title: S11_WS2
organism: Catharanthus roseus
description: Protocols: Whole stem tissue was collected between the mature leaves of greenhouse grown plants. Stem epidermis enriched tissue and peeled stems (pith) were obtained by peeling mature stems with a potato peeler. For every biological replicate (n=3), tissues from 5-7 leaves or stems from different plants were pooled together. All stem and leaf tissue samples were snap-frozen in liquid nitrogen. plants were grown in the greenhouse at 24°C under 16/8 hours light/dark conditions. About 100 mg of frozen ground plant material was used for RNA extraction using the Qiagen RNeasy mini kit. 450 μl Buffer RLT (supplemented with 10 μl of β-mercaptoethanol per ml Buffer RLT) was added and the mixture was vortexed vigorously. The lysate was transferred to a QIAshredder spin column placed in a 2 ml collection tube and centrifuged for 2 min at full speed. The supernatant was mixed with half a volume of ethanol and transferred to a an RNeasy spin column placed in a 2 ml collection tube followed by centrifugation for 15 seconds at full speed. 350 μl RW1 buffer was added and the column was centrifuged for 15s at max speed. Next an on column DNase digestion was performed and the column was washed again with 350 μl RW1 buffer. Next, the column was washed twice with 500 μl Buffer RPE. The RNA was eluated in a clean 1.5 mL eppendorf by adding 40 μl RNase-free water and centrifuging at maximum speed for 1 minute. Random primed cDNA library: Purification of poly-A containing mRNA molecules - mRNA fragmentation - Random primed cDNA synthesis - Adapter ligation and adapter specific PCR amplification.