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| Status |
Public on Mar 30, 2020 |
| Title |
∆npf N8003 H3K27me2/3 ChIP-seq |
| Sample type |
SRA |
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| Source name |
germinated conidia
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| Organism |
Neurospora crassa |
| Characteristics |
tissue: germinated conidia
genotype: mat a; Dnpf::hph
chip antibody: H3K27me2/3 (Active motif #39536)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 18 hours at 32oC. 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 15 min with a cycle of 30 sec on followed by 30 sec off, at high power. Lysates were cleared by centrifugation, 1/20th Input was saved, and H3K27me2/3 antibody was added (typically 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 was 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 QIAquick 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. “Invisible” fragments between 250-400 bp were excised and purified using the MinElute gel extraction kit (Qiagen, 28606). 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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| Description |
GCTACTCT_N8003_npf_H3K27me2me3_062419_S30_L007_R1_001
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| Data processing |
ChIP-seq processing: All sequencing reads were mapped to the corrected N. crassa OR74A (NC12 genome) (Galazka et al., 2016) using Bowtie2 (Langmead and Salzberg, 2012). ChIP-seq read coverage was averaged, normalized, and analyzed using tools available from deepTools2 on the open-source platform Galaxy (Afgan et al., 2016). Sequencing tracks are displayed as 25-nt-window bigWig files with the Integrative Genomics Viewer (IGV) (Robinson et al., 2011).
RNA-seq processing: Tools available on Galaxy (Afgan et al., 2018) were used to map mRNA-sequencing reads (intron size < 1kb) (Dobin et al, 2012) against the corrected N. crassa OR74A (NC12) genome (Galazka et al., 2016), count the number of reads per gene (Dobin et al., 2012) and determine differentially expressed genes with DESeq2 (Love et al, 2014).
Genome_build: Neurospora crassa assembly 12 Fixed (files: neurospora_crassa_or74a_12_genome_FIXED.fasta, and neurospora_crassa_or74a_12_transcripts_FIXED.gtf; files are found in GEO submission GSE71024)
Supplementary_files_format_and_content: ChIP-seq processed data files are bigwig files generated using DeepTools (Ramirez et al., 2016) using a window size of 25bp.
Supplementary_files_format_and_content: RNA-seq processed data files are txt files containing normalized counts for 2 previously published replicates of wild type and ∆set-7 strains (both from Klocko et al. 2016; GSE82222). In addition they contain normalized counts for ∆pas replicates and an additional set of wild type replicates (totalling 4 wild type samples). We have also included the pairwise comparisons of wild type to ∆set-7 and wild type to ∆pas from DESeq2 (Love et al., 2014).
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| Submission date |
Nov 21, 2019 |
| Last update date |
Apr 01, 2020 |
| Contact name |
Eric Selker |
| E-mail(s) |
[email protected]
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| Organization name |
University of Oregon
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| Department |
Biology
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| Street address |
1370 Franklin Blvd.
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| City |
Eugene |
| State/province |
Oregon |
| ZIP/Postal code |
97403 |
| Country |
USA |
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| Platform ID |
GPL23150 |
| Series (1) |
| GSE140787 |
Identification of a novel fungal PRC2 accessory subunit |
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| Relations |
| BioSample |
SAMN13343896 |
| SRA |
SRX7198282 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4185070_GCTACTCT_N8003_npf_H3K27me2me3_062419_S30_L007_R1_001.bigwig |
6.9 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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