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Sample GSM6965690

Query DataSets for GSM6965690

Status

Public on Feb 14, 2023

Title

𝚫ppdA/B conidia 2

Sample type

SRA

Source name

Conidia

Organism

Aspergillus fumigatus

Characteristics

tissue: Conidia
cell line: CEA17 delta_akuB::KU80
genotype: delta_ppdA/B
time: 0 h
growth condition: Aspergillus minimal media

Growth protocol

A. fumigatus strains were grown on Aspergillus minimal medium (AMM) agar plates (70 mM NaNO3, 11.2 mM KH2PO4, 7 mM KCl, 2 mM MgSO4, 1% (w/v) glucose and 1 μl/ml trace element solution (pH 6.5)). The trace element solution was composed of 1 g FeSO4 • 7 H2O, 8.8 g ZnSO4 • 7 H2O, 0.4 g CuSO4 • 5 H2O, 0.15 g MnSO4 • H2O, 0.1 g NaB4O7 • 10 H2O, 0.05 g (NH4)6Mo7O24 • 4 H2O, and ultra-filtrated water to 1000 ml. Spores were harvested after 5 days with 10 ml of sterile distilled water using a T-shaped inoculation spreader and spore suspensions were filtered through a 30-µm cell strainer (MACS, Miltenyi Biotec GmbH, Germany) to exclude mycelium.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was isolated from three biological replicates of CEA17ΔakuBKU80, ΔppdA/B, ΔdclA/B, and ΔrrpA/B knockout strains from conidia, 24-h-, and 48-h-old mycelium. For A. fumigatus conidia, the spores were first homogenized in a bead beater (0.5 mm beads; FastPrep-24, MP Biomedicals) in Trizol. Fungal mycelium was collected from liquid culture using Miracloth (Millipore) and disrupted in liquid nitrogen using a precooled mortar and pestle. Roughly 0.5 g of homogenized mycelium was transferred into a 2-ml Eppendorf tube. 800 µl TRIzol was added to the ground mycelia and vortexed vigorously. Tubes were frozen briefly for 5 sec in liquid nitrogen and allowed to thaw on ice. To all samples, chloroform was added to the fungal samples in TRIzol, vortexed, and centrifugated for 5 min at 4°C at full speed. The aqueous upper phase was transferred to a fresh 2-ml tube without disturbing the interphase. RNA extraction from aqueous phase was done with 1 volume of phenol/chloroform/isoamyl alcohol (25:24:1, v/v). Brief vortexing preceded centrifugation for 5 min at 4°C. RNA was precipitated using 400 µl isopropanol for 20 min, followed by pelleting by centrifugation for 20 min at 4°C. The pellet was washed with 700 µl 70% ethanol and air dried at 37°C for 5 min prior to resuspension in RNase free water. The RNA isolation was followed by a DNase treatment using 2 units of TURBO DNase (Thermo Fisher) per 10 µg RNA for 30 min at 37°C in 100 µl total volume. Total RNA was then collected using the RNA Clean and Concentrator-25 kit (Zymo Research) according to the manufacturer’s instructions.
Poly-(A) selection and directional sequencing of mRNA was performed by Novogene using paired-end 150-bp read sequencing on a NovaSeq 6000.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Biological Replicate 2
A8

Data processing

Preprocessing of raw reads including quality control and gene abundance estimation was done with the GEO2RNaseq pipeline (v0.9.12; (https://doi.org/10.1101/771063)) in R (version 3.5.1). Quality analysis was done with FastQC (v0.11.8) before and after trimming. Read-quality trimming was done with Trimmomatic (v0.36).
Reads were rRNA-filtered using SortMeRNA (v2.1) with a single rRNA database combining all rRNA databases shipped with SortMeRNA.
Reference annotation was created by extracting and combining exon features from corresponding annotation files. Reads were mapped against the reference genome of A. fumigatus (Af293, ASM265v1) using HiSat2 (v2.1.0, paired-end mode). Gene abundance estimation was done with featureCounts (v1.28.0) in paired-end mode with default parameters. MultiQC version 1.7 was finally used to summarize and assess the quality of the output of FastQC, Trimmomatic, HiSat, featureCounts and SAMtools. The count matrix with gene abundance data without and with median-of-ratios normalization (MRN; (https://doi.org/10.1186/gb-2010-11-10-r106)) were extracted.
Assembly: A. fumigatus (Af293, ASM265v1)
Supplementary files format and content: The processed data file is an excel .xlsx file containing four tabs, including read counts for each gene, MRN-normalized counts, RPKM-normalized counts, and TPM-normalized counts.

Submission date

Jan 24, 2023

Last update date

Feb 14, 2023

Contact name

Matthew George Blango

Organization name

Leibniz Institute for Natural Product Research and Infection Biology

Department

Junior Resaerch Group RNA Biology of Fungal Infections

Street address

Adolf-Reichwein-Str. 23