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Sample GSM7984655

Query DataSets for GSM7984655

Status

Public on Feb 20, 2024

Title

Wild-type mycelium_FC 1 [C1]

Sample type

SRA

Source name

CEA17𝚫akuB::KU80

Organism

Aspergillus fumigatus

Characteristics

tissue: mycelium
cell line: CEA17{delta}akuB::KU80
genotype: Wild type
treatment: 5-fluorocyctosine
replicate: Biological Replicate 1

Treatment protocol

CEA17∆akuBKU80 and CEA17∆akuBKU80∆mod5 fungal samples (5x107 spores) were cultured either for 24 h or for 16 h in AMM followed by an 8-h treatment with 80 ng/µL of 5-fluorocytosine.

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 and Δmod5 knockout from 24-h-old mycelium either unstressed, or stressed for the final 8 hours with 5-fluorocytosine. Afterwards, mycelia were harvested using Miracloth (EMD Millipore Corp., USA), rinsed with sterile water and same amounts of mycelium were transferred to 2-mL screw cap tubes for RNA isolation. All centrifugation steps were performed at 4°C and full speed (20,817 x g). 800 µL of TRIzol (Thermo Fischer Scientific, Dreieich) were added to each tube, along with glass beads (1/3 of the tube) and the mycelium was mechanically disrupted by 3 rounds of 30 sec, 4.0 m/sec homogenization using the FastPrep-24™ Homogenizer. Subsequently, the samples were incubated for 5 min on ice, followed by additional 5 min at room temperature. After addition of 160 µL of chloroform, the samples were vigorously vortexed for 10 sec and centrifuged for 5 min. The resulting aqueous phase containing the RNA was carefully transferred to a new RNase-free tube without disturbing the interphase. The RNA phase was then purified using the same amount of phenol/chloroform/isoamyl alcohol and centrifuged for 5 min. The purification step was repeated 2-3 times until the interphase remained clear. In the next step, RNA was further purified with 400 µL of chloroform and centrifugation for 5 min. The aqueous phase was again transferred to a new RNase-free 2-mL tube, precipitated by addition of 500 µL isopropanol for 20 min, and RNA was pelleted by 20 min centrifugation. The RNA pellet was washed with 70% ethanol (v/v) and air-dried at 37°C. Finally, RNA was dissolved in 30-50 µL DEPC water at 65°C. The RNA was purified using the RNA Clean and Concentrator™ kit (Zymo Research, USA) following the manufacturer’s instructions. Up to 10 µg of total RNA was afterwards treated with 2 U of DNase I endonuclease (Thermo Fisher) for 30-60 min at 37°C in a volume of 20 µL. RNA concentration was then quantified with the QubitTM RNA BR Assay kit (Thermo Fisher Scientific) on the Qubit Flex Fluorometer (Thermo Fisher Scientific). Quality was assessed either by agarose gel electrophoresis or QUBITTM Integrity and Quality (IQ) kit (Thermo Fisher).
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

Data processing

Pre-processing of raw reads, quality control and gene abundance estimation used GEO2RNaseq pipeline (v0.100.3, (80)) in R (version 3.6.3). Quality analysis was performed with FastQC (v0.11.5) before and after trimming. Read-quality trimming was done with Trimmomatic (v0.36) and 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 annotation files. Reads were mapped against reference genome of A. fumigatus (A1163, assembly GCA_000150145.1) using HiSat2 (v2.1.0, paired-end mode). Gene abundance estimation was done with featureCounts (v2.0.1) in paired-end mode with default parameters. MultiQC version 1.7 was used to summarize and assess quality of output from FastQC, Trimmomatic, HiSat, featureCounts and SAMtools. The count matrix with gene abundance data without and with median-of-ratios normalization (MRN, (81)) were extracted.
Assembly: A. fumigatus (A1163, assembly GCA_000150145.1)
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

Dec 20, 2023

Last update date

Feb 20, 2024

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