Data Analysis Using directDIA Approach in Spectronaut Software (see Note 7)
4 Notes
References
Chapter 10: Replication Timing of Gene Loci in Different Cell Cycle Phases
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffer and Solutions
2.3 Equipment
3 Methods
3.1 BrdU Labelling
3.2 Cell Cycle Fractionation by Flow Cytometry
3.3 Isolating BrdU Labelled DNA
3.3.1 Phenol Chloroform Extraction
3.3.2 Ethanol Precipitation
3.3.3 Sonication
3.3.4 Immunoprecipitation
3.3.5 Proteinase K and Purification
3.3.6 Quantitative PCR
4 Notes
References
Chapter 11: Polycomb Bodies Detection in Murine Fibromuscular Stroma from Skin, Skeletal Muscles, and Aortic Tissues
1 Introduction
2 Materials
2.1 Mice
2.2 Reagents
2.3 Solutions
2.4 Equipment and Supplies
3 Methods
3.1 Equipment Preparation
3.2 Tissue Collection I TIMING: 30 Min Per Mouse
3.3 Enzymatical Digestion and Preparation of a Single-Cell Suspension I TIMING: Murine Dermis-4 h
3.4 Enzymatical Digestion and Preparation of a Single-Cell Suspension I TIMING: Murine Aorta-2 h
3.5 Enzymatical Digestion and Preparation of a Single-Cell Suspension I TIMING: Murine Muscle-2 h
3.6 Antibody Staining and Cell Sorting|TIMING 90 min + 45 min Sorting (Per Mouse)
3.7 Immunofluorescence
4 Notes
References
Chapter 12: Segmentation, 3D Reconstruction, and Analysis of PcG Proteins in Fluorescence Microscopy Images in Different Cell ...
1 Introduction
2 Materials
3 Methods
3.1 2D Segmentation
3.2 3D Reconstruction
3.2.1 PcG Random Shuffling
3.3 Nuclei/PcG Features Analysis
4 Notes
References
Chapter 13: STORM Microscopy and Cluster Analysis for PcG Studies
1 Introduction
2 Materials
3 Methods
3.1 Cell Plating
3.2 Cell Fixation
3.3 Cell Labeling
3.4 STORM Imaging
3.5 Imaging Analysis
4 Notes
References
Chapter 14: Quantitative Analysis of PcG-Associated Condensates by Stochastic Optical Reconstruction Microscopy (STORM)
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffers
2.3 Equipment
2.4 Software and Bioinformatic Tools
3 Methods
3.1 Cell Plating and Fixation
3.2 Immunofluorescence
3.3 Data Acquisition
3.4 Image Reconstruction
3.5 Quantitative Clustering Analysis in STORM
3.6 Global Characterization-Based Methods
3.7 Segmentation-Based Methods
4 Notes
References
Chapter 15: Immunoelectron Microscopy Methods
1 Introduction
2 Materials
2.1 Buffers and Fixatives
2.2 Dehydration Media and Resin
2.3 Reagents for Immunocytochemistry (ICC)
2.4 TEM Sections Staining Media
3 Methods
3.1 Fixation
3.2 Dehydration and Resin Embedding
3.3 Sectioning
3.4 Immunocytochemistry (ICC)
3.5 Section Staining
4 Notes
References
Chapter 16: Differentiation of hPSCs to Study PRC2 Role in Cell-Fate Specification and Neurodevelopment
1 Introduction
1.1 Neural Induction
1.2 Patterning and Specification
1.3 Terminal Differentiation
2 Materials
2.1 Cell Culture Reagents
2.2 Cells
2.3 Cell Culture Medium
2.4 Plastic and Glass Consumable Items
2.5 Equipment
3 Method
3.1 Preparation of CultrexTM and Geltrex-Coated Plates
3.2 hES and iPS Cell Maintenance
3.3 Differentiation Protocol
3.3.1 Day-2: Cell Preparation and Plating
3.3.2 Day 0-Day 4: Neural Induction
3.3.3 Day 4: Split in Droplets
3.3.4 Day 5-Day 11-12: Patterning and Specification
3.3.5 Day 11-12: Replating
3.3.6 Day 25-Day 50: Terminal Differentiation
4 Notes
References
Chapter 17: Analysis of Polycomb Epigenetic Marks in HeLa Spheroids
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Acid Wash Coverslip Cleaning and Poly-Lysine (PLL) Coating
2.3 Preparation of Low-Adhesion Tissue Culture Dishes
2.4 Immunofluorescence
2.5 Working Solutions
3 Methods
3.1 Glass Coverslips Cleaning (Acid Wash)
3.2 Preparation of Poly-Lysine-Coated Coverslips
3.3 Preparation of Low-Adhesion-Coated Dishes
3.4 Spheroid Generation
3.5 Immunofluorescence
4 Notes
References
Chapter 18: Establishment and Maintenance of Human CRC-Derived Organoids for PcG Studies
1 Introduction
2 Materials
2.1 Human Colorectal Cancer (CRC) Patients Derived Organoids (PDOs) Culture
2.2 Chromatin Immunoprecipitation on CRC PDOs
3 Methods
3.1 Human Colorectal Cancer Crypts Isolation from Post-surgical CRC Resections
3.2 Human CRC PDO Maintenance and Fixation for Chromatin Immunoprecipitation (ChIP) Experiments
3.3 ChIP on Human CRC PDOs
3.3.1 Day 1: Lysis and Sonication
3.3.2 Day 2: DNA Immunocomplexes Capture
3.3.3 Day 3: Chromatin DNA Purification
4 Notes
References
Index