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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 18, 2020 |
| Title |
vWAT, 20 weeks, H3K27ac, high fat diet, rep 2 |
| Sample type |
SRA |
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| Source name |
visceral adipose tissue
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: visceral (epididymal) white adipose tissue (VWAT)
age: 26 weeks old
diet: High fat diet (D12492)
time: 20 weeks
replicate: 2
chip antibody: H3K27ac (Abcam, ab4729)
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| Treatment protocol |
All mice were fed with a 10% in fat chow diet (D12450J, Research Diet) for two weeks. At 6 weeks of age a total of 170 and 180 mice were put on HFD (D12492, Research Diet) and chow diet (D12450J, Research Diet, respectively. Mice were sacrified after 1, 8 or 20 week of diet.
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| Growth protocol |
C57/BL6 male, 4-wk-old (at time of sacrifice), mice were housed in a 12 h light/12 h dark (LD) regimen with water and food available ad libitum. All mice were fed with a 10% in fat chow diet (D12450J, Research Diet) for two weeks. At 6 weeks of age they were either shifted to a high fat diet containing 60% in fat (D12492, Research Diet) or kept in control diet for 1, 8 and 20 weeks. At each time time point, 20 mice were sacrified using CO2. Subcutaneous and viceral adipose tissue were immediately collected, snap-frozen in liquid nitrogen and kept at −80°C for RNA extraction and chromatin preparation. For chromatin preparation, aliquats of 4 grams of adipose tissue were homogenized on dry ice and immediately crosslinked in 45ml of PBS containing 0.5% formaldehyde. All animal care and handling was performed according to the Swiss law for animal protection
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The chromatin samples collected from different mice at the same time point were pooled, frozen in liquid nitrogen and stored at -80˚C. Chromatin was subjected to immunoprecipitation with an anti-POLR2B (Santa Cruz Biotechnology, H-201) antibody as described (Le Martelot et al. 2012). Immuno-precipitated complexes were collected by adsorption to ten µl of protein-A-Sepharose beads that had been pre-blocked with 10 µg/ml of salmon sperm DNA and BSA at 4˚C overnight. The beads were washed with dialysis buffer and ChIP wash buffer, as described. The immuno-precipitated complexes were eluted with 50 mM NaHCO3, 1% SDS, 1mM EDTA and 50 mM of Tris pH8 at 65°C, adjusted to 125 mM NaCl, and incubated at 65°C overnight to reverse the cross-links. After successive treatments with 10 µg/ml of RNase A and 20 µg/ml of proteinase-K, the samples were extracted with a NucleoSpin® Kit (Machery Nagel). The DNA concentration was determined by fluorometry on the Qubit system (Invitrogen). 5-10 ng of DNA was used for the preparation of the library. Libraries for ultra-high throughput sequencing were prepared using Diagenode kit (C05010013) according to the manufacturer’s instruction and subjected PE sequencing in a HiSeq 2500.
Libraries for ultra-high throughput sequencing were prepared with the using Diagenode kit (C05010013) according to the manufacturer’s instruction.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
20V_K27AC_H2
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| Data processing |
Basecalls performed using CASAVA version 1.82
ChIP-seq 100 bases Single-ended reads were aligned to mouse genome Ensemble GRCm38 (mm10) by Elandv2e
Tags were filtered by the threshold of 5 mismatchs and ambiguous mapping
ChiP-Cor (https://ccg.vital-it.ch/chipseq/chip_cor.php) was used to analyze the correlation between 5’ and 3’ tags position and determined the average ChIP fragment length for each sample
The mm10 genome was divided into 500 bases long consecutive, non-overlapping bins to calculate the ChIP signal. For each bin, each sample has its own value of tag density which is the AUC (area under the curve) created by the tags pilled-up , strand-shifted by half of the fragment length produced by ChIP-Cor. The bin values were scaled by the total tags for each sample and 30 pseudo-counts were added to stabilize the variance of low scores. The same quantifications were done for all the INPUT samples. All the values were then log2 converted.
The log2 scaled bin values of each ChIP sample and of the corresponding time-tissue-treatment INPUT sample were used to compute ratio-mean distribution. This distribution was sectioned into 200 step-wise proportions along the mean axis. Smoothing function lowess with smoother-span 0.2 was applied for only the negative-ratio bin population of each step-wise proportion. From the predicted distribution of smooth function, the mirrored bins were selected on the positive-ratio population. Distribution function was applied for this positive-ratio population with the mean and standard deviation derived from smooth function. The p-values were adjusted for false discovery rate (Benjamini–Hochberg). All bins with adjusted p-value less than 0.05 were considered significant. Comparison was done for each replicates of ChIP sample over each replicate of INPUT sample, reapeated with shifted of half bin size (250 bases).
The significant bins in both replicates and both bin sets (non-shift and 250-shifted) were pooled into consistency analysis. A bin was considered consistently significant if it was found in both replicates, either in the same or shifted position. The final bin set was determined for each sample. All the collected bins for all time points, tissues, diet treatments and IP treatments were pooled and merged with maximum distant of 250–two bins separated by 250 bases would be merged–into significant regions.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files were generated from remaining tags and scaled by the total tag number for each sample
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| Submission date |
Jun 13, 2019 |
| Last update date |
Mar 18, 2020 |
| Contact name |
Federica Gilardi |
| E-mail(s) |
[email protected]
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| Organization name |
University of Lausanne
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| Department |
CIG - Center of Integrative Genomics
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| Street address |
Lausanne 1015
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| City |
Lausanne |
| State/province |
Vaud |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE132726 |
Systemic approaches reveal anti-adipogenic signals at the onset of obesity–related inflammation in white adipose tissue [ChIP-seq] |
| GSE132885 |
Systemic approaches reveal anti-adipogenic signals at the onset of obesity–related inflammation in white adipose tissue |
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| Relations |
| BioSample |
SAMN12056155 |
| SRA |
SRX6069732 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3890859_20V_K27AC_H2.bw |
375.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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