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Bisulfite sequencing (BS-seq) remains the gold standard technique to quantitively map DNA methylation at a single-base resolution. However, BS-seq cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Oxidative bisulfite sequencing (oxBS-seq) was one of the first techniques that enabled absolute quantification of 5mC and 5hmC at single-base resolution. OxBS-seq uses chemical oxidation of 5hmC prior to bisulfite treatment to provide a direct readout of 5mC; comparison with BS-seq data can then be used to infer 5hmC levels. Here we describe in detail an updated version of our laboratory's oxBS-seq protocol, which uses potassium perruthenate (KRuO4) as an oxidant. We also describe a bioinformatics pipeline designed to handle Illumina short read sequencing data from whole-genome oxBS-seq.DNA cytosine modification is an important epigenetic mechanism that serves critical functions in a variety of biological processes in development and disease. 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two most common epigenetic marks found in the mammalian genome. 5hmC is generated from 5mC by the ten-eleven translocation (TET) family of dioxygenase enzymes. This modification can reach substantial levels in certain cell types such as embryonic stem cells and neurons. Standard bisulfite sequencing techniques cannot distinguish between 5mC and 5hmC. selleck compound Therefore, the method of TET-assisted bisulfite sequencing has been developed for detecting 5hmC specifically. The method is based on protection of 5hmC by glycosylation followed by complete oxidation of both 5mC and 5fC to 5caC, which converts to uracil after bisulfite treatment leaving only 5hmC remaining as a cytosine signal after PCR and sequencing. The method requires a highly active TET protein for the conversion steps. Here, we present an efficient TET protein purification method and a streamlined TAB-sequencing protocol for 5hmC analysis at single base resolution.DNA methylation (5-methylcytosine, 5mC) is involved in regulation of a wide range of biological processes. TET proteins can oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although both 5fC and 5caC serve as intermediates in active demethylation pathway, growing body of experimental evidence indicate that these DNA modifications may also interact with specific sets of reader proteins and therefore may represent bona fide epigenetic marks. Despite a number of single-base resolution techniques have recently been proposed for 5fC/5caC mapping, antibody-based approaches still represent a relatively simple and plausible alternative for the analysis of genomic distribution of these DNA modifications. Here, we describe a protocol for 5caC DNA immunoprecipitation (5caC DIP) that can be used for both locus-specific and genome-wide assessment of 5caC distribution. In combination with mass spectrometry-based techniques and single base resolution mapping methods, this approach may contribute to elucidating the role of 5caC in development, differentiation, and tumorigenesis.Methylated DNA immunoprecipitation is a large scale purification technique. It enables the isolation of methylated DNA fragments for subsequent locus-specific or genome-wide analysis. Here we describe an immunoprecipitation protocol using a monoclonal mouse anti 5-methyl-cytidine antibody followed by next-generation sequencing (MeDIP-Seq).Ligation of a hairpin oligonucleotide to genomic DNA prior to bisulfite conversion and PCR amplification physically links the two complementary DNA strands. This additional step in the conversion procedure overcomes the limitations of conventional bisulfite sequencing where information of the cytosine methylation status is only obtained from one of the two strands of an individual DNA molecule. Sequences derived from hairpin bisulfite PCR products reveal the dynamics of this epigenetic memory system on both strands of individual DNA molecules. The chapter describes a reliable step-by-step procedure to generate hairpin-linked DNA. It also provides a guide for efficient bisulfite conversion that is suitable for both conventional and hairpin bisulfite sequencing approaches.5-hydroxymethyluracil was originally identified as an oxidatively modified DNA base derivative. Recent evidence suggests that its formation may result from the oxidation of thymine in a reaction that is catalyzed by TET proteins. Alternatively, it could be generated through the deamination of 5-hydroxymethylcytosine by activation-induced cytidine deaminase. The standard method for evaluating 5-hydroxymethyluracil content is the highly sensitive and highly specific isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). Despite many advantages, this method has one great limitation. It is not able to measure compounds at a single-cell level. Our goal was to develop and optimize a method based on flow cytometry that allows the evaluation of 5-hydroxymethyluracil levels at a single cell level in peripheral leukocytes.Male infertility is associated with several causes affecting the paternal nucleus such as DNA lesions (breaks, deletions, mutations, ...) or numerical chromosome anomalies. More recently, male infertility has also been associated with changes in the sperm epigenome, including modification in the topology of chromatin (Olszewska et al., Chromosome Research 16875-890, 2008; Alladin et al., Syst Biol Reprod Med 59 146-152, 2013) ref with number 1, 2. Indeed, the positioning of chromosomes in the sperm nucleus is nonrandom and defines chromosome territories (Champroux et al., Genes (Basel) 9501, 2018) ref with number 3 whose optimal organization determines the success of embryonic development. In this context, the study of the spatial distribution of chromosomes in sperm cells could be relevant for clinical diagnosis. We describe here a in situ fluorescence hybridization (FISH) strategy coupled with a fluorescent immunocytochemistry approach followed by confocal analysis and reconstruction (2D/3D) as a powerful tool to analyze the location of chromosomes in the sperm nucleus using the mouse sperm as a model.
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