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| Status |
Public on Dec 12, 2014 |
| Title |
RNA hMADS white rep 1, 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 |
total RNA |
| Extraction protocol |
Total RNA from hMADS cells was purified using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA was treated with Ribo-ZeroTM rRNA removal kit (Epicentre) and subjected to fragmentation and cDNA-synthesis using the TruSeq2 RNA kit (Illumina).
cDNA-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 |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Alignment:
RNA reads were aligned to the human reference genome (version hg19) using Bowtie2 (Langmead and Salzberg 2012, Nat Met 9: 357-359) with standard parameters. Splice-junction reads were handled by creation of a pseudo-splice genome, similar to the strategy utilized in RSEQTools (Habegger et al. 2011, Bioinformatics 27: 281-283). Reads were filtered post-alignment for a MAPQ score greater than or equal to 30. The number of exon reads for all RefSeq genes were counted using Subread (Liao et al. 2013, Nucleic Acids Res 41: e108).
Differential expression:
Differential expression between three independent replicates of white and brite hMADS adipocytes (FDR<0.05) was determined using paired analysis with DESeq2 (Love et al. 2014, http://dx.doi.org/10.1101/002832).
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 |
GPL18460 |
| Series (1) |
| GSE59703 |
Browning of human adipocytes requires KLF11 and reprogramming of PPARγ super-enhancers |
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| Relations |
| BioSample |
SAMN02934501 |
| SRA |
SRX660095 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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