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single cell Bisulfite-seq

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https://www.readbyqxmd.com/read/29524144/whole-genome-bisulfite-sequencing-for-the-analysis-of-genome-wide-dna-methylation-and-hydroxymethylation-patterns-at-single-nucleotide-resolution
#1
Magali Kernaleguen, Christian Daviaud, Yimin Shen, Eric Bonnet, Victor Renault, Jean-François Deleuze, Florence Mauger, Jörg Tost
The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and disease-associated investigations. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered as the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29472610/scnmt-seq-enables-joint-profiling-of-chromatin-accessibility-dna-methylation-and-transcription-in-single-cells
#2
Stephen J Clark, Ricard Argelaguet, Chantriolnt-Andreas Kapourani, Thomas M Stubbs, Heather J Lee, Celia Alda-Catalinas, Felix Krueger, Guido Sanguinetti, Gavin Kelsey, John C Marioni, Oliver Stegle, Wolf Reik
Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation...
February 22, 2018: Nature Communications
https://www.readbyqxmd.com/read/29436922/breast-cancer-family-history-and-allele-specific-dna-methylation-in-the-legacy-girls-study
#3
Hui-Chen Wu, Catherine Do, Irene L Andrulis, Esther M John, Mary B Daly, Saundra S Buys, Wendy K Chung, Julia A Knight, Angela R Bradbury, Theresa H M Keegan, Lisa Schwartz, Izabela Krupska, Rachel L Miller, Regina M Santella, Benjamin Tycko, Mary Beth Terry
Family history, a well-established risk factor for breast cancer, can have both genetic and environmental contributions. Shared environment in families as well as epigenetic changes that also may be influenced by shared genetics and environment may also explain familial clustering of cancers. Epigenetic regulation, such as DNA methylation, can change the activity of a DNA segment without a change in the sequence; environmental exposures experienced across the life course can induce such changes. However, genetic-epigenetic interactions, detected as methylation quantitative trait loci (mQTLs; a...
April 2, 2018: Epigenetics: Official Journal of the DNA Methylation Society
https://www.readbyqxmd.com/read/29409498/dna-methylome-profiling-at-single-base-resolution-through-bisulfite-sequencing-of-5mc-immunoprecipitated-dna
#4
Zhen Jia, Yueyi Shi, Lei Zhang, Yipeng Ren, Tong Wang, Lejun Xing, Baorong Zhang, Guolan Gao, Rongfa Bu
BACKGROUND: Detection of DNA methylome at single-base resolution is a significant challenge but promises to shed considerable light on human disease etiology. Current technologies could not detect DNA methylation genome-wide at single-base resolution with small amount of sequencing data and could not avoid detecting the methylation of repetitive elements which are considered as "junk DNA". METHODS: In this study, we have developed a novel DNA methylome profiling technology named MB-seq with its ability to identify genome-wide 5mC and quantify DNA methylation levels by introduced an assistant adapter AluI-linker This linker can be ligated to sonicated DNA and then be digested after the bisulfite treatment and amplification, which has no effect of MeDIP enrichment...
February 6, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29224168/tet-assisted-bisulfite-sequencing-tab-seq
#5
Miao Yu, Dali Han, Gary C Hon, Chuan He
5-Hydroxymethylcytosine (5hmC) is a modified form of cytosine, which has recently been found in mammalian cells and tissues. 5hmC is derived from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) family protein-mediated oxidation and may regulate gene expression. Numerous affinity-based profiling methods have been developed to help understand the exact function of 5hmC in the genome. However, these methods have a relatively low resolution (~100 bp) without quantitative information of the modification percentage on each site...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29224149/nucleosome-occupancy-and-methylome-sequencing-nome-seq
#6
Fides D Lay, Theresa K Kelly, Peter A Jones
Various methodologies are available to interrogate specific components of epigenetic mechanisms such as DNA methylation or nucleosome occupancy at both the locus-specific and the genome-wide level. It has become increasingly clear, however, that comprehension of the functional interactions between epigenetic mechanisms is critical for understanding how cellular transcription programs are regulated or deregulated during normal and disease development. The Nucleosome Occupancy and Methylome sequencing (NOMe-seq) assay allows us to directly measure the relationship between DNA methylation and nucleosome occupancy by taking advantage of the methyltransferase M...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29224070/genome-wide-analysis-of-dna-methylation-in-single-cells-using-a-post-bisulfite-adapter-tagging-approach
