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| Status |
Public on Jul 21, 2020 |
| Title |
Cardiomyocytes_DNA_rep1-TCDD treated_24hrs |
| Sample type |
SRA |
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| Source name |
Cardiomyocytes
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| Organism |
Mus musculus |
| Characteristics |
cell type: ES-derived cardiomyocytes
strain: C57BL/6
time (hrs): 24
treatment: TCDD
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| Treatment protocol |
Cardiomyocytes were treated with TCDD at a concentration of 5 nM, (a concentration commonly used for tissue culture studies of the high-affinity AHR of C57BL/6 mice), for 24, 72, and 96 hours. TCDD was dissolved in DMSO, which never exceeded 0.05% of the volume of medium in either treatment or vehicle control. Undifferentiated, untreated parental ES cells and differentiated untreated cardiomyocytes served as controls.
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| Growth protocol |
Undifferentiated C57BL/6N-C2 ES cells were maintained in ES medium containing high-glucose Dulbecco’s minimal essential medium (DMEM; Gibcol Carlsbad, CA) supplemented with 15% ES cell qualified serum (Knockout Serum Replacement; Gibco), 2 mM glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 100U/mL penicillin, 100 µg/mL streptomycin and 0.1 mM β-mercaptoethanol, and 1,000 U/mL ESGRO leukemia inhibitory factor (LIF; Bioscience Research Reagents, Temucula, CA). Cells were seeded on 0.1% gelatin-coated plates, incubated at 37OC (95% humidity, 5% CO2) and passaged every second or third day. The establishment and maintenance of the G418-resistant Nkx2.5-ES cell line containing the pNkx2.5PuroIRES2eGFP plasmid were performed as follows. Briefly, cells were transfected by the pNkx2.5PuroIRES2eGFP plasmid bearing as well a G418-resistance marker regulated by the CMV promoter, seeded on 0.1% gelatin-coated plates and ES medium, and allowed to attach for 24 hours before adding 600 µg/ml G418. The transfected cells were selected for 3-5 passages to insure all cells not resistant to G418 were removed. The media was then replaced with ES medium without G418. After 24 hours, cells were trypsinized and seeded into wells of gelatin-coated 6-well plates with 5x104 cells in ES medium. After 24 hours, ES medium was replaced with differentiation medium containing LIF-free DMEM supplemented with 15% non-ES qualified fetal bovine serum and subjected to differentiation treatment sequentially as follows: 150 ng/ml Noggin for 1 day, 5 ng/ml BMP4 for the next 2 days, and then 3 µg/ml puromycin for 4 days. Thereafter, Nkx2.5¬ positive cells were selected by treatment with 3µg/ml puromycin every 2-3 days and examined by microscopy until day 14 for the presence of beating cardiomyocytes.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted after each treatment timepoint using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Methyl-seq was performed by Genomics, Epigenomics and Sequencing Core at the University of Cincinnati. SureSelect Methyl-Seq Target Enrichment System for mouse (Agilent, Santa Clara, CA) was used to prepare the sequencing library. A total of 1 μg high quality genomic DNA quantified by Qubit assay (Thermo Fisher, Waltham, MA) was sheared by Covaris S2 focused-ultrasonicator (Covaris, Woburn, MA) to about 200 bp under the recommended settings, and validated by 2100 Bioanalyzer (Agilent, Santa Clara, CA). Next, the DNA fragments were end prepared and ligated to the methylated adaptor. The size of the ligated libraries was then validated by Bioanalyzer, followed by hybridization with biotin-labeled RNA-baits to capture the regions where methylation is known to impact gene regulation. After hybridization, libraries were captured with streptavidin beads and bisulfite modified with EZ DNA Methylation-Gold kit (Zymo, Irvine, CA), and enriched with 8 cycles of PCR. These individually amplified libraries were labeled with unique indices by 6 cycles of PCR. After purification and size selection of the indexed libraries using AMPure XP beads (Beckman Coulter, Indianapolis, IN), the quality and quantity of the libraries were assessed by Bioanalyzer High Sensitivity DNA assay. To quantify the library concentration for the clustering generation, the libraries were qPCR analyzed by NEBNext Library Quant Kit (New England BioLabs, Ipswich, MA) using QuantStudio 5 Real-Time PCR System (Thermo Fisher, Waltham, MA). The sequencing was performed using Illumina Nextseq 550 sequencer under the sequencing setting of PE 2x150 bp to generate ~60M pass filter reads per sample.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
NextSeq 550 |
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| Description |
DNA submitted for methyl-seq
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| Data processing |
Quality control of sequencing reads was performed using FastQC
Reads were trimmed with trim_galore in pair-end mode, steps included: removing sequencing artifacts, trimmed 5’ or 3’ ends with low scores, removed adaptor contamination, and filtered low quality reads.
The cleaned reads were then aligned to the Mus musculus reference genome (Genome Reference Consortium 37, mm10) using bismark.
Bismark_methylation_extractorwere used to infer the methylation state of all cytosines in a CpG, CHH, or CHG context and to compute the percentage of methylation for each CpG site.
BedGraph files were generated using bismark_methylation_extractor module of bismark.
Genome_build: mm10
Supplementary_files_format_and_content: BedGraph files were generated using bismark_methylation_extractor module of bismark.
Supplementary_files_format_and_content: cov file - Bismark Coverage report with chromosome, start position, end position, methylation Percentage, count Methylated, and count Unmethylated.
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| Submission date |
Jul 16, 2020 |
| Last update date |
Jul 24, 2020 |
| Contact name |
Mario Medvedovic |
| Organization name |
University of Cincinnati
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| Department |
Department of Environmental Health
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| Lab |
Laboratory for Statistical Genomics and Systems Biology
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| Street address |
3223 Eden Av. ML 56
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| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45267-0056 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE154597 |
Dioxin Disrupts Dynamic DNA Methylation Patterns in Genes that Govern Cardiomyocyte Maturation [BiSulfite-seq] |
| GSE154599 |
Dioxin Disrupts Dynamic DNA Methylation Patterns in Genes that Govern Cardiomyocyte Maturation |
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| Relations |
| BioSample |
SAMN15562314 |
| SRA |
SRX8752304 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4675257_APM13B_bismark_bt2_pe.bedGraph.gz |
30.9 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM4675257_APM13B_bismark_bt2_pe.cov.gz |
31.9 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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