|
| Status |
Public on Nov 01, 2008 |
| Title |
ChIP-chip time-course of H3K4me3 during meiosis (WT, t=1h) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
IP of H3K4me3 (t=1h in meiosis)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain ORD7339
t=1h after meiosis induction
|
| Growth protocol |
For sporulation, cells were grown in rich medium (YPD) for 24h, then transferred into SPS pre-sporulation medium and grown overnight to ~4.107 cells/ml. Cells were then harvested by centrifugation, washed with one volume 1% potassium acetate and resuspended into sporulation medium (1% potassium acetate, 1/1000 polypropylene glycol 2000, 20µg/ml uracil) at a density of 2.107 cells/ml (t=1h).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
20ml (4*108 cells) were crosslinked with 1% freshly prepared formaldehyde for 15 min at room temperature, then the reaction was stopped by the addition of 125mM glycin for 5 min. Cells were washed twice with cold TBS, and then chromatin immunoprecipitation was performed as described (Robine et al., 2007), using 3µl of rabbit polyclonal anti-triMe H3K4 (Abcam #Ab8580) and 30 µl protein G magnetic beads suspension (Dynal).
|
| Label |
Cy5
|
| Label protocol |
two-thirds of the immunoprecipitated DNA was amplified by random primer extension followed by PCR amplification, incorporating amino-allyl dUTP for subsequent dye coupling
|
| |
|
| Channel 2 |
| Source name |
Whole-cell extract
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain ORD7339
t=1h after meiosis induction
|
| Growth protocol |
For sporulation, cells were grown in rich medium (YPD) for 24h, then transferred into SPS pre-sporulation medium and grown overnight to ~4.107 cells/ml. Cells were then harvested by centrifugation, washed with one volume 1% potassium acetate and resuspended into sporulation medium (1% potassium acetate, 1/1000 polypropylene glycol 2000, 20µg/ml uracil) at a density of 2.107 cells/ml (t=1h).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
20ml (4*108 cells) were crosslinked with 1% freshly prepared formaldehyde for 15 min at room temperature, then the reaction was stopped by the addition of 125mM glycin for 5 min. Cells were washed twice with cold TBS and lyzed in ChIP lysis buffer.
|
| Label |
Cy3
|
| Label protocol |
1/70 of the DNA from the whole-cell extract was amplified by random primer extension followed by PCR amplification, incorporating amino-allyl dUTP for subsequent dye coupling
|
| |
|
| |
| Hybridization protocol |
16 hours at 65°C in the 1X hybridization buffer supplied by Agilent
|
| Scan protocol |
Axon 4000B scanner. GenePix5.1 software
|
| Description |
Time-course of the trimethylation of the Lysine4 of Histone H3 during meiosis
|
| Data processing |
Log2Ratios of background-corrected Intensities, spatial bias normalized with MANOR (Neuvial et al. 2006 Bioinformatics) mean fitted to 0. Then, we calculated for each time-point the average value of the ratios for probes located between 0 and 500 bp (for trimethylation) of all the ORFs present on the array. Next, we calculated the average value of this peak during the whole time-course, and normalized the ratios of each array such that each array has now this average peak value. Finally, we averaged the processed values of the two replicates.
|
| |
|
| Submission date |
Mar 14, 2008 |
| Last update date |
Aug 22, 2008 |
| Contact name |
Nicolas Robine |
| Organization name |
New York Genome Center
|
| Department |
Bioinformatics
|
| Lab |
Bioinformatics
|
| Street address |
101 Avenue of the Americas
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10013 |
| Country |
USA |
| |
|
| Platform ID |
GPL4131 |
| Series (2) |
| GSE10840 |
Meiotic time course of the trimethylation of the Lysine 4 of Histone H3 (H3K4me3) |
| GSE11004 |
Transcriptomic regulation and methylation of the Lysine4 of Histone H3 during meiosis |
|
