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Sample GSM4417310

Query DataSets for GSM4417310

Status

Public on Mar 18, 2020

Title

sre1∆_pH4, 3

Sample type

SRA

Source name

Vegetative fungal cells

Organism

Cryptococcus neoformans

Characteristics

genotype: sre1delta
condition: pH4
replicate: 3

Treatment protocol

Approximately 1x10^9 cells from each strain were pelleted and resuspended in YPD media buffered to pH4 or pH8 and incubated at 30°C for 90 minutes with 150 rpm shaking.

Growth protocol

H99 and sre1∆ cells were incubated at 30°C with 150 rpm shaking in YPD media to mid-logarithmic growth phase.

Extracted molecule

total RNA

Extraction protocol

All cells were pelleted, flash frozen on dry ice, and lyophilized overnight. RNA was isolated using the Qiagen RNeasy Plant Minikit with optional on column DNase digestion (Qiagen, Valencia, CA). RNA concentration and quality were measured using the Agilent 2100 Bioanalyzer.
The NEBNext Poly(A) mRNA Magnetic Isolation Module was used to enrich for mRNA and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina was used to prepare libraries (New England Biolabs, Ipswich, MA). Libraries were sequenced on the Illumina NextSeq 500 with 75 base pair, single-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

sre1∆
9_2019_P_M1_S9_sort_dedup

Data processing

Quality control was assessed by FastQC.
RNA-Seq reads were trimmed for adaptor sequences and filtered using "fastq-mcf" with the following parameters: -q 20 , -x 0.5. Trimmed and filtered reads were aligned to the C. neoformans H99 genome using "STAR" with the following parameters: --quantMode GeneCounts, --alignIntronMax 5000, --outSJfilterIntronMaxVsReadN 500 1000 2000.
Bam files were sorted, indexed, and merged using "Samtools". Duplicate reads were removed from bam files using "picard".
Differential expression analyses were performed in R using an RNA‐Seq Bioconductor (Love 2015, F1000Research) workflow followed by the DESeq2 package with a false discovery rate (FDR) of 5% (Love 2014, Genome Biol).
Genome_build: CNA3.44
Supplementary_files_format_and_content: raw counts

Submission date

Mar 17, 2020

Last update date

Mar 18, 2020

Contact name

J Andrew Alspaugh

E-mail(s)

andrew.alspaugh@duke.edu

Organization name

Duke University

Department

Molecular Genetics and Microbiology