Validation of transcriptome data using qRT-PCR
RNA-Seq data quality was validated by measuring the relative expression
pattern of 12 genes functioning in C and N metabolism using qRT-PCR
(Figure S2). Briefly, target-specific qRT-PCR primers were designed
using the Primer5.0 software and synthesized, as presented in Table S1.
First-strand cDNA was synthesized with purified total RNA using the
PrimeScriptTM RT reagent Kit (Takara, Dalian, China).
qRT-PCR was carried out in Lightcycler 480II (Roche, Rotkreuz,
Switzerland) using 2X SG Fast qPCR Master Mix (B639271, BBI, Roche).
Each sample was represented by three biological and three technical
replicates. The comparison of qRT-PCR results with RNA-Seq revealed the
similar expression pattern between two datasets, thus validating the
transcriptomic data (Figure S2). In addition, transcript abundance
pattern of embryo (OSH1 and OsLEC1 ) and endosperm(GluD-1 and RPBF) specific genes also confirmed the
credibility of the gene expression data (Figure S3),
indicating that the embryo and
endosperm samples were processed well.