Ctr_7_2h_MeJA
Medicago truncatula
Protocols: About 100 mg of frozen ground root material was used for RNA extraction Sterilization of M. truncatula seeds (ecotype Jemalong J5), transformation of seedlings by Agrobacterium rhizogenes (strain LBA 9402/12) and generation of hairy roots was carried out as described (Pollier et al. 2011, J Nat Prod 74: 1462-1476). Hairy roots were grown for 21 days in liquid medium. Hairy roots were treated with 100 µM of methyl jasmonate (dissolved in ethanol) for 2h or 24 h before harvesting the roots. Control hairy roots were treated with an equal amount of ethanol. RNA extraction was done 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.