 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 15, 2011 |
| Title |
WT H3K4me3 on H3 exp1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
H3K4me3 (Abcam ab8580) ChIP DNA from WT (BY4741) yeast cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
antibody: H3K4me3
vendor: Abcam ab8580
|
| Growth protocol |
200ml of yeast cells were grown up to mid-log phase (o.d. 600 of 0.6-0.8) in YPD
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated with 1% formaldehyde directly added to the media at room temperature for 10 minutes. Formaldehyde was quenched with 0.125 M of glycine and cells were washed three times in cold water. Pellets (approximately 1.2x109 cells) were resuspended in 1.2 ml of lysis buffer (50mM HEPES-KOH pH 7.5, 140mM NaCl, 1% Triton X-100, 0.1% Na-deoxycholate, 1mM EDTA, 1mM PMSF, 30mM sodium butyrate, complete EDTA-free protease inhibitor cocktail, Roche), and split into three tubes. After adding one volume of glass beads to each sample, cells were disrupted using a bead beater (Disruptor Genie, Scientific Industries) at maximal speed for 2h at 4°C. After removing the glass beads, the lysates were transferred to fresh tubes and sonicated for 15 minutes (30 seconds ON, 30 seconds OFF) at high intensity in a Bioruptor (Diagenode) connected to a water cooler at 4°C. Lysates were clarified by centrifugation and pooled into a 2 ml tube. After addition of 800µl of fresh lysis buffer and mixing, lysates were clarified again by centrifugation. 4µl of lysate (1% of the whole cell extract, WCE) was kept aside as an input control. For immunoprecipitation, aliquots of 400µl were prepared for each ChIP and mixed with the appropriate antibody: either 5µl of anti-H3K4ac, 5µl of anti-H3-C (Abcam ab1791), 3µl of anti-H3K4me3 (Abcam ab8580) for at least 1h at 4°C. Before use, Dynabeads M-280 coupled to sheep anti-rabbit IgG (InVitrogen) (30µl per ChIP) were washed in lysis buffer containing 5 mg/ml bovine serum albumin. The beads were added to each lysate and incubated at 4°C for at least 1h. Beads were washed twice with lysis buffer, twice with lysis buffer containing 500mM NaCl, twice in wash buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 0.5% Nonidet-P40, 0.5% Na-deoxycholate, 1mM EDTA) and once in TE (10mM Tris-HCl pH 8.0, 1mM EDTA). To elute the DNA, 100µl of a 10% Chelex 100 (Biorad) suspension in water was added to the beads (as well as to the 1% WCE) and incubated at 100°C for 10 minutes. Tubes were then cooled and treated with 0.2 µg/µl ribonuclease A at 50°C for 30 min, followed by 0.2 µg/µl proteinase K at 50°C for 30 min. Samples were incubated again at 100°C for 10 min to inactivate the proteinase K and then cooled down and spun down to get rid of debris. 80µl of each supernatant was kept at ‑20°C for qPCR or ChIP-chip analysis.
|
| Label |
biotin
|
| Label protocol |
ChIP samples were amplified using random primers and Sequenase with the addition of dUTP following the protocol supplied from Affymetrix. Amplified samples (8μg of DNA) were enzymatically fragmented and labeled using the WT Double Stranded DNA Terminal Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments.
