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| Status |
Public on Nov 17, 2013 |
| Title |
UG_Female_5 |
| Sample type |
SRA |
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| Source name |
Mouse Liver
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| Organism |
Mus musculus |
| Characteristics |
levels of exposure: 50 ug BPA/kg
gender: Female
origin: liver tissue from post-natal day 22 mouse
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| Treatment protocol |
To avoid effects associated with parity, virgin wild-type a/a dams, 6 weeks of age, were randomly assigned to one of three phytoestrogen free AIN-93G diets (diet 95092 with 7% corn oil substituted for 7% soybean oil; Harlan Teklad, Madison, WI): 1) standard diet; 2) standard diet supplemented with 50 µg BPA/kg diet; or 3) standard diet supplemented with 50 mg BPA/kg diet. All diet ingredients were supplied by Harlan Teklad except BPA, which was supplied by NTP (National Toxicology Program, Durham NC). Wild-type a/a dams were provided with their respective diet two weeks prior to mating with 8-week-old Avy/a males and housed in polycarbonate-free cages with ad libitum access to diet and BPA-free water. The dams remained on the assigned diets throughout pregnancy and lactation, after which offspring were sacrificed at post-natal day 22 (PND22). For this study, liver DNA from a subset of a/a wild-type animals was analyzed for full methylome characteristics: 1) standard diet (Ctr, n = 4 offspring); 2) 50 µg BPA/kg diet (UG, n = 4 offspring); 3) 50 mg BPA/kg diet (MG, n = 4 offspring).
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| Growth protocol |
Mice were obtained from a colony that has been maintained with sibling mating and forced heterozygosity for the viable yellow agouti (Avy) and non-agouti (a) alleles for over 220 generations, resulting in a genetically invariant background. Wild-type a/a dams were housed in polycarbonate-free cages with ad libitum access to diet and BPA-free water.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total genomic DNA was isolated from liver tissue using buffer ATL, proteinase K, and RNase A (Qiagen Inc., Valencia, CA), followed by phenol-chloroform extraction and ethanol precipitation. DNA quality and concentration was assessed using a ND1000 spectrophotometer (NanoDrop Technology, Wilmington, DL).
MethylPlex library synthesis and GC-enrichment service was obtained through a commercial service at Rubicon Genomics Inc., Ann Arbor, MI. Briefly, fifty nanograms of genomic DNA were digested with a proprietary cocktail of methylation-sensitive restriction enzymes and then amplified by PCR with universal primers to create a MethylPlex library that is enriched for methylated DNA. MethylPlex DNA was then subjected to additional enzymatic treatment to deplete all non-GC-rich DNA sequences, purified and amplified in a second round of PCR. After purification, amplification adaptors were removed by a restriction enzyme digest, and the purified products were directly incorporated into the Illumina genomic DNA sequencing sample preparation kit procedure (Illumina Inc., San Diego, CA) at the end repair step, skipping the nebulization process. An adenine base was then added to the purified end repaired products using Klenow exo (3’ to 5’ exo minus) enzyme. The reaction product was purified, ligated to Illumina adaptors with DNA ligase and resolved on a 2% agarose gel. Gel pieces were excised at 400 base pair positions, and the DNA was extracted using Qiagen gel extraction kit (Qiagen Inc., Valencia, CA). The purified MethylPlex library was analyzed by Bioanalyzer (Agilent Technologies, San Diego, CA) before subjecting it to flow cell generation, where 10 nM of library was used to prepare flowcells with approximately 30,000 clusters per lane, with the sequencing performed by the University of Michigan DNA Sequencing Core.
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| Library strategy |
MRE-Seq |
| Library source |
genomic |
| Library selection |
Restriction Digest |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
CpG enriched DNA library
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| Data processing |
The raw sequencing image data obtained by Illumina GAIIx using 80 cycles of single ends were analyzed by the Illumina analysis pipeline.
Around 30 million reads per sample (ranging between 27 to 37 million reads) were obtained, and 12 bp-long adapter sequences were trimmed for each sample. Approximately 70% of these were mapped uniquely to the mouse mm9 reference genome using Burrows-Wheeler Aligner (BWA) tool.
We adopted a tiered-based profiling pipeline to identify regions of altered methylation (RAMs) by examining the locus-specific genome-wide methylation patterns associated with BPA exposure levels. We scanned the entire genome using a window size of 100bp with a 50bp moving shift, which accounts for over 53 million windows for each sample. The genomic regions containing at least 10 reads in 25% of the samples (~ 4 million windows) were then subjected to edgeR analysis, which we used to test for differences in each exposure group.
The edgeR analysis using R software was run using the glmFit function, which uses a negative binomial generated linear model, and identified the regions with differential methylation in three different comparisons; the methylation levels from the control group (n=4) against the 50 µg BPA/kg diet group (Ctr vs. UG, n = 4), control group against 50 mg BPA/kg diet group (Ctr vs. MG, n = 4), and 50 µg BPA/kg diet group against 50 mg BPA/kg diet group (UG vs. MG).
Using MACS chip-seq tool (v1.4.0), the wig type files are generated for visualization.
Genome_build: mm9
Supplementary_files_format_and_content: WIG files generated from MACS peak tool showing methylation reads pileup
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| Submission date |
Apr 18, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jung Kim |
| E-mail(s) |
junghk@med.umich.edu
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| Organization name |
University of Michigan
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| Street address |
1400 E. Medical Center Drive
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE46183 |
Perinatal bisphenol A exposure promotes dose-dependent alterations of the mouse methylome |
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| Relations |
| BioSample |
SAMN02048956 |
| SRA |
SRX268465 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1125775_5d350_macsR14_treat_afterfiting_all.wig.gz |
137.0 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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