TET Proteins and DNA Demethylation: Methods and Protocols (Methods in Molecular Biology, 2272)

Preface
Contents
Contributors
Part I: Detection and Quantification of DNA Methylation Turnover
	Chapter 1: Reduced Bisulfite Sequencing: Quantitative Base-Resolution Sequencing of 5-Formylcytosine
		1 Introduction
		2 Materials
			2.1 General Reagents
			2.2 Reduction and Bisulfite Reagents
			2.3 DNA Reagents
		3 Methods
			3.1 Generation of C, 5mC, 5hmC, and 5fC Control Double Stranded DNA (dsDNA)
			3.2 Preparation of Genomic  DNA
			3.3 General DNA Reduction and Bisulfite Treatment
			3.4 HPLC Analysis of Single Stranded DNA (ssDNA) Controls
			3.5 Sanger Sequencing Analysis of dsDNA Controls
			3.6 Illumina MiSeq Analysis of dsDNA Controls
			3.7 Sequencing Analysis of Genomic DNA
		4 Notes
		References
	Chapter 2: Aba-Seq: High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Equipment
		3 Methods
			3.1 Glucosylation of 5-Hydroxymethylcytosine
			3.2 Phenol Extraction and Ethanol Precipitation
			3.3 AbaSI Digestion
			3.4 Ligation of Biotinylated Adaptor (P1b)
			3.5 Cleanup of P1b Ligated DNA and Removal of Excessive Adaptors
			3.6 DNA Fragmentation
			3.7 Capture of Biotinylated DNA
			3.8 End Repair Reaction to Generate Blunt-Ended Fragments
			3.9 A-Tailing of End-Repaired DNA
			3.10 Ligation of Annealed P2 Adaptor to dA-Tailed DNA
			3.11 PCR Amplification
			3.12 Cleanup of PCR Products
			3.13 Determination of Library Quality, Quantity, and Size Using the Bioanalyzer Platform
			3.14 Library Sequencing
			3.15 Bioinformatics Analysis
		4 Notes
		References
	Chapter 3: Estimating Global Methylation and Erasure Using Low-Coverage Whole-Genome Bisulfite Sequencing (WGBS)
		1 Introduction
		2 Materials
			2.1 WGBS Library Construction Using PBAT
			2.2 Computing Requirements
		3 Methods
			3.1 WGBS Library Preparation by PBAT
				3.1.1 Cell Lysis and DNA Purification
					Option 1: BOMB Genomic DNA Extraction
					Option 2: Using Cell Lysate Directly for Bisulfite Conversion
				3.1.2 Bisulfite Conversion
				3.1.3 First Stand Synthesis
				3.1.4 Exonuclease Treatment and Purification
				3.1.5 Biotin Capture
				3.1.6 Second Strand Synthesis
				3.1.7 PCR Amplification and Analysis
				3.1.8 Pooling and Library Purification
			3.2 Theoretical Prediction of Margin of Error
			3.3 Empirical Bootstrap Sampling
		4 Notes
		References
	Chapter 4: ELISA-Based Quantitation of Global 5hmC Levels
		1 Introduction
		2 Materials
			2.1 Sample and Standards Preparation
			2.2 Reagents for Passive Adsorption DNA ELISA
			2.3 Plasticware and Equipment
		3 Methods
			3.1 General Considerations and Planning
			3.2 Preparation of DNA Samples and Standards
				3.2.1 DNA Samples
				3.2.2 5hmC Standard
				3.2.3 DNA Loading Standard (See Note 13)
			3.3 Denaturation of  DNA
			3.4 Passive Adsorption of DNA Samples and Standards
			3.5 Blocking
			3.6 Incubation with Primary Antibody
			3.7 Incubation with HRP-Conjugated Secondary Antibody
			3.8 Enzymatic Reaction and Data Acquisition
			3.9 Analysis
		4 Notes
		References
	Chapter 5: Avidin-Biotin ELISA-Based Detection of 5hmC
		1 Introduction
		2 Materials
			2.1 Assay Buffers and Components
			2.2 DNA and Antibodies
			2.3 Plasticware and Equipment
		3 Methods
			3.1 General Considerations and Planning
			3.2 Plate Coating
			3.3 DNA Binding
			3.4 Blocking
			3.5 Incubation with a 5hmC Reader (See Note 9)
			3.6 Incubation with Primary Antibody (See Note 10)
			3.7 Incubation with HRP-Conjugated Secondary Antibody
			3.8 Enzymatic Reaction and Data Acquisition
			3.9 Analysis
		4 Notes
