GSM4672121: ribo_30C_2; Candida albicans; OTHER - SRA

SRX8736971: GSM4672121: ribo_30C_2; Candida albicans; OTHER
1 ILLUMINA (Illumina HiSeq 3000) run: 66.6M spots, 3.4G bases, 1.2Gb downloads

Submitted by: NCBI (GEO)

Study: Global Translational Landscape of the Candida albicans Morphological Transition

PRJNA646470 • SRP272074 • All experiments • All runs

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Candida albicans is a major human fungal pathogen that represents the fourth leading cause of hospital-acquired bloodstream infections in the U.S. and is associated with high mortality and/or morbidity rates in a wide variety of immunocompromised individuals, including cancer and AIDS patients. While the C. albicans morphological transition from yeast to filamentous cells is required for virulence, considerably little is known about translational mechanisms important for controlling this transition as well as other virulence-related processes in C. albicans and other human fungal pathogens. Using ribosome profiling, we report the first global translational profile associated with C. albicans morphogenesis. Strikingly, many genes involved in pathogenesis, filamentation, response to stress and cell wall organization show reduced translational efficiency (TE). Several of these genes are known to be strongly induced at the transcriptional level, suggesting that a translational fine-tuning mechanism is in place. Using a recently developed ORF-calling method, we have identified a significant number of potential uORFs in genes associated with pathogenesis and at least 57 potential novel ORFs, several of which appear to show altered TE in response to filamentation. Finally, using a novel method for global analysis of ribosome pausing from Ribo-seq data, we demonstrate an enrichment of ribosome pausing sites in C. albicans genes associated with protein synthesis and cell wall functions. Altogether, our results suggest that the C. albicans morphological transition is associated with widespread global translational alterations that do not simply reflect transcriptional changes and affect the expression of many genes associated with virulence-related processes and pathogenesis. Overall design: Examination of gene expression by RNA-seq and Ribo-seq in 1 strain of Candida albicans grown under 2 conditions (YEPD + serum at 37°C (filament-inducing) and YEPD at 30°C (non filament-inducing)). There are three biological replicates for each strain grown under each condition (12 samples total).

Sample: ribo_30C_2

SAMN15543347 • SRS7009578 • All experiments • All runs

Organism: Candida albicans

Library:

Instrument: Illumina HiSeq 3000

Strategy: OTHER

Source: TRANSCRIPTOMIC

Selection: other

Layout: SINGLE

Construction protocol: Lysates were prepared as described previously (Spealman et al. 2016) with a few modifications. Flash frozen cells were thawed on an ice water slurry and cell suspensions were transferred into tubes with 0.5 mm diameter acid-washed glass beads and placed on ice for 5 min. The samples were lysed by vortexing 8 times for 30 sec. each with a 30 sec. rest on ice between each vortex. Samples were further processed according to instructions for the Illumina TrueSeq Ribo Profile (Yeast) Kit. Briefly, lysates were pre-cleared by centrifugation at 3000g for 5 min. at 4°C to remove cell debris and further clarified by centrifugation at 20000g for 10 min. at 4°C. The lysates were treated for 10 min. on ice with 10U/mL DNase I (Illumina) and quantified by measuring absorbance at 260 nm (A260). Finally, 100 uL aliquots were frozen in liquid nitrogen and stored at -80°C until further use. Ribosome profiling and library preparation were carried out according to the protocol described for the True Seq Ribo Profile (Yeast) Kit (Illumina). Lysates were digested with 15 U RNase I (Ambion) per A260 unit and incubated at room temperature for 45 min. with gentle rotation. The reactions were stopped by the addition of 1000U/mL SUPERase-IN (Ambion). 80S monosome fractions were purified from the cell lysate using MicroSpin S-400 HR columns (GE Healthcare). Ribosome protected fragments (RPFs) were further purified using the RNA Clean & Concentrator-25 Kit (Zymo Research) method. This kit was also used for total RNA purification. rRNA depletion was carried out using a Ribo-Zero Magnetic Gold (Yeast) kit (Illumina). An additional subtraction step was included to remove rRNA sequences from circularized cDNA as described previously (Spealman et al. 2016) with slight modifications. Subtractions were carried out in a 30 uL reaction volume, with 10 uL sample, 2 uL 20X SSC and 2 uL of a rRNA subtraction pool containing custom-designed biotinylated oligonucleotides (Dataset S8). The circularized DNA was next used as a template for amplification of library PCR products. PCR products of 140-160 bp were recovered from excised gel slices, quantified by Agilent Bioanalyzer/Fragment analyzer and deep sequencing was performed using an Illumina Hiseq3000 machine at the Greehey Children's Cancer Research Institute Genome Sequencing Facility (University of Texas Health Science Center at San Antonio). All ribosome profiling experiments were performed in biological triplicate.

Experiment attributes:

GEO Accession: GSM4672121

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 66.6M spots, 3.4G bases, 1.2Gb

Run	# of Spots	# of Bases	Size	Published
SRR12228382	66,648,535	3.4G	1.2Gb	2020-12-16

ID:: 11364354

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