Time-series RNA-seq on potato root tissue infected by two populations of Globodera pallida
Time-series RNA-seq on potato root tissue infected by two populations of Globodera pallida
To assess the transcriptome of pre-parasitic and parasitic G. pallida juveniles in vitro infection assays were performed as described before (Zheng et al., 2022). In brief, Rookmaker and AMpop02DS4 cysts were hatched according to Goverse et al. (2000). Pre-parasitic second stage juveniles (ppJ2s) were collected, cleaned by sucrose purification (Jenkins, 1964) and surface-sterilized in 0.008% (w/v) mercuric chloride. Fourteen-day-old stem cuttings of the potato cultivar Seresta, were inoculated with 100 surface-sterilized Rookmaker or AMPOP02 ppJ2s. After inoculation, plants were kept in the dark at 18 °C, and infected root segments were harvested 1-, 3-, 6-, and 9-days post inoculation (dpi). Each sample consisted of the infected root tissue pulled from 10 plants. At each batch, a subsample of the inoculum was used to assess transcription in ppJ2s. Four independent hatchings were used to conduct four time-separated biological replicates of the experiment. Total RNA was extracted with the Maxwell 16 LEV-plant RNA kit (Promega, USA), according to the manufacturer's instructions. 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.