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| Status |
Public on Aug 01, 2013 |
| Title |
MED1 ChIP-seq, 3T3-L1 Day7 1h Rosi |
| Sample type |
SRA |
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| Source name |
3T3-L1 adipocytes (Day 7)
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| Organism |
Mus musculus |
| Characteristics |
cell type: 3T3-L1 adipocytes
chip antibody: anti-MED1 (sc-8998; Santa Cruz)
treatment: 1 μM rosiglitazone / 0.1% DMSO for 1 hour
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| Treatment protocol |
Day 7 3T3-L1 adipocytes were treated directly in incubators, by supplementing the culture media with either 1 μM rosiglitazone or 0.1% DMSO (vehicle). Chromatin was harvested after 1 hour of treatment.
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| Growth protocol |
3T3-L1 cells were maintained in DMEM supplemented with calf serum. Two days post confluency (day 0), the cells were induced to differentiate into adipocytes using a cocktail of adipogenic inducers (1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxantine, 1 μg/mL insulin, 10% FCS). Two days post induction, medium was changed to DMEM supplemented with FCS and insulin, and four days post induction the medium was changed to DMEM supplemented with FCS. The medium was changed a final time at day 6 post induction and all experiments were performed on day 7.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared from 149 mm culture dishes first by crosslinking in 1% formaldehyde in PBS (10 minutes, RT). Cross-linking was stopped by adding glycine to a final concentration of 1 M (10 minutes, RT). The cells were washed twice in ice-cold PBS, and harvested in ice-cold lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0,5% NP-40). Nuclei were subsequently released using a Dounce homogenizer, and crude nuclei were resuspended at 10^7 nuclei / mL in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and sonicated at high setting in a Bioruptor-Twin (Diagenode) at a volume of 300 μL in 1.5-mL tubes for 40 cycles of 30 seconds on and 30 seconds off. The chromatin IPs were performed as described in (Siersbæk MS, et al. 2012. Mol Cell Biol. 32:3452-3463).
ChIP-seq libraries were constructed according to the manufacturer's instructions (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Quality of all libraries were assessed using FastQC (Babraham Bioinformatics). Reads were trimmed and/or filtered using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).
Sequenced reads were aligned to the genome (mm9) using bowtie (Langmead B, et al. 2009. Genome Biol. 10:R25) with the options: -m 1 --best --strata
Reads from paired Rosi and DMSO libraries were combined into one library and peaks were called using HOMER (Heinz S, et al. 2010. Mol. Cell 38:576–589) with the default settings, and using sonicated input samples as control.
Binding intensity was scored using HOMER for each factor and each condition by counting tags aligning within detected peaks, and normalizing to a total read depth of 10^7 aligned reads.
Expression was scored by counting RNAPII ChIP-seq reads mapping within RefSeq gene bodies longer than 1 kb (omitting the initial 250 bp) using HOMER. Differential expression was called using edgeR (Robinson MD, et al. 2010. Bioinformatics 26:139-140)
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files created using HOMER for visualization of binding profiles in the UCSC genome browser. HOMER peak files with tag counts were generated using the HOMER annotatePeaks.pl script using the -d option. The files contain read counts of PPARγ, C/EBPα, MED1, and CBP ChIP-seq libraries (from both conditions). The read counts were normalized to a total read depth of 10^7 aligned reads.
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| Submission date |
Jul 31, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Susanne Mandrup |
| E-mail(s) |
s.mandrup@bmb.sdu.dk
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| Phone |
+45 6550 2340
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| Organization name |
University of Southern Denmark
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| Department |
Biochemistry and Molecular Biology
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| Street address |
Campusvej 55
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| City |
Odense M |
| ZIP/Postal code |
5230 |
| Country |
Denmark |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE49423 |
Acute Genome-Wide Effects of Rosiglitazone on PPARγ Transcriptional Networks in Adipocytes |
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| Relations |
| BioSample |
SAMN02298462 |
| SRA |
SRX330320 |
| Named Annotation |
GSM1199133_SM184_MED1_BRL.ucsc.bigWig |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1199133_SM184_MED1_BRL.ucsc.bigWig |
13.5 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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