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Sample GSM5218544

Query DataSets for GSM5218544

Status

Public on Mar 30, 2021

Title

H99_aCSF_r2

Sample type

SRA

Source name

yeast cell

Organism

Cryptococcus neoformans

Characteristics

isolate: H99
strain: VNI
condition: Artificial CSF
day: Day1
repeat: Rep2

Growth protocol

(1) aCSF: yeast cells incubated in aCSF at 37oC for 24 hours. Cells were harvested by centrifugation at 3000 RPM and stored at -800C. (2) rCSF: yeast cells were inoculated intracisternally into 2-3 kg New Zealand White rabbits receiving daily cortisone acetate. Each yeast strain was inoculated into three separate rabbits. Rabbit CSF was withdrawn (1-2ml) after 24 and 96 hours in the rabbit subarachnoid space. rCSF containing the same strain were pooled, centrifuged to pellet the cells and the cell pellets were stored at -800C. (3) YPD / CAP: yeast cells were grown for 24 hours in yeast peptone dextrose broth (YPD) or capsule-inducing media (CAP), centrifuged to collect the cell pellet, and store at -800C. (4) Human CSF (hCSF): yeasts per ml of CSF (1-5mls) was directly withdrawn under a lumbar puncture as standard of care, centrifuged to pellet and stored at -80C until RNA isolation was performed.

Extracted molecule

total RNA

Extraction protocol

These lyophilized samples were treated with Trizol and total RNA was extracted using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions.
Library preparation was prepared by shearing the RNA and adapted for Illumina sequencing using the TruSeq RNA Library Preparation Kit (Illumina, San Diego, CA) for YPD and CAP conditions or the Tag-Seq Protocol (Shishkin et al Nat Methods 2015) for the in vivo CSF and aCSF samples.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

The quality filtering and adaptor trimming were performed using Cutadapt (v 1.12).
Paired-read alignment for RNA-seq data was carried out against the gene set for C. neoformans var. grubii H99 (CNA3) excluding non-coding RNAs and mitochondria genes, using STAR (v 2.5.3a).
The expected count, TPM, and FPKM for each gene were estimated with RSEM (v1.2.31).
Genome_build: CNA3 (GCA_000149245.3)
Supplementary_files_format_and_content: Tab-delimited text files include length, Effective length, Expected count, TPM, and FPKM For each gene.

Submission date

Mar 29, 2021

Last update date

Mar 30, 2021

Contact name

John R. Perfect

E-mail(s)

john.perfect@duke.edu

Organization name

Duke University School of Medicine

Department

Division of Infectious Diseases