Solexa 0

Approximately 1 μg RNA of an RNA integrity number (RIN) >7.0 from each sample was used for the subsequent cDNA library construction following the protocol of TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA). Single-end sequencing (36 bp) was then performed on an Illumina HiSeq 2500 at the LC-BIO (Hangzhou, China) according to the vendor’s recommended protocol.
Initially, the raw reads were subjected to the Illumina pipeline filter (Solexa 0.3), and the datasets were further processed with an in-house program, ACGT101-miR (LC Sciences, Houston, Texas, USA), to remove adapter dimers, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA) and repeats. Subsequently, the remaining clean reads were used to search against miRBase 21.0 (http://www.mirbase.org/search.shtml) for known miRNA prediction. During the alignment, length variation at both the 5’ and 3’ ends and one mismatch inside of the sequence were allowed. For the miRNAs identified in this study, the conservation analysis was conducted through comparisons with 40 species listed in S1 Table.
The unmapped sequences were further searched against P. olivaceus genomes [42 (link)] using RNAfold software (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) for novel miRNA prediction. The criteria for secondary structure prediction were similar to a previous study [34 (link)].

Approximately 2.5 μg of total RNA were used to prepare small RNA library according to the protocol of TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA). And then the libraries were sequenced by Illumina Hiseq2500 50SE (single end) at the LC-BIO (Hangzhou, China) following the vendor’s recommended protocol. Data processing followed the procedures as described in a previous study. Briefly, the raw reads were subjected to the Illumina pipeline filter (Solexa 0.3), and then the dataset was 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, 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 3′ and 5′ ends and one mismatch inside of the sequence were allowed in the alignment. Mapping methods for identification of conserved and novel miRNAs are listed in Table S6.

Data processing followed the procedures as described previously [23 (link),24 (link)]. The raw reads were subjected to the Illumina pipeline filter (Solexa 0.3), and then the dataset was further processed with an in-house program, ACGT101-V4.2 (LC Sciences, Houston, TX, USA) to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA), and repeats.