		References
	Chapter 6: Quantification of DNA Methylation and Its Oxidized Derivatives Using LC-MS
		1 Introduction
		2 Materials
			2.1 Nucleosides
			2.2 UHPLC-QQQ-MS Equipment
			2.3 UHPLC-QQQ-MS Setup
			2.4 Reagents and Equipment for Isolation of Genomic DNA (gDNA) of Mammalian Cells
			2.5 Enzymatic Digest of gDNA
		3 Methods
			3.1 External Calibration Curves
			3.2 Internal Calibration Curves
			3.3 gDNA Isolation
			3.4 Enzymatic Digest to Nucleoside Level and Sample Preparation
			3.5 UHPLC-QQQ-MS Measurement and Analysis
		4 Notes
		References
Part II: Bioinformatic Analysis of DNA Methylation and Hydroxymethylation
	Chapter 7: Distinguishing Active Versus Passive DNA Demethylation Using Illumina MethylationEPIC BeadChip Microarrays
		1 Introduction
		2 Materials
			2.1 Benchtop Assays to Detect DNA Modification Change
				2.1.1 Locus-Specific High-Resolution Melt (HRM) Analysis
					Equipment and Reagents
				2.1.2 Global Quantification of 5hmC
					ELISA-Based Assay
						Equipment and Reagents
					DNA Dot Blot
						Equipment and Reagents
			2.2 Modifications to Bisulfite Conversion Chemistry to Distinguish 5mC from 5hmC
				2.2.1 TET-Assisted Bisulfite (TAB) Array
					Equipment and Reagents
				2.2.2 Oxidative Bisulfite (OxBS) Array
					Equipment and Reagents
		3 Methods
			3.1 Benchtop Assays to Detect DNA Modification Change
				3.1.1 Locus-Specific High-Resolution Melt (HRM) Analysis
					General Procedure
				3.1.2 Global Quantification of 5hmC
					ELISA-Based Assay
						General Procedure
					DNA Dot Blot
						General Procedure
			3.2 Modifications to Bisulfite Conversion Chemistry to Distinguish 5mC from 5hmC
				3.2.1 Tet-Assisted Bisulfite (TAB) Array
					General Procedure
						Preparation of gDNA
						T4-β-glucosyltransferase (T4-βGT) Reaction
						TET Oxidation Treatments
						Bisulfite Sanger Sequencing of 5mC/5hmC Standards
				3.2.2 Oxidative Bisulfite (OxBS) Method
					General Procedure
			3.3 Bioinformatic Pipelines for EPIC Array Analysis
				3.3.1 TAB Array Processing
					General Pipeline
				3.3.2 OxBS Array Processing
					General Pipeline
				3.3.3 Comparison of TAB and OxBS Array Results
					General Pipeline
				3.3.4 Determining Active Versus Passive DNA Demethylation Using TAB and OxBS Arrays
					TAB Array Pipeline
					OxBS Array Pipeline
		4 Notes
		References
	Chapter 8: Bioinformatic Estimation of DNA Methylation and Hydroxymethylation Proportions
		1 Introduction
		2 Materials
		3 Methods
			3.1 TAB and BS Combination Using Array
				3.1.1 Loading the  Data
				3.1.2 Quality Control
				3.1.3 Preprocessing
				3.1.4 Filtering Probes
				3.1.5 Estimation of 5-hmC, 5-mC, and uC Proportions
			3.2 OxBS and BS Combination Using Array
				3.2.1 Loading the  Data
				3.2.2 Quality Control
				3.2.3 Preprocessing
				3.2.4 Filtering Probes
				3.2.5 Estimation of 5-hmC, 5-mC, and uC Proportions
		4 Notes
		References
	Chapter 9: TAB-seq and ACE-seq Data Processing for Genome-Wide DNA hydroxymethylation Profiling
		1 Introduction
		2 Software
		3 Methods
			3.1 Data Sources
			3.2 Data Download
			3.3 Quality Control
			3.4 Adapter Clipping
			3.5 Read Alignment
			3.6 5hmC Calling (CpG Context)
			3.7 Visualization of 5hmC  Data
			3.8 Sequencing Metrics
			3.9 Assessment of 5hmC Calling Accuracy
		4 Notes
		References
Part III: Precise Epigenome Manipulation Using TET Enzymes
	Chapter 10: Genomic Targeting of TET Activity for Targeted Demethylation Using CRISPR/Cas9
		1 Introduction
		2 Materials
			2.1 Plasmids
			2.2 Molecular Biology Reagents and Equipment for Cloning
