|
| Status |
Public on Feb 01, 2010 |
| Title |
nucleosomes_Fft3_1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Fft3 mutant S. pombe
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: HU1939
genome/variation: Fft3 mutant
fraction: nucleosomal DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared from formaldehyde crosslinked log phase cells and digested with micrococcal nuclease (MNase, Sigma-Aldrich) to yield about 90% mononucleosomes. DNA of mononucleosomal size was purified from agarose gels, fragmented to an average size of about 50 bp with DNaseI. DNAseI fragmented genomic DNA from S. pombe was used as hybridization control.
For full details, see publication PMID: 19233281
|
| Label |
biotin
|
| Label protocol |
Samples were processed at BEA Novum http://www.bea.ki.se/
The isolated mononucleosomal DNA fragments and genomic control DNA were fragmented with DNaseI and labelled with terminal deoxynucleotidyl transferase according to Affymetrix GeneChip Mapping 10K Xba Assay Kit (P/N 900441). The purified fragments were diluted to 20 ug of DNA in a volume of 45 ul with 1× fragmentation buffer and freshly prepared fragmentation reagent (DNaseI) was added to a final concentration of 0.048 U/ul. The samples were incubated for 1 h in a preheated thermal cycler at 37 °C. The DNaseI was inactivated by incubating 15 min at 95°C. The samples were immediately placed on ice and the fragments were checked to have a size of about 50 bp with an Agilent bioanalyzer. Next, the fragmented samples were incubated at 37 °C in a thermal cycler with a labeling master mix containing DNA Labeling Reagent (5 mM), terminal deoxynucleotidyl transferase (30 U/ul) and 1× Terminal Deoxynucleotidyl Transferase Buffer as described in the Affymetrix GeneChip Mapping Assay Manual.
See publication PMID: 19233281
|
| |
|
| Channel 2 |
| Source name |
wildtype and Mit1 mutant S. pombe
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: HU303
genome/variation: wildtype
strain: HU1295
genome/variation: Mit1 mutant
fraction: Genomic Input DNA (comprising the gDNA of wildtype and Mit1 mutant combined)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared from formaldehyde crosslinked log phase cells and digested with micrococcal nuclease (MNase, Sigma-Aldrich) to yield about 90% mononucleosomes. DNA of mononucleosomal size was purified from agarose gels, fragmented to an average size of about 50 bp with DNaseI. DNAseI fragmented genomic DNA from S. pombe was used as hybridization control.
For full details, see publication PMID: 19233281
|
| Label |
biotin
|
| Label protocol |
Samples were processed at BEA Novum http://www.bea.ki.se/
The isolated mononucleosomal DNA fragments and genomic control DNA were fragmented with DNaseI and labelled with terminal deoxynucleotidyl transferase according to Affymetrix GeneChip Mapping 10K Xba Assay Kit (P/N 900441). The purified fragments were diluted to 20 ug of DNA in a volume of 45 ul with 1× fragmentation buffer and freshly prepared fragmentation reagent (DNaseI) was added to a final concentration of 0.048 U/ul. The samples were incubated for 1 h in a preheated thermal cycler at 37 °C. The DNaseI was inactivated by incubating 15 min at 95°C. The samples were immediately placed on ice and the fragments were checked to have a size of about 50 bp with an Agilent bioanalyzer. Next, the fragmented samples were incubated at 37 °C in a thermal cycler with a labeling master mix containing DNA Labeling Reagent (5 mM), terminal deoxynucleotidyl transferase (30 U/ul) and 1× Terminal Deoxynucleotidyl Transferase Buffer as described in the Affymetrix GeneChip Mapping Assay Manual.
See publication PMID: 19233281
|
| |
|
| |
| Hybridization protocol |
Arrays were hybridized at BEA Novum http://www.bea.ki.se/
200 ul 1x hybridization mixture containing a final concentration of 7.5 ug of fragmented and labeled DNA target, 7% DMSO, 50 pM control oligo B2 (Affymetrix P/N 900301), BSA (0.5 mg/ml) and Herring sperm DNA (0.1 mg/ml) were prepared according to the Affymetrix Chromatin Immunoprecipitation Assay Protocol. The final DNA target mixture was hybridized onto Affymetrix S. pombe Tiling 1.0FR arrays and incubated in a hybridization oven at 45 °C and 60 rpm for 16 h.
See publication PMID: 19233281
|
| Scan protocol |
Arrays were scanned at BEA Novum http://www.bea.ki.se/
After hybridization of the arrays, the arrays were washed and stained with streptavidinâ€"phycoerythrin (SAPE, Invitrogen P/N S-866). The signals were amplified with biotinylated anti-streptavidin antibodies (Vector Laboratories, P/N BA-0500) in an Affymetrix Fluidics 450 wash station. Finally, the arrays were scanned in a 3000 7G scanner or similar, and the signal was quantified with the Affymetrix GeneChip Command Console Software (AGCC) to generate CEL files.
See publication PMID: 19233281
|
| Description |
Fft3 mutant S. pombe; replicate 1
Processed data file: bar
Raw data file(s)_nucleosomal fraction: mono*.CEL
Raw data file(s)_input fraction: genome*.CEL
|
| Data processing |
Raw data was processed using Affymetrix Tiling Array Software (TAS). We applied quantile normalization and a 50 bp averaging to obtain the log2 (nucleosomal fraction/input fraction) ratios for each sample.
|
| |
|
| Submission date |
May 11, 2009 |
| Last update date |
Jan 31, 2010 |
| Contact name |
Tobias Straub |
| E-mail(s) |
tstraub@med.uni-muenchen.de
|
| Organization name |
LMU Munich
|
| Department |
Biomedical Center, Bioinformatics
|
| Street address |
Großhadener Str. 9
|
| City |
Martinsried |
| ZIP/Postal code |
82152 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7715 |
| Series (1) |
| GSE16040 |
S. pombe genome-wide nucleosome mapping reveals positioning mechanisms distinct from S. cerevisiae |
|
