Postreplicative recruitment of Cohesin to double-strand breaks
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE1905 | GEO | 2004/12/22
SECONDARY ACCESSION(S): PRJNA90969
REPOSITORIES: GEO
Similar Datasets
Project description:This series is a supplementary data set for the manuscript titled "Postreplicative recruitment of Cohesin to double-strand breaks is required for DNA repair" All data shown in the paper plus duplicate experiments were registered (15 ChIP-chip data). Experimental design: 1. Type of experiment: ChIP (Chromatin immunoprecipitation) analysis hybridized to high density oligonucleotide arrays. The S. cerevisiae chromosome VI array described by Katou et al. (2003) Nature 424, 1078-83 and the newly developed S. cerevisiae chromosomes III-VIa have been used. 2. Experimental factors: Distribution of the sister chromatid cohesion protein Scc1, before and after induction of a DNA double strand break (dsb) on chromosome III, V and VI in G2/metaphase cells were analysed (Benomyl arrested cells). 3. Number of hybridizations performed: ChIP analysis: 15 4. Hybridisation design: ChIP analysis: comparison of ChIP fraction with SUP (supernatant) fraction 5. Quality control: Duplication of experiments, confirmation of recruitment of the analysed protein to a dsb by induction of breaks at three different positions in the genome. Mock hybridisation of samples immunoprecipitated from cells containing no tag recognized by antibody used. Checking of the ChIP fraction by Western blotting. 6. URL of supplemental website: GEO Accession number: GSE1905 Web site: http://chromosomedynamics.bio.titech.ac.jp Samples used, extract preparation and labeling: 1. Origin of the biological sample Budding yeast (Saccharomyces cerevisiae). 2. Manipulation of the samples and the protocols used: Mitotic budding yeast cells were synchronised in G2/M with Benomyl (80 ug/ml) Cells were fixed 30 minutes at room temperature plus over night in 1% formaldehyde at 4C. 3. Protocols for preparing extracts: ChIP analysis: Extract preparation was processed following the Shirahige lab protocol (http://chromosomedynamics.bio.titech.ac.jp ). 5x10^8 cells were disrupted using Multi-Beads Shocker (MB400U, YASUI KIKAI, Osaka). Whole cell extract was sonicated to obtain 400-600 bp genomic DNA fragments. Anti-Flag monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Dynal, protein A Dynabeads) were used for chromatin immunoprecipitation. The immunprecipates were eluted and incubated over night at 65¨¬C to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95¨¬C, fragments were labelled as detailed below. 4. Labeling protocol DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37C as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. 5. Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://chromosomedynamics.bio.titech.ac.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3â??biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100C for 10 minutes, and then put on ice before being hybridized for 16 hours at 42C in an hybridization oven (GeneChip Hybri. Oven 320, Affymetrix). Washing and scanning protocol (FlexMidi-euk2v3-450) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix). 6. Measurement data and specifications: S. cerevisiae arrays were scanned using the HP GeneArray Scanner (Affymetrix) at an emission wavelength of 560 nm, resolution 7.5 uM. Grids were aligned to scans following the library array description. Primary data analyses were carried out using Affymetrix Microarray Suite Ver.5.0 software to obtain signal intensity, fold change value, change p-value and detection p-value. The detailed information of this analysis is available at http://www.affymetrix.com/support/technical/whitepapers/sadd_whitepaper.pdf and http://www.affymetrix.com/support/technical/technotes/statistical_reference_ guide.pdf To discriminate significant signals for binding to DNA (dark grey signals), signals from the ChIP fraction scan were compared to the SUP (supernatant) fraction scan using the 3 following criteria: first, the signal reliability was judged by the detection p-value of each locus (p-value <=0.025); secondly, reliability of binding ratio was judged by change p-value (p- value <=0.025); thirdly, only clusters of at least 3 contiguous loci responding to the 2 previous criteria were selected as significantly enriched locus. The light grey signals represent the statistically not significantly enriched signals. The software to present the result (chr6viewer) is available at http://everythingchromovi.gsc.riken.go.jp. The raw and transformed data files are available at GEO database website and at our web site http://chromosomedynamics.bio.titech.ac.jp. 6. Array Design 1. General array design: in situ synthesized arrays by Affymetrix 2. Availability of arrays: commercially available from Affymetrix 3. Location and ID of each spot on arrays: available at our web site (http://chromosomedynamics.bio.titech.ac.jp) and from Affymetrix on request 4. Probe type: oligonucleotide 5. The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACF, P/N# 510636 Chromosome III,IV,V,VIa S.cerevisiae: SC3456a520015F, P/N# 520015 Keywords: other
2010-05-27 | E-GEOD-1905 | biostudies-arrayexpress
