 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 31, 2016 |
| Title |
05420-37C.CO2.90m-1-11.06.15-11.16.15-607 |
| Sample type |
SRA |
| |
|
| Source name |
cultured cells
|
| Organism |
Cryptococcus neoformans |
| Characteristics |
strain: KN99{alpha}
genotype: usv101(CNAG_05420)delta
condition description: capsule-inducing
condition: 37°C DMEM + 5% CO2 for 90mins
induction date: 11.06.15
library date: 11.16.15
|
| Treatment protocol |
Cells were transfered to grow for 90 minutes in capsule-inducing (DMEM, 37 °C, 5% CO2) conditions, 24 hours in capsule-inducing conditions, or 90 minutes in capsule non-inducing (YPD, 30 °C, room air) conditions prior to isolation of total RNA.
|
| Growth protocol |
All strains were constructed in the C. neoformans KN99α background. Cells were cultured overnight in YPD to a density of 1-2 OD600.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA isolation, the culture was treated with ice-cold stop solution (5% Tris-saturated phenol in ethanol) to prevent RNA degradation and then collected by centrifugation. The cells were suspended in TRIzol reagent (Life Technologies) and lysed by mechanical bead-beating at 4°C with 0.5-mm silica-zirconia beads (4 cycles of 3 min of beating followed by 2 min on ice). Following lysis, total RNA was extracted according to the manufacturer’s instructions. Residual DNA was removed from the RNA preparation with the TURBO DNA-free kit (Life Technologies).
Poly(A) RNA was selected from the total using the mRNA Catcher Plus Kit (Life Technologies) with an epMotion 5075 automated pipettor (Eppendorf). The poly(A) RNA was subsequently sheared by incubating in TURBO DNA-free buffer at 75°C for 10 minutes and purified with the QIAquick PCR Purification Kit (Qiagen). First strand cDNA synthesis was performed using random hexameric primers and SuperScript III Reverse Transcriptase, followed by treatment with E. coli DNA ligase, DNA polymerase I, and RNase H for second-strand synthesis, all using standard methods. The cDNA libraries were end-repaired with a Quick Blunting kit and A-tailed using Klenow exo- with dATP (New England Biolabs). Illumina adapters were ligated to the cDNA and fragments ranging from 150-250 bp in size were selected using gel electrophoresis. The libraries were enriched and indexed in a 10-cycle PCR using Phusion Hot Start II High-Fidelity DNA (Fermentas), purified, and pooled in equimolar ratios for multiplex sequencing on an Illumina HiSeq 2500 at the Genome Technology Access Center.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
usv101(CNAG_05420)Δ in 37°C DMEM + 5% CO2 for 90mins
|
| Data processing |
Sequenced reads were aligned to the C. neoformans H99 reference sequence (v2) using TopHat version 2.0.4 and Bowtie version 0.12.8
Reads that aligned uniquely to the reference sequence were considered for gene expression quantification with Cufflinks version 2.0.2. Gene boundaries were defined using the current C. neoformans genome annotation provided by the Broad institute. Gene expression was normalized using the Cufflinks provided option for quartile normalization.
After gene expression quantification, samples not passing the following quality control filters were removed from consideration. The expression of the deleted gene in mutant expression profiles was required to be less than 10% of its expression in WT. The mean and median expression of USV101 in deletion mutants was 0.3% and 0% of WT USV101 levels respectively. The low levels of expression that remained for some samples were likely the result of errors in the sequencing process itself, either due to contamination between samples or a low level of error in matching barcodes for multiplexed samples to reads. In addition, the mean and median expression of USV101 in overexpression mutants was 801% and 844% of WT USV101 levels respectively. Wild-type samples were required to express the resistance marker at less than 10% of its median level in all mutant expression profiles. In addition, to maximize the power to detect differentially expressed genes, a quality control filter was established to ensure that replicate expression profiles were in agreement. Specifically, within a replicate set the median of all gene coefficients of variation (CoV), defined as the standard deviation of gene expression over mean gene expression, was required to be less than or equal to 0.2. Any replicate set which did not pass this CoV filter was not used for differential expression analysis, and the mutant expression profiles were remade. However, replicate sets were rescued if the median CoV could be lowered below 0.2 by removing an outlier expression profile replicate.
Counts of reads mapping to each gene were quantitated using the htseq-count tool in version 0.5.3 of the HTSeq Python package.
Differential expression analysis comparing mutant and wildtype expression profiles sets in the same condition was performed by using LIMMA with the voom transformation applied to read counts.
Genome_build: C. neoformans H99 reference sequence (Cryptococcus neoformans var. grubii H99 Sequencing Project) – ASM14924v2
Supplementary_files_format_and_content: cuff: Gene expression was quantified processing tophat produced bam alignments with Cufflinks v2.0.2 with the current H99 annotation (v2) provided by the Broad institute's Fungal Genome initiative. Expression was normalized by applying the Cufflinks supplied quartile normalization routine. counts: Counts of reads mapping to each gene were quantitated using the htseq-count tool in version 0.5.3 of the HTSeq Python package (Anders et al. 2014).
|
| |
|
| Submission date |
Feb 13, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ezekiel Maier |
| E-mail(s) |
emaier@wustl.edu
|
| Organization name |
Washington University in Saint Louis
|
| Department |
Computer Science
|
| Lab |
Michael Brent
|
| Street address |
4515 McKinley Ave
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL19081 |
| Series (1) |
| GSE69532 |
Usv101 is a key regulator of cryptococcal virulence and determinant of host response |
|
| Relations |
| SRA |
SRX1584351 |
| BioSample |
SAMN04504716 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2061091_05420-37C.CO2.90m-1-11.06.15-11.16.15-607.counts.txt.gz |
30.0 Kb |
(ftp)(http) |
TXT |
| GSM2061091_05420-37C.CO2.90m-1-11.06.15-11.16.15-607.cuff.txt.gz |
185.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
