|
Status |
Public on Sep 10, 2015 |
Title |
BrdUIP_WT |
Sample type |
SRA |
|
|
Source name |
KYP123
|
Organism |
Schizosaccharomyces pombe |
Characteristics |
genotype/variation: Wild_Type epitope-tag: adh1-Thymidine kinase antibody: Anti-bromodeoxyuridine (MBL), Subclass; mouse IgG1, Clone; 2B1, Cat No.; MI-11-3, strain: KYP123
|
Treatment protocol |
Cells during G1 phase were fixed with 1% Formaldehyde at 20min after release. Cells were arrested at early S-phase in 25mM HU at 60min after release from M-phase arrest and then the early replicated regions were labeled with 200µg/ml BrdU.
|
Growth protocol |
The stains were grown until mid log phase in YES medium at 30ºC. Cells containing nda3-KM311 mutation were arrested at M-phase at 20ºC for 5 hr. Cells were released into sequential cell cycle at 30ºC.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
The cell wall was disrupted by zirconia beads and multi-beads shocker (Yasui Kikai Co.). Lysates were prepared by sonication and clarified by centrifugation. Protein and DNA complexes were immuno-precipitated by antibody conjugated with Protein G Dynabeads. BrdU-incorporated genomic DNA was purified by Qiagen Genomic-tip and genomic buffer set. Purified genomic DNA was sheared by sonication. The fragmented DNA was heat-denatured and BrdU-substituted DNA was immuno-precipitated by Anti-BrdU antibody conjugated with anti-IgG1a-dynabeads. ChIP, BrdUIP, and Input DNA was sheared to around 150bp by S220 Focused-ultrasonicators (COVARIS, INC.). The fragmented DNA were end-repaired, ligated to sequencing adapters and amplified according to the protocol of NEBNext® ChIP-Seq Library Prep Master Mix Set and NEBNext Multiplex Oligos for Illumina (New England Biolabs) to prepare library. The size and concentration of libraries were checked by Bioanalyzer 2000 and real-time PCR using KAPA Library Quantification Kits, respectively.
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|
|
Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina MiSeq |
|
|
Description |
Wild_Type BrdUIP_HU_WT.bedgraph
|
Data processing |
All fastq files were aligned to S.pombe reference genomic ASM294v2 from Pombase by Bowtie1.0.0 with defalt setting. The generated SAM files by Bowtie were converted to BAM and sorted by Samtools. Peaks were called with Model-based analysis of ChIP-seq (MACS2.0.10) using following parameters; macs2 callpeak -t ChIP.sam -c Input.sam -f SAM -g 1.4e107 -n result_file -B -q 0.01 --nomodel The generated treat_pileup.bedgraph files were loaded on Integrated Genome Browser. Genome_build: ASM294v2 Supplementary_files_format_and_content: bedgraph
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|
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Submission date |
Jan 26, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Yutaka Kanoh |
E-mail(s) |
kanou-yt@igakuken.or.jp
|
Organization name |
Tokyo Metropolitan Institute of Medical Science
|
Department |
Genome Medicine
|
Lab |
Genome Dynamics Projec
|
Street address |
2-1-6 Kamikitazawa, Setagaya-ku
|
City |
Tokyo |
ZIP/Postal code |
156-8506 |
Country |
Japan |
|
|
Platform ID |
GPL16192 |
Series (1) |
GSE65293 |
Rif1 binds to G-quadruplexes and suppresses replication over a long distances |
|
Relations |
BioSample |
SAMN03295234 |
SRA |
SRX853383 |