NextSeq 500 paired end sequencing; RNA-seq of Fusarium graminearum under predation by the springtail Folsomia candida against untreated controls
RNA-seq of Fusarium graminearum under predation by the springtail Folsomia candida against untreated controls
Mycelium was harvested and springtails were removed. Whole mycelium was used to extract the RNA. 5,000 fungal spores in 5 µL water were inoculated onto three rice kernels autoclaved with 200 µl demineralized water in 15 ml Falcon tubes and incubated at 15°C in dark for 7-8 days   20 mg (around 200 individual animal) of springtail Folsomia candida starved for two days were placed into mycelia and the incubated at 15°C in dark. Controls were incubated under the same conditions without animals. Each group consisted of 4 replicates.  Suspension of mycelia in 400 µl of RNA extraction solution [95% deionized formamide, 18 mM EDTA, 0.025% SDS, and 1% 2-mercaptoethanol] was disrupted by shaking with zirconia beads (2.0 mm diameter, Carl Roth, Karlsruhe, Germany) in reciprocal mill MM 200 (Retsch, Haan, Germany) for 2 min at maximum power, followed by incubation at 95°C for 7 min. Cells debris was removed by centrifugation at 16,000x g for 5 min at room temperature. The supernatant was transferred into RNA precipitation mixture consisting of 800 µl of isobutanol, 400 µl of 5 M guanidine thiocyanate and 5 µl of linear polyacrylamide used as co-precipitant (Co-Precipitant Pink, Bioline, London, UK). The mixture was centrifuged at 16,000x g for 5 min at room temperature and the pellet was washed with 75% ethanol, dried and dissolved in RNAase-free water. Strand-specific cDNA libraries were prepared using Illumina's TruSeq stranded mRNA kit (75 bp paired-end).