Project description:This series is a supplementary data set for the manuscript titled "Cohesin relocation from sites of chromosomal loading to places of convergent transcription" All data shown in the paper were registered (30 ChIP-chip and one transcription data). Experimental design: 1. Type of experiment: a. ChIP (Chromatin immunoprecipitation) analysis hybridised to a high density oligonucleotide array. The S. cerevisiae chromosome VI array described by Katou et al. (2003) Nature 424, 1078-83 has been used. Newly developed S. cerevisiae chromosomes III-V and S. pombe chromosomes 2-3 oligonucleotide arrays produced by Affymetrix have also been used. b. Gene expression profile analysis of S. cerevisiae. 2. Experimental factors: a. Distribution of different proteins related to sister chromatid cohesion or condensation during the cell cycle, mainly in G1 (alpha-factor arrested cells), S-phase (HU arrested cells) and metaphase (Nocodazole arrested cells). b. Gene expression profile analysis of logarithmically growing cells. 3. Number of hybridizations performed: a. ChIP analysis: 32 b. Gene expression: 1 4. Hybridisation design: ChIP analysis: comparison of ChIP fraction with SUP (supernatant) fraction 5. Quality control: Duplication, confirmation using different tags, different subunits of the same complex, and different yeast strain backgrounds (W303, BY4741, and SK1). Checking of the ChIP fraction by Western blotting. Checking amount of mRNA by RT-PCR. 6. URL of supplemental website: GEO Accession number: GSE1461 Web site: http://chromosomedynamics.bio.titech.ac.jp Samples used, extract preparation and labeling: 1. Origin of the biological sample Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) 2. Manipulation of the samples and the protocols used: a. Mitotic budding yeast cells were synchronised in G1 with alpha-factor (2uM) and then released at 23ºC into medium containing 200 mM HU or 7.5 ug/ml Nocodazole for 60 min and 150 min, respectively. scc2-4 cells were shifted to 37ºC 60 min before alpha-factor release. Heat-shock treatment has been done at 37ºC for 15 minutes. b. Meiotic budding yeast cells were collected 4 hours after meiosis induction at 23ºC. c. Mitotic fission yeast cells were grown logarithmically in minimum medium (MM). Cells were fixed over night in 1% formaldehyde at 4ºC. 3. Protocols for preparing extracts: a. ChIP analysis: Extract preparation was processed following the Shirahige lab protocol (http://chromosomedynamics.bio.titech.ac.jp ). 5x10^8 cells were disrupted using Multi-Beads Shocker (MB400U, YASUI KIKAI, Osaka). Whole cell extract was sonicated to obtain 400-600 bp genomic DNA fragments. Anti-HA monoclonal antibody (clone 16B12, CRP Inc., Denver, PA) and anti-Flag monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Dynal, protein A Dynabeads) were used for chromatin immunoprecipitation. The immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC, fragments were labelled as detailled below. b. Gene expression analysis: Total RNA was prepared using FastRNA Pro Red Kit (BIO 101 Systems) according to the manufacturer’s instructions. 10 ug of total RNA was reverse transcribed according to the protocol recommended by Affymetrix. In vitro transcription was performed on 1 ug of cDNA. cRNA was cleaned and fragmented by heating. 20 ug of fragmented cRNA were hybridised. 4. Labelling protocol DNA fragments were end-labelled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://chromosomedynamics.bio.titech.ac.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3’biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in an hybridization oven (GeneChip Hybri. Oven 320, Affymetrix). Washing and scanning protocol (FlexMidi-euk2v3-450) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix). Measurement data and specifications: S. cerevisiae arrays were scanned using the HP GeneArray Scanner (Affymetrix) at an emission wavelength of 560 nm, resolution 7.5 uM. S. pombe arrays were scanned by GeneChip Scanner 3000 (Affymetrix) at an emission wavelength of 570 nm, resolution 2.5 uM. Grids were aligned to scans following the library array description. Primary data analyses were carried out using Affymetrix Microarray Suite Ver.5.0 software to obtain signal intensity, fold change value, change p-value and detection p-value. The detailed information of this analysis is available at http://www.affymetrix.com/support/technical/whitepapers/sadd_whitepaper.pdf and http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf To discriminate significant signals for binding to DNA (dark grey signals), signals from the ChIP fraction scan were compared to the SUP (supernatant) fraction scan using the 3 following criteria: first, the signal reliability was judged by the detection p-value of each locus (p-value ≤0.025); secondly, reliability of binding ratio was judged by change p-value (p-value ≤0.025); thirdly, only clusters of at least 3 contiguous loci responding to the 2 previous criteria were selected as significantly enriched locus. The light grey signals represent the statistically not significantly enriched signals. The software to present the result (chr6viewer) is available at http://everythingchromovi.gsc.riken.go.jp. The raw and transformed data files are available at GEO database website and at our web site http://chromosomedynamics.bio.titech.ac.jp. Array Design 1. General array design: in situ synthesized arrays by Affymetrix 2. Availability of arrays: commercially available from Affymetrix 3. Location and ID of each spot on arrays: available at our web site (http://chromosomedynamics.bio.titech.ac.jp) and from Affymetrix on request 4. Probe type: oligonucleotide 5. The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACF, P/N# 510636 Chromosome III,IV,V S.cerevisiae: SC3456a520015F, P/N# 520015 Chromosome II,III S.pombe: S_pombea520106F, P/N# 520106 YGS98 GeneChipsT oligonucleotide arrays: P/N# 900256 Keywords = Yeast Cohesin Condensin Loading Distribution Keywords: other
2004-06-29 | GSE1461 | GEO
Project description:This series is a supplementary data set for the manuscript titled "Cohesin relocation from sites of chromosomal loading to places of convergent transcription" All data shown in the paper were registered (30 ChIP-chip and one transcription data). Experimental design: 1. Type of experiment: a. ChIP (Chromatin immunoprecipitation) analysis hybridised to a high density oligonucleotide array. The S. cerevisiae chromosome VI array described by Katou et al. (2003) Nature 424, 1078-83 has been used. Newly developed S. cerevisiae chromosomes III-V and S. pombe chromosomes 2-3 oligonucleotide arrays produced by Affymetrix have also been used. b. Gene expression profile analysis of S. cerevisiae. 2. Experimental factors: a. Distribution of different proteins related to sister chromatid cohesion or condensation during the cell cycle, mainly in G1 (alpha-factor arrested cells), S-phase (HU arrested cells) and metaphase (Nocodazole arrested cells). b. Gene expression profile analysis of logarithmically growing cells. 