|
Status |
Public on Nov 26, 2008 |
Title |
ES cells_untreated_Rabbit-Ig-IP |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
mouse embryonic stem (ES) cells
|
Organism |
Mus musculus |
Characteristics |
C57BL/6x129Sv F1 Input fraction
|
Treatment protocol |
no treatment
|
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 |
no treatment
|
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 |
|