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Sample GSM1566739

Query DataSets for GSM1566739

Status

Public on Jun 17, 2015

Title

DHS-seq TNF

Sample type

SRA

Source name

Adipocyte

Organism

Homo sapiens

Characteristics

cell type: SGBS adipocyte D10

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

DHS-seq was performed on ~10 mill. nuclei essentially as described in (Siersbaek et al. 2011).
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

DNase-Hypersensitivity

Library source

genomic

Library selection

DNAse

Instrument model

Illumina HiSeq 1500

Data processing

Library strategy: DHS-seq
Paired-end DHS-seq reads were mapped to hg19 with STAR (Dobin, A., et al., Bioinformatics, 2013. 29(1): p. 15-21) using default paired-end parameters. Prior to data processing, aligned paired-end sequencing reads were processed by unix awk commands to convert paired-end reads to single-end reads.
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