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| Status |
Public on May 14, 2024 |
| Title |
beige_H3K27me3_ChIP |
| Sample type |
SRA |
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| Source name |
primary adipose stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: primary adipose stem cells
subject: 6
time: day15
treatment: beige adipogenic differentiation
chip antibody: H3K27me3
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| Treatment protocol |
For adipogenic differentiation, confluent cells were cultivated for 72h in DMEM/F12/10% fetal bovine serum without growth factors. For white adipose differentiation ASCs were induced by addition of 10 µg/ml insulin, 200 µM indomethacin, 0.5 µM 1-methyl-3 isobutylxanthine and 1 µM dexamethasone. Medium was renewed every 3 days until day 9. Cells were then maintained in DMEM/F12/10% fetal bovine serum and 10 µg/ml insulin until day 15. For beige adipose differentiation, media were supplemented with 1 µM rosiglitazone until day 15.
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| Growth protocol |
Human normal adipose tissue-derived mesenchymal stem cells were isolated from subcutaneous liposuction material. Cells were expanded in DMEM/F12 containing 10% fetal calf serum, and 20 ng/ml basic fibroblast growth factor (FGF2; Sigma-Aldrich F5392). Cells at passage 5-8 were used in three independent differentiations into the adipocyte lineage.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Day 15 differentiated white and beige samples were trypsinized, resuspended in HBSS, 0,5% BSA, and centrifuged 200g for 5 min to isolate floating mature adipocytes. Purified nuclei were fixed with 1 % formaldehyde; lysed for 10 min in ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, proteinase inhibitors, 1 mM PMSF, 20 mM Na Butyrate) and sonicated for 30 sec ON/OFF for 10 min in a Bioruptor®Pico (Diagenode) into ~200 base-pair fragments. After sedimentation, the supernatant was diluted 10 times and chromatin incubated with anti-H3K27ac (Diagenode c15410174), anti-H3K4me3 (Diagenode c15410003), anti-H3K27me3 (Diagenode c15410069), or anti-H3K4me1 (Diagenode c15410037) antibodies, each at 2.5 μg/106 cells, for 2 h at 4°C. ChIP samples were washed, cross-links reversed, and DNA eluted for 2 h at 68°C. DNA was purified using phenol-chloroform isoamylalcohol and dissolved in H2O.
Libraries were prepared using a Microplex kit (Diagenode) and sequenced on a Nextseq 500 or Novaseq (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads were filtered with fastp, aligned to the genome with bowtie2, and deduplicated.
Log ratio sample/input tracks were generated using deeptools bwCompare from RPGC normalized tracks created with bamCoverage.
Peaks were called from aligned reads using macs2 with default parameters for H3K4me3 and H3K27ac or with the --broad option for H3K27me3 and H3K4me1.
Assembly: hg38
Supplementary files format and content: Coverage tracks of log ratio sample/input (.grep.norm.bw). Raw coverage tracks for input samples are also provided (.bw).
Supplementary files format and content: Peaks called with macs2.
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| Submission date |
Feb 21, 2024 |
| Last update date |
May 14, 2024 |
| Contact name |
Philippe Collas |
| Organization name |
University of Oslo
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| Department |
Institute of Basic Medical Sciences
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| Street address |
PO Box 1112 Blindern
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| City |
Oslo |
| ZIP/Postal code |
0317 |
| Country |
Norway |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE256260 |
ChIP-seq of active and repressive histone post-translational modifications in human beige and white adipocytes. |
| GSE256262 |
Alternative isoform expression of key thermogenic genes in human beige adipocytes |
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| Relations |
| BioSample |
SAMN40020990 |
| SRA |
SRX23685587 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8092137_32-22575_S86.grep.norm.bw |
920.1 Mb |
(ftp)(http) |
BW |
| GSM8092137_32-22575_S86_peaks.broadPeak.gz |
2.4 Mb |
(ftp)(http) |
BROADPEAK |
SRA Run Selector |
| Raw data are available in SRA |
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