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| Status |
Public on Aug 18, 2020 |
| Title |
∆lsd1 N5555 RNA-seq rep2 |
| Sample type |
SRA |
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| Source name |
germinated conidia
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| Organism |
Neurospora crassa |
| Characteristics |
genotype: mat a delta_lsd1::hph
tissue: germinated conidia
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32˚C. Mycelia were harvested and added to a tube containing ~350µL glass beads (260-300 micrometer, acid washed [Sigma Aldrich] and resuspended in sdH2O), 350µL NETS buffer (10mM Tris pH 7.5, 300mM NaCl, 1mM EDTA, 0.2% SDS) and 350µL phenol:chloroform:isoamyl alcohol (25:24:1) and lysed by a bead beater for 3 minutes at room temperature. Samples were cleared by centrifugation, and the aqueous (top) phase was mixed with 350µL of chloroform. After centrifugation, the aqueous phase was divided into two tubes containing 650µL cold 100% EtOH. Samples were precipitated at -20˚C, pelleted by centrifugation, washed twice with 1mL 70% EtOH, and resuspended in DEPC-treated dH2O. RNA was treated with DNAseI, amplification grade (Thermo Fisher Scientific), cleaned (Agencourt® RNAclean XP® beads, Beckman Coulter), and RNA-seq libraries were prepared (KAPA Stranded mRNA-seq kit, KAPA Biosystems).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
lsd1_sig_DOWNregulated_genes.txt
lsd1_sig_UPregulated_genes.txt
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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 |
GPL20660 |
| Series (1) |
| GSE137018 |
Loss of Lysine-Specific Demethylase 1 (LSD1) Drives Aberrant Heterochromatin Formation in Neurospora crassa |
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| Relations |
| BioSample |
SAMN12708219 |
| SRA |
SRX6811941 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4065472_CGAACA-N5555-2-lsd1-polyA-RNA-102215_S28_sort_counts.txt.gz |
37.5 Kb |
(ftp)(http) |
TXT |
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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