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| Status |
Public on Jun 06, 2018 |
| Title |
hda1-2 |
| Sample type |
SRA |
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| Source name |
fungal cells
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| Organism |
Cryptococcus neoformans var. grubii H99 |
| Characteristics |
genotype/variation: hda1delta
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| Growth protocol |
Three biological replicates of WT and hda1∆ mutant cells were incubated in YPD medium to mid-log phase and transferred to minimal medium for 2 hrs at 30˚C
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were flash frozen, lyophilized, and lysed by vortexing with glass beads. RNA was extracted using the Qiagen RNeasy Plant Mini Kit with optional on-column DNase digestion.
Libraries were prepared by the Duke Sequencing and Genomic Technologies Shared Resource using the Kapa Stranded mRNA-Seq library prep kit (Catalog # KK8421). A single 6 bp index strategy was used, followed by pooling all 6 libraries.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
FS5-clr3-2
hda1∆
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| Data processing |
Base-calling and demultiplexing were done using bcl2fastq
Data was processed using 2 separate analysis pipelines described in detail below
(1) TopHat2/Cufflinks
Reads were aligned to the C. neoformans genome using TopHat2 with default parameters
Transcriptomes were assembled for each sample using Cufflinks with default parameters. The accepted_hits.bam files from each sample were used as input.
Cuffmerge was used to merge the assembled transcriptomes with the reference transcriptome
Reads for each transcript were quantified using Cuffquant with default parameters and the accepted_hits.bam files as inputs
Cuffdiff was used to quantify differential expression between the samples with parameters -u -o cuffdiff_out/ -L hda1,H99 -FDR 0.1
Genome_build: The C. neoformans H99 reference genome used for this analysis was obtained from the Sequencing Project at the Broad Institute of MIT and Harvard (accessed 03/31/2015). The genome was indexed using Bowtie2 with default parameters.
Supplementary_files_format_and_content: *_abundances.cxb files were generated from Cuffquant for each individual sample. FS1-3 are H99 1-3, FS4-6 are hda1 1-3.
Supplementary_files_format_and_content: cuffdiff_101817_gene_exp.csv is the "gene_exp.diff" output file from Cuffdiff, which tests differences in the summed FPKM of transcripts sharing each gene_id for each strain, replicates included. This comma separated file includes FPKM, log2(fold change), and significance values for each locus by strain.
STAR/DeSeq2
Reads were aligned to the C. neoformans genome using STAR alignment software with default parameters
Differential expression analysis was performed in R with DESeq2 following an RNA-Seq Bioconductor workflow (Love, M. I., Anders, S., Kim, V. & Huber, W. RNA-Seq workflow: gene-level exploratory analysis and differential expression. F1000Res 4, 1070, doi:10.12688/f1000research.7035.1 (2015).)
Genome_build: The C. neoformans H99 reference genome used for this analysis was obtained from NCBI (assembly CNA3, accessed 07/2017)
Supplementary_files_format_and_content: H99_v_hda1_counts.csv is a comma separated file with read counts for each strain/biological replicate
Supplementary_files_format_and_content: DESeq_results.csv is a comma separated file with log2(fold change) and significance values for each gene
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| Submission date |
Jan 24, 2018 |
| Last update date |
Jun 06, 2018 |
| Contact name |
Shannon Esher |
| E-mail(s) |
shannon.esher@duke.edu
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| Phone |
(919) 684-5054
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| Organization name |
Duke University
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| Department |
Molecular Genetics and Microbiology
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| Lab |
Alspaugh
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| Street address |
337 Sands Building, 303 Research Drive
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
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| Platform ID |
GPL21548 |
| Series (1) |
| GSE109582 |
HDAC genes play distinct and redundant roles in Cryptococcus neoformans virulence |
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| Relations |
| BioSample |
SAMN08391217 |
| SRA |
SRX3596454 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2946800_FS5_abundances.cxb.gz |
1.5 Mb |
(ftp)(http) |
CXB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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