GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4672121

Query DataSets for GSM4672121

Status

Public on Dec 10, 2020

Title

ribo_30C_2

Sample type

SRA

Source name

fungal cells

Organism

Candida albicans

Characteristics

growth_condition: YEPD at 30°C
assay: Ribo
tissue: fungal cells

Treatment protocol

Next, cells were scraped off the filter paper with a cell scraper, placed in ice-cold lysis buffer (1X yeast polysome buffer (Illumina), 10% Triton X-100 (Sigma), 10 mg/mL GMPPNP (Sigma), 10 mg/mL Blasticidin S (InvivoGen) and homogeneously mixed prior to snap freezing in liquid nitrogen.

Growth protocol

Serum inductions were carried out in YEPD (yeast extract peptone dextrose) medium as described previously using C. albicans wild-type strain DK 318 (Banerjee et al. 2008) with the following exceptions: 1) the initial starting overnight culture volume was 150 mL and overnight cultures were diluted at OD600 ~ 4.0, 2) cells were diluted into a final culture volume of 450 mL of YEPD at 30°C or YEPD + 10% serum at 37°C. Cells were harvested at the 1-hour post-induction time point and recovered by rapid filtration.

Extracted molecule

total RNA

Extraction 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.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 3000

Data processing

Library strategy: Ribo-seq
Adaptor sequences and low-quality score (phred quality score < 5) bases were trimmed from RNA-Seq and Ribo-Seq datasets with TrimGalore (v 0.4.3) (Krueger, 2012). Only reads that were at least 20 nucleotide long were retained.
Trimmed sequences from RNA-seq and Ribo-seq were both mapped using STAR (v.2.5.2b) (Dobin, 2013) using Assembly 22 fasta as the reference and GTF s07-m01-r27 from CGD allowing a mismatch of at most two positions (--outFilterMismatchNmax 2). Only uniquely mapping reads were retained (-outFilterMultimapNmax 1). All the reads mapping to non-mRNA sequences were filtered out before downstream analysis. The corresponding non-mRNA sequences were also obtained from CGD (C_albicans_SC5314_version _A22-s07-m01-r27_other_features_no_introns.fasta). The periodicity analysis of Ribo-seq data was performed using ribotricer (v1.3.2) (Choudhary, 2020). Gene level counting was performed using featureCounts (v1.6.4) (Liao, 2014). Since Assembly 22 is a diploid assembly, the reads were counted for each copy of the gene separately (hapA and hapB) and then merged into a single read count per gene.
Differential expression analysis was performed using DESeq2 (Love, et al, 2014). Only genes with at least one read per replicate were selected for performing the library size normalization step and running the moderated t-statistic test. Genes were defined to be differentially expressed (DE) if the transcripts per million (TPM) was greater than 1 in at least 2 replicates and if their absolute fold-change on log2 scale is at least 1 with an FDR adjusted p-value of at least 0.05.
Differential translational efficiency was performed using riborex (Li, 2017) by retaining genes that had at least one read count in one replicate across the two conditions in both Ribo-seq and RNA-seq samples. Only genes that had a read count of at least one per replicate were used as input to riborex. We define genes to be exhibiting differential translational efficiency if their TPM was greater than 1 in at least 2 replicates while the absolute fold-change on log2 scale was at least 1 and the corresponding non-adjusted p-value at least 0.05. Gene ontology (GO) analysis was performed using clusterProfiler (Yu, 2012) using GO slim ontology file available from CGD.
Genome_build: ASM18296v3
Supplementary_files_format_and_content: raw counts table

Submission date

Jul 15, 2020

Last update date

Dec 10, 2020

Contact name

David Kadosh

E-mail(s)

[email protected]

Phone

(210) 567-3976

Organization name

University of Texas Health Science Center at San Antonio