|
| Status |
Public on Oct 11, 2012 |
| Title |
nucleosomes_snf21- ts swr1Δ_1 |
| Sample type |
genomic |
| |
|
| Source name |
snf21- ts swr1Δ mutant S.pombe
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
mutant: Hu2314
|
| Growth protocol |
Liquid cultures were grown in YES media. Temperature sensitive strains were grown at the permissive temperature (25 °C) either for ~30 h or for ~24 h followed by 6 h at the nonpermissive temperature (34 °C) before harvesting. All other strains were grown at 30 °C.
|
| 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.
|
| Label |
biotin
|
| Label protocol |
The isolated mononucleosomal DNA fragments 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.
|
| |
|
| Hybridization protocol |
Arrays were hybridized at Gene Center Munich, Research Group Cramer. 200 ul 1x hybridization mixture containing a final concentration of 7.5 ug of fragmented and labeled DNA target, 7% DMSO and 50 pM control oligo B2 (Affymetrix P/N 900301) 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.
|
| Scan protocol |
Arrays were scanned at Gene Center Munich, Research Group Cramer. After hybridization the arrays were washed and stained in an Affymetrix GeneChip Fluidics Station 450 according to the manual of the Affymetrix GeneChip hybridization, wash, and stain kit (#900720).Finally, the arrays were scanned in a Gene Chip 3000 7G scanner, and the signal was quantified with the Affymetrix GeneChip Command Console Software (AGCC) to generate CEL files.
|
| Description |
snf21- ts swr1Δ mutant S.pombe; replicate 1
|
| Data processing |
Raw data were processed using R/Bioconductor. Data were vsn normalized. Processed data: wig, raw data: CEL
|
| |
|
| Submission date |
Oct 10, 2012 |
| Last update date |
Oct 11, 2012 |
| Contact name |
Karl Ekwall |
| E-mail(s) |
karl.ekwall@ki.se
|
| Phone |
+46 8 6089133
|
| Organization name |
Karolinska Inst
|
| Street address |
Alfred Nobels Alle 7
|
| City |
Stockholm |
| ZIP/Postal code |
S-141 89 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL7715 |
| Series (1) |
| GSE41024 |
CHD1 remodelers space nucleosomes in vitro and link regular arrays to 5’ ends of genes in S. Pombe |
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