RNA-Seq libraries were constructed as in Tsuda et al.59 (link) except that 6 µg of total RNA was used rather than 3 µg. Three libraries were made of four pooled 9–11 mm tassels: two mutants and one wild type. Libraries were sequenced on a NextSeq Illumina platform with 75 bp paired-end reads. Raw reads were aligned to the maize B73 genome AGPv3.30 using HISAT260 (link) and counted to AGPv3.30 gene models using HTSeq-counts61 (link) using the cyberinfrastructure provided by Cyverse Atmosphere62 (link). Reads were visualized using the Integrative Genomics Viewer (Broad Institute)63 (link). Counted reads were tested for differential expression with edgeR using a generalized linear model (GLM) approach on transcripts with a raw count greater than 5 in at least one condition and FDR significance threshold of 0.0564 (link),65 (link). Differentially expressed genes (FDR ≤ 0.05) between Ts5 and WT siblings were separated by -log(fold-change) into upregulated and downregulated differentially expressed gene lists. Gene accessions from each list were tested for Gene Ontology term enrichment by singular Gene Ontology term enrichment analysis (SEA) with agriGO v2.066 (link). Quantitative reverse transcription PCR was performed as in Bolduc et al.67 (link).
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