title: small RNAseq - Plant parasitic cyst nematodes respond to viral infection through siRNA activation
design: small RNAseq - Plant parasitic cyst nematodes respond to viral infection through siRNA activation
library_construction_protocol: In this study, the greenhouse propagated Woensdrecht (also known as IRS) isolate of BCN Heterodera schachtii (Beet Cyst Nematode; BCN) was used (kindly provided by Stichting IRS, Dinteloord, the Netherlands). For Globodera rostochiensis (Potato Cyst Nematode; PCN) two populations/lines were used; Ro1-line-19 and Ro5-line-22 that are inbred lines of Ro1-Mierenbos and Ro5-Harmerz, respectively, and were 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. For BCN the same life stage was obtained by incubation of hydrated cysts in 3 mM ZnCl2.   H. schachtii J2s as well as PCN J2s were sucrose purified and washed extensively in tap water and transferred to 1.5 mL RNase free tubes. All nematode samples were 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 samples were stored at -80°C. Nematode and root material were homogenized as follows: To each sample 2 steel balls (diameter 3 mm) and 35 µL of homogenisation buffer (Maxwell LEV16 Plant RNA kit) were 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.  For small RNA-enriched RNAseq libraries total RNA from G. rostochiensis line 19 and line 22 and H. schachtii Woensdrecht were used for size selection, library preparation (NEBNext small RNA library - NEB).