RNA-Seq of Alligator sinensis: adult female hypothalamus
Twelve small RNA libraries were constructed to identify miRNAs involved in hibernation adaptability. And then we performed the single-end sequencing (36bp or 50bp) on an Illumina Hiseq2500 at the LC-BIO (Hangzhou, China) following the vendors recommended protocol. Raw reads were subjected to an in-house program, ACGT101-miR (LC Sciences, Houston, Texas, USA) to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA) and repeats. Subsequently, unique sequences with a length in 18~26 nucleotide were mapped to specific species precursors in miRBase 21.0 by BLAST 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. The unique sequences mapping to specific species mature miRNAs in hairpin arms were identified as known miRNAs. The unique sequences mapping to the other arm of known specific species precursor hairpin opposite to the annotated mature miRNA-containing arm were considered to be novel 5p-or 3p-derived miRNA candidates. The remaining sequences were mapped to other selected species precursors (with the exclusion of specific species) in miRBase 21.0 by BLAST search, and the mapped pre-miRNAs were further BLASTed against the specific species genomes to determine their genomic locations.