|
| Status |
Public on Mar 24, 2021 |
| Title |
CONTROL |
| Sample type |
genomic |
| |
|
| Source name |
CONTROL
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
treatment: None
antibody: none
|
| Treatment protocol |
First in small start-up culture, 1-2 ml culture of cdc25-22 was grown overnight at 25°C in shaking air incubator. Next day two cdc25-22 culture (biological replicates) were grown in 50 ml YES media in a 500 ml flask at 25°C to 2 x 106 cells/ml. 3. The cultures were transferred to a 37°C shaking air incubator for 3.5 hr to arrest. 4. 2 μl of the cultures were examined under the microscope for cell cycle arrest at 3 and 3.5 hr, more than 80% cells should be elongated and stuck in G2 phase after 3.5 hr. 5. Cell number was counted with a hemocytometer to 2 x 106 cells/ ml. 6. Cultures were removed from 25°C incubator at 60 min after release and fixed with formaldehyde for ChIP at S phase. Cell cycle synchrony was monitored every time by visualization under microscope before fixing the cells. Typically more than 80% of the cells should be in a specific cell cycle.
|
| Growth protocol |
2 ml pre-culture of tested strain was grown over night. Next day, appropriate amount of over night grown pre-culture was added to 100 ml of YES media, the volume of cells inoculated should be such that the culture reaches the desired cell density next morning
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Phenol chloroform
|
| Label |
biotin
|
| Label protocol |
amplified ChIP DNA was fragmented and labelled using the GeneChip® WT Double-Stranded DNA Terminal Labeling Kit (P/N 900812).
|
| |
|
| Hybridization protocol |
Fragmented and labelled DNA samples were hybridized according to the protocol described by the GeneChip® Hybridization, Wash, and Stain Kit according to Affymetrix specifications.
|
| Scan protocol |
Hybridization signal produced from tiling arrays was detected with a GeneChip® Scanner 3000 7G and .CEL files (The CEL file stores the results of the intensity calculations on the pixel values of the DAT file.
|
| Description |
Input DNA
|
| Data processing |
Data processed using custom scripts, initially a matlab script using the "Bioinformatics Toolbox" to process raw data, then R scripts to carry out noise correction, gene expression value calculation, normalisation and finally correlations with downloaded datasets.
Table with values corresopnding to correlation plots in figure 2A and 2B. Each row corresponds to a different gene and each column to different datasets (log(FPKM+1) values from downloaded processed RNA-seq data (GSM2803075 and GSM2803077), gene expression values from downloaded Pol2 ChIP-chip data (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-18/), assynchronous and S-phase Upf1 gene expression values from raw data described here, normalised using control files).
|
| |
|
| Submission date |
Mar 23, 2021 |
| Last update date |
Mar 24, 2021 |
| Contact name |
Saverio Brogna |
| E-mail(s) |
s.brogna@bham.ac.uk
|
| Organization name |
University of Birmingham
|
| Department |
School of Biosciences
|
| Street address |
School of Biosciences, University of Birmingham
|
| City |
Edgebaston |
| State/province |
West midlands |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10187 |
| Series (2) |
| GSE169422 |
Genome-wide chromosomal associationof Upf1 is linked toPol II transcriptionin Schizosaccharomyces pombe [ChIP-chip] |
| GSE169425 |
Genome-wide chromosomal associationof Upf1 is linked toPol II transcriptionin Schizosaccharomyces pombe |
|
