Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq) to Study DNA Methylation Using Low Amounts of DNA
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ABSTRACT:
PROVIDER: PRJNA932753 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23373000_1.fastq.gz | Fastqsanger.gz | |||
| SRR23373000_2.fastq.gz | Fastqsanger.gz | |||
| SRR23373001_1.fastq.gz | Fastqsanger.gz | |||
| SRR23373001_2.fastq.gz | Fastqsanger.gz | |||
| SRR23373002_1.fastq.gz | Fastqsanger.gz |
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Project description:DNA methylation is an epigenetic mark that has a crucial role in regulating gene expression. Aberrant DNA methylation results in severe diseases in humans, such as cancer, autoimmune disease, atherosclerosis, and cardiovascular diseases. Whole-genome bisulfite sequencing and methylated DNA immunoprecipitation are available to study DNA methylation changes, but they are typically used on a few samples at a time. Here, we developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), that can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis.
2024-12-31 | GSE224820 | GEO
Project description:BackgroundDNA methylation contributes to the regulation of gene expression during development and cellular differentiation. The recently developed Methylated DNA ImmunoPrecipitation (MeDIP) assay allows a comprehensive analysis of this epigenetic mark at the genomic level in normal and disease-derived cells. However, estimating the efficiency of the MeDIP technique is difficult without previous knowledge of the methylation status of a given cell population. Attempts to circumvent this problem have involved the use of in vitro methylated DNA in parallel to the investigated samples. Taking advantage of this stratagem, we sought to improve the sensitivity of the approach and to assess potential biases resulting from DNA amplification and hybridization procedures using MeDIP samples.FindingsWe performed MeDIP assays using in vitro methylated DNA, with or without previous DNA amplification, and hybridization to a human promoter array. We observed that CpG content at gene promoters indeed correlates strongly with the MeDIP signal obtained using in vitro methylated DNA, even when lowering significantly the amount of starting material. In analyzing MeDIP products that were subjected to whole genome amplification (WGA), we also revealed a strong bias against CpG-rich promoters during this amplification procedure, which may potentially affect the significance of the resulting data.ConclusionWe illustrate the use of in vitro methylated DNA to assess the efficiency and accuracy of MeDIP procedures. We report that efficient and reproducible genome-wide data can be obtained via MeDIP experiments using relatively low amount of starting genomic DNA; and emphasize for the precaution that must be taken in data analysis when an additional DNA amplification step is required.
| S-EPMC2949662 | biostudies-literature
Project description:DNA extraction from natural and spiked MX 80 bentonite
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Project description:We exposed Salmonella TA100 cells to 3 concentrations of MX that produced a linear concentration-response for mutagenesis with little cell killing. We measured mutagenesis, survival, and global gene expression. We used custom-spotted glass slides as our microarray platform and identified genes whose expressions were altered by MX treatment using three methods: (1) a Bayesian t-test, (2) an operon analysis that assumes if one gene in an operon is differentially expressed then all genes in that operon are differentially expressed, and (3) a monotonic-expression response to increasing doses of MX. The resulting list of genes was analyzed for functional and KEGG pathway representation. Keywords: dose response
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Project description:The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp™ Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies.
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Project description:Epigenetic changes have been implicated in pathogenesis of asthma. We sought to determine if IL13, a key cytokine in airway inflammation and remodeling, induced epigenetic DNA methylation changes in the airways in conjunction with its transcriptional gene regulation. For our studies, we used a well-characterized transgenic mouse model of allergic airway inflammation induced by IL13. In this model, IL13 is conditionally overexpressed in the mouse lung when treated with doxycycline. Upon IL13 induction, these mice showed inflammatory cell infiltration, pronounced emphysema, increased pulmonary compliance, lung volume enlargement, mucus metaplasia, and increased expression of matrix metalloproteinases and cathepsins in the lung. We performed MeDIP microarray to examine the changes in DNA promoter methylation during IL13-induced allergic airway inflammation.
2012-04-07 | GSE37079 | GEO
Project description:Epigenetic changes have been implicated in pathogenesis of asthma. We sought to determine if IL13, a key cytokine in airway inflammation and remodeling, induced epigenetic DNA methylation changes in the airways in conjunction with its transcriptional gene regulation. For our studies, we used a well-characterized transgenic mouse model of allergic airway inflammation induced by IL13. In this model, IL13 is conditionally overexpressed in the mouse lung when treated with doxycycline. Upon IL13 induction, these mice showed inflammatory cell infiltration, pronounced emphysema, increased pulmonary compliance, lung volume enlargement, mucus metaplasia, and increased expression of matrix metalloproteinases and cathepsins in the lung. We performed MeDIP microarray to examine the changes in DNA promoter methylation during IL13-induced allergic airway inflammation. The CC10-rtTA-IL13 transgenic (TG) and wildtype (WT) mice were treated with doxycycline for seven days. Mice were euthanized and the left lower lobes from all mice were removed for DNA extraction followed by MeDIP array analysis.
2012-04-06 | E-GEOD-37079 | biostudies-arrayexpress
Project description:Breast cancer is a leading cause of cancer-related deaths among women in the Western world. Anthracyclines, including doxorubicin (DOX), along with taxanes, cyclophosphamide and platinum compounds are the main chemotherapeutic agents applied for breast cancer treatment. However, chemoresistance is the major cause of the disease progression following chemotherapy. Drug resistance can be either inherent or acquired during the treatment, but most possibly the interaction of both mechanisms drives the rapid development of treatment refractory cancer. In the present study, the mechanisms of acquired cellular chemoresistance were studied in breast cancer cell line MX-1 exposed to gradually increasing concentration of the chemotherapeutic compound DOX (MX-1/D). Nongenotoxic phosphorganic compound tetraphenylphosphonium cation (TPP+) – a potent substrate and activator for ABCB1 transporter – was used in the cell line model of intrinsic chemoresistance (MX-1/T). Finally, DOX highly resistant cell subline was derived from ABCB1-activated, TPP+ pretreated cells (MX-1/TD). Chemoresistance in all cell sublines was closely associated with the EMT, and the ABCB1 hyperexpression was a possibly trigger of this process.
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