Project description:We describe XmaI-RRBS method for rapid and affordable genome-wide DNA methylation analysis, with library preparation taking only four days and sequencing possible within four hours. Small sizes of the XmaI-RRBS libraries allow their multiplexing and sequencing on the benchtop high-throughput machines. Described here is the first RRBS protocol validated for the Ion Torrent Personal Genome Machine. DNA from MCF7 cell line and 6 normal breast samples (total 7 samples) were subjected to reduced representation bisulfite sequencing analysis (XmaI-RRBS) by using Ion Torrent platform.
Project description:DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging due to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called Laser Capture Microdissection-Reduced Representation Bisulfite Sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of CpG Islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared to adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development. Laser capture microdissection-reduced representation bisulfite sequencing and reduced representation bisulfite sequencing on human blood leukocyte, human endometrial tumor, mouse liver tissue, and mouse normal and neoplastic adrenal tissue
Project description:To assess variation and inheritance of genome-wide patterns of DNA methylation simultaneously in humans, we applied reduced representation bisulfite sequencing (RRBS) to somatic DNA from six members of a three-generation family. Reduced representation bisulfite sequencing was applied to genomic DNA from leukocytes of 6 family members and two unrelated individuals.
Project description:DNA methylation plays critical roles in gene regulation and cellular specification without altering DNA sequences. The wide application of reduced representation bisulfite sequencing (RRBS) and whole genome bisulfite sequencing (bis-seq) opens the door to study DNA methylation at single CpG site resolution. One challenging question is how best to test for significant methylation differences between groups of biological samples in order to minimize false positive findings. Current methods to analyze genome-wide bisulfite sequencing data use a smoothing approach or a simple statistical test based on the binomial distribution. Comparative DNA methylation profiling in AML blasts and normal CD34(+) control cells
Project description:Estrogen receptor beta (ERβ) is a ligand inducible transcription factor regulating gene expression in response to the female sex hormone estrogen. Previously, we found that ERβ deficiency results in changes in DNA methylation patterns at two gene promoters, implicating an involvement of ERβ in DNA methylation. In this study, we set out to explore this involvement on a genome-wide level, and to investigate the underlying mechanisms of this function. Using reduced representation bisulfite sequencing (RRBS), we compared genome-wide DNA methylation in mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and ERβ knock-out (βerko) mice, and identified around 8000 differentially methylated positions (DMPs). This suggests that ERβ is involved in regulating DNA methylation at specific sites in the genome. Genome-wide DNA methylation was analysed in MEFs derived from wildtype and ERbeta null mice by educed representation bisulfite sequencing (RRBS) on an Illumina Genome Analyser IIx platform.
Project description:To assess variation and inheritance of genome-wide patterns of DNA methylation simultaneously in humans, we applied reduced representation bisulfite sequencing (RRBS) to somatic DNA from six members of a three-generation family.
Project description:DNA methylation plays critical roles in gene regulation and cellular specification without altering DNA sequences. The wide application of reduced representation bisulfite sequencing (RRBS) and whole genome bisulfite sequencing (bis-seq) opens the door to study DNA methylation at single CpG site resolution. One challenging question is how best to test for significant methylation differences between groups of biological samples in order to minimize false positive findings. Current methods to analyze genome-wide bisulfite sequencing data use a smoothing approach or a simple statistical test based on the binomial distribution.
Project description:We describe XmaI-RRBS method for rapid and affordable genome-wide DNA methylation analysis, with library preparation taking only four days and sequencing possible within four hours. Small sizes of the XmaI-RRBS libraries allow their multiplexing and sequencing on the benchtop high-throughput machines. Described here is the first RRBS protocol validated for the Ion Torrent Personal Genome Machine.
Project description:Reduced representation bisulfite sequencing (RRBS) was conducted on dorsolateral prefrontal cortex tissue samples taken from the brains of control individuals not affected by neurological disorder DNA methylation profiling was conducted using RRBS and the Illumina Genome Analyzer IIx
Project description:We used in ovo embryonic heat conditioning (EHC) of first-generation chicks and assessed the effect on their untreated offspring by measuring genome-wide alterations in DNA-methylation patterns. DNA was extracted from the APH and reduced representation bisulfite sequencing (RRBS) was performed
