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| Status |
Public on Mar 11, 2019 |
| Title |
WT_TopAplasmid_1hr_Supercoil_INPUT |
| Sample type |
genomic |
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| Source name |
INPUT fraction of Biotin tagged psoralean of wt TopA plasmid in GLU at 37 degree for 1hr15mins S Phase
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
category: BTMP DNA-Supercoil
cell cycle: S Phase
antibody: None
strain: W303
treatment: Glucose
temperature: 37 degree for 1hr 15mins
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| Growth protocol |
1X10e7 cells were synchronized at G1 phase using Alpha-factor and cells were harvested for "G1" samples. For "S" phase samples cells were released into fresh medium after washing cells with 2X YP-medium. After 15 mins of the realese from alpha factor arrest cells were harvested for Chip protocols
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from Qiagen Gneomic-tip 100/G (Cat no 13343) & Genomic DNA buffer Set (Cat no 19060). Purified DNA was sheared to an average size of 500 bp by 6X 15-second pulses using a Biorupter sonicator. bTMP was added to purified DNA and incubated in dark for 90 mins and crosslinked with UV at 365nm (800 mJ/cm2). DNA is precipitated using isopropanol and washed with 70% ethannol. Dried Pellate was dissolved in buffer (50mM tris Ph 8.0, 10mM EDTA, 0.1% SDS) and incuabted with Dynabeads MyOne streptavidin (Invitrogen cat no. 65001) overnight at 4C. The beads were washed 2X with each of the following buffers; wash Buffer I (20mM tris-HCL pH 8, 2mM EDTA, 150mM NaCl, 1% triton X, 0.1% SDS), wash buffer II (20mM tris-HCL pH 8, 2mM EDTA, 500mM NaCl, 1% triton X, 0.1% SDS) wash buffer III (250 mM LiCl, 10 mM Tris pH8.0, 0.5% Na deoxycholate, 0.5% NP40, 1mM EDTA) and 1x TE (20 mM Tris pH8.0, 2 mM EDTA). The bTMP-DNA complexes were eluted from the beads in 250 µl elution buffer (95% Formamide, 10mM EDTA ) at 90C for 20 min and eluted samples were cleaned with Qiagen PCR clean up kit. For Input, DNA was isolated with sheared chromatin input (1/100 of the material used for ChIP).
Cells were collected blocked using Sodium Azide (0.1%). bTMP is added and cells were incubated in dark for 90 mins and crosslinked with UV at 365nm (800 mJ/cm2). The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (50mM Hepes KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate) using Zirconia beads. The crosslinked chromatin was sheared to an average size of 500 bp by 6X 15-second pulses using a Biorupter sonicator. The lysate was then centrifuged to remove cell debris. The chromatin fraction is incuabted with Dynabeads MyOne streptavidin (Invitrogen cat no. 65001) overnight at 4C. The beads were washed 2X with each of the following buffers; wash Buffer I (20mM tris-HCL pH 8, 2mM EDTA, 150mM NaCl, 1% triton X, 0.1% SDS), wash buffer II (20mM tris-HCL pH 8, 2mM EDTA, 500mM NaCl, 1% triton X, 0.1% SDS) wash buffer III (250 mM LiCl, 10 mM Tris pH8.0, 0.5% Na deoxycholate, 0.5% NP40, 1mM EDTA) and 1x TE (20 mM Tris pH8.0, 2 mM EDTA). The bTMP-DNA complexes were eluted from the beads in 250 µl elution buffer (95% Formamide, 10mM EDTA ) at 90C for 20 min and eluted samples were cleaned with Qiagen PCR clean up kit. For Input, DNA was isolated with sheared chromatin input (1/100 of the material used for ChIP).
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| Label |
biotin
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| Label protocol |
Both IP and Input samples are amplified by using Whole genome amplificatio ki protocol ( Sigma-Aldrich WGA1-50RXN) pripor to labelling with Biotin-N11-ddATP (1nMole/ul).
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| Hybridization protocol |
Labelled probes were hybridized to Affymetrix S.cerevisiae whole genome tiling R array for 16 hours at 42 C in a hybridization oven at 60 rpm
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| Scan protocol |
Arrays were scanned on Affymetrix geneChip Scanner 3000 7G
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| Description |
Control data from naked genomic DNA used to eliminate the false positive binding of BTMP drug
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| Data processing |
The CEL files are processed using rMAT R package for normalization, MAT score calculation and calling enriched regions or peaks. The following commands and parameters are used; i) (Normalization) SetNorm = NormalizeProbes(Set, method="MAT", robust=T, all=T, standard=TRUE, verbose=FALSE), where 'Set' is the probe map object created using CEL file and probe annotation file (Sc03b_MR_v04.bpmap) ii) (MAT score calculation) RD = computeMATScore(SetNorm, dMax=300, verbose=FALSE, cName = InputColumnName), iii) (Calling Enriched Region) peak = callEnrichedRegions(RD, dMax=300, dMerge=300, nProbesMin=8, method=method, threshold=1.5, verbose=FALSE)
Bed file containing the enriched regions with respective MAT score and BedGraph file containing the score for short interval bases. Both bed and bedGraph files are generated using rMAT R package and these files are UCSC genome compatible with sacCer3 reference.
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| Submission date |
Mar 11, 2019 |
| Last update date |
Mar 11, 2019 |
| Contact name |
Mohamood Adhil Mohammed Iqbal |
| E-mail(s) |
[email protected]
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| Organization name |
IFOM - The FIRC Institute of Molecular Oncology
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| Street address |
Via Adamello 16
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| City |
Milano |
| State/province |
Lombardia |
| ZIP/Postal code |
20139 |
| Country |
Italy |
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| Platform ID |
GPL7250 |
| Series (1) |
| GSE114410 |
DNA topoisomerase and supercoil accumulation across yeast genome |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM3664889_WT_TopAplasmid_1hr_Supercoil_INPUT.CEL.gz |
29.6 Mb |
(ftp)(http) |
CEL |
| Processed data provided as supplementary file |
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