title: Illumina NovaSeq 6000 paired end sequencing; RNA-seq, G pallida population D383 batch 1-2A cyst and hatched J2 samples, exposed for 8h, 24h, 48h to 2nM Solanoeclepin A (dissolved in 1% EtOH), potato root diffusate from wild accession PNT 777-2 (diluted to 0,1x with tap water) or tap water with 1% EtOH (spontaneous hatching control)
design: RNA-seq, G pallida population D383 batch 1-2A cyst and hatched J2 samples, exposed for 8h, 24h, 48h to 2nM Solanoeclepin A (dissolved in 1% EtOH), potato root diffusate from wild accession PNT 777-2 (diluted to 0,1x with tap water) or tap water with 1% EtOH (spontaneous hatching control)
library_construction_protocol: G. pallida D383 1-2A was mass propagated in potato plants using the standard protocol of the Laboratory of Nematology of Wageningen University. Batch 1-2A of population D383 was removed from the -80C and brought to 4C where it is hatching competent between 2015 and 2018. Hatching stimlant production and application: Potato root exudates were collected from 6 weeks old plants of wild potato accessions PNT 777-2 (CGN, the Netherlands) according to the protocol described by Guerrieri et al., 2021. Briefly, 5-10 in vitro cuttings of this potato accessions were transplanted into a single 14cm (diameter) plastic pot and let grow for 6 weeks. Exudates were collected by flushing pots with 500ml of distilled water. Post extraction exudates were filtered in whatman filter paper to remove excess debris and soil particles. Exudate aliquots were stored at -20C.   For Solaneclepin A, the material used was pure, chemically synthesized SolA (as described and kindly provided by Tanino et al., 2011). Prior to exposure to treatments dry cysts of G. pallida D383 1-2A were hydrated for 7 days in tap water from the laboratory sink of the University of Amsterdam. The water used for hydration of the cysts was changed daily to prevent the growth of microorganisms on the surface of the cysts. After 7 days, approximately 13mg cysts were placed on each insert of a Netwell 12 well plate with insert with 74um mesh size. The distribution of the treatments over the netwell plates was randomized.Technical replicates, (n=5), were generated for each time point of harvest. The hatching stimulation treatment was performed in 12 well Netwell plates with 74um mesh size insert (Corning)(SolA) . The potato root diffusate used at 0.1x dilution in tap water for this experiment was collected from wild potato cuttings of accessions PNT 777-2 (WUR Plant breeding) using the method described by Guerrieri et al., 2021. Pure Solanoeclepin A was chemically synthesized by Tanino et al., 2018 and was kindly given to Dr. H. Bouwmeester. Solanoeclepin A was dissolved to a final concentration of 2nM in a 1% ethanol solution. One percent ethanol was used as a control. Cysts exposed to treatments or control were collected in 2ml safe-lock Eppendorf tube containing two 3mm metal beads and placed immediately in liquid nitrogen. Until all replicates and time points were collected, samples were placed at -80C. Once the time course was finished and all samples collected, tubes were brought to liquid nitrogen from their storage at -80C and were homogenized at room temperature with 75ul of RLB buffer using a Tissue-lizer by 2 pulses of 60s at a frequency of 25 cycles/s.  Hatching stimlant production and application: Potato root exudates were collected from 6 weeks old plants of wild potato accessions PNT 777-2 (CGN, the Netherlands) according to the protocol described by Guerrieri et al., 2021. Briefly, 5-10 in vitro cuttings of this potato accessions were transplanted into a single 14cm (diameter) plastic pot and let grow for 6 weeks. Exudates were collected by flushing pots with 500ml of distilled water. Post extraction exudates were filtered in whatman filter paper to remove excess debris and soil particles. Exudate aliquots were stored at -20C.   For Solaneclepin A, the material used was pure, chemically synthesized SolA (as described and kindly provided by Tanino et al., 2011). Prior to exposure to treatments dry cysts of G. pallida D383 1-2A were hydrated for 7 days in tap water from the laboratory sink of the University of Amsterdam. The water used for hydration of the cysts was changed daily to prevent the growth of microorganisms on the surface of the cysts. After 7 days, approximately 13mg cysts were placed on each insert of a Netwell 12 well plate with insert with 74um mesh size. The distribution of the treatments over the netwell plates was randomized.Technical replicates, (n=5), were generated for each time point of harvest. The hatching stimulation treatment was performed in 12 well Netwell plates with 74um mesh size insert (Corning)(SolA) . The potato root diffusate used at 0.1x dilution in tap water for this experiment was collected from wild potato cuttings of accessions PNT 777-2 (WUR Plant breeding) using the method described by Guerrieri et al., 2021. Pure Solanoeclepin A was chemically synthesized by Tanino et al., 2018 and was kindly given to Dr. H. Bouwmeester. Solanoeclepin A was dissolved to a final concentration of 2nM in a 1% ethanol solution. One percent ethanol was used as a control. Cysts exposed to treatments or control were collected in 2ml safe-lock Eppendorf tube containing two 3mm metal beads and placed immediately in liquid nitrogen. Until all replicates and time points were collected, samples were placed at -80C. Once the time course was finished and all samples collected, tubes were brought to liquid nitrogen from their storage at -80C and were homogenized at room temperature with 75ul of RLB buffer using a Tissue-lizer by 2 pulses of 60s at a frequency of 25 cycles/s.  RNA was isolated using Qiagens RNeasy micro kit with in-column DNase I treatment of the RNA according to the manufacturers intructions. RNA samples were analysed by Nanodrop, Qubit and were stored at -80  until further use. Approximately 0.5 microgram total RNA was used for RNA library preparation suitable for illumina HiSeq paired end sequencing using TruSeq Stranded mRNA Sample Prep kit (illumina). In short, after polyA based mRNA selection, RNA were further processed including subsequently fragmentation, first and second strand cDNA synthesis, adapter ligation and final library amplification resulting in RNAseq libraries including unique dual indexes, all following manufacturers protocol. Final libraries were eluted in 30 l elution buffer followed by library quality assessment using a Fragment Analyzer (Agilent Technologies) and quantified by Qubit fluorescence measurements (Invitrogen, Life Technologies).  Prepared libraries were pooled equimolar and combined with other indexed libraries for sequencing on an illumina NovaSeq 6000 system. Final sequencing was done using a S2 type flow cell, XP loading workflow and with settings specific for 2x150 nt paired end reads plus dual indexes reads. All steps for sequencing were carried out according to manufacturers protocol. Demultiplexing of reads per sample by  corresponding indexes was done using bcl2fastq v2.20.0.422 (illumina Inc, San Diego CA, USA).