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Sample GSM1261657 Query DataSets for GSM1261657
Status Public on Nov 09, 2013
Title RNA-seq_cotreat_rep3
Sample type SRA
Source name 3T3-L1_differentiated_Cotreat
Organism Mus musculus
Characteristics treatment: TNFalpha and hypoxia
cell line: 3T3-L1
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. Insulin resistance was induced with one of the following: 2.5 nM of TNF-α (R&D Systems) for 24 hr; incubation in a 1% oxygen chamber for 24 hr; treatment with both 2.5 nM TNF-α and 1% oxygen for 24 hr; 1 μM dexamethasone (Sigma-Aldrich) for 24 hr; 100 nM insulin (Sigma-Aldrich) in high-glucose (4.5 g/l) medium for 24 hr; or 800 μM of palmitate (dissolved in 70% ethanol) for 48 hr in DMEM containing 1% serum and 2% BSA.
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 or HiSeq.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Data processing Pair-end, 36-bp reads from each sample were aligned to the mouse genome (mm9 build) using TopHat (version 1.1.0). For the cotreatment, palmitate and palmiate control samples, single-end, 50-bp reads were aligned.
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
Genome Build: mm9
Submission date Nov 08, 2013
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 GPL13112
Series (1)
GSE35724 Comprehensive analysis of different in vitro insulin resistance models
Reanalyzed by GSE80797
BioSample SAMN02400871
SRA SRX375295

Supplementary file Size Download File type/resource 65.1 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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