3. Number of hybridizations performed: a. ChIP analysis: 32 b. Gene expression: 1 4. Hybridisation design: ChIP analysis: comparison of ChIP fraction with SUP (supernatant) fraction 5. Quality control: Duplication, confirmation using different tags, different subunits of the same complex, and different yeast strain backgrounds (W303, BY4741, and SK1). Checking of the ChIP fraction by Western blotting. Checking amount of mRNA by RT-PCR. 6. URL of supplemental website: GEO Accession number: GSE1461 Web site: http://chromosomedynamics.bio.titech.ac.jp Samples used, extract preparation and labeling: 1. Origin of the biological sample Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) 2. Manipulation of the samples and the protocols used: a. Mitotic budding yeast cells were synchronised in G1 with alpha-factor (2uM) and then released at 23ºC into medium containing 200 mM HU or 7.5 ug/ml Nocodazole for 60 min and 150 min, respectively. scc2-4 cells were shifted to 37ºC 60 min before alpha-factor release. Heat-shock treatment has been done at 37ºC for 15 minutes. b. Meiotic budding yeast cells were collected 4 hours after meiosis induction at 23ºC. c. Mitotic fission yeast cells were grown logarithmically in minimum medium (MM). Cells were fixed over night in 1% formaldehyde at 4ºC. 3. Protocols for preparing extracts: a. ChIP analysis: Extract preparation was processed following the Shirahige lab protocol (http://chromosomedynamics.bio.titech.ac.jp ). 5x10^8 cells were disrupted using Multi-Beads Shocker (MB400U, YASUI KIKAI, Osaka). Whole cell extract was sonicated to obtain 400-600 bp genomic DNA fragments. Anti-HA monoclonal antibody (clone 16B12, CRP Inc., Denver, PA) and anti-Flag monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Dynal, protein A Dynabeads) were used for chromatin immunoprecipitation. The immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC, fragments were labelled as detailled below. b. Gene expression analysis: Total RNA was prepared using FastRNA Pro Red Kit (BIO 101 Systems) according to the manufacturerâs instructions. 10 ug of total RNA was reverse transcribed according to the protocol recommended by Affymetrix. In vitro transcription was performed on 1 ug of cDNA. cRNA was cleaned and fragmented by heating. 20 ug of fragmented cRNA were hybridised. 4. Labelling protocol DNA fragments were end-labelled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://chromosomedynamics.bio.titech.ac.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3âbiotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in an hybridization oven (GeneChip Hybri. Oven 320, Affymetrix). Washing and scanning protocol (FlexMidi-euk2v3-450) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix). Measurement data and specifications: S. cerevisiae arrays were scanned using the HP GeneArray Scanner (Affymetrix) at an emission wavelength of 560 nm, resolution 7.5 uM. S. pombe arrays were scanned by GeneChip Scanner 3000 (Affymetrix) at an emission wavelength of 570 nm, resolution 2.5 uM. Grids were aligned to scans following the library array description. Primary data analyses were carried out using Affymetrix Microarray Suite Ver.5.0 software to obtain signal intensity, fold change value, change p-value and detection p-value. The detailed information of this analysis is available at http://www.affymetrix.com/support/technical/whitepapers/sadd_whitepaper.pdf and http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf To discriminate significant signals for binding to DNA (dark grey signals), signals from the ChIP fraction scan were compared to the SUP (supernatant) fraction scan using the 3 following criteria: first, the signal reliability was judged by the detection p-value of each locus (p-value â¤0.025); secondly, reliability of binding ratio was judged by change p-value (p-value â¤0.025); thirdly, only clusters of at least 3 contiguous loci responding to the 2 previous criteria were selected as significantly enriched locus. The light grey signals represent the statistically not significantly enriched signals. The software to present the result (chr6viewer) is available at http://everythingchromovi.gsc.riken.go.jp. The raw and transformed data files are available at GEO database website and at our web site http://chromosomedynamics.bio.titech.ac.jp. Array Design 1. General array design: in situ synthesized arrays by Affymetrix 2. Availability of arrays: commercially available from Affymetrix 3. Location and ID of each spot on arrays: available at our web site (http://chromosomedynamics.bio.titech.ac.jp) and from Affymetrix on request 4. Probe type: oligonucleotide 5. The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACF, P/N# 510636 Chromosome III,IV,V S.cerevisiae: SC3456a520015F, P/N# 520015 Chromosome II,III S.pombe: S_pombea520106F, P/N# 520106 YGS98 GeneChipsT oligonucleotide arrays: P/N# 900256 Keywords = Yeast Cohesin Condensin Loading Distribution Keywords: other
2010-06-09 | E-GEOD-1461 | biostudies-arrayexpress
Project description:The segregation of maternal centromeres away from the paternal ones during the first division of meiosis depends on the attachment of sister kinetochores to microtubules emanating from the same spindle pole. In budding yeast monopolar attachment requires the recruitment to kinetochores of a protein complex called monopolin. The biochemical function of monopolin was unknown. Here, we have identified the casein kinase I Hrr25 as a hitherto unknown subunit of monopolin. Hrr25 differs from other monopolin components by its enzymatic activity and strong evolutionary conservation. We demonstrate that Hrr25âs kinase activity and its interaction with monopolin are both required for monopolar attachment. Accordingly, Hrr25 is associated with centromeres in meiosis I. Our results revealed a surprising new role for casein kinases and provide a hypothesis for the mechanism of monopolar attachment during meiosis I in sexually reproducing organisms: casein kinase I-dependent phosphorylation of kinetochore proteins. Keywords: ChIP-chip, Meiosis, Cell cycle, Saccharomyces cerevisiae, Chromosome