ChIP-Seq experiments to analyse Rec8 and Zip1 localisation in Ctf19c mutants during meiotic prophase
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Alastair Kerr
PROVIDER: E-GEOD-70032 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Ctf19c mutants were shown to specifically impair cohesin localisation at the pericentromere during mitosis. The goal of this experiment is to confirm whether this mutant has the same effect on cohesin localisation during meiotic prophase. To do so, diploid S. cerevisiae SK1 cells were constructed containing Rec8-3HA in an ndt80-d background. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication) Diploid S. cerevisiae SK1 cells were constructed containing Rec8-3HA in an ndt80-d background. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication)
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Project description:Purpose: The Goal of this study is to look at 1) Differences in genome wide localization of Scc2 between wild type and scc4-m35 cells in S-phase 2) Differences in genome wide localisation of Scc1 cohesin between wild type and scc4-m35 in metaphase arrested cells. Method: 1) Wild-type (AM15307) and scc4-m35 (AM15311) cells carrying SCC2-3FLAG were fixed for 2 hr, 15 min following an a-factor arrest. Samples were harvested, anti-HA ChIP was performed and both input and IP samples were sequenced for both strains. 2) Wild-type (AM1145), wild type constructed in the same way as the mutant strain (carrying a HIS3 marker) (AM15540) and scc4-m35 mutant (AM15537) cells carrying SCC1-6HA were synchronised with a-factor and arrested in mitosis by treatment with nocodazole/benomyl for 2 hr. Samples were harvested, anti-HA ChIP was performed and input and IP samples were sequenced for the three strains. Results: 1) This experiment did not yield meaningful data. 2) All 16 centromeres were specifically depleted of Scc1 in the scc4-m35 background. Scc1 depletion extended roughly 10 kilobases to either side of the core centromere but not to chromosome arms 1) ChIP-seq for S. cerevisiae Scc2-3FLAG in S-phase cells in Wild type and in scc4-m35 mutant (samples VM1-VM4). 2) ChIP-seq for Scc1-6HA in metaphase-arrested cells in Wild type and in scc4-m35 mutant (samples VM5-VM10) Vasso Makrantoni: experiments and Alastair Kerr: ChIP Seq Data analysis https://github.com/AlastairKerr/Hinshaw2015
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