Name: Sample 90_p
Instrument: DNBSEQ-T7
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC SINGLE CELL
Selection: PolyA
Layout: PAIRED
Construction protocol: The whole root and whole terminal leaflet samples were harvested post 1, 2, 3, 4, 5 day of second stressor. Root were harvested after cleaning them in water and flash freezing them immediately in liquid nitrogen. Both root and leaf samples were harvested within 45 secs. Three biological replicates were collected, each biological replicate consisting of one individual plant. Tomato, Solanum lycopersicum (Moneymaker), seeds were sown in the tray in climate cabinet with 22 °C diurnal and 18 °C nocturnal temperatures, 16L:8D photoperiod and 70 ± 10 % relative humidity. Ten days later, plants were individually transplanted into 14cm diameter pots containing silver sand . Post transplanting into pots containing silver sand, the plants were fertilized with fertilized water . The fertilized water was given twice a week. Nematodes – First stressor Nematode treatment was performed when plants were nineteen (19) days old from sowing. The density of nematodes used for infestation was 1000 nematodes per plant. The pipette tips were already present on both sides of root which were taken off and then 250 ul of nematode suspension was inoculated on both sides.Second stressor was performed when plants were 24 days old. Aphids - Macrosiphum euphorbiae (the potato aphid), were reared on sweet pepper plants (Capsicum annuum (Mandy variety, Rijk Zwaan (De Lier, The Netherlands)) in a climate-controlled cabinet with 16L:8D photoperiod, 21±2 °C and 70 ± 10 % relative humidity to produce nymphs for the experiments. Twelve 2-3 days old aphid nymphs were used for infestation, confined in the clip-cage (3 cm diameter) on terminal leaflet of one of the first two true leaves. Empty clip-cages were used as mock treatment. Aphids with CMV - Twenty-four hours earlier, we transferred 1-day old nymphs to CMV infected sweet pepper plants (Capsicum annuum (Mandy variety, Rijk Zwaan (De Lier, The Netherlands)) in a climate-controlled cabinet with 16L:8D photoperiod, 21±2 °C and 70 ± 10 % relative humidity . Twelve 2-3 days old aphid nymphs were used for infestation, confined in the clip-cage (3 cm diameter) on terminal leaflet of one of the first two true leaves. Empty clip-cages were used as mock treatment. CMV - Some crystal powder was sprayed on tomato terminal leaflet. Then CMV infected tobacco leaves were grinded with some buffer followed by finger infection to terminal leaflet. In order to infect the plants with Cladosporium fulvum, the small paint brush was dipped into fungal suspension (12.5 x 10^6/ml) and was rubbed onto the terminal leaflet.In order to elevate the infection rate, the plants were covered with transparent plastic bags for 24hrs. The transparent plastic bags were taken off after one day. RNA was using a Maxwell® 16 AS2000 instrument with a Maxwell® 16 LEV Plant RNA Kit (both Promega Corporation, Madison, WI, USA) following the manufacturer's instructions with minor modifications. Namely, the homogenization step was performed by adding 600 μl of the chilled 1-thioglycerol/homogenization solution, and by vigorously vortex the sample for 1 minute. After the standard protocol was followed. RNA samples were stored at -80 ℃ until further use. After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. The suitable fragments are size-selected for the PCR amplification as templates.