VI tiling array, Hrr25, Mam1 ⢠Experimental factors Distribution of the Hrr25 and Mam1 at Meiosis I. Distribution of Hrr25 in Mam1 deletion mutant (S.cerevisiae). All experiments were performed in cells with the same genetic background (Saccharomyces cerevisiae SK1). ⢠Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Cerevisiae chromosome VI array was used. ⢠Quality control steps taken Confirmation using different subunits of the same complex. Confirmation of protein distribution using deletion mutant strain. Checking of the ChIP fraction by Western blotting. Mock hybridisation of samples immunoprecipitated from cells containing no tag recognized by antibody used. Swapping SUP fraction. Q-PCR. Samples used, extract preparation and labelling: ⢠The origin of each biological sample Saccharomyces cerevisiae (SK1). ⢠Manipulation of biological samples and protocols used Chromatin immunoprecipitation (ChIP) and hybridization to Affimetrix high-density oligonucleotide arrays of S. cerevisiae chromosome VI were performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). ⢠Technical protocols for preparing the hybridization extract The chromatin-immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. ⢠Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://everythingchromosomevi.gsc.riken.go.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3âbiotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix). ⢠Measurement data and specifications: Arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the raw data files can be downloaded from GEO database. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site at http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf. The analysis is available on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-valueâ¤0.025). Secondly, reliability of binding ratio was judged by change p-value (p-valueâ¤0.025). Thirdly, clusters consisting of at least 900bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. ⢠Array Design: General array design: in situ synthesized arrays by Affymetrix Availability of arrays: commercially available from Affymetrix Location and ID of each spot on arrays: available from Affymetrix on request Probe type: oligonucleotide The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACFC6, P/N# 510636
2010-06-10 | E-GEOD-4792 | biostudies-arrayexpress
Project description:Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres. Keywords: ChIP-chip, Mitosis, Meiosis, Cell cycle, Saccharomyces cerevisiae, Chromosome VI tiling array, Sgo1, Pp2A, Cse4, Ndc10, Rts1, Rec8 â?¢ Experimental factors Distribution of Cse4, Ndc10, Rts1 in mitotic G2 phase and meiosis I (S. cerevisiae). Distribution of Rec8 in meiosis I (S.cerevisiae). All experiments were performed in cells with the same genetic background (Saccharomyces cerevisiae SK1). â?¢ Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with SUP (supernatant) fraction. SUP fraction is same as WCE fraction. â?¢ Quality control steps taken Duplication, confirmation using different tags, and different subunits of the same complex. Confirmation of protein distribution using deletion mutant strain. Confirmation of several loci by q-PCR. Checking of the ChIP fraction by Western blotting. Checking of the ChIP fraction by swapping SUP fraction. Samples used, extract preparation and labelling: â?¢ The origin of each biological sample Saccharomyces cerevisiae (SK1) . â?¢ Manipulation of biological samples and protocols used Chromatin immunoprecipitation (ChIP) and hybridization to Affimetrix high-density oligonucleotide arrays of S. cerevisiae chromosome VI was performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). â?¢ Technical protocols for preparing the hybridization extract The chromatin-immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. â?¢ Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://everythingchromosomevi.gsc.riken.go.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3â??biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix). â?¢ Measurement data and specifications: Arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the raw data files can be downloaded from GEO database. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site at http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf. The analysis is available on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-valueâ?¤0.025 for cerevisiae). Secondly, reliability of binding ratio was judged by change p-value (p-valueâ?¤0.025 for cerevisiae). Thirdly, clusters consisting of at least 900bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. â?¢ Array Design: General array design: in situ synthesized arrays by Affymetrix Availability of arrays: commercially available from Affymetrix Location and ID of each spot on arrays: available from Affymetrix on request Probe type: oligonucleotide The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACFC6, P/N# 510636
2010-06-11 | E-GEOD-4791 | biostudies-arrayexpress
Project description:Cohesin is a multisubunit complex that mediates sister-chromatid cohesion. Its Smc1 and Smc3 subunits possess ABC-like ATPases at one end of 50 nm long coiled coils. At the other ends are pseudosymmetrical hinge domains that interact to create V-shaped Smc1/Smc3 heterodimers. N- and C-terminal domains within cohesin's kleisin subunit Scc1 bind to Smc3 and Smc1 ATPase heads respectively, thereby creating a huge tripartite ring. It has been suggested that cohesin associates with chromosomes by trapping DNA within its ring. Opening of the ring due to cleavage of Scc1 by separase destroys sister-chromatid cohesion and triggers anaphase. We show that cohesin's hinges are not merely dimerization domains. They are essential for cohesin's association with chromosomes, which is blocked by artificially holding hinge domains together but not by preventing Scc1's dissociation from SMC ATPase heads. Our results suggest that entry of DNA into cohesin's ring requires transient dissociation of Smc1 and Smc3 hinge domains. Keywords: Cohesin loading, Scc1, Smc1, Smc3, Hinge opening, ChIP-chip â?