M.musculus - Platelet, SC (Kruger,cell,2008)
Ontology highlight
ABSTRACT:
ORGANISM(S): 10090
SUBMITTER: 
Christian von Mering
PROVIDER: 3649033762 | PAXDB | 2008
REPOSITORIES: PAXDB
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| bioprojects-abundance-files-v4.0.zip | Other |
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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
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