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Series GSE9265 Query DataSets for GSE9265
Status Public on Nov 15, 2007
Title Whole genome recruitment of Rpb3 and Rpb4 in S.cerevesiae
Organism Saccharomyces cerevisiae
Experiment type Genome binding/occupancy profiling by genome tiling array
Summary ChIP on chip microarray experiment to decipher Rpb3 and Rpb4 recruitment in S.cerevesiae genome.
Keywords: ChIP-chip
Overall design TAP- tagged Rpb3 and Rpb4 strains were cultured in 100 ml YPD medium at 25°C till log phase (O. D. 600 ~ 0.8). Cells were crosslinked with formaldehyde to a final concentration of 1% for 20 minutes. Cells were collected by centrifugation, washed three times with 40 ml of ice-cold Tris-buffered saline buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl), and resuspended in 800ul of lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, Protease inhibitor cocktail supplied by Roche). Yeast cells were disrupted by shaking for 30 sec for 20 times in the presence of glass beads (diameter, 0.5 mm) using vortexer. Glass beads were removed, and samples were sonicated five times for 15 sec at 20% duty cycle on a sonicator in order to shear chromatin DNA into fragments of average size ~400- 500 bp. Samples were centrifuged 10 min at maximum speed in a micro centrifuge, and the supernatant (from now on referred to as whole-cell extract) was used as input material for immunoprecipitation. Two hundred microliters of whole-cell extract was incubated with Rabbit IgG agarose beads (Sigma) for 3 hrs at 4°C with agitation. Beads were washed twice with 1 ml of lysis buffer, twice with 1 ml of lysis buffer plus 500 mM NaCl, twice with 1 ml of wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1 mM EDTA) and once with 1 ml of Tris-EDTA (TE; 10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Bound material was eluted from the beads by resuspending beads in 100ul of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS) and incubating 10 min at 65°C with occasional agitation. Samples were centrifuged briefly and the cross-linking was reversed by incubating overnight at 65°C. Samples were treated with proteinase K and RNase A, extracted twice with phenol, extracted once with chloroform, precipitated with ethanol, and resuspended in 30ul of TE. These samples were further purified with Qiagen PCR product purification Kit.
Web link
Contributor(s) Sadhale PP, Verma Gaur J
Citation(s) 18441121
Submission date Oct 09, 2007
Last update date Feb 15, 2018
Contact name madavan vasudevan
Phone +91-9845093355
Organization name Theomics International Private Limited
Street address Kasturi Nagar
City Bangalore
State/province Karnataka
ZIP/Postal code 560043
Country India
Platforms (1)
GPL4131 Agilent-014810 Yeast Whole Genome ChIP-on-Chip Microarray 4x44K (G4493A)
Samples (4)
GSM234991 Whole genome recruitment of Rpb3 in S.cerevesiae
GSM235050 Whole genome recruitment of Rpb3 in S.cerevesiae Replicate
GSM235051 Whole genome recruitment of Rpb4 in S.cerevesiae
BioProject PRJNA102895

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE9265_RAW.tar 18.1 Mb (http)(custom) TAR (of TSV)
Processed data included within Sample table

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