¢ Experimental factors Distribution of the mutant and wild type form of cohesin in mitotic G2 phase. â?¢ Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with SUP (supernatant) fraction. SUP fraction is equal to WCE (Whole Cell Extract) fraction. Essentially WCE or SUP fraction of any cell cycle phase can be used for normalization, and there was no effect on protein binding profiles. Cerevisiae chromosome VI array was used. â?¢ Quality control steps taken Confirmation using different tags. Confirmation by q-PCR. Checking of the ChIP fraction by Western blotting. Mock hybridisation of samples immuno-precipitated from cells containing no tag recognized by antibody used. Samples used, extract preparation and labelling: â?¢ The origin of each biological sample Saccharomyces cerevisiae (W303). â?¢ Manipulation of biological samples and protocols used Chromatin immunoprecipitation (ChIP) and hybridization to Affimetrix high-density oligonucleotide arrays of S. cerevisiae chromosome VIwere performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). â?¢ Technical protocols for preparing the hybridization extract The chromatin-immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. â?¢ Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (Lengronne et al. Nature 2004). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3â??biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix). â?¢ Measurement data and specifications: Arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the cel files data and processed data files can be downloaded from GEO database. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site. The analysis is available on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-valueâ?¤0.025). Secondly, reliability of binding ratio was judged by change p-value (p-valueâ?¤0.025). Thirdly, clusters consisting of at least 900bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. â?¢ Array Design: General array design: in situ synthesized arrays by Affymetrix Availability of arrays: commercially available from Affymetrix Location and ID of each spot on arrays: available from Affymetrix on request Probe type: oligonucleotide The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACFC6, P/N# 510636
2010-06-09 | E-GEOD-4827 | biostudies-arrayexpress
Project description:BACKGROUND: Cells undergoing meiosis perform two consecutive divisions after a single round of DNA replication. During the first meiotic division, homologous chromosomes segregate to opposite poles. This is achieved by (1) the pairing of maternal and paternal chromosomes via recombination producing chiasmata, (2) coorientation of homologous chromosomes such that sister chromatids attach to the same spindle pole, and (3) resolution of chiasmata by proteolytic cleavage by separase of the meiotic-specific cohesin Rec8 along chromosome arms. Crucially, cohesin at centromeres is retained to allow sister centromeres to biorient at the second division. Little is known about how these meiosis I-specific events are regulated. RESULTS: Here, we show that Spo13, a centromere-associated protein produced exclusively during meiosis I, is required to prevent sister kinetochore biorientation by facilitating the recruitment of the monopolin complex to kinetochores. Spo13 is also required for the reaccumulation of securin, the persistence of centromeric cohesin during meiosis II, and the maintenance of a metaphase I arrest induced by downregulation of the APC/C activator CDC20. CONCLUSION: Spo13 is a key regulator of several meiosis I events. The presence of Spo13 at centromere-surrounding regions is consistent with the notion that it plays a direct role in both monopolin recruitment to centromeres during meiosis I and maintenance of centromeric cohesion between the meiotic divisions. Spo13 may also limit separase activity after the first division by ensuring securin reaccumulation and, in doing so, preventing precocious removal from chromatin of centromeric cohesin. Keywords: Meiosis, Cell cycle, Saccharomyces cerevisiae, Chromosome VI tiling array, Spo13, ChIP-chip ⢠Experimental factors Distribution of the Spo13 at Meiosis I. Experiment was performed in Saccharomyces cerevisiae SK1 strain. ⢠Experimental design ChIP analysis: Hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Cerevisiae chromosome VI array was used. ⢠Quality control steps taken Checking of the ChIP fraction by Western blotting. Mock hybridisation of samples immunoprecipitated from cells containing no tag recognized by antibody used. Sup swapping. Samples used, extract preparation and labelling: ⢠The origin of each biological sample Saccharomyces cerevisiae (SK1). ⢠Manipulation of biological samples and protocols used Chromatin immunoprecipitation (ChIP) and hybridization to Affimetrix high-density oligonucleotide arrays of S. cerevisiae chromosome VIwere performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). ⢠Technical protocols for preparing the hybridization extract The chromatin-immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. ⢠Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (Lengronne et al. Nature 2004). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3âbiotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix). ⢠Measurement data and specifications: Arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the cel files data and processed data files can be downloaded from GEO database. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site. The analysis is available on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-valueâ¤0.025). Secondly, reliability of binding ratio was judged by change p-value (p-valueâ¤0.0025). Thirdly, clusters consisting of at least 900bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. ⢠Array Design: General array design: in situ synthesized arrays by Affymetrix Availability of arrays: commercially available from Affymetrix Location and ID of each spot on arrays: available from Affymetrix on request Probe type: oligonucleotide The arrays used in this study can be purchased from Affymetrix: Chromosome VI S.cerevisiae: rikDACFC6, P/N# 510636 GSM108198 was used for normalization of GSM108511.
