|
| Status |
Public on Jun 12, 2008 |
| Title |
ES_E2f1 |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
E14 ES cells
Chromatin immunoprecipitation for E2f1 transcription factor
|
| Treatment protocol |
The ES cells were fixed in 1% formaldehyde for 10 minutes at room temperature. The formaldehyde was quenched using 0.1 M glycine before harvest for extract preparation.
|
| Growth protocol |
E14 mouse ES cells, cultured under feeder-free conditions were maintained in Dulbecco's Modified Eagle-Medium (DMEM, GIBCO), with 15% heat-inactivated ES qualified fetal bovine serum (FBS, GIBCO), 0.055 mM beta-mercaptoethanol (GIBCO), 2mM L-glutamine, 0.1 mM MEM non-essential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of LIF (Chemicon).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Whole cell extracts were sonicated to solubilize the chromatin. The chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using different antibodies. ChIP-enriched DNA from multiple ChIP experiments was pooled and quantified using PicoGreen dsDNA quantitation kit (Invitrogen). The ChIP-enriched DNA was processed for Solexa sequencing using ChIP-Seq Sample Prep Kit (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
05-379 (Upstate) antibody was used for ChIP.
|
| Data processing |
The raw images have been processed using the Solexa Pipeline and mapped to the reference genome (NCBI Build 36, mm8) using Eland software with maximal 2 mis-matches. The ChIP-seq data were processed in 3 steps: a) A peak-finding algorithm was applied to identify the candidate binding site, where the cut-off threshold of peak intensity was determined by false discovery rate approximated with a random model; b) The candidate binding sites were further filtered based on the criterion of 5-fold change in relative to negative control library (GFP); c) The cut-off threshold of peak intensity were refined by qPCR validation. The software for the ChIP-seq data processing is available at http://cmb.gis.a-star.edu.sg/ChIPSeq/tools.htm.
The final processed peak list is linked as a supplementary file at the foot of this record. The file contains Chromosome number, Peak_location (mm8), and Count (abundance). BED files are also available (mm8).
|
| |
|
| Submission date |
May 13, 2008 |
| Last update date |
May 15, 2019 |
| Contact name |
Chia-Lin Wei |
| E-mail(s) |
weicl@gis.a-star.edu.sg
|
| Phone |
65 64788074
|
| Fax |
65 64789059
|
| URL |
http://www.gis.a-star.edu.sg/internet/site/investigators.php?f=cv&user_id=9
|
| Organization name |
Genome Institute of Singapore
|
| Department |
Genome Technology & Biology
|
| Street address |
60 Biopolis Street #02-01
|
| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL9185 |
| Series (1) |
| GSE11431 |
Mapping of transcription factor binding sites in mouse embryonic stem cells |
|
| Relations |
| SRA |
SRX000541 |
| BioSample |
SAMN02195292 |