|
| |
|
| Channel 2 |
| Source name |
non modified H3 (Abcam 1791) ChIP DNA from WT (BY4741) yeast cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
antibody: H3
vendor: Abcam 1791
|
| Growth protocol |
200ml of yeast cells were grown up to mid-log phase (o.d. 600 of 0.6-0.8) in YPD
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated with 1% formaldehyde directly added to the media at room temperature for 10 minutes. Formaldehyde was quenched with 0.125 M of glycine and cells were washed three times in cold water. Pellets (approximately 1.2x109 cells) were resuspended in 1.2 ml of lysis buffer (50mM HEPES-KOH pH 7.5, 140mM NaCl, 1% Triton X-100, 0.1% Na-deoxycholate, 1mM EDTA, 1mM PMSF, 30mM sodium butyrate, complete EDTA-free protease inhibitor cocktail, Roche), and split into three tubes. After adding one volume of glass beads to each sample, cells were disrupted using a bead beater (Disruptor Genie, Scientific Industries) at maximal speed for 2h at 4°C. After removing the glass beads, the lysates were transferred to fresh tubes and sonicated for 15 minutes (30 seconds ON, 30 seconds OFF) at high intensity in a Bioruptor (Diagenode) connected to a water cooler at 4°C. Lysates were clarified by centrifugation and pooled into a 2 ml tube. After addition of 800µl of fresh lysis buffer and mixing, lysates were clarified again by centrifugation. 4µl of lysate (1% of the whole cell extract, WCE) was kept aside as an input control. For immunoprecipitation, aliquots of 400µl were prepared for each ChIP and mixed with the appropriate antibody: either 5µl of anti-H3K4ac, 5µl of anti-H3-C (Abcam ab1791), 3µl of anti-H3K4me3 (Abcam ab8580) for at least 1h at 4°C. Before use, Dynabeads M-280 coupled to sheep anti-rabbit IgG (InVitrogen) (30µl per ChIP) were washed in lysis buffer containing 5 mg/ml bovine serum albumin. The beads were added to each lysate and incubated at 4°C for at least 1h. Beads were washed twice with lysis buffer, twice with lysis buffer containing 500mM NaCl, twice in wash buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 0.5% Nonidet-P40, 0.5% Na-deoxycholate, 1mM EDTA) and once in TE (10mM Tris-HCl pH 8.0, 1mM EDTA). To elute the DNA, 100µl of a 10% Chelex 100 (Biorad) suspension in water was added to the beads (as well as to the 1% WCE) and incubated at 100°C for 10 minutes. Tubes were then cooled and treated with 0.2 µg/µl ribonuclease A at 50°C for 30 min, followed by 0.2 µg/µl proteinase K at 50°C for 30 min. Samples were incubated again at 100°C for 10 min to inactivate the proteinase K and then cooled down and spun down to get rid of debris. 80µl of each supernatant was kept at ‑20°C for qPCR or ChIP-chip analysis.
|
| Label |
biotin
|
| Label protocol |
ChIP samples were amplified using random primers and Sequenase with the addition of dUTP following the protocol supplied from Affymetrix. Amplified samples (8μg of DNA) were enzymatically fragmented and labeled using the WT Double Stranded DNA Terminal Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments.
|
| |
|
| |
| Hybridization protocol |
Approximately 7.5µg per sample of labeled/fragmented DNA was hybridized per array using the Affymetrix GeneChip Hybridization, Wash, and Strain kit. Hybridization was done using a GeneChip Hybridisation oven, washes were done in a GeneChip Fluidics Stations F450.
|
| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G with autoloader
|
| Data processing |
Data were processed with the Affymetrix Tiling Analysis Software - Version 1.1 using the Two sample comparison analysis in Intensities, exported in .BAR files, Signal scale was set to log2, and probe bandwidth was set to 200bp. PM values only were considered.
The corresponding RPT .txt files were converted to .WIG files for uploading into the UCSC Genome Browser and Galaxy for further analyses.
|
| |
|
| Submission date |
Feb 14, 2011 |
| Last update date |
Feb 15, 2011 |
| Contact name |
Benoit Guillemette |
| E-mail(s) |
benoit.guillemette@umontreal.ca
|
| Organization name |
Université de Montréal
|
| Department |
IRIC
|
| Lab |
Alain Verreault
|
| Street address |
2950, chemin de Polytechnique
|
| City |
Montréal |
| State/province |
Qc |
| ZIP/Postal code |
H3T 1J4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL7250 |
| Series (1) |
| GSE27307 |
H3 Lysine 4 Is Acetylated at Active Gene Promoters and Is Regulated by H3 Lysine 4 Methylation |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM675135_WT_H3K4me3_01.CEL.gz |
33.5 Mb |
(ftp)(http) |
CEL |
| GSM675135_WT_H3K4me3_02.CEL.gz |
33.6 Mb |
(ftp)(http) |
CEL |
| GSM675135_WT_H3K4me3_03.CEL.gz |
33.7 Mb |
(ftp)(http) |
CEL |
| GSM675135_WT_H3K4me3_on_H3.wig.gz |
14.6 Mb |
(ftp)(http) |
WIG |
| GSM675135_WT_H3_01_2.CEL.gz |
34.2 Mb |
(ftp)(http) |
CEL |
| GSM675135_WT_H3_02_2.CEL.gz |
33.7 Mb |
(ftp)(http) |
CEL |
| GSM675135_WT_H3_03_2.CEL.gz |
33.3 Mb |
(ftp)(http) |
CEL |
| Processed data provided as supplementary file |
|
|
|
|
 |
