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.