normalization data transformation protocol
These BAM files were loaded into Partek Genomics Suite 6.6 (Saint Louis, MO) for further analysis. Briefly, the reads per kilobase of exon model per million mapped reads (RPKM)-normalized reads (Nature, 2008; 5: 621-8.) were calculated and the expression levels of genes were estimated.
high throughpt sequence alignment protocol
The raw fastq files were aligned to the genome of VdLs.17 strain of Verticillium dahliae (Broad Institute, www.broadinstitute.org) following workflow of Galaxy instance installed at University of Alabama at Birmingham (UAB, http://galaxy.uabgrid.uab.edu). The BAM files were generated following RNA-seq workflow of tophat (Bioinformatics 25, 1105-1111, 2009), cufflinks and cuffcompare (Nature Biotechnology doi:10.1038/nbt.1621).
nucleic acid sequencing protocol
HiSeq 2000 (Illumina) at Centrillion Biosciences
nucleic acid extraction protocol
Total RNA was extracted from the pulverized fungal tissue using a Qiagen RNeasy Kit (Valencia, CA) and following the manufacturer’s instructions, and included an on-column DNase I digestion.
The cultures were of Verticillium dahliae, strain VdLs.17, that was producing (MS) or not producing microsclerotia (NoMS). The NoMS culture could be considered the control. Cultures were grown at 25 C on potato dextrose agar overlaid with a nitrocellulose membrane. All MS and NoMS cultures were ten days old.
nucleic acid library construction protocol
The sequncing library was constructed by using ScriptSeq_V2 RNA-Seq Library Preparation kit.