|
| Status |
Public on Jun 30, 2014 |
| Title |
IPO323_YMS |
| Sample type |
SRA |
| |
|
| Source name |
yeast cells
|
| Organism |
Zymoseptoria tritici |
| Characteristics |
strain: IPO323
tissue: yeast
age: freshly grown liquid culture (YMS medium)
genotype: wild type
|
| Growth protocol |
Cultures were inoculated onto solid YMS agar (4 g yeast extract, 4 g malt extract, 4 g sucrose, 20 g bacto agar, 1 l H2O) at 18°C. For transcriptional profiling of axenic culture, yeast-like cells were isolated from these plates after 72 h. For transcriptional profiling of plant infections, 15 day old wheat seedlings of the cultivar Obelisk and the Brachypodium distachyon accession Bd21 were inoculated as follows. A distinct area of the second leaf (10 - 15 cm) was marked and a spore solution of 1 x 107 yeast cells/ml containing 0.1% Tween 20 was brushed onto these areas. After an initial incubation for 48 h at 100% humidity and 22°C, inoculated plants were incubated with a 16 h light period at 75% humidity and 22°C for four days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To extract total RNA from fungal axenic culture or freeze-dried leaf tissue infected with Z. tritici, 100 mg of sample was crushed in liquid nitrogen and total RNA was extracted using the TRIZOL reagent (Invitrogen) following the manufacturer’s protocol. RNA samples were purified twice with Dynal oligo(dT) magnetic beads (Invitrogen), following the manufacturer's instructions.
RNA-seq libraries for plant infection replicates were prepared from an input of 4 µg total purified polyA RNA according to recommendations of the supplier (TruSeq RNA sample preparation v2 guide, Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Quality evaluation using FastQC
Trimming: 12 bp from left and 5 bp from right for: 234_A, 234_B, 234_C, 234_D, 234_E, 234_F
Quality filtering and Masking: Reads with an overall quality score below 20 as well as reads having less than 25% of nucleotides with a quality score above 20 were discarded. For the remaining reads nucleotides with quality scores below 20 were masked with Ns.
Filtering for plant reads: In order to account for incorrect mapping of plant reads with high similarity to fungal sequences, reads from infected plant tissue were mapped to both the plant genomes and the genome of Z. tritici IPO323. All plant reads were removed for further downstream analyses. To this end reads with 100% identity to the plant genomes and a maximum of 25% N positions were filtered out in fastq_screen v0.4.1 (www.bioinformatics.babraham.ac.uk/projects/fastq_screen) applying a sensitive local Bowtie2 alignment
Read Mapping: Mapping of single end RNAseq reads to the genome of the Z. tritici isolate IPO323 (Goodwin et al. 2011) was carried out with tophat v2.0.9 (Kim et al. 2013). Mapping was disabled for novel splice discovery and used the JGI transcript annotation of IPO323 as reference (Goodwin et al. 2011; http://genome.jgi.doe.gov/Mycgr3/Mycgr3.home.html). The maximum number of read mismatches and read gap length were set to 2. Reads that mapped to more than 10 locations were eliminated from the analysis. Relative abundances for predicted transcripts of IPO323 were estimated in cufflinks v2.1.1 (Trapnell et al. 2012). Total read counts per transcript were estimated in htseq-count (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html) applying the intersection-strict mode.
Supplementary_files_format_and_content: csv files with FPKM values and total counts for each transcript of IPO323 (Z. tritici)
|
| |
|
| Submission date |
Feb 11, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ronny Kellner |
| E-mail(s) |
ronny.kellner@mpi-marburg.mpg.de
|
| Organization name |
MPI for Terrestrial Microbiology
|
| Lab |
Fungal Biodiversity Group
|
| Street address |
Karl-von-Frischstrasse 10
|
| City |
Marburg |
| ZIP/Postal code |
35043 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18273 |
| Series (1) |
| GSE54874 |
Transcriptome sequencing at early stage infection of the wheat pathogen Zymoseptoria tritici reveals chromosomal differences in transcription patterns and host specific gene expression. |
|
| Relations |
| BioSample |
SAMN02640204 |
| SRA |
SRX469417 |
