Total RNA was isolated and purified using RNAprep pure Plant Kits (TIANGEN BIOTECH). Approximately 15 μg of total RNA was used for library construction following a standard procedure. Libraries were sequenced with a read length of 100 bp (paired-end) and an insertion size of 300 bp on an Illumina HiSeq 2000 at Berry Genomics, Beijing. Read quality was evaluated using FastQC software (Andrews 2010 ). 3′ reads with quality less than 20 were first trimmed by NGS QC Toolkit (v2.3) (Patel and Jain 2012 (link)). Only reads with a read length greater than 50 bp were kept for downstream analysis. The high-quality reads were then aligned to the B73 reference sequence (AGPv2) (Schnable et al. 2009 (link)) using Tophat2/Bowtie1 (Kim et al. 2013 (link)). Five mismatches, a minimum intron size of 5 bp and a maximum intron size of 60,000 bp were used for alignment. For each sample, the number of reads covering the gene model (filtered gene set 5b) was calculated using htseq-count with the intersection-strict option (Anders et al. 2014 (link)).
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