|
| Status |
Public on Oct 18, 2013 |
| Title |
RNA-seq Control rep2 |
| Sample type |
SRA |
| |
|
| Source name |
3T3-L1 differentiated untreated
|
| Organism |
Mus musculus |
| Characteristics |
cell line: 3T3-L1
treatment: differentiated untreated
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:
95-2_accepted_hits.bw: mm9
|
| |
|
| Submission date |
Feb 10, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Kinyui Alice LO |
| E-mail(s) |
lky@mit.edu
|
| 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 |
|
| Relations |
| Reanalyzed by |
GSE80797 |
| SRA |
SRX119577 |
| BioSample |
SAMN00788731 |
