Quantifying Rice DRP Gene Expression

Total RNA was extracted from SR segments including the earlier stage of LRP (Part 3 in Supplementary Figure 4A) in 5-day-old seedlings using the NucleoSpin^® RNA Plant Kit (Macherey-Nagel, Germany) following the manufacturer’s instructions. Quantitative reverse-transcription PCR (qRT-PCR) was performed using the One-Step SYBR PrimeScript RT-PCR Kit II (Perfect Real-Time) (TaKaRa Bio, Japan) and StepOnePlus Real-Time PCR (Life Technologies, United States). The expression was normalized to that of OsUBQ5 (Os03g0234200). All primers are listed in Supplementary Table 1. The expression of OsDRP family genes was compared using the RNA-seq data in S- and L-type LRP with four biological replications (Kawai et al., 2022a (link)). The read counts were normalized by coding sequence length (per 1 kb) for all genes in each replicate to calculate transcript per million (TPM). The normalized read counts were further normalized by summing the normalized counts of all genes (per million reads). Means of the calculated TPMs of four biological replications were used to compare the expression levels between S- and L-type LRP. The significance of the difference between S- and L-type LRP was determined by Padj values calculated by DESeq2 (Love et al., 2014 (link)).