#7
Heather J Lee, Sébastien A Smallwood
DNA methylation is an epigenetic mark implicated in the regulation of key biological processes. Using high-throughput sequencing technologies and bisulfite-based approaches, it is possible to obtain comprehensive genome-wide maps of the mammalian DNA methylation landscape with a single-nucleotide resolution and absolute quantification. However, these methods were only applicable to bulk populations of cells. Here, we present a protocol to perform whole-genome bisulfite sequencing on single cells (scBS-Seq) using a post-bisulfite adapter tagging approach...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29052184/bisulfite-sequencing-using-small-dna-amounts
#8
Susanne Edelmann, Stefan Scholten
Bisulfite sequencing (BS-seq) enables the detection of DNA methylation at cytosine residues (5mC) at single-nucleotide resolution. For many applications, a limiting factor of conventional BS-seq protocols is the high amount of DNA required, since the treatment with bisulfite causes severe DNA fragmentation. Here, we describe a post-bisulfite tagging method that accounts for this problem. Illumina-compatible BS-seq libraries can be obtained from as little as five single haploid maize cells, enabling whole genome BS-seq (WGBS) for the generation of genome-wide, cell-type specific DNA methylation profiles...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28923089/dna-epigenome-editing-using-crispr-cas-suntag-directed-dnmt3a
#9
Yung-Hsin Huang, Jianzhong Su, Yong Lei, Lorenzo Brunetti, Michael C Gundry, Xiaotian Zhang, Mira Jeong, Wei Li, Margaret A Goodell
BACKGROUND: DNA methylation has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression. RESULTS: Here, we utilize nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltransferase 3A (DNMT3A) (dCas9-SunTag-DNMT3A) to amplify the local DNMT3A concentration to methylate genomic sites of interest...
September 18, 2017: Genome Biology
https://www.readbyqxmd.com/read/28360182/simultaneous-mapping-of-active-dna-demethylation-and-sister-chromatid-exchange-in-single-cells
#10
Xiaoji Wu, Azusa Inoue, Tsukasa Suzuki, Yi Zhang
To understand mammalian active DNA demethylation, various methods have been developed to map the genomic distribution of the demethylation intermediates 5-formylcysotine (5fC) and 5-carboxylcytosine (5caC). However, the majority of these methods requires a large number of cells to begin with. In this study, we describe low-input methylase-assisted bisulfite sequencing (liMAB-seq ) and single-cell MAB-seq (scMAB-seq), capable of profiling 5fC and 5caC at genome scale using ∼100 cells and single cells, respectively...
March 1, 2017: Genes & Development
https://www.readbyqxmd.com/read/28182018/genome-wide-base-resolution-mapping-of-dna-methylation-in-single-cells-using-single-cell-bisulfite-sequencing-scbs-seq
#11
Stephen J Clark, Sébastien A Smallwood, Heather J Lee, Felix Krueger, Wolf Reik, Gavin Kelsey
DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28126923/bisulfite-independent-analysis-of-cpg-island-methylation-enables-genome-scale-stratification-of-single-cells
#12
Lin Han, Hua-Jun Wu, Haiying Zhu, Kun-Yong Kim, Sadie L Marjani, Markus Riester, Ghia Euskirchen, Xiaoyuan Zi, Jennifer Yang, Jasper Han, Michael Snyder, In-Hyun Park, Rafael Irizarry, Sherman M Weissman, Franziska Michor, Rong Fan, Xinghua Pan
Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells...
June 2, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28111014/tethered-oligonucleotide-primed-sequencing-top-seq-a-high-resolution-economical-approach-for-dna-epigenome-profiling
#13
Zdislav Staševskij, Povilas Gibas, Juozas Gordevičius, Edita Kriukienė, Saulius Klimašauskas
Modification of CG dinucleotides in DNA is part of epigenetic regulation of gene function in vertebrates and is associated with complex human disease. Bisulfite sequencing permits high-resolution analysis of cytosine modification in mammalian genomes; however, its utility is often limited due to substantial cost. Here, we describe an alternative epigenome profiling approach, named TOP-seq, which is based on covalent tagging of individual unmodified CG sites followed by non-homologous priming of the DNA polymerase action at these sites to directly produce adjoining regions for their sequencing and precise genomic mapping...
