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| Status |
Public on Dec 11, 2019 |
| Title |
RNAPII_pob3_1 |
| Sample type |
SRA |
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| Source name |
whole cell
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: pob3
chip antibody: RNAPIISer2P - Dirk Eick lab
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
100 ml yeast cultures were grown to mid-log phase to OD600=0.6. The cultures were cooled down at RT for 10 min and fixed with 1% FA for 20 min at RT on the shaker. Cross-linking was stopped with 150 mM Glycine for 10 min at RT on the shaker. Cells were spun down 2 min at 3000 rpm at 4°C, washed 2x with 30 ml cold dH2O, the pellet wash immediately frozen down in liquid nitrogen and kept at -80°C until further processing. Pellets were resuspended in 1 ml FA(1) buffer (50 mM Hepes-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (v/v) , 0,1% NaDeoxycholate (w/v) , 0,1% SDS (w/v)0 supplemented with Roche protease inhibitors and phosSTOP (Roche). Cells were broken in a bead beater (Precellys): 9x30s. After centrifugation, the supernatant and the pellet were sonicated for 15 min at 4°C. Chromatin extracts were spun down for 10 min at 14000 rpm at 4°C. Different amount of chromatin was used for different antibodies: 100 ul chromatin (for H3, Spt16, Pob3 ChIP) or 500 ul chromatin (for RNAPIISer2, H3K9me2, H2Bub ChIP). Sampels were incubated with antibodies O/N at 4°C on the wheel. 25 ul of FA(1) buffer washed Dynabeads were added to each sample and they were incubated for 2 hours at 4°C on the wheel. Samples were then washed 3x for 5 min at RT with FA(1) buffer, FA(2) buffer (FA(1) buffer with 500 mM NaCl), once with LiCl buffer(10 mM TrisHCl, pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0,5% NP-40 (v/v) , 0,5% NaDeoxycholate (w/v)) and once with TE buffer. DNA was eluted from the antibodies with ChIP Elution buffer (50 mM Tris HCl, pH 7.5, 10 mM EDTA, 1% SDS) for 15 min at 65°C in a thermomixer set to 1300 rpm. DNA was treated with RNAseA (Thermo Fisher) for 1 hr at 37°C, followed by Proteinase K digestion and de-crosslinking O/N at 65°C. DNA was purified with Zymo Research ChIP DNA Clean and Concentrator kit according to the manual instructions. To obtain enough material for the library preparation usually 3 technical IP replicates were pulled.
ChIP-seq libraries were prepared with 2 ng of DNA with NEBNext®Ultra™ II DNA Library Prep Kit for Illumina® according to the manual instructions. The libraries were barcoded and sequenced at LAFUGA at the Gene Center (Munich).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
ChIP-seq 50 bp single-end reads were aligned to the reference (Schizosaccharomyces pombe ASM294v2) using bowtie2 (version 2.2.9).
Reads were processed using the Homer software package (Heinz et al., 2010). Tag directories were created with the parameter -mapq 1 and bedgraph coverages were generated and normalized to the total number of reads and to the corresponding input using the makeUCSCfile command.
Genome_build: Schizosaccharomyces pombe ASM294v2
Supplementary_files_format_and_content: Bedgraph format. Normalized coverage (ChIP/Input).
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| Submission date |
Aug 13, 2019 |
| Last update date |
Dec 12, 2019 |
| Contact name |
Tamas Schauer |
| E-mail(s) |
tamas.schauer@helmholtz-munich.de
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| Organization name |
Helmholtz Zentrum München
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| Department |
Institute of Epigenetics and Stem Cells
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| Street address |
Feodor-Lynen-Straße 21
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| City |
Munich |
| ZIP/Postal code |
81377 |
| Country |
Germany |
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| Platform ID |
GPL13988 |
| Series (2) |
| GSE124092 |
The chaperone FACT and H2B ubiquitination maintain S. pombe genome architecture through genic and subtelomeric functions |
| GSE135766 |
The chaperone FACT and H2B ubiquitination maintain S. pombe genome architecture through genic and subtelomeric functions [ChIP-Seq] |
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| Relations |
| BioSample |
SAMN12566357 |
| SRA |
SRX6707763 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4029382_RNAPII_pob3_1.INPnorm.bedgraph.gz |
57.2 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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