|
| Status |
Public on Apr 15, 2015 |
| Title |
HS_Top2_ChIPSeq IP |
| Sample type |
SRA |
| |
|
| Source name |
Cells cultivated in rich media
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: Top2-3FLAG
cell type: Top2-FLAG strain after temperature-shift from 20C to 36C
passages: Treated by 36°C for 9min
chip antibody: anti-Flag M2 antibody
chip antibody vendor: Sigma-Aldrich
chip antibody cat. #: F3165
chip antibody lot #: 069K6006
molecule subtype: Immunoprecipitated genomic DNA
|
| Treatment protocol |
For temperature-shift (T-shift) conditions in asynchronously cultured cells, cells incubated at 20°C for 8 h were shifted to 36°C for 9 min.
|
| Growth protocol |
Temperature-shift experiments were performed as previously reported (Nakazawa et al. 2011), using nda3-KM311 mutant. Cs mutant cells were grown in YPD at 30°C (a permissive temperature). When cell concentrations reached 4×10^6 cells/mL, cultures were transferred to 20°C and incubated for 8 hr to (reversibly) block spindle formation and to arrest cells at a prometaphase-like stage (Hiraoka et al. 1984). Asynchronously cultured cells were also harvested at 20°C for 8 hr in the absence of nda3-KM311.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sonicated and cross-linked chromatin were prepared from S. pombe cell extracts (Nakazawa et al., 2008). DNA samples co-precipitated with anti-FLAG antibody were prepared along with input DNA (whole cell extract [WCE] DNA).
Prepared DNA libraries were processed by single-end sequencing (36 bp) on the iIlumina Genome Analyzer GA IIx, according to the manufacturer’s instructions.
ReferenceHiraoka et al., 1984 Cell 39, 349-358. Nakazawa et al., 2008 J Cell Biol 180, 1115-1131. Nakazawa et al., 2011 J Cell Sci 124, 1795-1807.
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
processed data file: HS_Top2-3FLAG.w500.b10.pseuduvalue10.Ratio.wig
|
| Data processing |
After each completed sequencing run, raw image files from the illumina sequencer were computationally processed to obtain nucleotide-base calls. The standard analytical pipeline provided by illumina (CASAVA 1.8.2) was used for base-calling and demultiplexing of samples with an index sequence that was attached to the template during sample preparation.
Subsequent to base-calling and demultiplexing, sequencing reads were aligned to the S. pombe genome sequence (downloaded from Ensemble genomes [ftp://ftp.ensemblgenomes.org/pub/fungi/current/fasta/schizosaccharomyces_pombe/dna/], Schizosaccharomyces_pombe.ASM294v1.16.dna.chromosome I, II, III & MT FASTA format) using Bowtie alignment software (bowtie-bio.sourceforge.net) with default parameters. To avoid artifacts associated with spuriously high read numbers, the 10x average read number was set as maximum at each location.
To compare precipitated DNA (IP) and input DNA (WCE) data, the total read number of each sample was normalized to 10 million. The whole genome was then scanned using a sliding window to smooth the data (window = 500 bp, step = 10 bp).
Before calculating a ratio, to avoid dividing by a small number caused by low WCE coverage, we added a pseudocount of 10 to all windows of each sample. ChIP enrichment was calculated as the ratio of IP to WCE for each window.
This ratio was used for visualization of the ChIP-seq profile along three chromosomes using an Integrated Genome Browser (IGB, ver. 7.0.3).
Genome_build: S. pombe genome sequence (downloaded from Ensemble genomes [ftp://ftp.ensemblgenomes.org/pub/fungi/current/fasta/schizosaccharomyces_pombe/dna/], Schizosaccharomyces_pombe.ASM294v1.16.dna.chromosome I, II, III & MT FASTA format)
Supplementary_files_format_and_content: wig files for ChIP enrichment calculated as the ratio of IP to WCE
|
| |
|
| Submission date |
Feb 16, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Norihiko Nakazawa |
| E-mail(s) |
nakazawa@oist.jp
|
| Organization name |
Okinawa Institute of Science and Technology Graduate University
|
| Department |
G0 Cell Unit
|
| Lab |
Yanagida lab
|
| Street address |
1919-1 Tancha
|
| City |
Onna-son |
| State/province |
Okinawa |
| ZIP/Postal code |
904-0495 |
| Country |
Japan |
| |
|
| Platform ID |
GPL15167 |
| Series (1) |
| GSE65956 |
RNA pol II transcript abundance controls condensin accumulation at mitotically upregulated and heat shock-inducible genes in fission yeast |
|
| Relations |
| BioSample |
SAMN03349705 |
| SRA |
SRX878638 |
