title: The transcriptome of motile stages of Pratylenchus zeae
design: Vermiform juveniles and adults of P. zeae were harvested from roots of infectedsugarcane using a mist apparatus (Tan et al., 2013): infected roots were cut into 5 mm pieces, placed on coffee filters and sprayed with a fine mist for 10 seconds at 10 minutes intervals for 24 hr.  Mixed stages of the nematodes moved through the filter and were collected at 4 hr intervals.  Total RNA was extracted from freshly collected active nematodes using Trizol (Invitrogen Life Technologies, Carlsbad, USA) and the RNA cleaned up with RNeasy Mini kit (Qiagen Inc, California, USA).  The quality and quantity of the RNA was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, Canada).  A cDNA library was prepared from 2.5 μg of total RNA (RIN of >7) using the Ovation RNA-Seq system (NuGEN Technologies, Inc., CA, USA) and the quality assessed using an Agilent2100 Bioanalyzer.  Half a picotitre plate was used to generate the 454 GS FLX dataset using a Roche 454 GS FLX.  Sequencing linkers were removed from all reads and de novo assembly was carried out using standard settings of the SoftGenetics NextGENe V2.16 software and the Condensation Tool (Manion et al. 2009).