|
| Status |
Public on Dec 14, 2012 |
| Title |
3Flag-Sox17-ERT_Promoter 1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Input
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: embryonic stem cell
sample type: reference
|
| Treatment protocol |
ES cells were treated with 1 % formaldehyde/PBS for 10 min at room temperature. Cells were washed with PBS, collected and resuspended in swelling buffer [20 mM Hepes (pH 7.8), 1.5 mM MgCl2, 10 mM KCl, 0.1 % NP-40, and 1 mM DTT] by pipetting and then kept on ice for 10 min. After Dounce homogenizing 10-20 times, the cells were centrifuged and then the pellets were resuspended in RIPA buffer [20 mM Tis-HCl (pH 8.0), 1 mM EDTA, 140 mM NaCl, 1 % Triton X-100, 0.1 % SDS, and 0.1 % deoxycholic acid] containing protease inhibitors and sonicated into fragments with an average length of 0.3-0.5 kb. After centrifugation, the supernatants were subjected to IP with specific antibodies as previously described (Orlando et al., 1997)
|
| Growth protocol |
ES cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)-F12 (Sigma) supplemented with 1x MEM non-essential amino acids (Gibco-Invitrogen), 1x GlutaMAX-I (Gibco-Invitrogen), 20% knockout serum replacement (KSR) (Gibco-Invitrogen), 0.1 mM 2-mercaptoethanol (2-ME)(Sigma), and 5 ng/mL basic fibroblast growth factor (bFGF)(ReproCELL) either on irradiated MEF as feeder layers.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ES cells were subjected to ChIP assay using a Rybp antibody. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification as described previously (van Bakel et al., 2008).
|
| Label |
Cy3
|
| Label protocol |
Agilent mammalian ChIP-on-chip protocol (ver.9.0)
|
| |
|
| Channel 2 |
| Source name |
Flag-IP
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: embryonic stem cell
antibody: anti-FLAG
vendor: M2 Monoclonal
catalog/lot#: Code F 3165
|
| Treatment protocol |
ES cells were treated with 1 % formaldehyde/PBS for 10 min at room temperature. Cells were washed with PBS, collected and resuspended in swelling buffer [20 mM Hepes (pH 7.8), 1.5 mM MgCl2, 10 mM KCl, 0.1 % NP-40, and 1 mM DTT] by pipetting and then kept on ice for 10 min. After Dounce homogenizing 10-20 times, the cells were centrifuged and then the pellets were resuspended in RIPA buffer [20 mM Tis-HCl (pH 8.0), 1 mM EDTA, 140 mM NaCl, 1 % Triton X-100, 0.1 % SDS, and 0.1 % deoxycholic acid] containing protease inhibitors and sonicated into fragments with an average length of 0.3-0.5 kb. After centrifugation, the supernatants were subjected to IP with specific antibodies as previously described (Orlando et al., 1997)
|
| Growth protocol |
ES cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)-F12 (Sigma) supplemented with 1x MEM non-essential amino acids (Gibco-Invitrogen), 1x GlutaMAX-I (Gibco-Invitrogen), 20% knockout serum replacement (KSR) (Gibco-Invitrogen), 0.1 mM 2-mercaptoethanol (2-ME)(Sigma), and 5 ng/mL basic fibroblast growth factor (bFGF)(ReproCELL) either on irradiated MEF as feeder layers.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ES cells were subjected to ChIP assay using a Rybp antibody. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification as described previously (van Bakel et al., 2008).
|
| Label |
Cy5
|
| Label protocol |
Agilent mammalian ChIP-on-chip protocol (ver.9.0)
|
| |
|
| |
| Hybridization protocol |
Agilent mammalian ChIP-on-chip protocol (ver.9.0)
|
| Scan protocol |
ChIP-v1_10_Apr08, Agilent DNA Microarray Scanner G2505C, Agilent Scan Control (Ver. A.8.4.1)
|
| Description |
ChIP-on-chip data from human ES cells-derived CD34+CD43+CD45low cells (hemogenic endothelium-like cells) overexpressing 3xFLAG-Sox17-ERT
|
| Data processing |
Scanned images were quantified with Agilent Feature Extraction software under standard conditions. Signals were normalized using rank consistency filter algorithm.
|
| |
|
| Submission date |
Apr 23, 2012 |
| Last update date |
Dec 14, 2012 |
| Contact name |
Takaho A. Endo |
| E-mail(s) |
takaho.endo@riken.jp
|
| Organization name |
RIKEN
|
| Department |
IMS
|
| Lab |
Laboratory for Integrative Genomics
|
| Street address |
1-7-22 Suehiro, Tsurumi
|
| City |
Yokohama |
| State/province |
Kanagawa |
| ZIP/Postal code |
230-0045 |
| Country |
Japan |
| |
|
| Platform ID |
GPL14622 |
| Series (2) |
| GSE37528 |
ChIP-on-chip data from human ES cells-derived CD34+CD43+CD45low cells (hemogenic endothelium-like cells) overexpressing 3xFLAG-Sox17-ERT |
| GSE38156 |
Critical role of SOX17 in the hematopoietic development from human embryonic stem cells |
|
