title: RNA-seq of Globodera rostochiensis: Ro5-line-22
design: RNA-seq of Globodera rostochiensis: Ro5-line-22
library_construction_protocol: PCN J2s were sucrose purified and washed extensively in tap water and transferred to 1.5 mL RNase free tubes. The sample was incubated in RNA-later (Invitrogen) for 30-45 minutes at room temperature (~20°C). Next, most of the RNA-later was removed by pipetting and the sample was stored at -80°C. The greenhouse propagated Globodera rostochiensis (Potato Cyst Nematode; PCN) a population/line was used; Ro5-line-22 these is a inbred line of Ro5-Harmerz, and was mass-propagated in the greenhouse.  Second stage pre-parasitic juveniles (J2's) of PCN were obtained by artificial hatching through rehydration in tap water for 7 days followed by incubation of cysts in root exudate from Solanum lycopersicum cv. Money Maker. Nematode and root material were homogenized as follows: To the sample 2 steel balls (diameter 3 mm) and 35 µL of homogenisation buffer (Maxwell LEV16 Plant RNA kit) was added and homogenized in a tissue homogenizer (Qiagen GmbH, Swingmühle Tissue Lyser 2) at 30/s for 2x 80 seconds. 100 µL of homogenisation buffer was then added and samples were again homogenized at 30/s for 80 seconds. The steel balls were removed immediately after homogenization. RNA extraction was performed according to the Maxwell LEV16 plant RNA kit. RNA concentration was measured in a Qubit Fluorometer with the Qubit™ RNA High Sensitivity (HS) Assay Kit, according to the manufacturer's instructions and RNA integrity was analysed on a 1% agarose-TAE gel stained with Midori green.  Library prep was generated by USeq using Truseq RNA stranded RiboZeroPlus (human, mouse, rat,viral, bacterial) kit from Illumina according to the manufacturer's instruction.