|
| Status |
Public on Sep 23, 2010 |
| Title |
ach4_wt_1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
AcH4 ChIP DNA, wild type cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
yeast strain: BY4741
growth media: YPD
antibody: anti-acetylated histone H4 antibody
type: ChIP
|
| Treatment protocol |
Chromatin immunoprecipitation (ChIP) assays were performed as previously described (Li et al., 2007 Genes & development 21, 1422-1430), except 50 uL of protein G Dynabeads (Invitrogen) was used for the immunoprecipitation.
|
| Growth protocol |
All yeast strains were grown in 200 mL YPD cultures to OD600=1.0-1.2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP and input samples were amplified using double T7 Linear Amplification
|
| Label |
Cy5
|
| Label protocol |
For the labeling reactions, 4-6 ug amino allyl-incorporated aRNA in a 5 uL volume of 0.1M Carbonate Buffer, pH 8.7, was mixed with 5 uL (0.01 nmol) monofunctional NHS-ester Cy3 or Cy5 dye in DMSO (Sigma) and incubated at 22°C for 2 hours. Reactions were quenched with 5 uL 4M hydroxylamine at 22°C for 15 minutes, cleaned with an RNeasy MinElute Cleanup Kit (Qiagen), and the efficiency of dye incorporation measured using the Nanodrop 2000 spectrophotometer (Thermo Scientific). Samples with a label incorporation efficiency of 2-4% were used for micorarray hybridization.
|
| |
|
| Channel 2 |
| Source name |
AcH4 ChIP DNA, wild type cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
yeast strain: BY4741
growth media: YPD
antibody: none
type: input
|
| Treatment protocol |
Chromatin immunoprecipitation (ChIP) assays were performed as previously described (Li et al., 2007 Genes & development 21, 1422-1430), except 50 uL of protein G Dynabeads (Invitrogen) was used for the immunoprecipitation.
|
| Growth protocol |
All yeast strains were grown in 200 mL YPD cultures to OD600=1.0-1.2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP and input samples were amplified using double T7 Linear Amplification
|
| Label |
Cy3
|
| Label protocol |
For the labeling reactions, 4-6 ug amino allyl-incorporated aRNA in a 5 uL volume of 0.1M Carbonate Buffer, pH 8.7, was mixed with 5 uL (0.01 nmol) monofunctional NHS-ester Cy3 or Cy5 dye in DMSO (Sigma) and incubated at 22°C for 2 hours. Reactions were quenched with 5 uL 4M hydroxylamine at 22°C for 15 minutes, cleaned with an RNeasy MinElute Cleanup Kit (Qiagen), and the efficiency of dye incorporation measured using the Nanodrop 2000 spectrophotometer (Thermo Scientific). Samples with a label incorporation efficiency of 2-4% were used for micorarray hybridization.
|
| |
|
| |
| Hybridization protocol |
Input was labeled with Cy3 dye, while immunoprecipitated samples were labeled with Cy5 dye. Samples were combined 1:1 based on quantity (ng). Before hybridization, labeled aRNA samples were fragmented (Fragmentation Reagent Kit, Ambion) according to manufacturer’s instructions. The hybridization mixture was set up for the Agilent yeast 4x44K platform (Agilent Technologies) according to manufacturer’s instructions with the addition of 20 ug of T7 blocking oligo.
|
| Scan protocol |
Scanning was performed on the Agilent DNA Microarray Scanner (Agilent Technologies, Model#G2505B)
|
| Description |
Biological repeat 1 of 3, anti-acetylated histone H4 antibody (Upstate #06-866)
|
| Data processing |
Agilent Feature Extraction Software (v 10.5.1.1) was used to quantify images. Data was read into R and normalized within arrays using median normalization and between arrays using Aquantile normalization from the limma package (Smyth, 2005)
|
| |
|
| Submission date |
Aug 05, 2009 |
| Last update date |
Sep 21, 2010 |
| Contact name |
Samantha Gail Pattenden |
| Organization name |
Stowers Institute for Medical Research
|
| Lab |
Jerry L. Workman Lab
|
| Street address |
1000 E 50th Street
|
| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64110 |
| Country |
USA |
| |
|
| Platform ID |
GPL4131 |
| Series (1) |
| GSE17521 |
Features of cryptic promoters and their varied reliance on bromodomain-containing factors |
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