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| Status |
Public on May 19, 2011 |
| Title |
Input_RC_ChIP-seq |
| Sample type |
SRA |
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| Source name |
YMC RC phase
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: CEN.PK a SPT7-FLAG::Nat
chip antibody: none
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| Treatment protocol |
OX and RC phase cells were collected and crosslinked for 15min at room temperature with 1% formaldehyde and quenched with 150mM glycine before flash frozen
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| Growth protocol |
Yeast cells from SPT7-FLAG strain were cultured in fermentor under low glucose continuous growth condition described in Tu., et al 2005.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP protocol: About 50 OD cycling cells collected from the appropriate time point were rapidly fixed in 1% formaldehyde for 15 min in 50 ml total volume and then quenched using 3 ml 2.5 M glycine for 10 min. The fixed cells were pelleted and washed twice with buffer containing 100 mM NaCl, 10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine-HCl before freezing. The frozen pellet was resuspended in 0.45 ml ChIP lysis buffer (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate (DOC), 0.1% SDS, 1 mM PMSF, 10 µM leupeptin, 5 µM pepstatin A, Roche protease inhibitor cocktail) and lysed by bead beating. Lysate from 50 OD cells were split into two tubes each containing 280 µl lysate and sonicated for 16 cycles (30 sec on, 1 min off, high output) using a Bioruptor (Diagenode). The supernatant of the sonicated lysate was pre-cleared. 50 µl lysate was saved as input.
For ChIP with histone antibodies, 50 µl whole cell extract (WCE) was diluted 1:10 and used for each ChIP. For SAGA ChIP, 500 µl WCE and 2 µg Flag M2 antibody was used. After incubation overnight, 20 µl magnetic beads resuspended in ChIP lysis buffer was added and incubated for 1.5 h. Beads were washed twice with ChIP lysis buffer, once with DOC buffer (10 mM Tris-Cl pH 8.0, 0.25 M LiCl, 0.5% DOC, 1 mM EDTA) and once with TE. 60 µl of TES buffer (50 mM Tris-Cl pH 8.0,10 mM EDTA, 1% SDS) was added to resuspend the beads. Supernatant was collected after incubation at 65°C for 10 min. A second round of elution was performed and the eluates were combined. 150 µl TES was added to the input sample. Reverse crosslinking was performed for the ChIP and input sample by incubating for 6 hours at 65°C. An equal volume of TE containing 1.25 mg/ml proteinase K was added to the samples after reverse crosslinking and samples were incubated for 2 hours at 37°C. DNA was then purified with a QIAquick PCR purification kit.
Chromatin was sheared with Bioruptor and precleared before immunoprecipitation with histone or Flag antibody, ChIP-seq library construction was following protocol from http://bioinfo.mbb.yale.edu/array/Solexa_LibraryPrep_20080229ge.pdf
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Input DNA
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| Data processing |
Alignment: Sequence reads were obtained and mapped to the saccharomyces cerevisiae (build sacCer1, Oct, 2003) genomes using the bowtie.
Peaks: Peak detection was performed with CisGenome
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| Submission date |
Apr 20, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Ling Cai |
| E-mail(s) |
[email protected]
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| Organization name |
UT Southwestern Medical Center at Dallas
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| Street address |
5323 Harry Hines Boulevard
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platform ID |
GPL9134 |
| Series (1) |
| GSE28734 |
Genome wide H3K9ac and SAGA occupancy at OX growth phase and RC quiescent phase of yeast metabolic cycle |
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| Relations |
| SRA |
SRX058456 |
| BioSample |
SAMN00260254 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM711903.wig.gz |
1.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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