Rneasy plus mini kit

One-month-old midribs from plants homozygous for five of the bm4-Mu alleles, bm4-ref, and B73 (Table S2) were collected according to the method described above for RNA-Seq analysis. Single pools of 3–4 midribs from separate plants were prepared for each genotype. RNA was extracted from each pool using Qiagen's RNeasy Plus Mini kit and reversed transcribed with Bio-Rad's iScript cDNA Synthesis kit. Thus, one cDNA library was prepared for each genotype. Two technical replicates of each cDNA sample were run with bm4 gene-specific primers and two technical replicates of each cDNA sample were run with GAP primers. Quantitative real-time PCR was performed on a Roche Light Cycler 480II instrument using Bio-Rad's iQ SYBR Green Supermix. Relative expression for each sample was quantified using the 2^−ΔΔCt method (Yuan et al., 2006 (link)). A single expression value was calculated for each genotype by averaging across all technical replicates. bm4 primers: (forward) bm4CE11L10.2 = 5′-TTGGCTAGTACGTGGCTTGA-3′ and (reverse) bm4C1E12R10.1 = 5′-GCGCTCCATCCAAATAAAAA-3′. GAP primers: (forward) gap987f = 5′-CTGAACGACCACTTCGTCAA-3′ and (reverse) gap1143r = 5′-TTCTCGGCATACACAAGCAG-3′.