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Sample GSM4185073

Query DataSets for GSM4185073

Status

Public on Mar 30, 2020

Title

wild type N3753 mRNA-seq

Sample type

SRA

Source name

germinated conidia

Organism

Neurospora crassa

Characteristics

tissue: germinated conidia
genotype: mat a; WT

Extracted molecule

polyA 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

TCTTCGAC_N3753_WT_polyARNAseq_080818_S223_L007_R1_001

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).

Submission date

Nov 21, 2019

Last update date

Mar 30, 2020

Contact name

Eric Selker

E-mail(s)

[email protected]

Organization name

University of Oregon

Department

Biology