Transcriptome sequencing on a time series of potato infected with Globodera pallida to explore the sex determination
Transcriptome sequencing on a time series of potato infected with Globodera pallida to explore the sex determination
In vitro infection assays were performed as described by Goverse et al. (2000) with additions made by Zheng et al. (2022) and Schaveling et al., (2025). In brief, we used cuttings of the susceptible potato cultivar Desirée, which were grown on Gamborg B5 medium supplemented with either 20 g/L or 1.5 g/L of sucrose. After 14 days, cuttings were inoculated with 100 G. pallida Rookmaker J2s per plate.  We harvested infected roots at 1, 3, 6, and 9 days post inoculation (dpi). At 1 and 3 dpi, infected root segments of 15 plants were pooled per sample and at 6 and 9 dpi, infected root segments of 10 plants were pooled per sample.  Total RNA was extracted with the Maxwell 16 LEV-plant RNA kit (Promega, USA), according to the manufacturer's instructions. RNA samples were send for mRNA sequencing to BGI Genomics (Hong Kong, China). 1. Take a certain amount of total RNA samples, and use oligo dT beads to enrich mRNA with poly A tail; 2. mRNA molecules were fragmented into small pieces; 3. The fragmented mRNA was synthesized into first strand cDNA using random primers; 4. The second strand cDNA was synthesized with dUTP instead of dTTP; 5. The synthesized cDNA was subjected to end-repair and 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments; 6. Perform PCR amplification; 7. Library quality control; 8. Library circularization; 9. The library was amplified to make DNA nanoball (DNB); 10. Sequencing on DNBSEQ (DNBSEQ Technology) platform. 