5-Methylcytosine (5mC) is one of the most abundant and well-studied chemical DNA modifications of vertebrate genomes. 5mC plays an essential role in genome regulation including silencing of retroelements, X chromosome inactivation, and heterochromatin stability. Furthermore, 5mC shapes the activity of cis-regulatory elements crucial for cell fate determination. TET enzymes can oxidize 5mC to form 5-hydroxymethylcytosine (5hmC), thereby adding an additional layer of complexity to the DNA methylation landscape dynamics. The advent of techniques enabling genome-wide 5hmC profiling provided critical insights into its genomic distribution, scope, and function. These methods include immunoprecipitation, chemical labeling and capture-based approaches, as well as single-nucleotide 5hmC profiling techniques such as TET-assisted bisulfite sequencing (TAB-seq) and APOBEC-coupled epigenetic sequencing (ACE-seq). Here we provide a detailed protocol for computational analysis required for the genomic alignment of TAB-seq and ACE-seq data, 5hmC calling, and statistical analysis.Simultaneous measurement of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) at the single-nucleotide level can be obtained by combining data from DNA processing methods including traditional bisulfite (BS), oxidative bisulfite (oxBS), or Tet-assisted (TAB) bisulfite conversion. Array-based technologies have been widely used in this task, due to their time and cost efficiency. For methylation studies using BS data, many protocols and related packages have been suggested in the literature to deal with limitations and confounders that arise from array data. In this chapter, we illustrate how the reader can make small adjustments to these protocols to obtain estimates of methylation and hydroxymethylation proportions.The 5-carbon positions on cytosine nucleotides preceding guanines in genomic DNA (CpG) are common targets for DNA methylation (5mC). DNA methylation removal can occur through both active and passive mechanisms. Ten-eleven translocation enzymes (TETs) oxidize 5mC in a stepwise manner to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC can also be removed passively through sequential cell divisions in the absence of DNA methylation maintenance. In this chapter, we describe approaches that couple TET-assisted bisulfite (TAB) and oxidative bisulfite (OxBS) conversion to the Illumina MethylationEPIC BeadChIP (EPIC array) and show how these technologies can be used to distinguish active versus passive DNA demethylation. We also describe integrative bioinformatics pipelines to facilitate this analysis.The amount of 5-methyl-2′-deoxycytidine (m5dC) and its oxidized derivatives 5-hydroxymethyl-dC (hm5dC), 5-formyl-dC (f5dC), and 5-carboxy-dC (ca5dC) inside mammalian cells provides valuable information concerning cellular state and fate. LC-MS methods enable reliable quantification of these noncanonical DNA modifications in the low femtomolar range. Here, we describe a broadly applicable protocol to quantify m5dC, hm5dC, f5dC, and ca5dC in vertebrate-derived cells using ultra-HPLC triple quadrupole MS (UHPLC-QQQ-MS).The enzyme-linked immunosorbent assay (ELISA) technique has been developed half a century ago, and yet its role in molecular biology remains significant. Among the most sensitive of immunoassays, it offers high throughput, combined with affordability and ease of use. This chapter provides the procedure of a highly reproducible indirect sandwich ELISA protocol, which can be applied to a variety of semi-quantitative assays for the investigation of the molecular biology of 5-hydroxymethylcytosine (5hmC) or TET enzymes. Three variations of this protocol are described assessment and validation of 5hmC-binding proteins, screening and validation of anti-5hmC antibodies, or a readout of TET catalytic activity in in vitro experiments. The assay principle is based on the use of a high affinity avidin-biotin system for efficient immobilization of DNA fragments for further detection by high specificity antibodies. A colorimetric enzymatic reaction is ultimately developed with intensity correlating with the amount of attached antigen.5-Hydroxymethylcytosine (5hmC) is an abundant DNA modification in human and mouse brain, as well as in embryonic stem cells, while severely depleted in multiple types of cancer. Assays for 5hmC detection and quantification, both on a locus-specific and global level, are limited in number and often resource-intensive. Immunodetection of 5hmC through antibodies remains a cost-effective and widely accessible approach. This chapter describes an ELISA-based protocol for 5hmC detection and quantification in genomic or in vitro modified DNA. It is based on the passive adsorption of DNA onto a solid polystyrene surface and the specific detection of 5hmC, which generates a measurable chemiluminescent signal, proportional to the amount of immobilized 5hmC. The assay utilizes a standard curve for interpolation of 5hmC percentage and a loading standard for monitoring loading precision.Whole-genome bisulfite sequencing (WGBS) is a popular method for characterizing cytosine methylation because it is fully quantitative and has base-pair resolution. While WGBS is prohibitively expensive for experiments involving many samples, low-coverage WGBS can accurately determine global methylation and erasure at similar cost to high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assays (ELISA). Moreover, low-coverage WGBS has the capacity to distinguish between methylation in different cytosine contexts (e.g., CG, CHH, and CHG), can tolerate low-input material ( less then 100 cells), and can detect the presence of overrepresented DNA originating from mitochondria or amplified ribosomal DNA. compound library chemical In addition to describing a WGBS library construction and quantitation approach, here we detail computational methods to predict the accuracy of low-coverage WGBS using empirical bootstrap samplers and theoretical estimators similar to those used in election polling. Using examples, we further demonstrate how non-independent sampling of cytosines can alter the precision of error calculation and provide methods to improve this.