Transcriptomic analysis of Medicago truncatula hairy roots overexpressing 35S::MtHSFA9::GFP compared to 35S::GUS::GFP
For ectopic overexpression of MtHSFA9 ,35S::MtHSFA9::GFP/35::GUS and the control (plasmid containing the GUS  gene alone) plasmids were obtained by the gateway cloning method according to Verdier et al (2013) and used forAgrobacterium rhizogenes transformation for subsequent hairy root transformation in Medicago truncatula following the protocol of (Boisson-Dernier et al. 2001). MtHSFA9 and GUSamplicons were obtained by PCR using primers listed in Table S1 and cloned in pK7GW2D,1 (Karimi, Inzé & Depicker 2002) containing a 35S cauliflower mosaic virus promoter and GFP reporter that was used as transformation marker. Seedlings were grown in square Petri dishes with Fahraeus medium two months at 20°C, 16h light, at an angle of 45°. Transformed roots were selected by GFP fluorescence under UV microscope (stereomicroscope, Olympus U-RFL-T) and harvested in liquid nitrogen. RNA was extracted from three replicates of similar amounts of roots using Nucleospin Extract II, Total RNA Purification Kit (Macherey Nagel). RNA amplification, labeling, and hybridization of Nimblegen Medtr_v1.0 12x135K arrays were performed according to Terrassonet al. (2013). Three biological replicates were analyzed per comparison using the dye-switch method, and statistical analysis on the gene expression data was performed according to Verdier et al. (2013). Transcriptome data has been submitted to the Gene Expression Omnibus as GSExxxxx.