title: miRNA-Seq of body wall: sea cucumber
design: Raw reads were subjected to an in-house program, ACGT101-miR (LC Sciences, Houston, Texas, USA) to remove adapterdimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA) and repeats. Subsequently, uniquesequences with length in 18~26 nucleotide were mapped to specific species precursors in miRBase 22.0 by BLAST searchto identify known miRNAs and novel 3p- and 5p- derived miRNAs. Length variation at both 3 and 5 ends and one mismatchinside of the sequence were allowed in the alignment. The unique sequences mapping to specific species mature miRNAs inhairpin arms were identified as known miRNAs. The unique sequences mapping to the other arm of known specific speciesprecursor hairpin opposite to the annotated mature miRNA-containing arm were considered to be novel 5p- or 3pderivedmiRNA candidates. The remaining sequences were mapped to other selected species precursors (with the exclusion ofspecific species) in miRBase 22.0 by BLAST search, and the mapped pre-miRNAs were further BLASTed against the specificspecies genomes to determine their genomic locations. The above two we defined as known miRNAs. The unmappedsequences were BLASTed against the specific genomes, and the hairpin RNA structures containing sequences werepredicated from the flank 80 nt sequences using RNAfold software (http://rna.tbi.univie.ac. at/cgi-bin/RNAfold.cgi). The criteriafor secondary structure prediction were: (1) number of nucleotides in one bulge in stem (12) (2) number of base pairs in thestem region of the predicted hairpin (16) (3) cutoff of free energy (kCal/mol -15) (4) length of hairpin (up and down stems +terminal loop 50) (5) length of hairpin loop (20). (6) number of nucleotides in one bulge in mature region (8) (7) number ofbiased errors in one bulge in mature region (4) (8) number of biased bulges in mature region (2) (9) number of errors inmature region (7) (10) number of base pairs in the mature region of the predicted hairpin (12) (11) percent of mature instem (80).