RNA-seq of Zea mays: mature husk
High-quality RNA from each biological replicate was submitted to the DNA Services laboratory of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign and quantified by Qubit (Life Technologies). Using the TruSeq Stranded RNA Sample Preparation Kit (Illumina), PolyA+ RNA was selected from 1 µg of total RNA per sample. PolyA+ RNA was fragmented for 4 min at 94 ˚C, then first-strand cDNA was synthesized with a random hexamer and SuperScript II (Life Technologies). Double-stranded DNA was blunt-ended, 3'-end A-tailed and ligated to unique dual-indexed adaptors. The adaptor-ligated double-stranded cDNA was amplified by PCR for 10 cycles with the Kapa HiFi polymerase (Kapa Biosystems). The final libraries were quantified on Qubit and the average size determined on the AATI Fragment Analyzer (Advanced Analytics) and diluted to 5 nM final concentration. The 5 nM dilution was further quantitated by qPCR on a BioRad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc.). The final stranded RNASeq library pool consisting of 6 libraries was sequenced on one lane of an Illumina NovaSeq 6000 SP flowcell as paired-reads with 150 nt length. Raw data was adaptor-trimmed and demultiplexed using bcl2fastq v2.20 Conversion Software (Illumina).