|
| Status |
Public on Mar 21, 2013 |
| Title |
Rad51_ChIP_HoChromosomeIII_MATalpha_DSB1hr_DeltaRad54_rep1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Input DNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
chip antibody: Input DNA
strain: JoR03
treatment: Addition of 2% Galactose (Gal-driven HO endonuclease expression) at 0hr
|
| Treatment protocol |
Addition of 2% Galactose (Gal-driven HO endonuclease expression) at 0hr
|
| Growth protocol |
YP lactate
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin extract preparation and chromatin immunoprecipitation were performed as described in (Kalocsay et al., 2009). Briefly, 200ml cultures of every time point were cross-linked by the addition of 1% (final concentration) formaldehyde for 16 min, and subsequent quenching with 375 mM (final concentration) glycine for at least 10 min. Cells were lysed using Silica beads, the chromatin was enriched by centrifugation and sonicated (water bath sonification: Bioruptor UCD-200, Diagenode) to shear the DNA to an average size of 250-500 bp. After RNase treatment and ligation of linker oligonucleotides, DNA was amplified two times for 14 PCR cycles (using the GenomePlex Whole Genome Amplification (WGA) and reamplifications kits (Sigma)), as described in the Farnham Lab protocol for WGA amplification of DNA (O'Geen et al., 2006). Subsequent labeling of input and IP samples (either Cy3 or Cy5), hybridization to custom-made high-density whole S. cerevisiae genome NimbleGen arrays, array scanning and raw data extraction was done by imaGenes (www.imaGenes-bio.de).
|
| Label |
Cy3
|
| Label protocol |
standard NimbleGen protocol
|
| |
|
| Channel 2 |
| Source name |
Rad51 ChIP DNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
chip antibody: Rad51
strain: JoR03
treatment: Addition of 2% Galactose (Gal-driven HO endonuclease expression) at 0hr
|
| Treatment protocol |
Addition of 2% Galactose (Gal-driven HO endonuclease expression) at 0hr
|
| Growth protocol |
YP lactate
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin extract preparation and chromatin immunoprecipitation were performed as described in (Kalocsay et al., 2009). Briefly, 200ml cultures of every time point were cross-linked by the addition of 1% (final concentration) formaldehyde for 16 min, and subsequent quenching with 375 mM (final concentration) glycine for at least 10 min. Cells were lysed using Silica beads, the chromatin was enriched by centrifugation and sonicated (water bath sonification: Bioruptor UCD-200, Diagenode) to shear the DNA to an average size of 250-500 bp. After RNase treatment and ligation of linker oligonucleotides, DNA was amplified two times for 14 PCR cycles (using the GenomePlex Whole Genome Amplification (WGA) and reamplifications kits (Sigma)), as described in the Farnham Lab protocol for WGA amplification of DNA (O'Geen et al., 2006). Subsequent labeling of input and IP samples (either Cy3 or Cy5), hybridization to custom-made high-density whole S. cerevisiae genome NimbleGen arrays, array scanning and raw data extraction was done by imaGenes (www.imaGenes-bio.de).
|
| Label |
Cy5
|
| Label protocol |
standard NimbleGen protocol
|
| |
|
| |
| Hybridization protocol |
standard NimbleGen protocol
|
| Scan protocol |
standard NimbleGen protocol
|
| Data processing |
Raw signals of all channels in all replicate arrays were normalized and log2 transformed using the 'vsn' package in R/Bioconductor as described ((Prestel et al., 2010) and Tobias Straub Epigenome project PROT43, http://www.epigenesys.eu/).
|
| |
|
| Submission date |
Mar 04, 2013 |
| Last update date |
Mar 21, 2013 |
| Contact name |
Jörg Renkawitz |
| E-mail(s) |
renkawit@biochem.mpg.de
|
| Organization name |
Max Planck Institute of Biochemistry
|
| Department |
Molecular Cell Biology
|
| Lab |
Stefan Jentsch
|
| Street address |
Am Klopferspitz 18
|
| City |
Martinsried/Munich |
| ZIP/Postal code |
82152 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16746 |
| Series (1) |
| GSE44844 |
Monitoring Homology Search during DNA Double-Strand Break Repair in vivo |
|
