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| Status |
Public on Jan 03, 2019 |
| Title |
WT_RDN1-150_INPUT1 |
| Sample type |
SRA |
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| Source name |
MAT a exponentially growing yeast cells
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| Organism |
Saccharomyces cerevisiae BY4741 |
| Characteristics |
strain name: YTT121
mating type: MATa
growth phase: exponential
chip antibody: None
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| Growth protocol |
Exponentially growing yeast cells in YPD media at 30°C were fixed with 1% formaldehyde (Wako, 064-00406) for 30 minutes at 30°C, followed by an additional 5 min with glycine added to a final concentration of 125 mM. Fixed cells were washed with PBS, pelleted by centrifugation at 20,000 g in a 2 ml screw-capped tube at 4°C and stored at -80°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Exponentially growing yeast cells in YPD media at 30°C were fixed with 1% formaldehyde (Wako, 064-00406) for 30 minutes at 30°C, followed by an additional 5 min with glycine added to a final concentration of 125 mM. Fixed cells were washed with PBS, pelleted by centrifugation at 20,000 g in a 2 ml screw-capped tube at 4°C and stored at -80°C. Cells were lysed with 1.5 ml yttrium stabilized zirconia grinding beads with a diameter of 0.5 mm (Nikkato, YTZ-0.5) for 45 seconds twice at 4 °C and 2,500 rpm on a horizontal tube-holder attached to a micro tube mixer (Eyla, CM-1000). Cell lysate was recovered in 800 µl of lysis buffer (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton-X100, 0.1% Na-deoxicholate, 1 mM PMSF and 1x Complete protease inhibitor cocktail (Roche, 11873580001)). The lysate (850 µl) was sonicated at 4°C in a 1ml milliTube with an AFA Fiber (Covaris, 520130) using a Covaris S220 as follows: 3 cycles of peak power 200, duty factor 20, cycle/burst 200 and duration 5 minutes. The sheared DNA fragments (with an average length of 200 bp) were separated from the cell-debris by centrifugation at 20,000g, 5 minutes at 4°C, and the pellet was re-extracted with 375 µl of lysis buffer to completely collect the chromatin. From the collected chromatin (1115 µl) 50 µl samples were kept as controls for input and DNA content and a 15 µl sample to check proteins. The remainder (1 ml) was incubated with 10 µg anti-HA F-7 antibody at 4°C overnight on a rotation mixer. Subsequently, a 100 µl Dynabeads Protein G (Thermo Fisher, 10004D) suspension, masked with 5 mg BSA/ml, was incubated with the IP mixture for 1 hour at 4°C. The beads were washed rigorously with 1 ml lysis buffer (twice), 1ml lysis buffer with 500 mM NaCl (twice), 1ml wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1 mM EDTA) (twice) and 1 ml of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The material captured on the beads was eluted by 10 minutes long incubations at 65°C with 50 µl, and 2 x 100 µl TES (10 mM Tris-HCl pH8.0, 1 mM EDTA, 1% SDS), after which the eluates were combined. The recovered material and input extract (50 µl combined with 200 µl TES) were incubated with 2 mg/ml Proteinase K (Merck Millipore, 124568) at 56 °C overnight to reverse the crosslinking and to digest the protein. After phenol/chloroform/isoamylalcohol (pH 8.0) extraction and ethanol precipitation with Ethachinmate (Nippongene, 312-01791), the precipitates were dissolved in 50 µl of TE containing 200 µg/ml RNase A and incubated at 37°C for 1 hour. The DNA sample was purified using 2 volumes (100 µl) of AMpureXP and eluted with 20 µl 5 mM Tris-HCl pH8.5.
Sequencing libraries for NGS sequencing were prepared with a Kapa Hyper Prep kit (Kapa Biosystems, KK8502) with 250 ng input DNA, or 1~5 ng ChIP DNA and Illumina TruSeq HT compatible adaptors according to the manufacturer’s protocol. The adaptor-ligated libraries were amplified by 6 cycles of PCR (98°C 15s, 60°C 30 s and 72°C 40 s) in the case of the input-library and by 11 cycles in the case of the ChIP sample using the Kapa kit and primer set TruHTAmp5Fw/TruHTAmp7Rv. Library purification was performed by double size-selection using 0.5 volume of AMpureXP beads (Beckman Coulter, A63881) per PCR reaction in each step: 25 µl of AMpureXP beads was added to 50 µl of PCR reaction to remove long library, after removal of 1st beads, and another 25 µl of AMpureXP beads was added to the supernatant and the library DNA was recovered from the 2nd bead fractions with 12.5 µl 5 mM Tris-HCl pH8.5. Equimolar mixtures of the libraries were prepared for paired-end sequencing (75 bp) with a MiSeq Reagent Kit v3 (Illumina, MS-102-3001) and sequenced on a MiSeq (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Description |
UAF_ChIP001
Replicate sample_1. Parental yeast strain with normal rDNA copy, YPD culture cross-linked by 1% formaldehyde at 30degree Celsius for 30 minutes. 2x10^9 cells were lysed by YTZ-beads, and the extracts were sheared by Covaris S220 to 100bp average. After pelleting, soluble fractions were used for INPUT samples. After reverse-crosslinking and DNA purification, sequencing libraries were prepared by KAPA Hyperprep kit and TruSeqHT-compatible adaptors.
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| Data processing |
ChIP-seq: The paired-end reads were merged to a set of single reads.
Low quality 15 bases were trimmed from the 3'-end of each read with Seqtk (https://github.com/lh3/seqtk).
Reads were mapped to the S. cerevisiae reference genome EF4.68 by means of bowtie-1.0.0 using the single-read mapping mode: “bowtie -S --best -k 1 -n 3 EF4.68.index Sample_trim.fastq > Sample_trim.sam”.
Mapped reads were counted by samtools-0.1.19.
The pileup data was generated by means of MACS2 (http://liulab.dfci.harvard.edu/MACS/).
ChIP-seq profile data were normalized by the number of mapped reads as fragment per million reads (fpm).
Whole genome sequencing: To identify causative rcc-mutations, FASTQ files were analyzed with the webtool Mudi (version2, http://naoii.nig.ac.jp/mudi_top.html).
Genome_build: Saccharomyces_cerevisiae.EF4.68
Supplementary_files_format_and_content: BedGraph files were generated by macs2 and normalized by mapped read number as fragment count per million (fpm).
Supplementary_files_format_and_content: *.tsv: Tab-delimited text files were generated by the webtool "mudi". Mutations called by the analysis were listed.
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| Submission date |
Jul 05, 2018 |
| Last update date |
Jan 03, 2019 |
| Contact name |
Tetsushi Iida |
| E-mail(s) |
iidatessi@gmail.com
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| Phone |
+81-3-5841-7860
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| Organization name |
The Institute for Quantitative Biosciences (IQB), The University of Tokyo
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| Department |
Research Center for Biological Visualization
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| Lab |
Laboratory of Genome Regeneration
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| Street address |
1-1-1 Yayoi
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| City |
Bunkyo-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
113-0032 |
| Country |
Japan |
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| Platform ID |
GPL21372 |
| Series (1) |
| GSE116661 |
RNA polymerase I activators count and adjust ribosomal RNA gene copy number |
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| Relations |
| BioSample |
SAMD00119009 |
| SRA |
DRX123807 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3244974_WT_RDN1_150_INPUT_1sort_macs2_fpm.bedgraph.gz |
28.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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