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| Status |
Public on Dec 12, 2014 |
| Title |
DHS hMADS white, 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
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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 |
DNase I digestion was performed in mature hMADS adipocytes on ~10,000,000 million nuclei, essentially as previously described (Siersbæk et al., 2011, EMBO J, 30(8):1459-72), except that hMADS cells were harvested directly in Buffer A (15 mM Tris–HCl pH=8, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine) and 120 U/ml DNase I (Roche, cat# 11 284 932 001) was used for digestion.
DHS-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 |
DNase-Hypersensitivity |
| Library source |
genomic |
| Library selection |
DNAse |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Alignment:
Sequence reads were trimmed and 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 DHS 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 >10 fold (DHS) over the matching input control were kept for further analyses. Merged peak files were generated from the individual DHS 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 10 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.
Genome_build: hg19
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| Submission date |
Jul 23, 2014 |
| Last update date |
May 15, 2019 |
| 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 |
GPL10999 |
| Series (1) |
| GSE59703 |
Browning of human adipocytes requires KLF11 and reprogramming of PPARγ super-enhancers |
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| Relations |
| BioSample |
SAMN02934481 |
| SRA |
SRX660075 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1443801_WhiteDHS.ucsc.bedGraph.gz |
40.0 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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