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Sample GSM5029536

Query DataSets for GSM5029536

Status

Public on Jan 23, 2021

Title

LN30B - replicate 2

Sample type

SRA

Source name

Yeast cells

Organism

Cryptococcus neoformans

Characteristics

strain: H99
cell type: Yeast cells
protocol: Low nitrogen, 30°C

Growth protocol

Ammonium chloride (NH4Cl) was used as a nitrogen source in all experimentation and was supplemented as follows: 0.53 g/L NH4Cl for a higher nitrogen concentration (HN) and 0.21 g/L NH4Cl for a lower nitrogen concentration (LN). These nitrogen concentrations were selected based on the lowest ammonium-nitrogen (NH4-N) concentration (ca. 0.16 g NH4-N/kg) of the woody debris from which pathogenic cryptococci were previously isolated. C. neoformans cells were inoculated into 100 mL yeast carbon base (contained in 1L conical flasks) supplemented with HN or LN at a final concentration of 1×106 cells/mL. Following agitated incubation for either 38 h or 28 h at 30°C or 37°C, respectively, total RNA was extracted.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using an Ambion RiboPureTM-Yeast kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Four biological repeats were performed for each culture condition.
RNA libraries were prepared for sequencing using standard protocols for Ion Torrent sequencing

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent S5

Data processing

Torrent Suite Version 5.12.0 Software used for basecalling
Data were quality filtered (Q>20) using FastX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/), and length-filtered (<10 bp) using the awk command (Yalamanchili, Wan and Lui 2017)
Read quality assessed with FastQC, whereafter reads were aligned to the reference genome of C. neoformans H99 (Janbon et al. 2014) using HISAT2 v2.1.0 (Kim, Langmead and Salzberg 2015)
Alignment quality assessed using Qualimap2 v2.2.1 (Okonechnikov, Conesa and García-Alcalde 2016) and transcript assembly performed using StringTie v1.3.3b (Pertea et al. 2015)
Differential gene expression analysis was performed using DeSeq2 v1.20.0 (Love, Huber and Anders 2014)
Genome_build: C. neoformans H99
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

Submission date

Jan 22, 2021

Last update date

Jan 23, 2021

Contact name

Caylin Bosch

E-mail(s)

18344666@sun.ac.za

Organization name

Stellenbosch University

Department

Microbiology