|
| Status |
Public on Dec 01, 2007 |
| Title |
Mononucleosome_Degron_Before_replicate3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Genomic DNA derived from YBC2192
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Yeast strain: YBC2192
relevant genotype: Sth1td ubr1(delta)
|
| Growth protocol |
Cells were grown in liquid YP containing 2% raffinose and/or 2% galactose and 0.05 mM Cu(II)SO4.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were spheroplasted in spheroplastiing solution (1 M sorbitol, 10 mM Tris-HCl pH 7.4, 10 mM 2-Mercaptoethanol) using zymolyase at 37 C. Spheroplasting was stopped when the OD600 absorbance of an aliquot of cells in 1% SDS dropped to < 10% of the starting value. The cell suspension was adjusted to 0.5% SDS and 5 mM EDTA. Proteinase K was added to 50 ug/ml concentration and the suspension incubated at 65 C for overnight. The suspension was adjusted to 1.2 M potassium acetate and incubated in water/ice bath to precipitate proteins/SDS/salts. The samples were centrifuged at 20K x g for 10 minutes and the pellet discarded. Nucleic acids were then ethanol precipitated from the supernatant. The nucleic acids were resuspended in TE, RNase added, and incubated at 37 C for 10 minutes. The samples were then extracted once with phenol/chloroform, once with chloroform, and ethanol precipitated again. The yield of genomic DNA was measured by OD260. An aliquot of genomic DNA was then fragmented by three rounds of sonication for 25 seconds at 90% duty cycle at power setting 4. An aliquot of 2 ug of fragmented genomic DNA was used as template in the labeling reaction.
|
| Label |
Cy3
|
| Label protocol |
The template DNA was diluted in 25 ul volume containing 1.2X New England Biolab Buffer 2 and 1 ug of random primers (Invitrogen). The DNA was denatured by incubating at 94 C for 2 minutes and rapid cooled to 8 C. To the reaction 1 nM dATP, 1 nM dTTP, 1 nM dGTP, 0.5 nM dCTP, 1 nM labeled-dCTP, and 5 units of Exo-Klenow (New England Biolabs) was added for a final volume of 30 ul. The reaction was incubated at 37 C for 3 hours. The labeled products were purified using Qiagen PCR cleanup columns (not recommended) with an additional 35% guanidine HCl wash. DNA was eluted in 30 ul Elution Buffer. Incorporation and yield was checked using a Nano-Drop spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Mononucleosomal DNA from YBC2191 biological replicate 3 before Sth1 degradation.
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Yeast strain: YBC2191
relevant genotype: Sth1td pGAL1-UBR1
|
| Growth protocol |
Cells were grown in liquid YP containing 2% raffinose and 0.05 mM Cu(II)SO4 at 27 C until OD600 of 0.4Ð0.6. An aliquot of cells were removed and crosslinked in 1% formaldehyde for 5 minutes.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell pellets were resuspended in 1 ml of spheroplasting solution (1 M sorbitol, 10 mM Tris-HCl pH 7.4, 10 mM 2-Mercaptoethanol). The density of cell suspension was measured by absorbance at OD600 and an aliquot equal in cell amount to the other replicates (20-30 OD600 units) was taken. The aliquot of cells were pelleted and resuspended in 1 ml of fresh spheroplasting solution. Zymolyase was added to the samples and incubated at 37 C. Spheroplasting was stopped when the OD600 absorbance of an aliquot of cells in 1% SDS dropped to < 10% of the starting value. Spheroplasts were pelleted and resuspended in 500 ul MNase Digestion Buffer (1 M sorbitol, 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP-40). To the suspension 3.75 units of Micrococcal Nuclease (USB) was added, followed by incubation at 37 C for 25 minutes. The digestion was stopped by adjusting to 0.5% SDS and 5 mM EDTA. Proteinase K was added to 50 ug/ml concentration and incubated at 65 C overnight. The suspension was adjusted to 1.2 M potassium acetate and incubated in water/ice bath to precipitate proteins/SDS/salts. The samples were centrifuged at 20K x g for 10 minutes and the pellet discarded. Nucleic acids were then ethanol precipitated from the supernatant. The nucleic acids were resuspended in TE, RNase added, and incubated at 37 C for 10 minutes. Samples were loaded on a 1.2% agarose gel and DNA fragments separated by electrophoresis. Mononucleosome DNA was excised from the gel and purified using the Qiagen Gel Extraction Kit. DNA yield was measured by OD260. An aliquot of 0.5-1.0 ug DNA was used as template in the labeling reaction.
|
| Label |
Cy5
|
| Label protocol |
refer to Sample_label_protocol_ch1.
|
| |
|
| |
| Hybridization protocol |
An aliquot of 250 ng of labeled DNA for each sample (channel) was used. Eight experiments were prepared for simultaneous hybridization to each array on the 8 pack slide using the 8 pack gasket coverslip. Hybridizations and washes were performed according to manufacturer's recommended procedures (Agilent Technologies).
|
| Scan protocol |
Slides were scanned using an Agilent Technologies Scanner G2505B.
|
| Description |
Biological replicate 3 of 3 for mononucleosome DNA hybridization for the degron strain before Sth1 degradation.
|
| Data processing |
Data was extracted from the scanned images using Agilent Feature Extraction software version 8.5.1.1. The 22K 8 pack array format requires a minimum list of 50 pre-identified probes to use as median normalization controls. A list of 312 probes representing genomic loci previously reported as having low RSC occupancy was used as the normalization list in this experiment. Ratios between ChIP and Input channels were determined for probes that were not saturated or below background in either channel. Data from probes that had multiple genomic targets as determined by BLAST analysis were not included.
|
| |
|
| Submission date |
Aug 23, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Timothy J Parnell |
| E-mail(s) |
timothy.parnell@hci.utah.edu
|
| Organization name |
Huntsman Cancer Institute
|
| Street address |
2000 Circle of Hope
|
| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
| |
|
| Platform ID |
GPL5637 |
| Series (1) |
| GSE8862 |
RSC Regulates Nucleosome Positioning at Pol II Genes and Density at Pol III Genes |
|
