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| Status |
Public on Apr 20, 2015 |
| Title |
Hu mrc1∆ Mcm2-7 2 |
| Sample type |
SRA |
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| Source name |
Yeast culture
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
treatment: Hu
genotype: mrc1-delta
strain: UPY722
chip antibody: Mcm2-7
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For G1 experiments, strains were arrested in α-factor for 3 hours at 23C then processed for ChIP. For S-phase experiments, strain were similarly arrested with α-factor, then released into fresh media containing 400 µg/ml BrdU and 0.2 M HU until cell budding reached 75% (100-110 minutes at 23°C), harvested, and processed for ChIP-seq. Replica 1 of wild type was arrested in HU for 90 min, where 2DENQ and mrc1∆ were arrested in HU for 100 min; replica 2 of wild type, 2DENQ, and mrc1∆ were arrested in HU for 100 min; mcm2-1 and chaos3 were arrested in HU for 100 minutes. To assay Mcm-bound DNA, ChIP-seq DNA preparation was performed as previously described (Aparicio et al., 1997). Formaldehyde was added to 50 ml of α-factor arrested cells to a final concentration of 1% and mixed for 15 minutes at room temperature. Crosslinking was quenched by the addition of glycine to a final concentration of 125 mM and room temperature incubation for 5 minutes. Cells were washed twice with 20 ml of ice-cold TBS (20 mM Tris-HCl, pH 7.6, and 150 mM NaCl) and the resulting cell pellet was frozen in liquid nitrogen and stored at -80C. The cell pellet was thawed on ice, resuspended in 250 µl of 1X lysis buffer (50 mM HEPES/KOH, pH 7.5, 140 mM NaCl, 1mM EDTA, 1% Triton X-100, and 0.1% Na-Deoxycholate) with fresh protease inhibitors (1 mM PMSF, 10 mM benzamidine, 0.1 mg/ml TPCK, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 0.5 mg/ml bacitracin), glass beads were added, and the sample vortexed (cycles of 30 seconds on ice and 30 seconds vortexing for a total of 30 min) . 250 µl of additional 1X lysis buffer was added and briefly mixed. Using a hot 27-gauge needle, a hole was poked into the bottom of the tube, and the cell lysates were separated from the glass beads by centrifugation into a fresh tube). The DNA in the lysate was then sheared using a Branson 250 model sonicator to yield an average DNA size of 700 bp. 6 µl of anti-Mcm2-7 antibody (UM174, Stephen P Bell, MIT) or 4 µl of anti-Mcm2 (sc6680, Santa Cruz,) was added to cell lysates and incubated at 4C for 2 hours. 30 µl of protein G beads pre-equilibrated in 1X PBS, 0.1% BSA (Gamma bind, GE Healthcare) was then added and incubated for an additional hour at 4°C. Following incubation, beads were washed twice with 1 ml of lysis buffer with fresh PMSF; then once each with 1 ml of lysis buffer containing 500 mM NaCl once, 1 ml of TLNNE (10 mM Tris/HCl, pH 8.0, 0.25 M LiCl, 0.5% NP-40, 0.5% Na-Deoxycholate, and 1 mM EDTA), then 1 ml of TE. Each wash was performed with 5 minutes of room temperature incubation with mixing. To elute the DNA, 100 µl of elution buffer (50 mM Tris/HCl, pH 8.0, and 10 mM EDTA/1% SDS) was then added to the beads and incubated at 65°C for 15 min. Beads were removed by centrifugation, and the supernatant was transferred to a fresh tube. Beads were resuspended in 150 µl of TE containing 0.67% SDS and spun down again. The supernatants were pooled and incubated at 65°C overnight to reverse the proteinDNA crosslinks. The next morning, 250 µl of TE, 5 µl of 20 mg/ml proteinase K, and 0.25 µl of 20 mg/ml glycogen were added and incubated at 37°C for 30 minutes. After incubation, 55 µl of 4 M LiCl was added, and the DNA was phenol/chloroform extracted and precipitated with 10/24/11 6 ammonium acetate and ethanol. The dried DNA pellet was processed to construct the library for sequencing (see Materials and Methods). To assay BrdU incorporation, genomic DNA was prepared using standard procedures (Ryba et al., 2011), boiled, and immunoprecipitated with anti-BrdU antibodies (555627, BD Bioscience). The resulting DNA pellet was subjected to sequencing library preparation.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Genome assembly: For each sample sequenced reads were aligned to the S. cerevisiae S288C reference genome (SacCer2, SGD June 2008) (Kent et al., 2002) using MAQ (Li et al., 2008) allowing up to three mismatched bases. Reads with a mapping quality of >= 35 were used in downstream analysis.
Data normalization: Reads were binned across the genome, and RPKM (reads per kilobase per million mappable reads) was calculated for each bin. For all of the BrdU ChIP-Seq experiments, bins of 5000 bp stepping every 1250 bp were used, and for all the Mcm ChIP-Seq experiments, bins of 1000 bp stepping every 250 bp were used. All in silico analyses were done in the R programming environment using the Bioconductor suite of packages (Gentleman et al., 2004; RDevelopmentCoreTeam, 2010). Experimental replicates were quantile normalized and combined by calculating the mean score within each bin.
Peak determination: For each bin along a chromosome, a probability was assigned according to that chromosome’s empirical cumulative distribution of RPKM. A threshold was determined, and bins with a probability greater than or equal to the cutoff were taken as peaks (the ranges of overlapping peaks were merged such that the resulting peaks represented the union of all enriched bins). For BrdU ChIP-Seq in HU arrested cells, the thresholds used were as follows: WT strains p ≥ 0.95, Mrc1 p ≥ 0.75, and Mcm2DENQ p ≥0.75. For ChIP-Seq experiments in G1 arrested cells, a threshold of p ≥ 0.96 was used. Replicates were combined by taking the intersection of peak calls. When possible, peaks were assayed for concordance against analogous data previously reported (Crabbe et al., 2010).
Genome_build: SacCer2
Supplementary_files_format_and_content: .wig files represent RPKM across the genome; .bed files indicate regions of enrichment.
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| Submission date |
May 17, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
David M MacAlpine |
| E-mail(s) |
david.macalpine@duke.edu
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| Phone |
919 681 6077
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| Organization name |
Duke University Medical Center
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| Department |
Pharmacology and Cancer Biology
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| Street address |
Box 3813 DUMC
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27714 |
| Country |
USA |
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| Platform ID |
GPL9377 |
| Series (1) |
| GSE38032 |
Mcm2-7 is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of its DNA Gate |
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| Relations |
| SRA |
SRX148618 |
| BioSample |
SAMN00996556 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM932795_Hu-MCM-Rep2-Norm-MRC1-1000-250.wig.gz |
291.4 Kb |
(ftp)(http) |
WIG |
| GSM932795_Hu-MCM-Rep2-Norm-Peaks-MRC1.bed.gz |
2.2 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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