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Sample GSM873966 Query DataSets for GSM873966
Status Public on Oct 18, 2013
Title RNA-seq TNF rep2
Sample type SRA
Source name 3T3-L1 differentiated TNF
Organism Mus musculus
Characteristics cell line: 3T3-L1
treatment: differentiated TNF
passage: less than 10
Treatment protocol Before starting any insulin resistance-inducing treatment, cells were washed with PBS and changed to serum-free, low-glucose (1g/L) DMEM with 0.5% BSA. For the TNFα treatment, cells were treated with 2.5µM of TNFα (R & D Systems) for 24h. For hypoxia treatments, cells were incubated in a 1% oxygen chamber (Powers, Millman et al. 2010) for 24h. Two types of hormonal stress were used to induce insulin resistance: dexamethasone treatment consisted of 24h incubation with 1µM dexamethasone (Sigma); high-insulin treatment consisted of 24h incubation of 100nM insulin (Sigma) in high glucose (4.5g/L) medium.
Growth protocol 3T3-L1 cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine serum (Invitrogen), 100 units/ml penicillin, and 100µg/ml streptomycin. Two days after confluence (Day 0), cells were induced to differentiate with DMEM containing 10% fetal bovine serum (FBS) , 1µM dexamethasone, 10 µg/ml insulin and 0.5mM 3-isobutyl-1-methyxanthine for 2 days. Cells were then incubated in DMEM containing 10% FBS and 10 µg/ml insulin for 2 more days. After Day 4, Cells were maintained in DMEM containing 10% FBS, with medium change every other day. Experiments were done on Day 8 to Day12 mature adipocytes.
Extracted molecule polyA RNA
Extraction protocol For each insulin resistance-inducing condition and the control condition, RNA-Seq experiments on biological triplicates were performed. 10ug of total RNA was used for each RNA-Seq library preparation according to the manufacturer's instructions (Illumina). Quality of RNA was verified using bioanalyzer (Agilent); only RNA with a RIN > 9 was used for RNA-Seq. cDNA libraries were prepared according to Illumina's instructions and sequenced by Illumina GAII.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer II
Data processing Pair-end, 36-bp reads from each sample were aligned to the mouse genome (mm9 build) using TopHat (version 1.1.0).
Differential expression: Differential expression of each insulin resistance model versus the control model was quantified using Cuffdiff (Trapnell, Williams et al. 2010) (version 1.0.3). Differentially expressed genes are those that have a log2 fold change of > 0.58 or < -0.58 and a q-value < 0.05 when compared to the control condition. We also required that the differentially expressed genes used for downstream analysis have a FPKM greater than 0.1 in the control condition.
Genome Build: mm9
Submission date Feb 10, 2012
Last update date May 15, 2019
Contact name Kinyui Alice LO
Phone 617-253-2042
Organization name MIT
Department Biological Engineering
Lab Fraenkel
Street address 77 Massachusetts Ave.
City Cambridge
State/province MA
ZIP/Postal code 02139
Country USA
Platform ID GPL9250
Series (1)
GSE35724 Comprehensive analysis of different in vitro insulin resistance models
Reanalyzed by GSE80797
SRA SRX119580
BioSample SAMN00788734

Supplementary file Size Download File type/resource 125.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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