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| Status |
Public on Jan 18, 2013 |
| Title |
Yeast Mif2 ChIP-Seq |
| Sample type |
SRA |
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| Source name |
Yeast cell
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: W303 Mif2-9xMyc-KanMX4
antibody: anti-Myc EZview affinity gel (Sigma)
antibody manufacturer: Sigma
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| Growth protocol |
Yeast cells were grown in 500 mL rich medium supplemented with adenine and uracil, in the presence of dextrose (YPADU) to mid-log phase (OD = 0.5-0.7).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-Seq experiments were performed at least in independent biological duplicates, as described previously (Aparicio et al., Curr Protoc Cell Biol (2004); Lefrançois et al., BMC Genomics (2009); Lefrançois et al., Methods Enzymology (2010)). Yeast strains were grown in 500 mL YP media supplemented with adenine and uracil, in presence of glucose or galactose/raffinose, to mid-log phase (OD 600=0.5-0.7). Proteins were crosslinked to DNA by treating cells with formaldehyde (1% final concentration) for 15 minutes, then quenched with glycine. Cells were collected by filtration after two washes. After cell lysis using a FastPrep machine (MP Biomedical), chromatin was sheared by sonication using a Branson Digital 450 sonifier (Branson). Clarified, sonicated lysates were taken at this step for Sono-Seq, prior to immunopreciptation. Immunoprecipitations of Myc-tagged and HA-tagged strains, as well as those of the respective control untagged strains, were carried out overnight with EZ-View anti-Myc or anti-HA affinity gels (Sigma). For native RNA Polymerase II ChIP, cell lysates were incubated with Pol II 8WG16 mouse monoclonal antibody (Covance) and pulled down using Protein G agarose beads (Millipore). After several washes and reversal of protein-DNA crosslinks, ChIP DNA was purified through a Qiagen MinElute PCR purification column (Qiagen). Illumina sequencing libraries were generated using adapters for multiplexing as described (Lefrançois et al., BMC Genomics (2009); Lefrançois et al., Methods Enzymology (2010)), and following Illuminaâs manufacturer recommendations. Four barcoded libraries were mixed in equimolar ratios and processed on an Illumina Genome Analyzer II. We have also used previously published Cse4 ChIP-Seq data, with normal Cse4 levels, which were deposited in the Gene Expression Omnibus (GEO) database under GSE13322.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Mif2 ChIP-Seq, normal Cse4 levels
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| Data processing |
Raw sequencing data were first processed by the built-in Illumina analysis pipeline. Prior to the actual sequence mapping, reads were parsed according to the 4-bp index, and barcodes were then removed. Those reads lacking an intact index were discarded. The remaining bases were aligned against the S. cerevisiae S288c reference genome version 2 (SGD/UCSC sacCer2, June 2008) by the ELAND algorithm (Illumina). The peak scoring algorithm PeakSeq (Rozowsky et al., Nat Biotech (2009)) was used to identify statistically significant binding sites, changing only the following parameters to account for the compact S. cerevisiae genome: window size of 10 kb during the normalization step and bin size of 1 kb during the linear regression step (PeakSeqOutput files, with Q-value < 0.05). ChIP-Seq data from epitope-tagged strains were scored against ChIP-Seq data from their matching untagged strains. In both cases, anti-Myc antibodies were used during ChIP. Scoring reference sets were created by pooling uniquely-mapping reads from biological replicates of untagged control strains. As a reference sample marking open chromatin (Auerbach et al., PNAS (2009)), two lists of Sono-Seq significant regions were generated, obtained after scoring against either anti-Myc or anti-HA control sets. To uncover Centromere-like Regions (CLRs), we took a conservative, stringent approach to minimize false positives lacking functional significance or failing qPCR validation. For each biological replicate of a particular kinetochore component, only putative binding regions with Q-value < 10-5 were considered (Target bed files). Several other criteria were then applied. First, binding sites called in two biological replicates from Cse4, Mif2, Ndc10 and Ndc80 ChIP-Seq data, in either a Cse4 overexpression strain or in a wild-type strain, were overlapped with maxgap=150 (maximum gap between non-overlapping peaks). Next, to identify a binding region as a CLR, 1) all four kinetochore proteins must be present at the putative site given the q-value threshold; and 2), for proteins in direct contact with DNA, mean PeakSeq ratios between both replicates at a particular target site should be above 2.00 for open chromatin marker Cse4 (same for PolII and Sono-Seq regions), and 1.50 for direct DNA binders Mif2 and Ndc10. Several other filters were used to distinguish between lower confidence regions and higher confidence regions for subsequent functional analyses, including comparison of PeakSeq experimental reads and PeakSeq background reads between CLRs and CENs, inspection of normalized signal tracks (good tagged/untagged signal ratio and low background in the appropriate untagged control desired), binding over a highly PolII-occupied ORF, and presence in a HOT region. Regions that passed those criteria and filters were termed CLRs (Centromere-Like Regions). Other binding sites that did not pass the aforementioned filters were referred to as LCNCRs (for low-confidence, negative control regions).
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| Submission date |
Aug 18, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Raymond K Auerbach |
| Organization name |
Yale University
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| Street address |
266 Whitney Ave
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06511 |
| Country |
USA |
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| Platform ID |
GPL9377 |
| Series (1) |
| GSE31466 |
Centromere-Like Regions in Budding Yeast |
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| Relations |
| SRA |
SRX092424 |
| BioSample |
SAMN00710370 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM781935_Mif2_Rep1_CATT.eland.txt.gz |
4.5 Mb |
(ftp)(http) |
TXT |
| GSM781935_Mif2_Rep1_CATT.sgr.gz |
1.3 Mb |
(ftp)(http) |
SGR |
| GSM781935_Mif2_Rep1_CATT_PeakSeqOutput.txt.gz |
7.1 Kb |
(ftp)(http) |
TXT |
| GSM781935_Mif2_Rep1_CATT_targets.bed.gz |
237 b |
(ftp)(http) |
BED |
| GSM781935_Mif2_Rep2_TGCT.eland.txt.gz |
7.9 Mb |
(ftp)(http) |
TXT |
| GSM781935_Mif2_Rep2_TGCT.sgr.gz |
2.2 Mb |
(ftp)(http) |
SGR |
| GSM781935_Mif2_Rep2_TGCT_PeakSeqOutput.txt.gz |
8.3 Kb |
(ftp)(http) |
TXT |
| GSM781935_Mif2_Rep2_TGCT_targets.bed.gz |
256 b |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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