RNA-seq of Medicago truncatula inoculated roots
Medicago truncatula Jemaolong A17 composite plants with transgenic roots expressing an empty vector (control) or the MtEPP1(Wt)-Ox or MtEPP1(S-D)-Ox constructs were grown in Fahräeus medium plates for three weeks under sterile conditions. Transgenic roots showing TDT fluorescence were harvested, immediately frozen in liquid nitrogen and stored at -80°C until used. Total RNA was isolated from transgenic roots from ten different composite plants using the ZR Plant RNA miniprep kit (Zymo Research, USA). Stranded messenger RNA-seq (mRNA-seq) libraries were generated from 1 μg of genomicDNA-free total RNA from each experimental condition and prepared using the TruSeq RNA Sample Prep kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. For each experimental condition, three biological replicates containing transgenic roots from 20 independent composite plants were included. Nine libraries for each genetic background were sequenced on an Illumina NextSeq 500 platform with a 150-cycle sequencing kit and a configuration of pair-end reads with a 75 bp read length. Library construction and sequencing were performed by the Unidad Universitaria de Secuenciación Masiva y Bioinformática (Instituto de Biotecnología, UNAM, México).