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Sample GSM1443827 Query DataSets for GSM1443827
Status Public on Dec 12, 2014
Title RNA hMADS, Scramble rep1, Day 16
Sample type SRA
Source name human adipose-derived stem cells
Organism Homo sapiens
Characteristics cell line: hMADS-3
cell type: human adipose-derived stem cells
lentiviral transfection: Mature hMADS adipocytes (day 10) were incubated with lentivirus expressing shRNA against Scramble for 24 hours.
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.
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).
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).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 1500
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 two independent replicates of scramble and KLF11 knockdown samples (FDR<0.1) was determined using paired analysis with DESeq2 (Love et al. 2014,
Genome_build: hg19
Submission date Jul 23, 2014
Last update date May 15, 2019
Contact name Anne Loft
Organization name University of Southern Denmark
Department Biochemistry & Molecular Biology
Street address Campusvej 55
City Odense M
ZIP/Postal code 5230
Country Denmark
Platform ID GPL18460
Series (1)
GSE59703 Browning of human adipocytes requires KLF11 and reprogramming of PPARγ super-enhancers
BioSample SAMN02934508
SRA SRX660101

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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