February 2, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28055034/applying-the-intact-method-to-purify-endosperm-nuclei-and-to-generate-parental-specific-epigenome-profiles
#14
Jordi Moreno-Romero, Juan Santos-González, Lars Hennig, Claudia Köhler
The early endosperm tissue of dicot species is very difficult to isolate by manual dissection. This protocol details how to apply the INTACT (isolation of nuclei tagged in specific cell types) system for isolating early endosperm nuclei of Arabidopsis at high purity and how to generate parental-specific epigenome profiles. As a Protocol Extension, this article describes an adaptation of an existing Nature Protocol that details the use of the INTACT method for purification of root nuclei. We address how to obtain the INTACT lines, generate the starting material and purify the nuclei...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28052964/whole-genome-bisulfite-sequencing-of-human-pancreatic-islets-reveals-novel-differentially-methylated-regions-in-type-2-diabetes-pathogenesis
#15
Petr Volkov, Karl Bacos, Jones K Ofori, Jonathan Lou S Esguerra, Lena Eliasson, Tina Rönn, Charlotte Ling
Current knowledge about the role of epigenetics in type 2 diabetes (T2D) remains limited. Only a few studies have investigated DNA methylation of selected candidate genes or a very small fraction of genomic CpG sites in human pancreatic islets, the tissue of primary pathogenic importance for diabetes. Our aim was to characterize the whole-genome DNA methylation landscape in human pancreatic islets, to identify differentially methylated regions (DMRs) in diabetic islets, and to investigate the function of DMRs in islet biology...
April 2017: Diabetes
https://www.readbyqxmd.com/read/27924045/a-novel-method-for-the-simultaneous-identification-of-methylcytosine-and-hydroxymethylcytosine-at-a-single-base-resolution
#16
Yuki Kawasaki, Yukiko Kuroda, Isao Suetake, Shoji Tajima, Fumitoshi Ishino, Takashi Kohda
Since the discovery of oxidative demethylation of methylcytosine (mC) by Tet enzymes, an analytical method has been urgently needed that would enable the identification of mC and hydroxymethylcytosine (hmC) at the single base resolution level, because their roles in gene regulation are quite different from each other. However, the bisulfite sequencing method, the gold standard for DNA methylation analysis at present, does not distinguish them. Recently reported alternative methods, such as oxBS-seq and TAB-seq, are not even capable of determining mC and hmC simultaneously...
October 24, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27895806/genome-wide-epigenomic-profiling-for-biomarker-discovery
#17
REVIEW
René A M Dirks, Hendrik G Stunnenberg, Hendrik Marks
A myriad of diseases is caused or characterized by alteration of epigenetic patterns, including changes in DNA methylation, post-translational histone modifications, or chromatin structure. These changes of the epigenome represent a highly interesting layer of information for disease stratification and for personalized medicine. Traditionally, epigenomic profiling required large amounts of cells, which are rarely available with clinical samples. Also, the cellular heterogeneity complicates analysis when profiling clinical samples for unbiased genome-wide biomarker discovery...
2016: Clinical Epigenetics
https://www.readbyqxmd.com/read/27848892/single-cell-sequencing-for-drug-discovery-and-drug-development
#18
REVIEW
Hongjin Wu, Charles Wang, Shixiu Wu
Next-generation sequencing (NGS), particularly single-cell sequencing, has revolutionized the scale and scope of genomic and biomedical research. Recent technological advances in NGS and singlecell studies have made the deep whole-genome (DNA-seq), whole epigenome and whole-transcriptome sequencing (RNA-seq) at single-cell level feasible. NGS at the single-cell level expands our view of genome, epigenome and transcriptome and allows the genome, epigenome and transcriptome of any organism to be explored without a priori assumptions and with unprecedented throughput...
2017: Current Topics in Medicinal Chemistry
https://www.readbyqxmd.com/read/27718505/optimization-of-techniques-for-multiple-platform-testing-in-small-precious-samples-such-as-human-chorionic-villus-sampling
#19
Margareta D Pisarska, Marzieh Akhlaghpour, Bora Lee, Gillian M Barlow, Ning Xu, Erica T Wang, Aaron J Mackey, Charles R Farber, Stephen S Rich, Jerome I Rotter, Yii-der I Chen, Mark O Goodarzi, Seth Guller, John Williams
BACKGROUND: Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established. RESULTS: Leftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg...
November 2016: Prenatal Diagnosis
https://www.readbyqxmd.com/read/27172195/histone-h1-limits-dna-methylation-in-neurospora-crassa
#20
Michael Seymour, Lexiang Ji, Alex M Santos, Masayuki Kamei, Takahiko Sasaki, Evelina Y Basenko, Robert J Schmitz, Xiaoyu Zhang, Zachary A Lewis
Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein...
July 7, 2016: G3: Genes—Genomes—Genetics
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