NextSeq 500 sequencing; The effect of loss of Makibishi1-(MKB10) function on the transcriptome of Medicago truncatula hairy roots
The effect of loss of Makibishi1-(MKB10) function on the transcriptome of Medicago truncatula hairy roots
Collection of Medicago truncatula hairy root lines grown in vitro at the VIB-UGent center for Plant Systems Biology in Gent, Belgium. Sterilization of M. truncatula seeds (ecotype Jemalong J5), transformation of seedlings by Agrobacterium rhizogenes (strain LBA 9402/12), and the subsequent generation of hairy roots were carried out as described previously (Pollier et al., 2011, J. Nat. Prod. 74, 1462-1476.). Hairy roots were cultivated for 21 days in liquid medium to provide proper amounts for RNA extraction. For elicitation of triterpene saponin pathway gene expression, 100 µM MeJA was added to the medium for 4 h (or an equivalent of the solvent EtOH for mock treatment) prior to sampling Frozen hairy roots were ground and the material was used to prepare total RNA using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Library preparation was done using the TruSeq RNA Sample Preparation Kit v2 (Illumina). In brief, poly(A)-containing mRNA molecules were reverse transcribed, double-stranded cDNA was generated, and adapters were ligated. After quality control using the 2100 Bioanalyzer, clusters were generated through amplification using the TruSeq kit (Illumina) followed by sequencing on Illumina NextSeq 500 with the TruSeq SBS Kit v3-HS (Illumina). Sequencing was performed in single-end mode with a read length of 75nt.