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| Status |
Public on Aug 18, 2020 |
| Title |
∆lsd1; ∆dim-2 H3K9me3 ChIP-seq |
| Sample type |
SRA |
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| Source name |
germinated conidia
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| Organism |
Neurospora crassa |
| Characteristics |
chip antibody: H3K9me3 (Active Motif 39161; lot #13509002
genotype: mat a delta_lsd1::hph; delta_dim-2::hph
tissue: germinated conidia
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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 H3K9me3 antibody was added (3µL) 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 4˚C, and washed twice in cold ChIP Lysis buffer, once with ChIP Lysis buffer + 0.5M NaCl, once with LiCl 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 65˚C. Samples (IP and input) were decrosslinked at 65˚C overnight. The next day, samples were proteinase K treated for 2 hours at 50˚C, 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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| 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).
Bisulfite-seq processing: The BRAT-BW software package (compbio.cs.ucr.edu/brat/; Harris et al, 2012) was used to prepare and map the reads to the N. crassa OR74A (annotation NC12) genome, which was converted to a four stranded reference genome to permit bisulfite mapping. BRAT-BW acgt-count “-B” option cytosine-only files produced for the forward and reverse strand reads were merged. The average 5mC level was determined for 25bp step-wise window size across the genome using the Methpipe program (http://smithlabresearch.org/software/methpipe/). The resulting file was renamed with a “.igv” file extension to allow display on the Integrated Genome Viewer (IGV; www.broadinstitute.org/igv/)
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 replicates each of wild type (Klocko et al. 2016; GSE82222) and ∆lsd1 and the output file from DESeq2 (Love et al., 2014) with pairwise comparisons of 2 biological replicates from each genotype.
Supplementary_files_format_and_content: Bisulfite-seq processed data files are .igv files displaying the % methylation over 25bp windows where a score of 0 is a DNA region that has no cytosine methylation and a score of 1.0 is DNA where all of the cytosines are methylated in the region. Values in between 0 and 1.0 represent partial methylation of a region.
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| Submission date |
Sep 06, 2019 |
| Last update date |
Aug 18, 2020 |
| Contact name |
Eric U Selker |
| E-mail(s) |
[email protected]
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| Organization name |
University of Oregon
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| Department |
Biology, Institute of Molecular Biology
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| Lab |
Selker
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| Street address |
1229 University of Oregon; 1318 Franklin Blvd.
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| City |
Eugene |
| State/province |
OR |
| ZIP/Postal code |
97403 |
| Country |
USA |
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| Platform ID |
GPL23150 |
| Series (1) |
| GSE137018 |
Loss of Lysine-Specific Demethylase 1 (LSD1) Drives Aberrant Heterochromatin Formation in Neurospora crassa |
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| Relations |
| BioSample |
SAMN12708222 |
| SRA |
SRX6811939 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4065469_TACTGCTC_N6337_H3K9me3_051019_S55_L008_R1_001.bigwig |
7.0 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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