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| Status |
Public on Sep 12, 2022 |
| Title |
Cgset4Δ grown in caspofungin-containing CAA medium: Replicate-2 |
| Sample type |
SRA |
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| Source name |
Cgset4-delta knockout
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| Organism |
Nakaseomyces glabratus |
| Characteristics |
strain: Cgset4-delta knockout
treatment: Caspofungin-treated
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| Treatment protocol |
Log-phase cultures of wild-type and Cgset4Δ strains were either treated with 0.25 µg/ml caspofungin for 1 h or left untreated. During caspofungin treatment, cultures were incubated at 30⁰C under shaking conditions (200 rpm).
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| Growth protocol |
C. glabrata wild-type and Cgset4Δ strains were inoculated in CAA medium and incubated for 16 h at 30⁰C under shaking conditions (200 rpm). These cultures were re-inoculated in the fresh CAA medium at an initial OD600 of 0.1 and grown for 4-5 h at 30⁰C to obtain logarithmic (log)-phase cultures.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the acid-phenol method. Briefly, caspofungin-treated and untreated cultures of C. glabrata wild-type and Cgset4Δ stains were pelleted down and washed twice with chilled DEPC-treated water. The cell pellet was suspended in 350 μl of chilled AE solution (50 mM NaOAc and 10 mM EDTA) and lysed by addition of 40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) and vortexing for 10 sec. Next, the samples were incubated at 65oC for 15 min on a thermomixer followed by incubation on ice for 5 min. The samples were centrifuged at 4oC at 13,000 rpm for 10 min, and the upper aqueous layer was collected in a new 1.5 ml microcentrifuge tube. 400 μl of chloroform was added to the aqueous layer and centrifuged at 3,000 rpm for 10 min at 4⁰C. The aqueous layer was transferred to a 1.5 ml microcentrifuge tube, and RNA was precipitated with 40 µl of 3 M sodium acetate (pH 5.3) and 400 µl of ethanol, following incubation at -20oC for 20 min. Next, RNA was pelleted down by centrifugation at 13,000 rpm for 10 min at 4oC, and the pellet was washed with chilled 70% ethanol, air dried and suspended in 100 μl DEPC-treated water. To remove DNA contamination, 1 μl of DNase I was added to 1 µg of total RNA and incubated for 20 min at 37°C.
RNA-Seq library was prepared with 1 μg of total RNA using TruSeq RNA Sample Prep Kits (Illumina), as recommended by the manufacturer. In brief, total RNA was subjected to poly-A tail-based mRNA purification using magnetic beads attached with poly-T oligos. After purification, mRNA was fragmented using divalent cations at high temperature. The cleaved RNA fragments were used to synthesize cDNA first strand using reverse transcriptase and random primers. The synthesis of the second strand cDNA was done using DNA polymerase I and RNase H. These cDNA fragments underwent to end repair process followed by addition of a single ‘A’ base and ligation of the adaptors. The product was finally purified and enriched with PCR to create final cDNA library. Bioanalyzer plots were used to assess mRNA quality, enrichment success, fragment size, and the final library size. Both qubit and qPCR methods were used to measure the quantity of library before sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Gene expression analysis
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| Data processing |
The constructed libraries were sequenced on the Hiseq 2500 Illumina platform and 2x100 bp paired-end sequence was obtained, which contained 40-60 million high-quality reads per sample.
FASTQ files were generated from Illumina RNA-seq data. Based on the FASTQ file quality report, the sequence was trimmed, and only high-quality sequences were retained for further analysis.
The pre-processed reads were aligned pairwise with the C. glabrata CBS138 genome using Hisat2 program (Version-2.0.5).
The differential expression analysis was performed using Cuffdiff program of Cufflinks package (version 2.2.1) with default settings.
Further, the number of up and down regulated genes were filtered using Cuffdiff with q-value cut off ≤ 0.05 and ≥ 1.5-fold change in expression.
Assembly: Candida glabrata CBS138 genome
Supplementary files format and content: Excel file; GeneID, FPKM values of all samples, Gene name, Aliases, Feature type, Chromosome, Start Coordinate, Stop Coordinate, Strand, Primary CGDID, Secondary CGDID, Description, Name of S. cerevisiae ortholog(s)
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| Submission date |
May 10, 2022 |
| Last update date |
Sep 12, 2022 |
| Contact name |
Rupinder Kaur |
| Organization name |
Centre for DNA Fingerprinting and Diagnostics
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| Lab |
Laboratory of Fungal Pathogenesis
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| Street address |
Inner Ring road
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| City |
Hyderabad |
| State/province |
Telangana |
| ZIP/Postal code |
500039 |
| Country |
India |
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| Platform ID |
GPL25492 |
| Series (1) |
| GSE202654 |
RNA-Seq analysis of Candida glabrata wild-type and Cgset4Δ mutant in the presence and absence of the cell wall targeting echinocandin antifungal drug, caspofungin. |
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| Relations |
| BioSample |
SAMN28177782 |
| SRA |
SRX15224934 |
| 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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