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Sample GSM1566745

Query DataSets for GSM1566745

Status

Public on Jun 17, 2015

Title

RNA-seq shScr TNF 3

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

total RNA

Extraction protocol

Following Isol extraction and column purification of total RNA, ribosomal RNAs were removed using the Ribo-Zero Human/Mouse/Rat kit (Epicentre).
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

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1500

Data processing

RNA-seq reads were mapped to hg19 with STAR (Dobin, A., et al., Bioinformatics, 2013. 29(1): p. 15-21) using default parameters.
Total RNA-seq coverage within introns were quantified using the iRNA-seq pipeline (Madsen et al, Nucl. Acids Res. 43 (6): e40), to asses primary transcript levels. Log2 (Reads pr. kilobase pr. mio reads) are provided in RNA.KD.counts.txt
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