Small RNA sequencing and analysis of differentially expressed miRNAs
35S:SlmiR-168a , 35S:rSlAGO1 , and JZ18 were used as small RNAs. In total, nine samples (35S:SlmiR-168a , 35S:rSlAGO1 , and JZ18, each with three replicates) were harvested. Approximately 2.5 μg total RNA was used to prepare a small RNA library using a TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The libraries were then sequenced using an Illumina Hiseq2500 50SE (single end) at LC-BIO (Hangzhou, China) following the vendor’s recommended protocol. Briefly, raw reads were subjected to the Illumina pipeline filter (Solexa 0.3), and the dataset was then further processed with an in-house program, ACGT101-miR (LC Sciences, Houston, TX, USA), to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, and snoRNA), and repeats. Subsequently, unique sequences 18–26 nt in length were mapped to specific species precursors in miRBase 21.0 by Bowtie search to identify known miRNAs and novel 3p- and 5p-derived miRNAs. Length variation at both the 3′ and 5′ ends and one mismatch inside of the sequence were allowed in the alignment. Mapping methods for the identification of conserved and novel miRNAs are listed in Table S2.