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| Status |
Public on Mar 08, 2022 |
| Title |
H3K36me3_iswi_N6171 |
| Sample type |
SRA |
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| Source name |
H3K36me3_iswi_N6171
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| Organism |
Neurospora crassa |
| Characteristics |
antibody: H3K36me2
sample type: nuclei
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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 before chromatin was sheared using a Bioruptor (Diagenode) for 3x 10 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
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were cleared by centrifugation and 1/20th input was saved. H3K27me2/3 antibody was added (Active Motif, 2uL) 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 NovaSeq 6000 |
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| Data processing |
MNase-seq
Paired-end sequence reads were aligned to the neurospora crassa genome (OR74A) using Bowtie2 (version 2.3.3) with the option “-q -p 4 -X 250 --no-discordant --no-mixed --no-unal” (Paired-end alignment reads with maximum 250bp distance gap between them were used in subsequent analysis. This length correspond to mono-nucleosome)
Only correctly aligned paired-end alignment reads were filtered using samtools (version 1.5) commands “samtools view –hf 0x2 input.bam | grep –v “XS:i:” “
Dyad Coverage was calculated using the scripts (03_PNA_SDE.R) from Baldi et al (Baldi, S. et al. Genome-wide Rules of Nucleosome Phasing in Drosophila. Mol. Cell 72, 661-672.e4 (2018))
The spectral density (SD) score correspond to periods of 182bp was calculated using the scripts (cov2spec.R) from Baldi et al. SD score was normalized as Z-score : (log2(SD score) – average) / standard deviation.
Regions with the average Z-score threshold of 2 were defined as the domain of a regular nucleosome array.
Genome_build: neurospora crassa genome (OR74A)
Supplementary_files_format_and_content: bigwig of dyad coverage and spectral density
ChIP-seq
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.
Genome_build: neurospora crassa genome (OR74A)
Supplementary_files_format_and_content: bigwig of pileup graph
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| Submission date |
Feb 04, 2022 |
| Last update date |
Mar 08, 2022 |
| Contact name |
Hideki Tanizawa |
| E-mail(s) |
hidekit@uoregon.edu
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| Organization name |
University of Oregon
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| Department |
Institute of Molecular Biology
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| Street address |
1370 Franklin Blvd
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| City |
Eugene |
| State/province |
OR |
| ZIP/Postal code |
97403 |
| Country |
USA |
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| Platform ID |
GPL26551 |
| Series (1) |
| GSE168277 |
The ACF chromatin remodeling complex is essential for Polycomb repression |
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| Relations |
| BioSample |
SAMN25648819 |
| SRA |
SRX14042110 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5860093_H3K36me3_iswi_N6171.bw |
61.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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