|
| Status |
Public on Oct 24, 2023 |
| Title |
H99 CO2 3 |
| Sample type |
SRA |
| |
|
| Source name |
H99
|
| Organism |
Cryptococcus neoformans |
| Characteristics |
cell line: H99
genotype: wild-type
treatment: 5% CO2
time: 24h
|
| Growth protocol |
Overnight cultures of indicated strains were washed and diluted to 7.5x105 cells/mL, 3 mL cultured per condition in a 6-well plate in RPMI 1640 medium with 165 mM MOPS, pH 7 for 4, 8, or 24 hours at 37°C in ambient air or 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected via centrifugation from growth medium, lyophilized overnight and RNA harvested according to manufacturer protocol for Invitrogen PureLink RNA Mini Kit.
Total RNA (>2 ug per sample) was submitted to Azenta Life Sciences for standard RNA-Seq next-generation sequencing. The RNA samples were quantified using Qubit 2.0 Fluorometer (ThermoFisher) and RNA integrity was checked using TapeStation (Agilent Technologies). The RNA sequencing libraries were prepared using the NEB Next Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (New England Biolabs). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 min at 94°C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies) and quantified by using Qubit 2.0 Fluorometer (ThermoFisher) as well as by quantitative PCR (KAPA Biosystems). The sequencing libraries were multiplexed and clustered onto a flowcell. After clustering, the flowcell was loaded onto the Illumina HiSeq instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mismatch was allowed for index sequence identification.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
H99 CO2 3
Supplemental Table 5 - RIM101 CO2 vs H99 CO2 RNA-Seq.xlsx
gene_count_matrix_H99_RIM101.xlsx
|
| Data processing |
Paired-end Illumina sequence read files were evaluated for quality and the absence of adaptor sequence using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Read files were mapped to C. neoformans reference genome H99 v48 (FungiDB).
Gene transcript expression was quantified using HISAT2 and Stringtie.
Differential expression fold change, Wald test p values, and Benjamini-Hochberg adjustment for multiple comparisons were determined using DESeq2.
Principle component analysis was performed on regularized log transformed gene counts to confirm the absence of batch effects.
Assembly: FungiDB-48_CneoformansH99
Supplementary files format and content: Supplemental Table 3 - H99 CO2-Amb RNA-Seq time course.xlsx; abundance measurements relative to ambient conditions for each time point
Supplementary files format and content: Supplemental Table 5 - RIM101 CO2 vs H99 CO2 RNA-Seq.xlsx; abundance measurements for RIM101 relative to H99
|
| |
|
| Submission date |
Aug 28, 2023 |
| Last update date |
Oct 24, 2023 |
| Contact name |
Damian J Krysan |
| E-mail(s) |
damian-krysan@uiowa.edu
|
| Phone |
319-335-3066
|
| Organization name |
University of Iowa Hospitals and Clinics
|
| Department |
Division of Pediatrics & Infectious Disease
|
| Lab |
Krysan
|
| Street address |
25 South Grand, 2040 ML
|
| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
| |
|
| Platform ID |
GPL21073 |
| Series (1) |
| GSE241788 |
The fungal pathogen Cryptococcus neoformans adapts to the host environment through TOR-mediated remodeling of phospholipid asymmetry |
|
| Relations |
| BioSample |
SAMN37165615 |
| SRA |
SRX21487196 |