2010-06-11 | E-GEOD-4813 | biostudies-arrayexpress
Project description:The SMC protein complexes safeguard genomic integrity through their functions in chromosome segregation and repair. The chromosomal localization of the budding yeast Smc5/6 complex here determined reveals that the complex works specifically on the duplicated genome in differently regulated pathways. One controls the association to centromeres and chromosome arms in unchallenged cells, the second regulates the association to DNA breaks, and the third directs the complex to the chromosome arm that harbors the ribosomal DNA arrays. The chromosomal interaction pattern predicts a function that becomes more important with increasing chromosome length, and that the complex's role in unchallenged cells is independent of DNA damage. Additionally, localization of Smc6 to collapsed replication forks indicates an involvement in their rescue. Altogether this shows that the complex maintains genomic integrity in multiple ways, and evidence is presented that the Smc5/6 complex is needed during replication to prevent the accumulation of branched chromosome structures. Keywords: ChIP-chip analysis ⢠The goal of the experiment - Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways. ⢠Keywords, for example, time course, cell type comparison, array CGH (the use of MGED ontology terms is recommended). Cell cycle, Double Strand Break Induction, time course after break induction, Saccharomyces cerevisiae, Chromosome VI array, Whole Genome Tiling Array, Smc5, Smc6, Nse1, Nse4, Nse5, Scc1, Scc2, Mre11, Rad53 ⢠Experimental factors Distribution of the Smc5/6 complex in the cell cycle, distribution of Smc5/6 complex before and after induction of a DNA double strand break on chromosome VI in G2/metaphase cells, distribution of Smc6 in the absence of functional Scc2, Mre11 or Rad53, distribution of Smc6 at collapsed replication forks, and distribution of Cohesin subunit Scc1 in G2/metaphase. Distribution was analyzed on Chromosome VI and/or whole genome arrays. Cells were grown in rich yeast cell extract media. All experiments were performed in cells with the same genetic background (Saccharomyces cerevisiae W303; ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3 RAD5) ⢠Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Cerevisiae chromosome VI array or Whole genome arrays were used. Total number of hybridization was 69. All description about hybridized samples is attached as the separate sheet. ⢠Quality control steps taken Duplication, confirmation using different tags, different subunits of the same complex, and time course after double-strand break induction. Checking of the ChIP fraction by Western blotting. Mock hybridisation of samples immunoprecipitated from cells containing no tag recognized by antibody used. ⢠Links to the publication, any supplemental websites or database accession numbers. http://tilingarray.bio.titech.ac.jp/smc56dsb Samples used, extract preparation and labelling: ⢠The origin of each biological sample Saccharomyces cerevisiae W303 (ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3 RAD5). ⢠Manipulation of biological samples and protocols used Strains containing flag-tagged versions of Smc6, Smc5, Nse1, Nse4, or Nse5 proteins were used. For synchronization in G1, alpha factor was added (Zymo research and Innovagen AB) at 3 µg/ml final concentrations to logarithmically growing cells, and G1 arrest was obtained at indicated temperatures for 2.5 h. For S-phase arrest, cells were grown to log phase and arrested in G1 with alpha factor for 2 h, and then incubated in the presence of 200 mM hydroxyurea (HU) (Sigma) for 30 min. Cells were then washed twice with rich medium containing 100µg/ml pronase (Calbiochem). Finally cells were resuspended in medium containing HU and pronase, and growth continued for indicated time periods. G2/M arrest was achieved 2.5 h after addition of benomyl (Sigma, 80µg/ml), to logarithmically growing cells. G1, S and G2/M arrests were checked microscopically and by FACS. DSBs were induced by activation of the HO endonuclease from the GAL1-10-promoter by addition of 2% galactose to cell cultures initially grown in 2% raffinose. ⢠Technical protocols for preparing the hybridization extract Extract preparation was processed following the Shirahige lab protocol (http://chromosomedynamics.bio.titech.ac.jp ). 5x108 cells were disrupted using Multi-Beads Shocker (MB400U, YASUI KIKAI, Osaka). Whole cell extract was sonicated to obtain 400-600 bp genomic DNA fragments. Anti-Flag monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Dynal, protein A Dynabeads) were used for chromatin immunoprecipitation. The immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. ⢠Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://chromosomedynamics.bio.titech.ac.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3'biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix). ⢠Measurement data and specifications: S. cerevisiae arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the cel files data and processed data files can be downloaded from GEO database or from our Web sites. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site at http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf. The analysis is available from K.S. on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp and for whole genome arrays to 100bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-value<=0.01 for chromosome VI and pvalue?0.0025 for whole genome array). Secondly, reliability of binding ratio was judged by change p-value (p-value<=0.01 for chromosome VI and p-value<=0.0025 for whole genome array). Thirdly, clusters consisting of at least 500bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. Under these conditions both types of arrays give essentially the same results for the binding profile. Repeated sequences were masked out and not included in the calculation of protein binding profile. The correlation coefficient of Smc6 binding profiles in G2/M from two independent experiments was 0.955, showing that the whole genome ChIP on chip data was reproducible. For comparison of the localization of Smc6 in different experiments and that of Smc6 and Scc1 on whole genome maps, peaks with signal to log ratio higher than 0.6 were chosen, and peaks which were distributed within a 5kb region was recognized as one peak. In the analysis of the number of Smc6 localization sites on each chromosome a 40 kb region spanning all centromeres were excluded. This region in addition to that downstream the rDNA repeats was also excluded from the analysis when comparing Smc6 localization in wild type, scc2-4 and scc1-73 cells. ⢠Array Design: General array design: in situ synthesized arrays by Affymetrix Chromosome VI S.cerevisiae: rikDACF, P/N# 510636 Whole Genome Array S.cerevisiae: S.cerevisiae Tiling1.0F Arrays, Part#520286 and Part#520280
