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Sample GSM8259070

Query DataSets for GSM8259070

Status

Public on May 13, 2024

Title

C. albicans_60min_SPB00525 _R2

Sample type

SRA

Source name

WT control C. albicans cells in SC medium treated with SPB00525 for 60 min_rep2

Organism

Candida albicans SC5314

Characteristics

tissue: Yeast cells
strain: SC5314
cell type: Yeast cells
genotype: WT
treatment: 60 min in SC medium + 6 ug/ml SPB00525

Treatment protocol

For RNA-seq, cultures were then either left untreated or exposed to 6 µg/ml SPB00525 and incubated at 30°C for 15- and 60-min. Cells were harvested by centrifugation and were flash-frozen and stored at -80°C. For each condition, a total of two biological replicates were considered for RNA-seq analysis. For the drug-induced haploinsufficiency profiling, the double-barcoded heterozygote C. albicans (DBC) mutant pools were diluted to OD600 = 0.062 and exposed or not to 6 µg/ml SPB00525 in either SC or YPD and grown at 30°C under agitation for 15 hours. 1 mL of the sub-cultured pool was distributed into triplicate culture tubes, each containing 1 mL of YPD or SC medium supplemented with either 6 µg/ml SPB00525 or DMSO solvent. These cultures were incubated at 30°C under shaking conditions for 24 hours.

Growth protocol

For RNA-seq profiling, overnight cultures of C. albicans SC5314 and C. auris 381 strains were diluted to an OD600 of 0.1 in 50 ml of fresh SC medium and grown at 30°C under agitation (200 rpm) to early logarithmic phase (OD600=0.6).

Extracted molecule

total RNA

Extraction protocol

For RNA-seq, total RNA was extracted using an RNAeasy purification kit (Qiagen) and glass bead lysis in a Biospec Mini 24 bead-beater. For the drug-induced haploinsufficiency profiling, cells were harvested by centrifugation and genomic DNA was extracted using YeaStar kit (Zymo Research).
For RNA-seq, the NEBNext UltraTM II RNA Library Prep Kit for Illumina was used to construct the RNA-seq library. For the drug-induced haploinsufficiency profiling, the Up-tag DNA-barcodes were amplified using 25 ng genomic DNA with DBC-F1 and DBC-R1 primers that recognize the common region of up-tag barcode and contain the multiplexing tag (DBC-F1) and sequences required for hybridization to the Illumina ﬂow cell. PCR amplification products were purified form an agarose gel using the QIAquick Gel Extraction kit (Qiagen) and quantiﬁed by QuantiFluor dsDNA System (Promega). Equal quantity of DNA from the SPB00525-treated and non-treated pools were combined prior to NGS sequencing using Illumina MiSeq platform and DBC1 and DBC2 sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

For RNA-seq, sequencing reads were filtered with fastp version 0.23.2.
For RNA-seq, filtered reads were mapped to the reference genome sequence of strain SC5314 and C. auris 381 with STAR version 2.7.9a
For RNA-seq, the number of reads mapping on each of the genes was calculated by featurecount version 2.0.1. For the drug-induced haploinsufficiency profiling , unique uptag DNA barcodes were extracted and counted using SeqKit
Differential gene expression and barcode abundance were calculated with DESeq2 version 1.26.0.
Assembly: Candida albicans assembly 21 and Candida auris B8441
Supplementary files format and content: "raw_counts_SPB00525_C.albicans.txt" file contains read counts for each experimental condition and replicate
Supplementary files format and content: "raw_counts_SPB00525_C.auris.txt" file contains read counts for each experimental condition and replicate
Supplementary files format and content: "Mutant fitness scores.txt" file contains barcode counts of each mutant in each experimental condition and replicate

Submission date

May 08, 2024

Last update date

May 13, 2024

Contact name

Adnane Sellam

E-mail(s)

adnane.sellam@gmail.com

Organization name

University Laval

Street address

2705 Laurier Blvd.