RNA-Seq of Medicago truncatula: roots inoculated with rhizobia and non-inoculated controls
Total RNA was extracted using TRIzol Reagent according to the manufacturers instructions and assessed for quality via Bioanalyser and NanoDrop, with only high-quality RNA used for sequencing. RNA-seq libraries were prepared and sequenced by Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd., using Illumina Stranded mRNA Prep, where mRNA was isolated by oligo(dT) beads and then fragmented by fragmentation buffer, and converted to cDNA using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA). Then the synthesized cDNA was subjected to end-repair, phosphorylation and A base addition according to Illuminas library construction protocol. Libraries were size-selected, PCR-amplified, and sequenced on a NovaSeq 6000 sequencer (2  150bp read length). The raw paired end reads were trimmed and quality controlled by fastp  and aligned to reference genome with HISAT2 software. Transcript assembly was done via StringTie (Pertea et al., 2015), and gene expression quantified using RSEM .