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| Status |
Public on Nov 09, 2013 |
| Title |
RNA-seq_cotreat_rep2 |
| Sample type |
SRA |
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| Source name |
3T3-L1_differentiated_Cotreat
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| Organism |
Mus musculus |
| Characteristics |
treatment: TNFalpha and hypoxia
cell line: 3T3-L1
passage: less than 10
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| 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.
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| 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.
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| 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| 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:
cotreatment4_accepted_hits.bw: mm9
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| Submission date |
Nov 08, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Kinyui Alice LO |
| E-mail(s) |
lky@mit.edu
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| Phone |
617-253-2042
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| Organization name |
MIT
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| Department |
Biological Engineering
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| Lab |
Fraenkel
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| Street address |
77 Massachusetts Ave.
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE35724 |
Comprehensive analysis of different in vitro insulin resistance models |
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| Relations |
| Reanalyzed by |
GSE80797 |
| BioSample |
SAMN02400868 |
| SRA |
SRX375294 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1261656_cotreatment4_accepted_hits.bw |
65.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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