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Sample GSM3332306

Query DataSets for GSM3332306

Status

Public on Aug 07, 2019

Title

D8ANA-2 hypoxia 8 d replicate 2

Sample type

SRA

Source name

Yeast

Organism

Cryptococcus neoformans

Characteristics

strain: H99 (ATCC 208821)

Growth protocol

Cryptococcus neoformans H99 strain stored in 20% glycerol at -80°C was cultured on Sabouraud agar plate at room temperature (step 1). After 2 to 5 days of culture, 10^7 cells were suspended in 10 mL of YPD in a T25cm3 flask and cultured for 22 hours at 30 °C, 150 rpm with lateral shaking until stationary phase (final concentration=2×10^8/mL) (step 2). 3×10^7 cells of this first culture was suspended in 10mL YPD (second culture) under the same conditions until stationary phase (Step 3). Then, flasks were incubated in hypoxia generated using a GENbag® anaer (Biomérieux) atmosphere generator in a dedicated plastic bag for 8 days in the dark at 30°C. A control sample always performed in parallel is incubated under the same conditions but in normoxia (Step 4). Logarithmic phase for Cryptococcus neoformans or Saccharomyces cerevisiae were grown during 17h in YPD.

Extracted molecule

total RNA

Extraction protocol

The RNA protocol extraction used was adapted from (Moyrand et al., Eukaryotic Cell, 2008). Briefly, the samples were lyophilized overnight. RNAs were extracted following a protocol divided in 3 parts: i) an extraction step was performed in Trizol reagent (Invitrogen), ii) a second step by adding Chloroform (Invitrogen) iii) the last step was an overnight Isopropanol precipitation at -20°C.
We used 1 μg of total RNA to purify polyadenylated mRNAs and to build an RNA library, using TruSeq Stranded mRNA Sample Prep Kit (Illumina, #RS-122-9004DOC) as recommended by the manufacturer. Directional library are checked for concentration and quality on DNA chips with the Bioanalyser Agilent. More precise and accurate quantification is performed with sensitive fluorescent-based quantitation assays ("Quant-It" assays kit and QuBit fluorometer, Invitrogen).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Samples are normalized to 2nM concentrations and then multiplexed 4 samples per lane. Then they have been denatured at a concentration of 1nM using 0.1N NaOH, 5’at room temperature to be finally diluted at 9pM. Each sample is loaded on the flowcell at 9pM. Sequencing of the 24 samples was performed on the HiSeq 2500 sequencer (Illumina) in 65 bases single-end mode.
Reads were cleaned of adapter sequences and low-quality sequences using cutadapt version 1.11.Only sequences at least 25 nt in length were considered for further analysis.
STAR version 2.5.0a, with default parameters, was used for alignment on the reference genome
Genes were counted using featureCounts version 1.4.6-p3 from Subreads package (parameters: -t gene -s 1 )
Count data were analyzed using R version 3.3.1 and the Bioconductor package DESeq2 version 1.12.3 The normalization and dispersion estimation were performed with DESeq2 using the default parameters but statistical tests for differential expression were performed without applying the independent filtering algorithm. A generalized linear model was set in order to test for the differential expression between D0, D8AE, D8ANA and Log. For each pairwise comparison, raw p-values were adjusted for multiple testing according to the Benjamini and Hochberg (BH) procedure and genes with an adjusted p-value lower than 0.01 were considered differentially expressed.
Genome_build: Cryptococcus neoformans var. grubii H99 from Broad Institute
Supplementary_files_format_and_content: Matrix of count from featureCounts

Submission date

Aug 14, 2018

Last update date

Aug 07, 2019

Contact name

Rachel Legendre

E-mail(s)

[email protected]

Organization name

Institut Pasteur

Department

Research and Resource Center for Scientific Informatics