|
| Status |
Public on Nov 25, 2013 |
| Title |
MN_pcr1D |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Mononucleosomal DNA_pcr1D
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: h+ ura4.d18 leu1.32 ade6M210 pcr1D::kanMX4
culture type: Asynchronous mitosis
|
| Growth protocol |
Rich medium (YES) at 32ºC until OD595=0.8
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared from formaldehyde crosslinked log phase cells and digested with micrococcal nuclease (Fermentas #ENO181) to a final concentration of 180 units/ml for 40 minutes at 37 ºC 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 |
4.5 mg of the fragmented double-stranded DNA samples resuspended in 45 mg were incubated with a labeling master mix containing DNA Labeling Reagent (5 mM), terminal deoxynucleotidyl transferase (30 U/ml) and 1× Terminal Deoxynucleotidyl Transferase Buffer in a thermal cycler as described in the Affymetrix GeneChip Whole Transcript (WT) Double-Stranded Target Assay Manual.
|
| |
|
| Channel 2 |
| Source name |
Naked DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: 972 h-
culture type: Asynchronous mitosis
|
| Growth protocol |
Rich medium (YES) at 32ºC until OD595=0.8
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared from formaldehyde crosslinked log phase cells and digested with micrococcal nuclease (Fermentas #ENO181) to a final concentration of 180 units/ml for 40 minutes at 37 ºC 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 |
4.5 mg of the fragmented double-stranded DNA samples resuspended in 45 mg were incubated with a labeling master mix containing DNA Labeling Reagent (5 mM), terminal deoxynucleotidyl transferase (30 U/ml) and 1× Terminal Deoxynucleotidyl Transferase Buffer in a thermal cycler as described in the Affymetrix GeneChip Whole Transcript (WT) Double-Stranded Target Assay Manual.
|
| |
|
| |
| Hybridization protocol |
200 ml 1x hybridization mixture containing 4.5 mg of fragmented and labeled DNA target, 7% DMSO and 50 pM control oligonucleotide B2 (Affymetrix P/N 900720) were prepared according to the Affymetrix hibridization protocol for single tiling arrays described in the Affymetrix GeneChip Whole Transcript (WT) Double-Stranded Target Assay Manual. 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 |
Affymetrix standard protocol using Affymetrix Fluidics 450 and Scanner 3000
|
| Description |
Control CEL: DNA_naked.CEL
Mononucleosomal DNA_pcr1D
|
| Data processing |
The test and ctrl CEL files were quantile normalized (PMID:12538238). After this, individual log2(test/control) fold changes were reported in WIG files.
All the results files are in Wiggle Track Format (WIG) containing: log2(mononucleosomal fraction/input fraction) for MN* samples. The WIG file coordinates are mapped against the Sanger September 2008 Schizosaccharomyces pombe genome version.
For all the genomic analyses, we have used as a reference the S. pombe genome version of 23/08/07 (http://www.pombase.org/ , ftp://ftp.sanger.ac.uk/pub/yeast/pombe/Chromosome_contigs/).
|
| |
|
| Submission date |
Oct 23, 2012 |
| Last update date |
Nov 25, 2013 |
| Contact name |
Luis Quintales |
| Organization name |
Instituto de Biología Funcional y Genómica (IBFG)
|
| Department |
Universidad de Salamanca / CSIC
|
| Street address |
Calle Zacarías González, 2
|
| City |
Salamanca |
| ZIP/Postal code |
37007 |
| Country |
Spain |
| |
|
| Platform ID |
GPL7715 |
| Series (2) |
| GSE41770 |
Clustered regulatory signals at nucleosome-depleted regions punctuate a constant nucleosomal landscape in Schizosaccharomyces pombe [Affymetrix] |
| GSE41773 |
Clustered regulatory signals at nucleosome-depleted regions punctuate a constant nucleosomal landscape in Schizosaccharomyces pombe |
|
