|
| Status |
Public on Dec 31, 2011 |
| Title |
ChIP_Orc4 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Orc4 ChIP DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: 972 h-
antibody: Rabbit polyclonal
culture type: Asynchronous Mitosis
sample type: IP DNA
|
| Treatment protocol |
Yeast extract (YE) medium at 32 ºC until OD595=0.8
|
| Growth protocol |
Yeast extract (YE) medium at 32 ºC until OD595=0.8
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Orc4 was immunoprecipitated with 5 microgrames of polyclonal anti Orc4 raised against the last 14 carboxy-terminal aminoacids of the protein. Ten microliters of immunoprecipitate and control DNA from whole cell extract (WCE) were amplified 4 times with Sequenase (70775Y, USB) and primer A (GTTTCCCAGTCACGGTC(N)9), followed by 20 rounds of amplification with Taq polymerase (Biotools) in the presence of 8 mM dUTP and primer B (GTTTCCCAGTCACGGTC).
|
| 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 |
Whole cell extract (WCE)
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: 972 h-
antibody: None
culture type: Asynchronous Mitosis
|
| Treatment protocol |
Yeast extract (YE) medium at 32 ºC until OD595=0.8
|
| Growth protocol |
Yeast extract (YE) medium at 32 ºC until OD595=0.8
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Orc4 was immunoprecipitated with 5 microgrames of polyclonal anti Orc4 raised against the last 14 carboxy-terminal aminoacids of the protein. Ten microliters of immunoprecipitate and control DNA from whole cell extract (WCE) were amplified 4 times with Sequenase (70775Y, USB) and primer A (GTTTCCCAGTCACGGTC(N)9), followed by 20 rounds of amplification with Taq polymerase (Biotools) in the presence of 8 mM dUTP and primer B (GTTTCCCAGTCACGGTC).
|
| 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 |
Orc4 ChIP DNA
All the results files are in Wiggle Track Format (WIG) containing: log2(IP fraction/input fraction) for ChIP* samples, log2(mononucleosomal fraction/input fraction) for NUC* samples, and RNA raw signal for TM* samples (transcription mapping). The WIG file coordinates are mapped against the Sanger September 2008 Schizosaccharomyces pombe genome version.
|
| Data processing |
Data was quantile normalized (PMID:12538238). log2(test/control) fold changes were reported in WIG files.
|
| |
|
| Submission date |
Apr 27, 2011 |
| Last update date |
Dec 31, 2011 |
| 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 (1) |
| GSE28879 |
Nucleosomal organization of replication origins and meiotic recombination hotspots in fission yeast |
|
