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| Status |
Public on Oct 18, 2013 |
| Title |
DHS_seq high insulin |
| Sample type |
SRA |
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| Source name |
3T3-L1 differentiated HighIns
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| Organism |
Mus musculus |
| Characteristics |
cell line: 3T3-L1
treatment: differentiated HighIns
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. 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 |
genomic DNA |
| Extraction protocol |
Intact nuclei were isolated from differentiated 3T3-L1 using a nuclei isolation kit (Sigma: NUC201). Nuclei digestion and the DNA fraction isolation were done as described (Sabo, Kuehn et al. 2006). In brief, at least 30 million nuclei were used for each experiment. For each digestion, 50U/ml of DNase I (Promega RQ1 RNase-free DNase; lot number: 25308616) was used for digesting 10M cells at 37°C. DNase I enzymatic reaction was stopped after 2 min by a SDS- and EDTA-based stop buffer. Digested nuclei were incubated at 55°C overnight with proteinase K. Digested DNA was then extracted using phenol chloroform, and the “2-hit” DNA fragments were isolated using a sucrose gradient. Isolated DNA fragments were purified, subjected to the standard Illumina library preparation, and sequenced using Illumina GA II.
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| Library strategy |
DNase-Hypersensitivity |
| Library source |
genomic |
| Library selection |
DNAse |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Alignment: Sequence reads were obtained and mapped to NCBI37/mm9(July, 2007) genomes using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained.
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS version 1.4) algorithm (http://liulab.dfci.harvard.edu/MACS/), with insulin resistant samples as foreground and control sample as background
Genome Build:
DHS_seq_52-7.bed: mm9
DHS_seq_52-7_vs_52-1_1e-10_peaks.txt: mm9
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| Submission date |
Feb 10, 2012 |
| 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 |
GPL9250 |
| Series (1) |
| GSE35724 |
Comprehensive analysis of different in vitro insulin resistance models |
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| Relations |
| SRA |
SRX119596 |
| BioSample |
SAMN00788750 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM873982_DHS_seq_52-7.bed.gz |
206.2 Mb |
(ftp)(http) |
BED |
| GSM873982_DHS_seq_52-7_vs_52-1_1e-10_peaks.txt.gz |
95.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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