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| Status |
Public on Oct 18, 2013 |
| Title |
ChIP-seq TNF p65 |
| Sample type |
SRA |
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| Source name |
3T3-L1 differentiated TNF
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| Organism |
Mus musculus |
| Characteristics |
cell line: 3T3-L1
treatment: differentiated TNF
passage: less than 10
antibody: p65
vendor/catalog/lot: abcam 7970, lot number GR51446-1
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| Treatment protocol |
For the TNFα treatment, cells were treated with 2.5µM of TNFα (R & D Systems) for 24h.
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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 |
Cells were lysed and chromatin was sonicated.The sonicated chromatin was incubated with antibody-coupled beads overnight. The beads were then washed and the chromatin was eluted, reverse-crosslinked at 65°C overnight, treated with RNase and Proteinase K and extracted with phenol-chloroform. The DNA was ethanol precipitated, purified and subjected to sequencing preparation using the Illumina sample preparation protocol. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments around 200-300bp were cut from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Sequencing was done using the Illumina Genome Analyzer II. Antibody used: p65 (abcam 7970, lot number GR51446-1), IgG (sc-2027, lot number C0411)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| 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 p65 ChIP-Sea samples as foreground and IgG sample as background
Genome Build:
ChIP_seq_p65.bed: mm9
ChIP_seq_p65_vs_IgG_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 |
SRX119597 |
| BioSample |
SAMN00788751 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM873983_ChIP_seq_p65.bed.gz |
165.7 Mb |
(ftp)(http) |
BED |
| GSM873983_ChIP_seq_p65_vs_IgG_peaks.txt.gz |
4.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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