2010-06-11 | E-GEOD-4808 | biostudies-arrayexpress
Project description:Type of experiment: ChIP-chip (Chromatin immunoprecipitation on DNA chip) analyses of replication related, checkpoint related proteins and BrdU incorporated regions at 300bp resolution. Newly developed S.cerevisiae chromosome VI tiling array produced by affymetrix was used. Experimental factors:Distribution of various replication and checkpoint related proteins on chromosome VI during S-phase (with or w/o hydroxy urea). Distribution of BrdU incorporated regions on chromosome VI during S-phase with hydroxy urea. Wild type, tof1 deletion mutant, mrc1 deletion mutant, rad9 deletion mutant, sml1 deletion mutant, and sml1 mec1 tel1 triple deletion mutant were also investigated. The results of 74 hybridization files presented in the paper are shown. The type of reference used for the hybridizations, if any: For the analysis of FLAG tagged protein, we usually used Cdc45-3XHA as an internal reference. For the analysis of HA tagged protein, POL1-3XFLAG was usually analyzed simultaneously as a reference. Hybridization design: Comparison of ChIP fraction with SUP fraction for protein binding profile. For the analyses of BrdU incorporated regions, anti BrdU antibody bound fraction was compared with SUP (Sup of purified DNA from cells at G1 phase) fraction. During a course of these experiments, we found that S-phase Sup fractions were essentially the same among different genetic backgrounds and did not affect the binding profiles. Based on this observation we use standardized Sups for all experiments. No locus was scored as significantly enriched in the control ChIP experiment using HU treated untagged cells. Quality control steps taken: Confirmation of ChIP fraction by Western blotting. Confirmation by conventional ChIP PCR methods for selected locus. Or Duplication. The origin of the biological sample: Budding Yeast (Saccharomyces cerevisiae) Manipulation of biological samples and protocols used:For the preparation of HU-arrested cells, we synchronized yeast cells with alpha-factor (2mM) and then released cells into 200mM HU for 60 min. at 23°C. For the preparation of S-phase cells, cells were released into S-phase without HU and at 16°C collected at the time indicated. These cells were fixed by 1% Formaldehyde and then used for ChIP of protein of interest. For the analyses of BrdU incorporation, the same fraction of cells used for ChIP were fixed by ice cold buffer containing 0.1% Azide, and then used for detection of BrdU incorporated regions. Protocol for preparing the hybridization extract:We disrupted 1.5X10^8 cells by MULTI-BEADS SHOCKER (MB400U, YASUI KIKAI, Osaka), which was able to keep cells precisely at lower than 6°C during disruption by glass beads. This enabled us to retrieve more than 60% of tagged proteins to soluble fraction routinely. Chromosomal DNA was shared into the average size of 400-600bp by sonication. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc., Denver, PA) and anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) were used for chromatin immuno-precipitation. Immunoprecipitated chromosomal DNA was subsequently purified following the protocol of Youngâs lab (http://inside.wi.mit.edu/young/pub/locationanalysis.html). Purified DNA was random primed and amplified by the protocol of Brownâs lab (http://www.microarrays.org). 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). For the analysis of BrdU incorporated regions, total DNA from 3X10^8 cells was purified by Qiagen Genomic-tip system (P/N10223, QIAGEN, Germany). DNA was sheared to 300bp by sonication, denatured, and mixed with 2ug of anti-mouse IgG1 dynabeads (P/N110.12, Dynal AS, Norway) bound anti-BrdU monoclonal antibody (2B1D5F5H4E2, MBL, Japan). Immunoprecipitation and purification of antibody bound DNA fraction was carried out following the protocol of Cimbora et al. (Mol. Cel. Biol. 20, 5581-5591, 2000). Purified DNA was random primed and amplified by the Brownâs lab protocol. 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling. Labeling protocol:DNA was end-labelled with Biotin-N6ddATP by Terminal Transferase as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998) The protocol and conditions used during hybridization, blocking and washing: Hybridization, blocking and washing steps were carried out as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). Each sample was hybridized to the array in 150ul containing 6XSSPE, 0.005% TritonX-100, 15ug fragmented denatured salmon sperm DNA (Gibco-BRL), and 1nmole of a 3â-biotin control oligonucleotide (oligo B2 probes) that hybridized to the border features on the array. Samples were heated to 100°C for 10 min., and then cooled on ice. Samples were hybridized for 16h at 42°C in a hybridization oven (GeneChip hybri oven 320, Affymetrix, CA). Washing and staining protocol (Mini_euk1 ver2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix, CA). Measurement data and specifications:Each array was scanned by HP GeneArray Scanner (Affymetrix, CA) at an emission wavelength of 560nm at 7.5uM resolution. Grids were aligned to the scanned images using the know feature of the array. Primary data analyses were carried out using the Affymetrix Microarray Suite Ver.5.0 software to obtain hybridization intensity, fold change value, change p-value, and detection p-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIP fraction with Sup (supernatant) fraction by using three criteria as follows. First, the reliability of strength of signal was judged by detection p-value of each locus (p-value lower than 0.025 was considered as significant). Secondly, reliability of binding ratio was judged by change p-value (p-value lower than 0.025 was considered as significant). Thirdly, clusters consisted of at least three contiguous loci which filled above two criteria were selected as significantly enriched locus, because it is apparent that a single site of protein-DNA interaction will result in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. This third criterion is very unique, make the data highly reliable and can be only applicable to the chip data obtained by our high-resolution tiling array. In the case of BrdU experiment, loci c6-462 to c6-479 (Ty element) and c6-591 to c6-601 (YFR016c) were excluded from calculation, because those loci gave high background in IP fraction even without BrdU treatment. The software to present the result of discriminant analyses (makeps) is available at our web site. Keywords = DNA replication checkpoint binding profile Keywords: other
