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| Status |
Public on Sep 25, 2019 |
| Title |
RRM3_Hybrid_S_28_IP |
| Sample type |
genomic |
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| Source name |
IP fraction of RNA-DNA Hybrid in rrm3 deletion at 28 degree S Phase
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
category: RNA-DNA Hybrid
cellcycle: S Phase
antibody: S9.6 Monoclonal Antibody
strain: W303
treatment: Glucose
temperature: 28
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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 |
Cells were crosslinked with 1% formaldehyde in culture medium for 30 min at room temperature followed by quenching with 0.125 M glycine for 5 min. 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 was incuabted with magnetic beads coated with anti-DNA:RNA hybrid monoclonal mouse antibody S9.6 overnight at 4C. The immune complexes were washed with the following buffers 2X; Chip-lysis buffer, high-salt lysis buffer (Chip-lysis buffer + 360mM NaCl), Chip-wash buffer (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 protein-DNA complexes were eluted from the beads in 250 µl elution buffer (1% SDS, 50 mM Tris pH8.0, 10mM EDTA) at 65C for 20 min followed by the addition of proteinaseK to 500 µg/ml and overnight incubation at 65C. Input DNA was isolated from 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 |
RNA-DNA hybrid mapping of across the genome
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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 |
Sep 24, 2019 |
| Last update date |
Sep 25, 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 |
| GSM4094804_RRM3_Hybrid_S_28.bed.gz |
105.2 Kb |
(ftp)(http) |
BED |
| GSM4094804_RRM3_Hybrid_S_28.bedGraph.gz |
34.8 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM4094804_RRM3_Hybrid_S_28_IP.CEL.gz |
24.3 Mb |
(ftp)(http) |
CEL |
| Processed data provided as supplementary file |
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