			2.3 Mammalian Cell Culture Reagents
			2.4 Sorting
			2.5 DNA Methylation Analysis
		3 Methods
			3.1 Design sgRNAs
			3.2 Design Targeted Bisulfite PCR Primers
			3.3 Construction of dC9Sun-TET1mut
			3.4 Preparation of Double Stranded sgRNA Oligonucleotides
			3.5 Preparation of Vector and Cloning of sgRNA by Gibson Assembly
			3.6 Plasmid Sterilization
			3.7 Transfection of Plasmid into Mammalian Cells
			3.8 FACS and Bisulfite Conversion
			3.9 Targeted Bisulfite PCR
			3.10 Targeted Bisulfite PCR Pooling and Purification
			3.11 Phosphorylation and Adapter Ligation
			3.12 Library Quantitation and Sequencing
		4 Notes
		References
	Chapter 11: High-Fidelity CRISPR/Cas9-Based Gene-Specific Hydroxymethylation
		1 Introduction
		2 Materials
			2.1 Web Tools for Designing sgRNAs
			2.2 Construction of Customized All-in-One sgRNA Vectors
			2.3 Cell Culture and Plasmid DNA Transfection
			2.4 RNA Purification and First-Strand cDNA Synthesis
			2.5 Gene Reactivation Assessment
			2.6 Methylated DNA Detection by MeDIP-qPCR
		3 Methods
			3.1 Design CRISPR sgRNAs
			3.2 Preparation of pLenti-dHFCas9-TET3CD-Myc-DDK-sgRNA
			3.3 Annealing of Complementary sgRNA Oligos
			3.4 Ligation and Transformation of Competent Cells
			3.5 Midi-Prep DNA Purification and Positive Clone Screening
			3.6 Cell Transfection
			3.7 RNA Purification and cDNA Synthesis
			3.8 qRT-PCR
			3.9 Genomic DNA Purification and MeDIP-qPCR
		4 Notes
		References
Part IV: TET and DNA Hydroxymethylation Biochemistry
	Chapter 12: Identifying Protein-(Hydroxy)Methylated DNA Interactions Using Quantitative Interaction Proteomics
		1 Introduction
		2 Materials
			2.1 Nuclear Protein Extraction
			2.2 DNA Probe Immobilization on Streptavidin-Coated Sepharose Beads
			2.3 DNA Affinity Purification
			2.4 On StageTip Dimethyl Labeling
				2.4.1 Protein Elution and On-Bead Digestion to Peptides
				2.4.2 Desalting and Peptide Purification on Stop-and-Go-Extraction (Stage) Tips
				2.4.3 Dimethyl Labeling of Peptides
			2.5 Mass Spectrometry
			2.6 Data Analysis
		3 Method
			3.1 Nuclear Protein Extraction
				3.1.1 Extraction of Nuclear Proteins from Cells/Tissues
				3.1.2 Determination of Protein Concentration
			3.2 DNA Probe Immobilization on Beads
				3.2.1 Annealing of DNA Oligonucleotides
				3.2.2 Immobilization of DNA Probes on Streptavidin-Coated Sepharose Beads
			3.3 DNA Affinity Purification
			3.4 On StageTip Dimethyl Labeling of Peptides
				3.4.1 Protein Elution and On-Bead Trypsin Digestion
				3.4.2 StageTipping and Dimethyl Labeling
			3.5 Mass Spectrometry
				3.5.1 Elution of Peptides for Mass Spectrometry
				3.5.2 Mass Spectrometry
			3.6 Data Analysis
		4 Notes
		References
	Chapter 13: Purification of TET Proteins
		1 Introduction
		2 Materials
			2.1 TET Protein Purification from Insect Cells
			2.2 TET Protein Purification from Mammalian Cells
			2.3 5caC/5fC-Sequencing
		3 Methods
			3.1 TET2-Catalytic Domain Protein Purification from Insect Cells
				3.1.1 Baculovirus Production
				3.1.2 TET Protein Purification from Insect Cells
			3.2 TET2-FL Protein Purification from Mammalian Cells
			3.3 TET3-FL and TET3-S Protein Purification
			3.4 Sequencing Protocol for Detecting 5caC and 5fC
		4 Notes
		References
	Chapter 14: Uncovering Sequence-Specific Transcription Factors Interacting with TET2
		1 Introduction
		2 Materials
			2.1 Immunoprecipitation
			2.2 His-Tag Affinity Purification and In Vitro Pulldown
		3 Methods
			3.1 Immunoprecipitation
				3.1.1 Conjugating Antibodies to Magnetic Beads
				3.1.2 Cell Lysis
				3.1.3 Immunoprecipitation
