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| Status |
Public on Dec 12, 2014 |
| Title |
PPARgamma hMADS brite rep2, day 19 |
| Sample type |
SRA |
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| Source name |
human adipose-derived stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: hMADS-3
cell type: human adipose-derived stem cells
chip antibody: anti-PPARγ (H-100, sc-7196; Santa Cruz)
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| Treatment protocol |
Two days post confluence (day 0) hMADS cells were induced to differentiate in DMEM/Ham`s F12 (Lonza) supplemented with 10 μg/ml transferrin, 1 μM dexamethasone, 500 μM 3-isobutyl-1-methylxanthine (MIX), 0.85 μM insulin, and 0.2 nM T3. At day 3 of differentiation, MIX and dexamethasone were omitted from the media and 0.5 μM rosiglitazone was added until day 9 of differentiation and again from day 13-16 for induction of brite adipocytes. Control white adipocytes were treated with DMSO from day 13-16. From day 16 and forth the differentiation medium of both white and brite adipocytes was depleted of rosiglitazone. White and brite hMADS adipocytes were harvested on day 19, unless stated otherwise.
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| Growth protocol |
Human multipotent adipose-derived stem (hMADS-3) cells were cultured in DMEM (Lonza, low glucose) supplemented with 10% fetal bovine serum (Lonza), 10 mM Hepes, 2 mM L-glutamine (Lonza), penicillin (62.5 μg/ml), streptomycin (100 μg/ml), and 2.5 ng/ml hFGF2 (Peprotech).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP experiments were performed in mature hMADS adipocytes essentially as previously described (Siersbæk et al. 2012, MCB, 32: 3452-3463). For cross-linking of chromatin, 0.2 mM disuccinimidyl glutarate (DSG) was applied (Proteochem, Denver, CO) for 45 min followed by cross-linking using 1% formaldehyde for 10 min.
ChIP-seq libraries were constructed according to the manufacturer's instructions (Illumina) as described in (Nielsen R, Mandrup S, 2014 Genome-Wide Profiling of Transcription Factor Binding and Epigenetic Marks in Adipocytes by ChIP-seq. Methods in Enzymology 2014, Vol. 537, pp. 261-279).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Alignment:
Sequence reads were collapsed using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Bowtie (Langmead B, et al., Genome Biol. 10:R25) was used to align each collapsed library to the human reference genome (version hg19) with the following parameters: “-m 3 –best –strata”, with all other parameters default.
Peak finding and visualization:
Regions enriched for PPARγ signal in white and brite hMADS adipocytes were identified using HOMER (Heinz S, et al. 2010. Mol. Cell 38:576–589) with the “-factor” and “-size given” settings and a 0.1% FDR threshold. An additional stringency filter was applied, so only peaks >15 fold over the matching input control were kept for further analyses. Merged peak files were generated from the individual PPARγ peak files if the center of two or more peaks were within 250 bp. Peaks from these merged peak files were only included if having more than 30 tags per 10 M tags in a 400 bp window around the center of each merged peak. BedGraph files were created using HOMER for visualization of binding profiles.
Differential signal intensity in PPARγ binding sites and super-enhancers:
Differential signal intensity in PPARγ binding regions in white and brite hMADS adipocytes was determined using paired analyses with EdgeR (Robinson et al. 2010, Bioinformatics 26: 139-140). A FDR<0.1 was used to determine differential PPARγ occupancy in identified PPARγ peak regions, whereas a FDR<0.01 was applied to test PPARγ super-enhancers for differential H3K27ac occupancy.
Genome_build: hg19
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| Submission date |
Jul 23, 2014 |
| Last update date |
Feb 21, 2024 |
| Contact name |
Anne Loft |
| E-mail(s) |
anlo@bmb.sdu.dk
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| Organization name |
University of Southern Denmark
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| Department |
Biochemistry & Molecular Biology
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| Street address |
Campusvej 55
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| City |
Odense M |
| ZIP/Postal code |
5230 |
| Country |
Denmark |
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| Platform ID |
GPL18460 |
| Series (1) |
| GSE59703 |
Browning of human adipocytes requires KLF11 and reprogramming of PPARγ super-enhancers |
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| Relations |
| Reanalyzed by |
GSE256261 |
| BioSample |
SAMN02934492 |
| SRA |
SRX660086 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1443812_SM550_PPARG_Brite_rep2.bedGraph.gz |
57.2 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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