|
| Status |
Public on Jun 08, 2012 |
| Title |
wtFLAGH3overH3 bio rep 1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
WT_FlagH3_IP
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: GHY2315
antibody: Anti-Flag M2
antibody vendor/catalog#: Sigma/A2220
genotype/variation: wild type
|
| Treatment protocol |
Cells were fixed in 1% formaldehyde.
|
| Growth protocol |
Strains transformed with pGAL-H4-FlagH3 were grown to ~1.2x107 cells/ml in SC-Ura media with raffinose as the carbon source. Cells were G1 arrested with alpha factor and Flag-H3/H4WT expression was induced by addition of galactose (2% final concentration) for 60 minutes.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were disrupted by bead beating and chromatin was sonicated using a Diagenode Bioruptor to obtain an average size of 500 bp. Chromatin was immunoprecipitated using 40 Microliter (1:2 slurry) Anti-Flag M2 Affinity gel (A2220; Sigma) or 1 ug of a rabbit polyclonal antibody against the C- terminus of H3 (ab1791; Abcam). Chelex 100 resin (BioRad) was added to the immunoprecipitated material and Input-DNA samples, and the suspensions were placed at 100°C for 10 minutes to reverse crosslinks. Samples were treated with proteinase K and DNA was recovered. The immunoprecipitated DNA was initially PCR amplified using random hexamer primers as described in [1]. The number of cycles used to amplify the samples was adjusted to between 28 and 37 so that there was equal amplification of DNA in the IP vs. Flag-tagged H3 and the IP vs. total H3 samples. Amplified DNA was visualized on a 1% agarose gel and checked for a visible smear of DNA between 500 and 1.2 kB.
|
| Label |
Cy5
|
| Label protocol |
DNA samples were amplified, with a starting amount of up to 75 ng for ChIP samples, using the DNA linear amplification method described previously [1]. 2.5 micrograms of aRNA produced from the linear amplification were labeled via the amino-allyl method as described on www.microarrays.org.
|
| |
|
| Channel 2 |
| Source name |
WT_TotalH3_IP
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: GHY2315
antibody: polyclonal antibody against the C- terminus of H3
antibody vendor/catalog#: Abcam/ab1791
genotype/variation: wild type
|
| Treatment protocol |
Cells were fixed in 1% formaldehyde.
|
| Growth protocol |
Strains transformed with pGAL-H4-FlagH3 were grown to ~1.2x107 cells/ml in SC-Ura media with raffinose as the carbon source. Cells were G1 arrested with alpha factor and Flag-H3/H4WT expression was induced by addition of galactose (2% final concentration) for 60 minutes.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were disrupted by bead beating and chromatin was sonicated using a Diagenode Bioruptor to obtain an average size of 500 bp. Chromatin was immunoprecipitated using 40 Microliter (1:2 slurry) Anti-Flag M2 Affinity gel (A2220; Sigma) or 1 ug of a rabbit polyclonal antibody against the C- terminus of H3 (ab1791; Abcam). Chelex 100 resin (BioRad) was added to the immunoprecipitated material and Input-DNA samples, and the suspensions were placed at 100°C for 10 minutes to reverse crosslinks. Samples were treated with proteinase K and DNA was recovered. The immunoprecipitated DNA was initially PCR amplified using random hexamer primers as described in [1]. The number of cycles used to amplify the samples was adjusted to between 28 and 37 so that there was equal amplification of DNA in the IP vs. Flag-tagged H3 and the IP vs. total H3 samples. Amplified DNA was visualized on a 1% agarose gel and checked for a visible smear of DNA between 500 and 1.2 kB.
|
| Label |
Cy3
|
| Label protocol |
DNA samples were amplified, with a starting amount of up to 75 ng for ChIP samples, using the DNA linear amplification method described previously [1]. 2.5 micrograms of aRNA produced from the linear amplification were labeled via the amino-allyl method as described on www.microarrays.org.
|
| |
|
| |
| Hybridization protocol |
Labeled probes were hybridized onto an Agilent yeast 4x44 whole genome array. The arrays were scanned at 5 micron resolution with the Agilent array scanner. Image analysis and data normalization were performed as previously described [1].
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000B fluorescent scanner at 5 micron resolution and image intensity data were extracted and analyzed with GenePix Pro 5.1 analysis software.
|
| Description |
biological replicate
|
| Data processing |
After background correction and removal of flagged values, data was block normalized by dilation and log base 2 ratios (FlagH3/TotalH3) were calculated to produce the values given in the data table.
|
| |
|
| Submission date |
Jun 05, 2012 |
| Last update date |
Jun 08, 2012 |
| Contact name |
Grant A Hartzog |
| E-mail(s) |
[email protected]
|
| Phone |
8314595826
|
| Fax |
8314593139
|
| Organization name |
UC Santa Cruz
|
| Department |
MCD Biology
|
| Street address |
349 Sinsheimer Labs
|
| City |
Santa Cruz |
| State/province |
CA |
| ZIP/Postal code |
95064 |
| Country |
USA |
| |
|
| Platform ID |
GPL4131 |
| Series (2) |
| GSE38491 |
A Key Role for Chd1 in Histone H3 Dynamics at the 3' Ends of Long Genes in Yeast (Flag-tagged histone H3 to total histone H3) |
| GSE38540 |
A Key Role for Chd1 in Histone H3 Dynamics at the 3' Ends of Long Genes in Yeast |
|
