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| Status |
Public on Mar 30, 2023 |
| Title |
CAU30 Glucose I |
| Sample type |
SRA |
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| Source name |
Planktonic cells
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| Organism |
[Candida] auris |
| Characteristics |
cell type: yeast cells
genotype/variation: B8441 clade I isolate
medium type: 0.17% YNB w/o ammonium sulfate and w/o amino acids; 0.5% ammonium sulfate; 1% glucose; pH 5
time point: 4 h
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| Treatment protocol |
Cells were separated from test medium by centrifugation 4 min at 4000g and 4ºC. Cells were shock frozen in liquid nitrogen and stored at -80°C until further processing.
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| Growth protocol |
C. auris was pre-grown overnight at 37°C in YPD (1% yeast extract, 2% peptone, 2% glucose) and used to set log-cultures in SD (0.17 yeast nitrogen base w/o ammonium sulfate and w/o amino cids, 0.5% ammonium sulfate, 2% glucose) at OD600 0.2. Log-cultures were incubated at 37°C until OD600 of ~1 was reached. Cells were collected, washed twice with PBS and resuspended in PBS and used to set OD600 to 0.2 in the respective test medium and incubated for 4 h at 37°C and shaking at 180 rpm.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For total RNA isolation cells were resuspended in 440 µl AE buffer with 10% SDS and 440 µl of Acid-phenol:Chloroform with isoamyl alcohol (Ambion, Germany) was added. The suspension was thoroughly mixed and incubated for 5 min at 65°C, frozen at -80°C for 30 min and subsequently thawed at 65°C for 5 min. The phases were separated via 4 min centrifugation at 13,000 g, the upper aqueous phase transferred and 1/10 of the volume sodium acetate (pH 5.3) and 1 volume of 2 propanol was added to precipitate the RNA overnight at -20°C. The supernatant was discarded after 10 min centrifugation at 12,000 g, the RNA pellet washed twice with 70 % ethanol and finally solved in 300 µl nuclease free H2O. Quality and quantity of the RNA was assessed using 2100 Bioanalyzer (Agilent Technologies, California, USA) and Nanodrop (Thermo Fischer Scientific, Massachusetts, USA) measurements.
A total amount of 0.5 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA. Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA by mRNAusing poly-T oligo-attached magnetic beads. Fragmentation was carried out by Covaris sonication. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using dTTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality and quantity are assessed by Qubit and real-time PCR. Sequencing platform was Illumina Novaseq 6000 PE150.
Paired end RNA sequencing of cDNA fragments of preferentially 150~200 bp in length.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Sample_49
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| Data processing |
Fastq files were assessed for sequence quality using FastQC (version 0.11.9) and a genome index from file C_auris_B8441_version_s01-m01-r21_chromosomes.fasta fom http://www.candidagenome.org/ was created with bowtie2 (version 2.3.5.1). Mapping of reads to the index was performed with bowtie2. Reads were counted and assigned to the corresponding genome using function featureCounts of R package Rsubread (version 2.2.6). Contrasts were calculated using R package DESeq2 (version 1.28.1). R version 4.0.4 was used.
Assembly: C_auris_B8441_version_s01-m01-r21_chromosomes.fasta
Supplementary files format and content: Raw.counts.B8441.S49.69.csv, Comma seperated text file containing counts of all samples
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| Submission date |
Jan 20, 2023 |
| Last update date |
Mar 30, 2023 |
| Contact name |
Philipp Brandt |
| Organization name |
Friedrich Schiller University
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| Department |
ZIK Septomics
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| Street address |
Albert-Einstein-Straße 10
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| City |
Jena |
| State/province |
Thuringia |
| ZIP/Postal code |
07745 |
| Country |
Germany |
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| Platform ID |
GPL28368 |
| Series (2) |
| GSE223318 |
Transcriptional profiling of Candida auris B8441 (CAU30) grown on different carbon and nitrogen sources |
| GSE223412 |
High-throughput profiling of Candida auris isolates reveals clade-specific metabolic differences |
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| Relations |
| BioSample |
SAMN32807676 |
| SRA |
SRX19098445 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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