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| Status |
Public on May 25, 2024 |
| Title |
đťš«ppdA/B_conidia_2 |
| Sample type |
SRA |
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| Source name |
conidia
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| Organism |
Aspergillus fumigatus |
| Characteristics |
tissue: conidia
cell line: CEA17 delta_akuB::KU85
growth condition: Aspergillus minimal media agar plates
genotype: {delta}ppdA/B
treatment: 0 h
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| Growth protocol |
A. fumigatus was 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.
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| Extracted molecule |
total 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. These samples were collected simultaneously as matched samples with those reported in GEO Series GSE223618.
Libraries were constructed by Novogene. According to the manufacturer, 3' and 5' adaptors were ligated to 3' and 5' ends of small RNA, repsectively. Then the first strand cDNA was synthesized after hybridization with reverse transcription primer. The double-stranded cDNA library was generated through PCR enrichment. After purification and size selection, libraries with insertions between 18-40 bp were ready for sequencing with SE50.
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
50-bp single read sequencing
ppdAB_conidia_R2
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| Data processing |
The sequencing results were analyzed with Unitas (https://doi.org/10.1186/s12864-017-4031-9) using default parameters.
A. fumigatus nuclear tRNAs were referenced from GtRNAdb, mitochondrial tRNAs were scanned from the mitochondrial genome using the 27 tRNAs predicted by tRNAscan-SE. All analyses were performed using “R” version 4.3.1 (2023-06-16) and Rstudio (2022-02-02), an integrated development environment (IDE) for R.
Assembly: Aspergillus fumigatus A1163 ASM15014v1
Supplementary files format and content: A single csv file (sRNA_processed) contains the abundance of each target from each sample replicate.
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| Submission date |
May 14, 2024 |
| Last update date |
May 25, 2024 |
| Contact name |
Matthew George Blango |
| Organization name |
Leibniz Institute for Natural Product Research and Infection Biology
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| Department |
Junior Resaerch Group RNA Biology of Fungal Infections
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| Street address |
Adolf-Reichwein-Str. 23
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| City |
Jena |
| ZIP/Postal code |
07745 |
| Country |
Germany |
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| Platform ID |
GPL33056 |
| Series (1) |
| GSE267454 |
sRNA-seq of Aspergillus fumigatus RNAi double knockouts |
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| Relations |
| BioSample |
SAMN41396808 |
| SRA |
SRX24549862 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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