|
Status |
Public on Jun 12, 2008 |
Title |
ES_Smad1 |
Sample type |
SRA |
|
|
Source name |
Embryonic stem cells
|
Organism |
Mus musculus |
Characteristics |
E14 ES cells Chromatin immunoprecipitation for Smad1 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 |
sc-7965 (Santa Cruz) 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 |
SRX000548 |
BioSample |
SAMN02195291 |