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| Status |
Public on Oct 06, 2011 |
| Title |
H3-T7 (new) in G2/M |
| Sample type |
SRA |
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| Source name |
H3-T7, wt, G2/M
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: wt
epitope tag: T7
histone h3: new
sampling time: after 5 hrs of G2/M arrest
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| Treatment protocol |
ChIP was performed as described previously (PMID 20018668) with the following modifications. All steps were done at 4°C unless otherwise indicated. Following cell lysis by bead beating, the insoluble chromatin of 1x10^9 cells was washed, resuspended in 400 μl FA lysis buffer (50 mM HEPESKOH [pH 7.6], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), and sheared using a Bioruptor (Diagenode) for 6 minutes with 30 seconds intervals at high. The 16 soluble fraction was diluted three fold in buffer 15mM Tris-HCl pH 7.4, 50mM NaCl, 1.5mM CaCl2, 5mM β-meracptoethanol, 5mM MgCl2, after which 25 units of micrococcal nuclease (MNase; Worthington) were added. The digestion reaction was incubated 20 minutes at 37°C and stopped by the addition of 10mM EDTA and 10mM EGTA, and tubes were placed on ice.
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| Growth protocol |
For tag switch experiments, yeast cells were grown overnight in YPD in the presence of Hygromycin B (200 μg/mL, Invitrogen). The cells were then diluted 1:10 into fresh YPD and incubated for 30–36 h. Recombination was induced by the addition of 1 μM β-estradiol (E-8875, Sigma-Aldrich). Subsequently, cells were diluted 1:25 in fresh YPD media to release the cells back into the cell cycle and kept in log phase by 1:2 dilutions into fresh media after each population doubling. Samples were taken after 1, 3, and 6 cell divisions or after 5 hours of G2/M arrest. The number of population doublings was determined by microscopy and OD. G2/M arrest was induced by addition of 15 μg/ml Nocodazole (Sigma-Aldrich) and confirmed by FACS analysis.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The majority of obtained fragments was around 150bp, as determined on a 2% TAE agarose gel stained with ethidium bromide. The isolated chromatin of the equivalent of 3x10^8 cells was immunoprecipitated overnight at 4°C using magnetic Dynabeads (Invitrogen), which were previously incubated with antibody O/N at 4°C.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
H3-T7-G2_M
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| Data processing |
Mapped Reads: Unique reads were mapped to the yeast genome (S288C_reference_genome_R61-1-1_20080605; 05-Jun-2008) with minimal errors (as described in Yassour et al. PNAS, 2009 (PMID 19208812)).
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| Submission date |
Oct 06, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Assaf Weiner |
| E-mail(s) |
assafwe@cs.huji.ac.il
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| Organization name |
The Hebrew University of Jerusalem
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| Department |
Computer Science
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| Street address |
Givat Ram
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| City |
Jerusalem |
| ZIP/Postal code |
91904 |
| Country |
Israel |
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| Platform ID |
GPL9134 |
| Series (1) |
| GSE28269 |
Patterns and mechanisms of ancestral histone protein inheritance in budding yeast |
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| Relations |
| SRA |
SRX100280 |
| BioSample |
SAMN00738232 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM811178_nocT7wt.FR.bedgraph.gz |
9.9 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM811178_nocT7wt_sequence_aligned.txt.gz |
43.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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