|
| Status |
Public on Aug 09, 2024 |
| Title |
A7-35-23 Amb 24h 3 |
| Sample type |
SRA |
| |
|
| Source name |
A7-35-23
|
| Organism |
Cryptococcus neoformans |
| Characteristics |
strain: A7-35-23
genotype: wild-type
treatment: Ambient Air
time: 24h
|
| Growth protocol |
Overnight cultures of indicated strains were washed and diluted to 7.5x10^5 cells/mL, 3 mL cultured per condition in a 6-well plate in RPMI 1640 medium with 165 mM MOPS, pH 7 for 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 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 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 |
| |
|
| 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.
Assembly: FungiDB-48_CneoformansH99
Supplementary files format and content: Normalized_counts.xlsx - normalized gene counts
Supplementary files format and content: Raw_gene_counts.xlsx - unnormalized gene counts
|
| |
|
| Submission date |
Mar 05, 2024 |
| Last update date |
Aug 09, 2024 |
| 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) |
| GSE260932 |
QTL Mapping and Bulk Segregant Analysis identifies CO2 tolerance genes associated with virulence in the global pathogen Cryptococcus neoformans |
|
| Relations |
| BioSample |
SAMN40269311 |
| SRA |
SRX23842424 |
