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| Status |
Public on Apr 17, 2014 |
| Title |
MED1 ChIP-seq, 3T3-L1 4h, Scr_4h_exp2_MED1 |
| Sample type |
SRA |
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| Source name |
3T3-L1 pre-adipocytes (4h)
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| Organism |
Mus musculus |
| Characteristics |
cell line: 3T3-L1
cell type: pre-adipocytes (4hr after induction)
transfected with: Scr. Lentivirus expressing shRNA against a scrambled sequence
chip antibody: MED1 (M-255)
chip antibody vendor: Santa Cruz
chip antibody cat. #: sc-8998
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| Treatment protocol |
3T3-L1 pre-adipocytes (day 0) were induced to differentiate using a cocktail of adipogenic inducers (1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxantine, 1 μg/mL insulin, 10% FCS). Cells were harvested four hours after induction.
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| Growth protocol |
3T3-L1 pre-adipocytes were maintained in DMEM supplemented with 10% calf serum. Two days post confluency (day 0), the cells were induced to differentiate using a cocktail of adipogenic inducers (1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxantine, 1 μg/mL insulin, 10% FCS).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq libraries were constructed according to the manufacturer's instructions (Illumina) as described in (Nielsen R, Mandrup S, 2014 Genome-Wide Profiling of Transcription Factor Binding and Epigenetic Marks in Adipocytes by ChIP-seq. Methods in Enzymology 2014, Vol. 537, pp. 261-279).
ChIP-seq: Chromatin was prepared from 150 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 0.125 M (10 minutes, RT). The cells were washed twice in ice-cold PBS, and harvested in ice-cold lysis buffer (0.1% SDS, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM Tris pH=8) and sonicated at high setting in a Bioruptor-Twin (Diagenode) at a volume of 1.5 ml in 15-mL tubes for 40 cycles of 30 seconds on and 30 seconds off. The chromatin IPs were performed as described in (Nielsen R, Mandrup S, 2014 Genome-Wide Profiling of Transcription Factor Binding and Epigenetic Marks in Adipocytes by ChIP-seq. Methods in Enzymology 2014, Vol. 537, pp. 261-279). Chromatin used for ChIP-seq of p300 and MED1 was also cross-linked in 2 mM disuccinimidyl glutarate (DSG) for 45 minutes at RT prior to cross-linking by formaldehyde.
RNA-seq: RNA was purified using TRIzol (Invitrogen) according to the manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Quality of all libraries were assessed using FastQC (Babraham Bioinformatics). Reads were trimmed using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).
ChIP-seq reads were aligned to the genome (mm9) using Bowtie (Langmead B, et al., Genome Biol. 10:R25) with the following parameters: –best –strata -m 3, with all other parameters set at default. Only one read was kept at locations where multiple reads align to the exact same position. 4sU-RNA-seq reads were aligned to the mm9 genome and to a pseudo genome containing all possible exon-exon junctions for all genes using Bowtie with the following parameters: --best --strata –m 1. Mapped tags were subsequently combined.
Regions enriched for transcription factor binding were identified using HOMER (Heinz S, et al. 2010. Mol. Cell 38:576 –589). Peaks were called for each transcription factor if they were four times enriched relative to the local background (20 kb around the peak), four times enriched relative to a 3T3-L1 input control sample (Siersbæk et al., 2011, EMBO J, 30, 1459-1472), and significant at a false discovery rate of 0.01%. A master set of peaks containing all the identified peaks for all transcription factors (if the center of two or more peaks were within 200 bp, the peaks were merged) was defined. All peaks from this master set containing at least 20 tags per 10M tags in a 250 bp window around the center of each peak for a given transcription factor were called as high-confidence transcription factor binding sites.
Genome_build: mm9
Supplementary_files_format_and_content: BedGraph files were created using HOMER for visualization of binding profiles and expression data in the UCSC genome browser.
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| Submission date |
Apr 17, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Susanne Mandrup |
| E-mail(s) |
[email protected]
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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 |
GPL18480 |
| Series (1) |
| GSE56872 |
Transcription factor cooperativity in early adipogenic hotspots and super-enhancers |
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| Relations |
| BioSample |
SAMN02729247 |
| SRA |
SRX520205 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1370460_SM623_Scr_4h_exp2_MED1.TD.bedgraph.gz |
52.2 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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