Transcriptome analyses and transcription factor-binding motif searches

The total RNA of transgenic soybean hairy roots was extracted using TRIzol™ Reagent (15596026, ThermoFisher Scientific) according to the manufacturer’s protocol. The isolation of total mRNA using oligo(dT) beads, generation of strand-specific libraries, and RNA sequencing were performed by a commercial service (BGI-Hong Kong, Hong Kong). The libraries were sequenced using the Illumina Hi-Seq 4000 paired end platform to generate >80 million raw reads of 101 bases from each library. Cleaned reads were aligned to the reference genome of soybean (version Wm82v2a1) (Schmutz et al., 2010) by HISAT (version 2.0.4) (Kim et al., 2015). The alignment result was then subjected to StringTie (version 1.3.0) (Pertea et al., 2015) for transcript assembly, and the assembled transcripts were used to generate a gene list. Ballgown (Frazee et al., 2015) was used to compute the expression levels of genes and to identify differentially expressed genes among the sequencing libraires. Selected genes were subjected to Gene Ontology (GO) enrichment analyses by agriGO (Tian et al., 2017). All protein-coding genes from the soybean reference genome (version Wm82v2a1) (Schmutz et al., 2010) were regarded as the background for the analyses. A hypergeometric test was performed with the threshold P<0.05. After that, hierachical diagrams were generated according to the internal relationships among the GO terms. Genomic sequences 3 kb upstream of the translational start site of putative target genes were extracted from our in-house genome sequence of wild soybean W05. The extracted sequences were subjected to MEME-Suite (version 4.11.3) (Machanick and Bailey, 2011) to discover putative transcription factor-binding motifs (E-value ≤0.05). The best-match motif from the genomic sequence upstream of the translational start site of the putative target gene was then subjected to homology search against known transcription factor-binding motifs by JASPAR (Khan et al., 2018).