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Sample GSM8282297

Query DataSets for GSM8282297

Status

Public on Oct 06, 2024

Title

MAY7 Control 5P-Seq biol rep3

Sample type

SRA

Source name

Clinical isolate; fluconazole resistant

Organism

Candida albicans

Characteristics

cell type: Clinical isolate; fluconazole resistant
genotype: MAY7
treatment: DMSO control

Treatment protocol

Fluconazole treatment: cultures were pre-grown overnight at 30 °C with constant shaking. The following day cultures were diluted to OD600 of 0.1. The starting culture was split into two tubes (2 mL per tube) and subsequently grown at 30 °C aerated until an OD600 of 1.5 – 2.2. One tube was treated with 2% DMSO (diluent control), while the second tube was treated for 30 minutes with 1 μg/ml (1x MIC (24)) fluconazole. Cells from three biological replicates were harvested by centrifugation and flash frozen in liquid nitrogen. Cycloheximide treatment: cultures were pre-grown as for fluconazole treatment. The following day cultures were used to inoculate two flasks (150 ml per flask) at an OD600 of 0.1. When the cultures reached an OD = 1, one culture was treated for 15 minutes with 1 mL cycloheximide working solution in ethanol (final 0.067 mg/ml concentration in media), whereas the control culture received 1 mL of 90% Ethanol. Cells from three biological replicates were harvested by centrifugation and flash frozen in liquid nitrogen. Amino acid deprivation: strain SC5314 was pre-grown overnight in 3 mL YPD shaking at 30 °C. Cells were harvested by centrifugation and resuspended in either proline starvation media or replete media. Proline starvation media is YNB (Yeast Nitrogen Base without amino acids and ammonium sulfate; 0.17%) supplemented with 2% glucose and all amino acids (0.2 g/L) except Proline, Arginine and Ornithine.

Growth protocol

C. albicans reference strain SC5314 and fluconazole resistant clinical isolates PLC124, MAY7 and MAY478 were maintained on YPD (yeast extract 1%, peptone 2%, and glucose 2%).

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from frozen cell pellets using the RiboPure-Yeast RNA Isolation Kit (Thermo Fisher) and RNA concentrations were measured using a Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific).
HT-5’P sequencing library preparation was performed as described in PMID: 35474692 and PMID: 33870233

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

NextSeq 2000

Description

5Pseq

Data processing

Base calls (BCL) were converted to fastq files and subsequently demultiplexed using the Illumina bcl2fastq v2.20.0.422 tool with option –barcode-mismatches 1. Sequencing adapter read trimming at 3’ end was applied using cutadapt V3.1 with Python 3.7.2 (https://cutadapt.readthedocs.io/en/v3.1/installation.html). The 5’ end 8-nt random barcodes were extracted and added as UMI to the header of fastq files using UMI-tools (27). Reads were mapped to the SC5314 reference genome (ASM18296v3) for Candida albicans using STAR version 2.7.9a (28) with parameter --alignEndsType Extend5pOfRead1 to exclude 5’end soft-clipped bases. In addition, reads were mapped to mRNA, rRNA, tRNA, snRNA, snoRNA and ncRNA separately with corresponding indexes generated by STAR 2.7.9a to calculate RNA content composition (see Supplementary Table 1). Duplicated 5’ read ends introduced by PCR during library preparation were collapsed and removed based on random barcode sequences with UMI-tools (27). Low complexity samples (may478_fluc_rep01, may478_ut_rep01, plc124_ut_rep01) were removed from further analyses ( see Supplementary Table 12). To compare differences in 5P’ read coverage at gene level, reads per CDS were counted using Subread package (featureCounts) using the options -t CDS -g ID -s 0 -T 8 (29). Differential gene expression analysis was performed using DeSeq2 (11). The threshold for differentially expressed genes was defined as p-value < 0.05 and log2FoldChange > 0.5. We adjusted the alpha parameter to 0.5, corresponding to FDR threshold 50% to increase the number of Differentially Expressed Genes (DEG) to 61. Analysis of 5’ ends positions was done using a modified version of the Fivepseq package (30) that accounts for the CUG codon reassignment from Leucine to Serine in C. albicans (31). Briefly, the 5’ mRNA reads are summed in biological samples and normalized to reads per million (rpm). The relative position of 5’ reads with respect to all codons of all open reading frames (ORF) were summed at each position. Metagene plots are generated by plotting the summed reads within a window of each annotated start and termination site, or codon.
Assembly: SC5314 reference genome (ASM18296v3)
Supplementary files format and content: tab-delimited text files with transcript id, raw count, preferred frame, preferred frame raw count, percentage, frame protection index

Submission date

May 20, 2024

Last update date

Oct 06, 2024

Contact name

Irene Stevens

E-mail(s)

irene.stevens@scilifelab.se

Phone

0046728774966

Organization name

Karolinska Institutet