title: Sample 19
organism: Brassica nigra
description: Protocols: Leaf disks were collected using a cork-borer (Ø 1.8 cm) from six plants from the same treatment group and pooled (=one replicate sample). Eighteen plants were used to obtain a total of three replicates for each treatment-time point combination. Samples were harvested at 3, 6, 24 and 48 hours post-infestation (hpi) of the herbivores, and a different set of plants was used for each treatment-time point combination. Leaf tissues were flash-frozen in liquid nitrogen and stored. Seeds of Brassica nigra (Brassicales: Brassicaceae) were collected from a wild population in Wageningen, The Netherlands (51°57′32′′N, 5°40′23′′E). Plants were grown from seeds in potting soil (Lentse potground no.4, Lent, The Netherlands) in a greenhouse compartment (22 ± 3 °C, 60-70% relative humidity [RH], light: dark regime [L:D] 16:8 h). After one-week from sowing, seedlings were transferred to 1 L pots (1 plant per pot) containing potting soil. Plants were placed on rolling tables (675 × 170 cm) that were automatically flooded every two days (for 20 minutes) with a nutrient solution (NH4 1.2, K 7.2, Ca 4.0, Mg 1.82, NO3 12.4, SO4 3.32, P 1.0, Fe 35.0, Mn 8.0, Zn 5.0, B 20.0, Cu 0.5, Mo 0.5 in mmol/L). The aphid Brevicoryne brassicae (Hemiptera: Aphididae) and caterpillar Plutella xylostella (Lepidoptera: Plutellidae) were reared on Brussels sprouts plants (Brassica oleracea var. gemmifera cv. Cyrus) in a greenhouse or a climate room (22 ± 2 °C, 60-70 % RH and 16:8 h L:D) at the Laboratory of Entomology, Wageningen University. We characterized plant responses to simultaneous and single herbivory by B. brassicae (Bb) aphids and P. xylostella (Px) as compared to transcript profiles of undamaged plants (control). Plants were exposed to herbivores 30 days after sowing (4-weeks-old and 3-6 expanded leaves). Four Px (L1-L2) caterpillars and 10 adults of Bb were placed on the youngest fully expanded leaf alone, or in combination (Bb + Px). To confine herbivores to the induced leaf, a mesh bag (0.04 x 0.05 cm mesh size) was placed around it. The leaf petiole was wrapped with cotton wool to avoid the leaf form breaking, and a wooden stick was present as support for the mesh. In addition, the whole plant was covered by another mesh bag, secured by a rubber band around the pot, to prevent movement of herbivores between plants. The plants that served as control were treated in a similar way as plants receiving herbivores and received mesh bags. Insects fed on the plants during four different exposure times and were removed only before sampling. RNA isolation was performed using the Maxwell® 16 LEV Plant RNA Kit (Promega) and the Maxwell® 16 AS2000 Instrument (Promega), following the manufacturer' s instructions. To run the Plant RNA protocol, the Maxwell® 16 firmware version ≥4.97 was installed on the Maxwell® 16 AS2000 Instrument. After the total RNA extraction poly(A) enrichment was performed. After fragmentation cDNA was created by reverse transcription and the library was prepared.