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| Status |
Public on Oct 11, 2022 |
| Title |
sen1_Sup_BrdU_G2M |
| Sample type |
genomic |
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| Source name |
yeast culture
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
antibody: Anti-BrdU_MBL_MI-11-3
treatment(s): Noco_G2/M_BrdU-Chip
strain: sen1
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| Growth protocol |
For BrdU-Chip_G2/M: Log in YPG, G1 cell cyle arrest in YPD+Alpha factor and release in S phase,23oC, YPD+Nocodazole to arrest cells in G2/M. For RNA:DNA hybrids Log in YPG, G1 cell cyle arrest in YPD+Alpha factor and release in S phase,23oC, at 45'-50' time point harvested as S phase time point
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
BrdU-Chip. Yeast culture was grown O/N as log at 23oC in SC-URA medium till up to 1x107 cells/ml. The synchronization was done in YPD or YPG medium using α-Factor at 23°C. After synchronization, BrdU 200μg/ml was added for 20 min before the release. Cells were released from G1 into YPD medium containing BrdU 200μg/ml. Cell were blocked using 0.1% of Sodium Azide and kept on ice for at least 5 minutes. Cells were pellet centrifuged and washed with 20ml of cold and sterilized 1xTE. Genomic DNA was isolated as mentioned in the genomic DNA extraction section and the DNA was re-suspended in 250 μl of 1xTE pH8. For each 200ml culture genomic DNA, 20μl Protein A dynabeads (Invitrogen) were washed twice in a costar prelubricated tube with 1ml of 1xPBS, 5mg/ml BSA, 0.1% Tween20. The beads were re-suspended in 20μl of 1x PBS, 5mg/ml BSA, 0.1% Tween20 and add 4μg of anti-BrdU antibody (MBL M1-11-3). This complex was incubated O/N at 4oC. BrdU containing genomic DNA was fragmented to 200-500bp using the bandelin UW2070 sonicator with following parameters as 20% power, 20 second/pulse for 6 times. Fragmented DNA was quantified and antibody-bead complex was washed two times with 1ml of 1xPBS, 5mg/ml BSA, 0.1% Tween20. Beads were resuspended in 20μl and split into 2 costar prelubricated tubes. The DNA was denatured at 100°C for 10 minutes and immediately put on ice and 100μl of 2xPBS and 200μl of ice cold 1xPBS, 2%BSA, 0.2%Tween-20 was added. 10μl of antibody-beads complex was added in denatured DNA and incubated O/N at 4°C rotating. Beads containing tubes were kept on magnetic grid and washed as following, 2 times with ChIP lysis buffer (50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate), 2 times with (ChIP Lysis buffer +500mM NaCl), 2 times with ChIP Washing buffer (10mM Tris-HCl pH8.0, 250mM LiCl, 0.5% NP-40, 0.5% Sodium deoxycholate, 1mM EDTA) and one wash with 1xTE pH8.0 and all residual liquid was removed with vacuum pump. The beads were re-suspended in 50μl of ChIP Elution buffer (50mM Tris-HCl pH8.0, 10mM EDTA, 1%SDS) and incubated at 65°C for 10 minutes. To the eluted material (IP) and Input, 49μl of 1xTE, 1μl of Proteinase K (Stock 50mg/mL) was added and incubated at 37oC for 1hr and the DNA was purified by Qiagen PCR purification Kit and eluted with EB buffer. IP and Input samples proceeded with WGA (Whole Genome Amplification) using WGA2 GenomePlex Complete Genome Amplification (WGA) Kit as per manufacturer’s instructions. DRIP-Chip. DRIP-ChIP was performed using anti-RNA:DNA hybrid monoclonal mouse antibody S9.6 as previously described (Achar et al., 2020; Chan et al., 2014). The method was adopted with a few changes; cells were cross-linked by shaking with 1% formaldehyde in culture medium for 20 min at room temperature and the reaction was quenched with 0.125 M glycine for 5 min at RT. Cells were centrifuged and washed twice with ice-cold 1xPBS and lysed in 1 ml of lysis buffer (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1%TritonX-100, 0.1%Na-deoxycholate) using Zirconia beads. Cross-linked chromatin was sheared to an average size of 300-500 bp by 6x15-s pulses using a Biorupter sonicator. The lysate was then centrifuged to remove cell debris and collected as chromatin fraction at 4oC. The chromatin fraction was incubated with Protein-A magnetic beads coated with anti-DNA:RNA hybrid-S9.6 antibody (Boguslawski et al., 1986) overnight at 4oC . The immune complexes were washed twice with the following buffers- ChIP-lysis buffer (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate), lysis buffer+ high salt (ChIP lysis buffer + 360mM NaCl), ChIP-wash buffer (250mM LiCl, 10mM Tris pH8.0, 0.5% Na-deoxycholate, 0.5%NP-40, 1mM EDTA) and 1xTE (20mM Tris pH8.0, 2mM EDTA). The RNA:DNA hybrid complexes were eluted from the beads in 250μl elution buffer (1%SDS, 50mM Tris pH8.0, 10mM EDTA) at 65oC for 20 min followed by the addition of proteinase K at 500μg/ml and incubated overnight at 65oC. Input DNA was isolated from sheared chromatin input (1/100 of the material used for ChIP). Both IP and input samples were processed as mentioned in the section below ‘Microarray and data processing’.
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| Label |
biotin
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| Label protocol |
In case of ChIP-chip, BrdU-chip and DRIP-Chip, samples were measured using nanodrop and 5000ng of Input and IP was used for the further steps. Both IP and input DNA were amplified using the GenomePlex complete whole-genome amplification kit, they were then biotin-labelled. Biotin labelling was performed using 4.85μl of 10XOne-Phor-All Buffer, 25mM CoCl2, 2.9μl DNAase reaction mix 1.5μl and 5μg of DNA (IP or Input) with ddH2O in 40.75μl. Samples were vortexed, pulse-spun and incubated in thermocycler at 37oC for 30 seconds and then transferred to 95oC for 15 minutes. Samples were transferred to new 1.5ml Eppendorf tube and DNA labelling was performed using 5μl of TdT reaction buffer, 1μl Biotin-N11-ddATP (1nMole/μl) and 1μl terminal transferase (400U/μl). Samples were incubated at 37oC for 1hr.
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| Hybridization protocol |
Hybridized to Affymetrix GeneChip S. cerevisiae Tiling 1.0R Array (Sc03b_MR) according to the Affymetrix standard protocol
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| Scan protocol |
Standard Affymetrix Protocol.
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| Description |
BrdU-Chip, Replication fork fusion at G2/M stage of cell cycle by Nocodazole.
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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 |
Oct 06, 2022 |
| Last update date |
Oct 12, 2022 |
| 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 (2) |
| GSE214908 |
Sen1 and Rrm3 ensure permissive topological conditions for replication termination [ChIP-chip] |
| GSE214930 |
Sen1 and Rrm3 ensure permissive topological conditions for replication termination |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6617809_sen1_Sup_BrdU_G2M.CEL.gz |
31.2 Mb |
(ftp)(http) |
CEL |
| Processed data provided as supplementary file |
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