Next Generation Sequencing for Genome-wide Maps of DNA Methylaton in mES Cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE107079 | GEO | 2018/11/17
REPOSITORIES: GEO
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Project description:Whole Genome Bisulfite Sequencing (WGBS) is the current standard for DNA methylation profiling; however, WGBS is costly as it requires sequencing coverage over the entire genome. Here we introduce Anchor-Based Bisulfite Sequencing (ABBS). We show that ABBS captures accurate DNA methylation information in Escherichia coli and mammalian cells, while requiring approximately 20 times fewer sequencing reads than WGBS. The ABBS protocol is simple and can be performed in a single day.
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Project description:The development of whole-genome bisulfite sequencing (WGBS) has led to a number of exciting discoveries about how genomes utilize DNA methylation and has led to a plethora of novel testable hypotheses. Methods for constructing sodium bisulfite-converted and amplified libraries have recently excelled to the point that the bottleneck for experiments that use WGBS has shifted to data analysis and interpretation. Here we present empirical evidence for an over-representation of methylated DNA from WGBS. This enrichment for methylated DNA is exacerbated by higher cycles of PCR and is influenced by the type of uracil-insensitive DNA polymerase used for amplifying the sequencing library. Future efforts to computationally correct for this enrichment bias will be essential to increasing the accuracy of determining methylation levels for individual cytosines. MethylC-Seq of Arabidopsis thaliana
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Project description:Cannabis is commonly used amongst reproductive age males. Our group and others have shown that chronic delta-9-tetrahydrocannabinol (THC) use adversely impacts male fertility, but less is known about the potential reversibility of these changes. Our study’s objective was to determine if THC discontinuation mitigates THC-associated changes in male reproductive health using a rhesus macaque (RM) model of daily THC edible consumption over a 280-day period (4 spermatogenic cycles). Testicular volume, serum male hormones, semen parameters, sperm DNA fragmentation, seminal fluid proteomics, and whole genome bisulfite sequencing (WGBS) of sperm DNA were assessed at pre-THC, moderate-THC, and heavy-THC dosing, and at 70 and 140 days after THC discontinuation. Chronic THC use resulted in significant testicular atrophy, increased gonadotropins, decreased serum sex steroids, and increased DNA fragmentation. THC discontinuation led to partial recovery of testicular volume, sex steroids and sperm DNA integrity. Seminal fluid proteome analysis revealed differential expression of proteins enriched for processes related to cellular secretion, immune response, and fibrinolysis. WGBS identified significant differential methylation in heavy-THC versus pre-THC sperm, with partial restoration of methylation after discontinuation of THC. Genes associated with differentially methylated regions (DMRs) were enriched for pathways involved in nervous system development and function. This is the first study demonstrating that chronic THC use in RMs adversely impacts male reproductive health, methylation of genes involved in development, and expression of proteins important for male fertility. THC discontinuation improves impacts to male fertility, including partial restoration of THC-associated sperm DMRs in genes important for development.
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Project description:The goals of this study are to compare genome-wide DNA methylation levels in young and aged oocytes,and to investigate the transgenerational inheritance of methylome profiles in oocytes during natural aging. We apply a novel protocol of rapamycin to overcome the DNA methylation drift associated with oocyte aging. 8-week-old female mice were injected intraperitoneally with rapamycin or vehicle for 40 weeks. At the end of the experiment, females (48 weeks, F0) were paired with young adult (~4 mo old) males to produce F1 offspring (OF1 and ORaF1). An F2 generation (OF2 and ORaF2) resulted from mating F1 female at 44~48 weeks of age with young adult males. To generate YF1 and YF2 as normal control (offspring of young mother), we mated females (~8 weeks) with young adult males. Then we collect oocytes (F0,F1 and F2 generations)，sperm ( F1 and F2 generations) and hippocampus (F1 female offspring) from different groups to investigate the transgenerational inheritance of DNA methylome profiles associated with oocyte aging by the single cell whole-genome methylation sequencing (sc-WGBS). We found that oocytes from aged mother exhibited increased DNA methylation levels in CpG sites. Maternal aging related methylome changes can be inherited transgenerationally though oocyte to the germ line of F1 and F2 offspring. The application of rapamycin during the course of oocyte aging could reverse these DNA methylation alterations, and it can ameliorate several neurobehavioral aging trails that were in observed in aged oocyte offspring. WGBS-seq on DNA from hippocampal tissue revealed a number of differentially methylated (P<0.05) genes in OF1 and ORaF1 compared with YF1, and some of the enriched pathways were associated with aging process, such as PI3K-Akt signaling pathway (akin to transcriptional alterations above), MAPK signaling and Ras signaling pathway .
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