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Sample GSM1566734

Query DataSets for GSM1566734

Status

Public on Jun 17, 2015

Title

p65 ChIP-seq TNF 1

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.
hg19

Submission date

Dec 16, 2014

Last update date

May 15, 2019

Contact name

Susanne Mandrup

E-mail(s)

s.mandrup@bmb.sdu.dk

Phone

+45 6550 2340

Organization name

University of Southern Denmark