|
| Status |
Public on May 12, 2010 |
| Title |
MSC_EGFP-H3.3_rep2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
H3.3-EGFP ChIP DNA from mesenchymal stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Mesenchymal stem cells
tissue: Adipose tissue
chip antibody: EGFP (Clontech)
|
| Treatment protocol |
The mesenchymal stem cells were transfected with H3.3-EGFP and sorted by FACS 168 hrs post transfection.
|
| Growth protocol |
The mesenchymal stem cells were cultured for seven passages in DMEM/F12 containing 20% FCS and 1 % PenStrep.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Histone ChIPs were done as described (Dahl, Collas, 2007) using 0.5 A260 units chromatin per ChIP. In short, cells were cross-linked in suspension with 1% formaldehyde for 8 min and quenched with glycine. Lysis buffer was added to ~120 µl and samples incubated for 5 min on ice. Cells were sonicated for 14 x 30 sec on ice with 30 sec pauses using a probe sonicator to produce fragments of ~400-500 bp. The lysate was centrifuged, the supernatant collected, chromatin diluted to 0.5 A260 units and 100 µl was incubated with 2.4 μg antibody coupled to Dynabeads Protein A (Invitrogen) for 2 h at 4oC. ChIP material was washed as described [46]. Elution buffer containing 1 % SDS and 50 µg/ ml Proteinase K was added and samples incubated for 2 h at 68oC on a Thermomixer (Eppendorf); after a second extraction both supernatants were pooled. ChIP DNA was purified by phenol-chloroform isoamylalcohol extraction, ethanol-precipitated and dissolved in 50 μl TE buffer. EGFP and H3.3-EGFP were immunoprecipitated from transfected and sorted cells as above, using 50 µl Dynabeads Protein G (Invitrogen) and 5 µl Living Colors® anti-EGFP antibody and 2 A260 units of chromatin per ChIP. For ChIP-on-chip 5 µg (10 µl) RNase A (Roche) was added before ChIP DNA elution. ChIP DNA samples were dissolved in 10 μl MilliQ water.
|
| Label |
Cy5
|
| Label protocol |
Labeling done by Nimblegen as per normal service protocol
|
| |
|
| Channel 2 |
| Source name |
Input DNA from H3.3-EGFP transfected mesenchymal stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Mesenchymal stem cells
tissue: Adipose tissue
chip antibody: none (input DNA)
|
| Treatment protocol |
The mesenchymal stem cells were transfected with H3.3-EGFP and sorted by FACS 168 hrs post transfection.
|
| Growth protocol |
The mesenchymal stem cells were cultured for seven passages in DMEM/F12 containing 20% FCS and 1 % PenStrep.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Histone ChIPs were done as described (Dahl, Collas, 2007) using 0.5 A260 units chromatin per ChIP. In short, cells were cross-linked in suspension with 1% formaldehyde for 8 min and quenched with glycine. Lysis buffer was added to ~120 µl and samples incubated for 5 min on ice. Cells were sonicated for 14 x 30 sec on ice with 30 sec pauses using a probe sonicator to produce fragments of ~400-500 bp. The lysate was centrifuged, the supernatant collected, chromatin diluted to 0.5 A260 units and 100 µl was incubated with 2.4 μg antibody coupled to Dynabeads Protein A (Invitrogen) for 2 h at 4oC. ChIP material was washed as described [46]. Elution buffer containing 1 % SDS and 50 µg/ ml Proteinase K was added and samples incubated for 2 h at 68oC on a Thermomixer (Eppendorf); after a second extraction both supernatants were pooled. ChIP DNA was purified by phenol-chloroform isoamylalcohol extraction, ethanol-precipitated and dissolved in 50 μl TE buffer. EGFP and H3.3-EGFP were immunoprecipitated from transfected and sorted cells as above, using 50 µl Dynabeads Protein G (Invitrogen) and 5 µl Living Colors® anti-EGFP antibody and 2 A260 units of chromatin per ChIP. For ChIP-on-chip 5 µg (10 µl) RNase A (Roche) was added before ChIP DNA elution. ChIP DNA samples were dissolved in 10 μl MilliQ water.
|
| Label |
Cy3
|
| Label protocol |
Labeling done by Nimblegen as per normal service protocol
|
| |
|
| |
| Hybridization protocol |
Hybridization done by Nimblegen as per normal service protocol
|
| Scan protocol |
Scanning done by Nimblegen as per normal service protocol
|
| Description |
ChIP-chip H3.3 EGFP transfected mesenchymal stem cells, Antibody: EGFP (Clontech)
|
| Data processing |
log2 (ChIP/Input) with biweight mean of values subtracted
|
| |
|
| Submission date |
Jul 10, 2009 |
| Last update date |
May 12, 2010 |
| Contact name |
Philippe Collas |
| Organization name |
University of Oslo
|
| Department |
Institute of Basic Medical Sciences
|
| Street address |
PO Box 1112 Blindern
|
| City |
Oslo |
| ZIP/Postal code |
0317 |
| Country |
Norway |
| |
|
| Platform ID |
GPL7408 |
| Series (1) |
| GSE17053 |
Epigenetic environment of histone H3.3 on promoters revealed by integration of imaging, ChIP-chip, and MeDIP-chip data |
|
