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| Status |
Public on Jun 30, 2019 |
| Title |
clr4D T0, H3K9me2, rep2 |
| Sample type |
genomic |
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| Source name |
clr4D T0, H3K9me2, rep2
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: PB2428
genotype: clr4D
antibody: anti-H3K9me2 (abcam 1220)
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| Treatment protocol |
2x10^8 cells at each time point were cross-linked with 1% formaldehyde at 25C for 30min. Crosslinking was quenched with 0.125M glycine for 5min and the cells were washed 2 times with cold PBS. Cell pellets were resuspended in 400μl lysis buffer (50mM HEPES(pH 7.5), 140mM NaCl, 1mM EDTA, 1% Triton-X, 0.1% Na-Deoxycholate and ) containing a protease inhibitor cocktail (cOmplete EDTA-free, Merck) and lysed with glass beads. Whole cell extracts (WCEs) were sonicated for 10min (20 cycles of 30 sec on 30 sec off) using a Bioruptor Pico sonicator (diagenode) producing chromatin 100-500bps in size. The extract was then incubated for 10 minutes at 4°C, and centrifuged for 5 minutes, 4°C at 14,000 × g and the supernatants were used for immunoprecipitation.
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| Growth protocol |
Cells grown in PMG (OD600 ~0.25) at 30C, and were washed twice with PMG-N. Cells were resuspended in PMG-N and incubated at 30C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For immunoprecipitation, 30 µL whole cell extracts were incubated with 4 µl anti-H3K9me2 (abcam 1220) and 10 µL Dynabeads conjugated to protein-A (New England BioLabs) for 2 hours at 4°C with agitation. The supernatant was removed and the beads were washed twice with Lysis buffer, followed by two washes each with Wash Buffer 1 (50 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% TritonX-100, and 0.1% Na-deoxycholate), Wash Buffer 2 (10 mM Tris (pH 8.0), 250 mM LiCl, 0.5% NP-40, and 0.5% Na-deoxycholate) and TE buffer (10 mM Tris (pH 8.0), 1 mM EDTA). Beads were resuspended in 100 μg/ml RNase A containing TE buffer incubated for 10 min at 37°C. Then, Proteinase K (0.5 mg/ml) was added and the mixture digested for 1 hour at 42°C. The crosslinks in samples were reversed by incubating for 4 hours at 65°C. After de-cross-linking, DNA was purified using a QIAquick PCR purificationkit (Qiagen).
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| Label |
biotin
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| Label protocol |
Samples were prepared and hybridized to GeneChip S.pombe Tiling 1.0FR Array according to Affymetrix specifications.
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| Hybridization protocol |
Samples were prepared and hybridized to GeneChip S.pombe Tiling 1.0FR Array according to Affymetrix specifications.
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| Scan protocol |
The tiling arrays were scanned using an Affymetrix GeneChip Scanner 3000 7G according to manufacturer's instruction.
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| Data processing |
The signal intensity data was analyzed with the Affymetrix Tiling Analysis Software (v. 1.1). Duplicate data were normalized with input using quantile normalization plus scaling and run with a bandwidth of 100. Data obtained from TAS were then directly loaded into Seqmonk, antibody background were next removed using ∆clr4 mutant and visualized using the following parameters (number of classes 30, Min probes count 30, Max probes count 100).
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| Submission date |
Jun 20, 2018 |
| Last update date |
Jun 30, 2019 |
| Contact name |
Eriko Oya |
| Organization name |
Karolinska Institutet
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| Street address |
Blickagången 16
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| City |
Huddinge |
| ZIP/Postal code |
141 83 |
| Country |
Sweden |
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| Platform ID |
GPL7715 |
| Series (1) |
| GSE116038 |
Leo1 is essential for dynamic regulation of heterochromatin and gene expression during cellular quiescence [ChIP microarray] |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM3207803_clr4D_T0_H3K9me2_rep2.CEL.gz |
11.1 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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