|
| Status |
Public on Dec 01, 2007 |
| Title |
Rsc8-ChIP_Mock-After_replicate2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Chromatin Input derived from YBC1547 mock-after
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Yeast strain: YBC1547 (yHN4)
relevant genotype: RSC8-9myc
|
| Growth protocol |
Cells were grown in liquid YP containing 2% raffinose at 27 C until OD600 of 0.4Ð0.6. The 'before' aliquot was removed from the culture and galactose was added to the remaining culture to 2% final and incubated for 60 minutes. The culture was then placed at 37 C for 120 minutes. An aliquot of cells were removed and crosslinked in 1% formaldehyde for 30 minutes.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin preparation: The washed cell pellets were resuspended in 500 ul of lysis buffer (50 mM Tris-HCl pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.4 mM DTT, 1X protease inhibitors) and mechanically lysed using 0.7 mm zirconia beads in a mechanical bead beater with four 2 minute rounds. The samples were centrifuged at 20K x g for 5 minutes and the supernatant discarded. The chromatin containing pellet was resuspended in 500 ul of fresh lysis buffer and sonicated five times for 25 seconds each at 90% duty cycle at power setting 4.5. The solubilized chromatin was separated from insoluble material by centrifugation at 20K x g for 10 minutes. The protein concentration was determined by Bradford assay.
Input: An aliquot of 400 ug was reserved for input material. Input chromatin was incubated at 65 C overnight to reverse cross links. The DNA was purified using Qiagen PCR cleanup columns and eluted in 50 ul Elution Buffer. An aliquot of 2 ul was used in subsequent amplification and labeling procedures.
|
| Label |
Cy3
|
| Label protocol |
Round A Amplification: The DNA sample was diluted in 10 ul reaction containing 1X of Sequenase Buffer and 40 pM ChIP-A primer (5'-GTTTCCCAGTCACGATCNNNNNNNNN-3'), denatured at 94 C for 2 min, and rapid cooled to 8 C. To the reaction 5 nM dNTP, 75 nM DTT, 0.5 ug BSA, and 4 units of Sequenase enzyme (USB) was added for a final volume of 15 ul. The temperature of the reaction was slowly raised to 37 C and incubated for 8 minutes. The DNA products were denatured again by incubating at 94 C for 2 minutes, followed by rapid cooling to 8 C. An additional 4 units of Sequenase enzyme was added, the temperature slowly raised to 37C and incubated for 8 minutes. The Round A products were diluted with water to 25 ul final volume.
Round B Amplification: A 7.5 ul aliquot of round A products was used as template in a PCR reaction using 2.5 units of Platinum Taq (Invitrogen), 2 mM MgCl2, and 5 uM ChIP-B primer (5'-GTTTCCCAGTCACGATC-3'). The amplification consisted of 27 rounds of 30 seconds at 94 C, 30 seconds at 40 C, 30 seconds at 50 C, and 60 seconds at 72 C. The Round B DNA products were purified using a Qiagen PCR cleanup column and eluted in 50 ul Elution Buffer. An aliquot was checked by agarose electrophoresis and the concentration determined by OD260.
Round C labeling: An aliquot of Round B product containing 2 ug of DNA was used as template in the labeling reaction. The template DNA was diluted in 25 ul volume containing 1.2X New England Biolab Buffer 2 and 10 pM of ChIP-B primer. 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.
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| |
|
| Channel 2 |
| Source name |
Chromatin precipitated from YBC1547 mock-after replicate 2 using anti-Myc 9E11
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Yeast strain: YBC1547 (yHN4)
relevant genotype: RSC8-9myc
|
| Growth protocol |
Refer to Sample_growth_protocol_ch1.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin preparation: Refer to Sample_extract_protocol_ch1.
Immunoprecipitation: An aliquot of 400 ug was used for the immunoprecipitation. Extracts were incubated with ~40 ul of anti-mouse beads Dynabeads (Dynal Biotech) pre-bound with 1.7 ul of anti-Myc 9E11 in 500 ul of lysis buffer supplemented with 250 ug BSA Input chromatin overnight. Beads with captured immunocomplexes were washed sequentially with 1 ml of lysis buffer twice, lysis buffer adjusted to 500 mM NaCl once, LiCl wash buffer (10 mM Tris-HCl pH 8, 250 mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate, 1 mM EDTA) once, and TE (10 mM Tris-HCl pH 8, 1 mM EDTA) once. Immunocomplexes were released and cross links reversed by incubating the beads with 200 ul ChIP Elution Buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 1% SDS) at 65 C overnight. The DNA was purified using Qiagen PCR cleanup columns and eluted in 50 ul Elution Buffer. An aliquot of 7 ul was used in subsequent amplification and labeling procedures.
|
| 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 2 of 3 for Rsc8-9myc ChIP at mock-after conditions.
|
| 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 200 probes which had the lowest raw (un-processed) ratio values was used as the normalization list in this experiment. The rationalization is that these probes and their corresponding values should represent the majority of the genome which is not enriched for the occupancy of RSC. 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.
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| |
|
| 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 |
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