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Sample GSM4667905 Query DataSets for GSM4667905
Status Public on Aug 18, 2020
Title catalytic null lsd1 H3K9me3 ChIP-seq rep2
Sample type SRA
Source name germinated conidia
Organism Neurospora crassa
Characteristics tissue: germinated conidia
chip antibody: H3K9me3 (Active Motif 39161; lot #13509002
genotype: his-3; lsd1*-3xFLAG::nat-1 (*=NK972,973AA)
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.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 4000
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).
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 tdf or bigwig files generated using IGV or DeepTools (Ramirez et al., 2016) respectively, using a window size of 25bp.
Submission date Jul 12, 2020
Last update date Aug 19, 2020
Contact name Eric U Selker
Organization name University of Oregon
Department Biology, Institute of Molecular Biology
Lab Selker
Street address 1229 University of Oregon; 1318 Franklin Blvd.
City Eugene
State/province OR
ZIP/Postal code 97403
Country USA
Platform ID GPL23150
Series (1)
GSE137018 Loss of Lysine-Specific Demethylase 1 (LSD1) Drives Aberrant Heterochromatin Formation in Neurospora crassa
BioSample SAMN15512215
SRA SRX8713243

Supplementary file Size Download File type/resource
GSM4667905_N7943_K9me3_051520_S2_L007_R1_001.bigwig 7.3 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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