|
| Status |
Public on Nov 26, 2008 |
| Title |
ES cells_H2O2_H1AcK26-IP |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
mouse embryonic stem (ES) cells
|
| Organism |
Mus musculus |
| Characteristics |
C57BL/6x129Sv F1
Input fraction
|
| Treatment protocol |
1h with 2 mM H2O2
|
| Growth protocol |
standard growth conditions
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mouse embryonic stem (ES) cells were fixed in 1% formaldehyde for 20 min at 37 degree Celsius, cells were than lysed and processed using the Upstate Chromatin IP Kit following the manufacturer's instructions. Samples were sonicated 5 times for 10 s and subjected to 1h preclear and o/n IP using Rabbit anti Sir2 (Upstate), Rabbit Ig (Upstate) or Rabbit anti-acetyl H1K26 (Vaquero et al., Mol Cell 2004). Input and IP samples were amplified using LM-PCR following the NImblegen protocol.
|
| Label |
Cy3
|
| Label protocol |
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming per manufacturer's protocol. Input DNA was labeled with Cy3, IP DNA was labeled with Cy5 (http://www.nimblegen.com/products/lit/lit.html).
|
| |
|
| Channel 2 |
| Source name |
mouse embryonic stem (ES) cells
|
| Organism |
Mus musculus |
| Characteristics |
C57BL/6x129Sv F1
IP fraction
|
| Treatment protocol |
1h with 2 mM H2O2
|
| Growth protocol |
standard growth conditions
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mouse embryonic stem (ES) cells were fixed in 1% formaldehyde for 20 min at 37 degree Celsius, cells were than lysed and processed using the Upstate Chromatin IP Kit following the manufacturer's instructions. Samples were sonicated 5 times for 10 s and subjected to 1h preclear and o/n IP using Rabbit anti Sir2 (Upstate), Rabbit Ig (Upstate) or Rabbit anti-acetyl H1K26 (Vaquero et al., Mol Cell 2004). Input and IP samples were amplified using LM-PCR following the NImblegen protocol.
|
| Label |
Cy5
|
| Label protocol |
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming per manufacturer's protocol. Input DNA was labeled with Cy3, IP DNA was labeled with Cy5 (http://www.nimblegen.com/products/lit/lit.html).
|
| |
|
| |
| Hybridization protocol |
The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in a total volume of 35 ul using 2X Hybridization Buffer (composition is proprietary) and hybridization component A (composition is priorietary). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
|
| Scan protocol |
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| Description |
no additional information
|
| Data processing |
Arrays were processed using Nimblegens standard protocol for Nimblescan 2.4 ChIP data extraction.
|
| |
|
| Submission date |
Oct 16, 2008 |
| Last update date |
Nov 26, 2008 |
| Contact name |
Philipp Oberdoerffer |
| E-mail(s) |
philober@hms.harvard.edu
|
| Organization name |
Harvard Medical School
|
| Department |
Pathology
|
| Lab |
Sinclair
|
| Street address |
77 Avenue Louis Pasteur
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL7496 |
| Series (2) |
| GSE13075 |
SIRT1 and H1AcK26 promoter-association in mouse ES cells changes upon oxidative stress |
| GSE13121 |
SIRT1 redistribution on chromatin promotes genome stability but alters gene expression during aging |
|
