PMC:4502367 / 8727-10653 JSONTXT

{"project":"2_test","denotations":[{"id":"25917918-24920004-43386536","span":{"begin":305,"end":309},"obj":"24920004"},{"id":"25917918-23935534-43386537","span":{"begin":865,"end":869},"obj":"23935534"},{"id":"25917918-16169926-43386538","span":{"begin":1546,"end":1550},"obj":"16169926"},{"id":"25917918-24920004-43386539","span":{"begin":1920,"end":1924},"obj":"24920004"}],"text":"RNA-seq data\nTwo previously published RNA-seq datasets were used for de novo transcript assembly of Z. tritici IPO323 including one dataset obtained from RNA extracted from infected Triticum aestivum (cultivar Obelisk) seedlings and one dataset obtained from axenically grown fungal cells (Kellner et al. 2014). For Z. pseudotritici, Z. ardabiliae, and Z. brevis, RNA-seq data were obtained from axenically grown cultures. Total RNA was extracted from fungal cells grown in YMS (4 g yeast extract, 4 g malt extract, 4 g sucrose, 20 g bacto agar, 1 liter H2O) agar in a shaking incubator at 200 rpm at 18° using the TRIZOL reagent (Invitrogen, Darmstadt, Germany) following the protocol of the manufacturer. Illumina RNA-seq libraries for two axenic culture replicates per species were prepared from an input of 10 µg total purified polyA RNA (Palma-Guerrero et al. 2013). Libraries were quantified by fluorometry, immobilized, and processed onto a flow cell with a cBot (Illumina), followed by sequencing-by-synthesis with TruSeq v3 chemistry on a HiSeq2000 at the Max Planck Genome Center (Cologne, Germany). RNA-seq data for Z. pseudotritici, Z. ardabiliae, and Z. brevis are respectively available in the NCBI BioProjects (PRJNA277173, PRJNA277174, PRJNA277175). For data processing, the sequence read quality was first evaluated using FASTQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Subsequently, reads were filtered using tools from the Galaxy server, including grooming, trimming, filtering, and masking steps (Giardine et al. 2005). Reads with an overall quality score less than 20 were removed. For the remaining reads, all nucleotides with a quality score less than 20 were masked with Ns. For Z. tritici, reads from the host (T. aestivum) transcriptome were initially filtered out using fastq_screen v0.4.1 (www.bioinformatics.babraham.ac.uk/projects/fastq_screen) as described by Kellner et al. (2014)."}