|
| Status |
Public on Jan 01, 2010 |
| Title |
Cdk9-4 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Cdk9-IP genomic DNA from wild-type S. pombe carrying Cdk9-HA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: "361 h+ cdk9-3HA-kanR ade6-216 ura4-D18 leu1-32"
|
| Growth protocol |
A culture of 100ml was grown at 32°C in YES medium until mid-log phase. Formaldehyde was added to a final concentration of 3% and cells were crosslinked for 10 mins. The crosslink was stopped by adding 20 ml of glycin 2.5 M and cells were further incubated for 5 min in a shaker.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After formaldehyde crosslinking, cells were lysed using glass beads and chromain extracts were sonicated on a Branson Sonifier 450 to shear the chromatin to an average size of ~500 bp. A single pulldown was performed with magnetic beads coated with appropriate antibodies and following decrosslinking and LM-PCR amplification of purified IP DNA, the sample was labeled and hybridized on a Agilent 4x44k array.
|
| Label |
Cy5
|
| Label protocol |
Fluorophores were resuspended in 9 ul of NaHCO3 0.1 M and incubated with the pelleted DNA for 1 hour. The reaction was stopped by the addition of 4.5 ul of Hydroxylamine 4M and incubation for 15 minutes. The labelled samples were purified using the Qiagen PCR purification kit.
|
| |
|
| Channel 2 |
| Source name |
Input genomic DNA from wild-type S. pombe carrying Cdk9-HA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: "361 h+ cdk9-3HA-kanR ade6-216 ura4-D18 leu1-32"
|
| Growth protocol |
A culture of 100ml was grown at 32°C in YES medium until mid-log phase. Formaldehyde was added to a final concentration of 3% and cells were crosslinked for 10 mins. The crosslink was stopped by adding 20 ml of glycin 2.5 M and cells were further incubated for 5 min in a shaker.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After formaldehyde crosslinking, cells were lysed using glass beads and chromain extracts were sonicated on a Branson Sonifier 450 to shear the chromatin to an average size of ~500 bp. A small fraction of the chromatin extract was decrosslinked and following LM-PCR amplification of purified input DNA, the sample was labeled and hybridized on an Agilent 4x44k array.
|
| Label |
Cy3
|
| Label protocol |
Fluorophores were resuspended in 9 ul of NaHCO3 0.1 M and incubated with the pelleted DNA for 1 hour. The reaction was stopped by the addition of 4.5 ul of Hydroxylamine 4M and incubation for 15 minutes. The labelled samples were purified using the Qiagen PCR purification kit.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized according to standard Agilent protocols.
|
| Scan protocol |
Microarrays were scanned using a GenePix 4000B laser scanner (Axon Instrument)
|
| Description |
Cdk9-IP vs. Input genomic DNA in a wild-type background, Replicate 4
|
| Data processing |
Spot quantification was carried out using GenePix (Axon Instruments). Following quantification, the ChIP microarray data was lowess-normalized and a binding ratio (ChIP enrichment) was calculated for each microarray feature by dividing the signal intensity of the immunoprecipitated DNA sample by that of the background control (input DNA).
|
| |
|
| Submission date |
Jun 08, 2009 |
| Last update date |
Nov 07, 2012 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
harm.vanbakel@mssm.edu
|
| Organization name |
Mount Sinai School of Medicine
|
| Department |
Genetics and Genomic Sciences
|
| Lab |
Bakel Lab
|
| Street address |
One Gustave L. Levy Place, Box 1498
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL16218 |
| Series (1) |
| GSE16498 |
A gene-specific requirement of RNA polymerase II CTD phosphorylation on serine 2 for sexual differentiation in fission yeast |
|
