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Sample GSM3671336

Query DataSets for GSM3671336

Status

Public on Apr 15, 2020

Title

∆epr-1 N7491 RNA-seq

Sample type

SRA

Source name

germinated conidia

Organism

Neurospora crassa

Characteristics

genotype: mat a; {delta}epr-1::hph

Extracted molecule

total RNA

Extraction protocol

Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32oC. Mycelia were harvested and added to a tube containing ~350uL glass beads (260-300 micrometer, acid washed [Sigma Aldrich] and resuspended in sdH2O), 350uL NETS buffer (10mM Tris pH 7.5, 300mM NaCl, 1mM EDTA, 0.2% SDS) and 350uL 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 350uL of chloroform. After centrifugation, the aqueous phase was divided into two tubes containing 650uL cold 100% EtOH. Samples were precipitated at -20oC, 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).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

AACAACCG_7491_deltaNCU07505_RNAseq_120117

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 TDF or bigWig files with the Integrative Genomics Viewer (IGV) (Robinson et al., 2011).
RNA-seq processing: Tools available on Galaxy {Afgan:2018gf} were used to map mRNA-sequencing reads (intron size < 1kb) {Dobin:2012fg} against the corrected N. crassa OR74A (NC12) genome {Galazka:2016cm}, count the number of reads per gene {Dobin:2012fg} and determine differentially expressed genes with DESeq2 {Love:2014ka}.
DamID-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 TDF or 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 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, ∆set-7 (both from Klocko et al. 2016; GSE82222) and ∆epr-1 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: DamID-seq processed data files are bigwig files generated using DeepTools (Ramirez et al., 2016) using a window size of 25bp

Submission date

Mar 14, 2019

Last update date

Apr 15, 2020

Contact name

Elizabeth Toomey Wiles

E-mail(s)

[email protected]

Organization name

University of Oregon

Department

Biology, Institute of Molecular Biology