Reduced Bisulfite-Sequencing: Quantitative Base-Resolution Sequencing of 5-Formylcytosine -- Aba-Seq: High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine -- Estimating Global Methylation and Erasure Using Low-Coverage Whole Genome Bisulfite Sequencing (WGBS) -- ELISA-Based Quantitation of Global 5hmC Levels -- Avidin-Biotin ELISA-Based Detection of 5hmC -- Quantification of DNA Methylation and Its Oxidized Derivatives using LC-MS -- Distinguishing Active Versus Passive DNA Demethylation using Illumina MethylationEPIC BeadChip Microarrays -- Bioinformatic Estimation of DNA Methylation and Hydroxymethylation Proportions -- TAB-seq and ACE-seq Data Processing for Genome-Wide DNA Hydroxymethylation Profiling -- Genomic Targeting of TET Activity for Targeted Demethylation using CRISPR/Cas9 -- High-Fidelity CRISPR/Cas9-Based Gene-Specific Hydroxymethylation -- Identifying Protein-(hydroxy)methylated DNA Interactions using Quantitative Interaction Proteomics -- Purification of TET Proteins -- Uncovering Sequence Specific Transcription Factors Interacting with TET2 -- ChIP-Sequencing of TET Proteins -- Harnessing Alternative Substrates to Probe TET Family Enzymes -- Generation and Molecular Characterization of Transient tet1/2/3 Zebrafish Knockouts.

This volume explores the latest methods used to study various aspects of TET proteins and their biology. Chapters in this book are divided into five parts. Part One describes technologies aimed at detecting and quantifying DNA methylation turnover using massively parallel sequencing, ELISA, and mass spectrometry approaches. Part Two looks at data analyses protocols for distinguishing acting versus passive DNA demethylation and estimation of 5mC and 5hmC levels. Part Three deals with a new topic that takes advantage of modified CRISPR/Cas9 genome editing systems to target DNA demethylation activity to genomic loci of interest. Part Four discusses protocols that detail how to purify TET proteins and unravel their protein interactions, and Part Five looks at the assessment of TET protein function and activity in vivo and in vitro. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, TET Proteins and DNA Demethylation: Methods and Protocols is a valuable resource that aims to help research scientists at all levels working in the fields of DNA demethylation dynamics. Chapters 3, 7 and 17 are available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.