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| Status |
Public on Aug 21, 2024 |
| Title |
ChIP-seq H3K27me2me3 ∆rlp1 (∆NCU09007) strain: N8186 |
| Sample type |
SRA |
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| Source name |
N8186
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| Organism |
Neurospora crassa |
| Characteristics |
strain: N8186
antibody: H3K27me2me3
genotype: [delta]rlp1 ([delta]NCU09007)
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| Treatment protocol |
Mycelia were washed in PBS prior to crosslinking. Mycelia were cross-linked for 10 min in 0.5% formaldehyde, glycine (125mM final concentration) was added for 5 minutes and tissue was washed with PBS. Tissue was added to ChIP lysis buffer (50mM Hepes pH 7.5, 90mM NaCl, 1mM EDTA, 1% triton + proteinase inhibitors) and disrupted by sonication for thirty pulses. Sonicated chromatin was lysed for 2 x 90s on a Mini-beadbeater-16 (Biospec Products) and then sheared using a Bioruptor (Diagenode) for 2 x 5 min with a cycle of 30 sec on followed by 30 sec off, at high power.
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| Growth protocol |
5 ml cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 18 hours at 32oC (supplemented with histidine for strain N6279).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were cleared by centrifugation and 1/20th input was saved. H3K27me2/3 antibody (Active Motif, 2uL), H3K9ac antibody (Diagenode, 2 uL), or H3K14ac antibody (Millipore, 2 uL) was added and samples were rotated overnight at 4oC. The next day, equilibrated Protein A/G agarose (Santa Cruz Biotechnology) was added to bind the antibody, incubated for 3 hours at 4oC, and washed twice in cold ChIP Lysis buffer, once with ChIP Lysis buffer + 0.5M NaCl, once with LiCl wash buffer (10mM Tris pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% NP40), and DNA/protein was eluted with TES buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS) and incubated at 65oC. Samples (IP and input) were decrosslinked at 65oC overnight. The next day, samples were proteinase K treated for 2 hours at 50oC, and DNA was purified using the Qiagen MinElute PCR purification kit and eluted in 30 μl of water.
Approximately 10 ng of DNA was used to generate ChIP-Seq libraries. Each library was prepared using the NEB Next DNALibrary Prep Kit for Illumina according to the manufacturer’s instructions. AMPure XP beads (Beckman Coulter A63881) were used for size selection of ~200-800 bp fragments. Final libraries were PCR-amplified using one cycle at 98 °C for 30 sec, 10 cycles at 98 °C for 10 sec, 60 °C for 30 sec and 72 °C for 30 sec and a final extension at 72 °C for 5 min.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Sequence reads were aligned to the neurospora crassa genome (OR74A) using Bowtie2 (version 2.3.3) with default option.
Reads with low mapping quality (MapQ < 10) and from repetitive seuqnces were discarded.
Pileup score were calculated using Homer software (version 4.10.1).
ChIP-seq scores correspond to the total number of mapped tags after normalizing to 10 million reads.
Assembly: neurospora crassa genome (OR74A)
Supplementary files format and content: bigwig of pileup graph
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| Submission date |
Mar 11, 2024 |
| Last update date |
Aug 21, 2024 |
| Contact name |
Hideki Tanizawa |
| E-mail(s) |
[email protected]
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| Phone |
011-706-5035
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| Organization name |
Hokkaido University
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| Department |
Institute for Genetic Medicine
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| Lab |
Division of Genome Biology
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| Street address |
Kita 15, Nishi 7, Kita-ku
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| City |
Sapporo |
| State/province |
Hokkaido |
| ZIP/Postal code |
060-8615 |
| Country |
Japan |
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| Platform ID |
GPL23150 |
| Series (1) |
| GSE261327 |
The RPD3L deacetylation complex is required for facultative heterochromatin repression in Neurospora crassa [ChIP-seq] |
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| Relations |
| BioSample |
SAMN40378678 |
| SRA |
SRX23901245 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8140590_H3K27me2me3_8186.bw |
18.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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