				3.1.4 Washes
				3.1.5 Western Blot Analysis
			3.2 His-Tag Pulldown Assays
				3.2.1 Recombinant Transcription Factors Production in E. coli
				3.2.2 Recombinant TET2 Production in Insect Cells
				3.2.3 Transcription Factor-TET2 Pulldown
				3.2.4 Analysis of the His-Tag Pulldown by Western Blot
		4 Notes
		References
	Chapter 15: ChIP-Sequencing of TET Proteins
		1 Introduction
		2 Materials
			2.1 Chromatin Preparation and Immunoprecipitation
				2.1.1 General Materials
				2.1.2 Buffers
			2.2 Library Preparation
		3 Methods
			3.1 Chromatin Preparation and Immunoprecipitation
				3.1.1 Introduction of Endogenous FLAG Epitope tag on TET2 C-Terminus
				3.1.2 Cell Culture (See Note 2)
				3.1.3 Crosslinking (See Note 3)
				3.1.4 Cell Lysis and Sonication of Chromatin
				3.1.5 Preclearing
				3.1.6 Immunoprecipitation and Washing Steps (See Note 4)
				3.1.7 Elution and Decrosslinking of Immunoprecipitated Protein Complexes
			3.2 Quality Control and DNA Library Preparation
				3.2.1 Quality Control
				3.2.2 DNA Library Preparation for Illumina Sequencing
		4 Notes
		References
Part V: Assessing TET Protein Activity and Function
	Chapter 16: Harnessing Alternative Substrates to Probe TET Family Enzymes
		1 Introduction
		2 Materials
			2.1 PCR Syntheses of Modified DNA Substrates
			2.2 Fluorometric Endpoint Read-out of TET Activity with vC-Containing DNA Substrates
			2.3 Activity-Based Crosslinking of TET Enzymes with eyC-Containing DNA Substrates
				2.3.1 Overexpression of TET2 or Variants in HEK293T Cells
				2.3.2 Preparation of Cell Lysates
				2.3.3 Activity-Based TET Crosslinking
		3 Methods
			3.1 PCR Syntheses of Modified DNA Substrates
			3.2 Fluorometric Endpoint Read-out of TET Activity with vC-Containing DNA Substrates
			3.3 Activity-Based Crosslinking of TET Enzymes with eyC-Containing DNA Substrates
				3.3.1 Overexpression of TET2 or Variants in HEK293T Cells
				3.3.2 Preparation of Cell Lysates
				3.3.3 Activity-Based TET Crosslinking Assay
		4 Notes
			4.1 PCR Syntheses of Modified DNA Substrates
			4.2 Fluorometric Endpoint Read-out of TET Activity with vC-Containing DNA Substrates
			4.3 Activity-Based Crosslinking of TET Enzymes with eyC-Containing DNA Substrates
				4.3.1 Overexpression of TET2 or Variants in HEK293T Cells
				4.3.2 Preparation of Cell Lysates
				4.3.3 Activity-Based TET Crosslinking Assay
		References
	Chapter 17: Generation and Molecular Characterization of Transient tet1/2/3 Zebrafish Knockouts
		1 Introduction
		2 Materials
			2.1 Zebrafish Husbandry
			2.2 Equipment
			2.3 Consumables
			2.4 Reagents
		3 Methods
			3.1 CRISPR/Cas9 Guide Oligo Design
			3.2 Annealing and Amplification of the DNA Guide Oligo and Universal Oligo
			3.3 gRNA In Vitro Transcription
			3.4 Microinjection Chambers and Needles
			3.5 Zebrafish Egg Collection and Preparation of the tet1/2/3 KO Microinjection Mixture
			3.6 Microinjections
			3.7 Embryo Collection
			3.8 gDNA and RNA Extraction
			3.9 Amplicon Sequencing
			3.10 RRBS: MspI Digestion
			3.11 RRBS: End Repair and A-Tailing
			3.12 RRBS: Adaptor Ligation
			3.13 RRBS: Bisulfite Conversion
			3.14 RRBS: PCR Amplification
			3.15 RRBS: Library Quantification and Multiplexing
			3.16 qPCR Validation of Knockout Efficiencies
			3.17 Software
			3.18 File Names
			3.19 Quality Control
			3.20 Adapter Trimming
			3.21 Amplicon Sequencing Analysis
			3.22 Read Mapping
			3.23 Methylation Calling
			3.24 Data Visualization
			3.25 Methylation Phenotype Analysis
		4 Notes
		References
Correction to: TET Proteins and DNA Demethylation
Index