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Sample GSM1566735 Query DataSets for GSM1566735
Status Public on Jun 17, 2015
Title p65 ChIP-seq TNF 2
Sample type SRA
Source name Adipocyte
Organism Homo sapiens
Characteristics cell type: SGBS adipocyte D10
chip antibody: RELA (C-20, sc-372, Santa Cruz)
Treatment protocol Mature SGBS adipocyte D10 were treated with 10ng/ml TNF or vehicle for 90/60 min before harvest of total RNA or chromatin for ChIP-seq respectively. p65 knockdown was achieved by transduction of SGBS adipocytes at day 6 of differentiation with shRNA-expressing lentivira. For JQ1 experiments, vehicle and TNF treated adipocytes were cotreated with 500nM JQ1 or DMSO for 90 min.
Growth protocol SGBS cells were grown to confluence in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham’s supplemented with 10% fetal bovine serum, 33 µM biotin, 17 µM pantothenate, 100 µg/ml streptomycin, 62.5 µg/ml penicillin, 1ng/µl fibroblast growth factors (FGF) 1, and 90 µg/µl heparin. At two days postconfluency, SGBS cells were stimulated to differentiate with serum-free growth medium supplemented with 10 nM insulin, 200 pM triiodothyronine, 1 µM cortisol, 2 µM BRL 49653, 0.115 mg/ml MIX, 0.25 mmol/L DEX, and 0.01 mg/ml human transferrin. After 3 days, the medium was replaced with the differentiation medium without FGF1 and heparin, and after 6 days Rosiglitazone/BRL49653, MIX, and DEX was removed from the medium.
Extracted molecule genomic DNA
Extraction protocol ChIP experiments were performed according to standard protocol as described in (Siersbæk et al. 2012, MCB, 32: 3452-3463)
Libraries were constructed according to the manufacturer's instructions (Illumina) as described in (Nielsen R, Mandrup S, 2014, Methods in Enzymology 2014, Vol. 537, pp. 261-279).
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 1500
Data processing ChIP-seq reads were mapped to hg19 with STAR (Dobin, A., et al., Bioinformatics, 2013. 29(1): p. 15-21) specifying --alignIntronMax 1 to avoid potentially aligning across exon-exon junctions.
Regions enriched for p65 ChIP-seq signal in TNF treated adipocytes were identified using HOMER (Heinz S, et al. 2010. Mol. Cell 38:576 –589) with the “-factor” setting and a 0.1% FDR threshold. Merged peak files were generated from the individual p65 peak files if the center of two or more peaks were within 200 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 in both replicates of the TNF condition. BedGraph files were created using HOMER for visualization of binding profiles.
Submission date Dec 16, 2014
Last update date May 15, 2019
Contact name Susanne Mandrup
Phone +45 6550 2340
Organization name University of Southern Denmark
Department Biochemistry and Molecular Biology
Street address Campusvej 55
City Odense M
ZIP/Postal code 5230
Country Denmark
Platform ID GPL18460
Series (1)
GSE64233 Acute TNF-induced repression of cell identity genes is mediated by NFkB-directed redistribution of cofactors from super-enhancers
BioSample SAMN03266585
SRA SRX813775

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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