2010-06-05 | E-GEOD-486 | biostudies-arrayexpress
Project description:Type of experiment: ChIP-chip (Chromatin immunoprecipitation on DNA chip) analyses of replication related, checkpoint related proteins and BrdU incorporated regions at 300bp resolution. Newly developed S.cerevisiae chromosome VI tiling array produced by affymetrix was used. Experimental factors:Distribution of various replication and checkpoint related proteins on chromosome VI during S-phase (with or w/o hydroxy urea). Distribution of BrdU incorporated regions on chromosome VI during S-phase with hydroxy urea. Wild type, tof1 deletion mutant, mrc1 deletion mutant, rad9 deletion mutant, sml1 deletion mutant, and sml1 mec1 tel1 triple deletion mutant were also investigated. The results of 74 hybridization files presented in the paper are shown. The type of reference used for the hybridizations, if any: For the analysis of FLAG tagged protein, we usually used Cdc45-3XHA as an internal reference. For the analysis of HA tagged protein, POL1-3XFLAG was usually analyzed simultaneously as a reference. Hybridization design: Comparison of ChIP fraction with SUP fraction for protein binding profile. For the analyses of BrdU incorporated regions, anti BrdU antibody bound fraction was compared with SUP (Sup of purified DNA from cells at G1 phase) fraction. During a course of these experiments, we found that S-phase Sup fractions were essentially the same among different genetic backgrounds and did not affect the binding profiles. Based on this observation we use standardized Sups for all experiments. No locus was scored as significantly enriched in the control ChIP experiment using HU treated untagged cells. Quality control steps taken: Confirmation of ChIP fraction by Western blotting. Confirmation by conventional ChIP PCR methods for selected locus. Or Duplication. The origin of the biological sample: Budding Yeast (Saccharomyces cerevisiae) Manipulation of biological samples and protocols used:For the preparation of HU-arrested cells, we synchronized yeast cells with alpha-factor (2mM) and then released cells into 200mM HU for 60 min. at 23°C. For the preparation of S-phase cells, cells were released into S-phase without HU and at 16°C collected at the time indicated. These cells were fixed by 1% Formaldehyde and then used for ChIP of protein of interest. For the analyses of BrdU incorporation, the same fraction of cells used for ChIP were fixed by ice cold buffer containing 0.1% Azide, and then used for detection of BrdU incorporated regions. Protocol for preparing the hybridization extract:We disrupted 1.5X10^8 cells by MULTI-BEADS SHOCKER (MB400U, YASUI KIKAI, Osaka), which was able to keep cells precisely at lower than 6°C during disruption by glass beads. This enabled us to retrieve more than 60% of tagged proteins to soluble fraction routinely. Chromosomal DNA was shared into the average size of 400-600bp by sonication. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc., Denver, PA) and anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) were used for chromatin immuno-precipitation. Immunoprecipitated chromosomal DNA was subsequently purified following the protocol of Young’s lab (http://inside.wi.mit.edu/young/pub/locationanalysis.html). Purified DNA was random primed and amplified by the protocol of Brown’s lab (http://www.microarrays.org). 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). For the analysis of BrdU incorporated regions, total DNA from 3X10^8 cells was purified by Qiagen Genomic-tip system (P/N10223, QIAGEN, Germany). DNA was sheared to 300bp by sonication, denatured, and mixed with 2ug of anti-mouse IgG1 dynabeads (P/N110.12, Dynal AS, Norway) bound anti-BrdU monoclonal antibody (2B1D5F5H4E2, MBL, Japan). Immunoprecipitation and purification of antibody bound DNA fraction was carried out following the protocol of Cimbora et al. (Mol. Cel. Biol. 20, 5581-5591, 2000). Purified DNA was random primed and amplified by the Brown’s lab protocol. 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling. Labeling protocol:DNA was end-labelled with Biotin-N6ddATP by Terminal Transferase as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998) The protocol and conditions used during hybridization, blocking and washing: Hybridization, blocking and washing steps were carried out as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). Each sample was hybridized to the array in 150ul containing 6XSSPE, 0.005% TritonX-100, 15ug fragmented denatured salmon sperm DNA (Gibco-BRL), and 1nmole of a 3’-biotin control oligonucleotide (oligo B2 probes) that hybridized to the border features on the array. Samples were heated to 100°C for 10 min., and then cooled on ice. Samples were hybridized for 16h at 42°C in a hybridization oven (GeneChip hybri oven 320, Affymetrix, CA). Washing and staining protocol (Mini_euk1 ver2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix, CA). Measurement data and specifications:Each array was scanned by HP GeneArray Scanner (Affymetrix, CA) at an emission wavelength of 560nm at 7.5uM resolution. Grids were aligned to the scanned images using the know feature of the array. Primary data analyses were carried out using the Affymetrix Microarray Suite Ver.5.0 software to obtain hybridization intensity, fold change value, change p-value, and detection p-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIP fraction with Sup (supernatant) fraction by using three criteria as follows. First, the reliability of strength of signal was judged by detection p-value of each locus (p-value lower than 0.025 was considered as significant). Secondly, reliability of binding ratio was judged by change p-value (p-value lower than 0.025 was considered as significant). Thirdly, clusters consisted of at least three contiguous loci which filled above two criteria were selected as significantly enriched locus, because it is apparent that a single site of protein-DNA interaction will result in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. This third criterion is very unique, make the data highly reliable and can be only applicable to the chip data obtained by our high-resolution tiling array. In the case of BrdU experiment, loci c6-462 to c6-479 (Ty element) and c6-591 to c6-601 (YFR016c) were excluded from calculation, because those loci gave high background in IP fraction even without BrdU treatment. The software to present the result of discriminant analyses (makeps) is available at our web site. Keywords = DNA replication checkpoint binding profile Keywords: other
2003-08-27 | GSE486 | GEO