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| Status |
Public on Jul 26, 2023 |
| Title |
khd4D yeast RNAseq replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
yeast
|
| Organism |
Mycosarcoma maydis |
| Characteristics |
strain: AB33 khd4D
cell type: yeast
genotype: khd4 deletion
|
| Treatment protocol |
For the induction of hyphal growth of lab strain AB33 and derivates, 50ml cultures were grown to an OD600 of 0.5 in CM-glucose medium and shifted to nitrate minimal medium supplemented with 1% glucose. HyperTRIBE analyses was performed 6 hours post induction.
|
| Growth protocol |
The conditions used for cultivation of U. maydis are described in Brachmann et al., Mol Genet Genomics, 2004 (DOI: 10.1007/s00438-004-1047-z). Starting cultures were grown in CM medium supplemented with glucose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested using RNeasy Plant Mini Kit (74904; Qiagen, Hilden, Germany) following the manufacturer’s instructions. 500 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using VAHTS® Universal V6 RNA-seq Library Prep Kit for Illumina (NR604-01/02; Vazyme) following manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Data processing |
Briefly, RNA-seq reads were trimmed based on sequencing quality (Phred score) using Trimmomatic (version 0.39; Bolger et al., 2014). Specifically, reads with the Phred score of less than 25 were trimmed and removed when the read length fell below 19 nt.
RNAseq data analysis was carried out in Galaxy, an open-source platform (Afgan et al., 2018).
The raw sequencing reads were quality filtered based on their quality score and length with Trimmomatic (Version 0.36; Bolger et al., 2014). Specifically, the reads were trimmed 20 nt from the start, trimmed from the end when the Phred score dropped below 30, and discarded if the read length is less than 20 nt.
STAR (version 2.7.2b) was used to align the trimmed reads to the U. maydis genome (see HyperTRIBE experiment and data processing; Dobin et al., 2013), allowing up to 4% mismatches in the mapped read length while limiting the mapping to one locus.
Uniquely mapped reads on each gene were counted using htseq-count (version 0.9.1; Anders et al., 2014).
The resulting raw counts of mRNA libraries were used as input for the subsequent differential gene expression analysis. All differential gene expression analysis was performed using the R/Bioconductor package DESeq2 (Love et al., 2014). Genes with an absolute fold change > 1.5 (after fold change shrinkage) and an adjusted p-value < 0.05 (Benjamini-Hochberg correction) were considered differentially expressed.
Assembly: The bioinformatics analyses were based on the U. maydis 521 genome sequence (Ustilago_maydis.Umaydis521_2.0.dna_rm.chromosome.1) and the associated gene annotation (Ustilago_maydis.Umaydis521_2.0.41.gff3; both downloaded from http://ftp.ensemblgenomes.org/pub/fungi/release-53/fasta/ustilago_maydis/dna/; Kämper et al., 2006). We manually extended all genes by 300 nt on each side to include potential 5´ UTR and 3´ UTR regions which are not currently annotated in the U. maydis genome. The extended GTF file was converted to refFlat format using the UCSC gtfToGenePred tool.
Supplementary files format and content: tab-delimited text files include raw read counts for each Sample
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| |
|
| Submission date |
Feb 03, 2023 |
| Last update date |
Jul 26, 2023 |
| Contact name |
Prof. Dr. Michael Feldbrügge |
| E-mail(s) |
[email protected]
|
| Phone |
+49 211 81-15475
|
| Organization name |
Heinrich Heine University of Düsseldorf
|
| Department |
Department of Biology
|
| Lab |
Institute of Microbiology
|
| Street address |
Universitätsstraße 1
|
| City |
Düsseldorf |
| State/province |
North Rheine-Westphalia |
| ZIP/Postal code |
40225 |
| Country |
Germany |
| |
|
| Platform ID |
GPL33088 |
| Series (2) |
| GSE224486 |
The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [RNA-Seq] |
| GSE224487 |
The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis |
|
| Relations |
| BioSample |
SAMN33057052 |
| SRA |
SRX19277549 |
