Overview of smRNA and mRNA analysis workflow. Cultures of Symbiodinium microadriaticum were subjected to 9 experimental treatments (noon: 12 h/12 h day/night cycle, sampled at noon; 4°C: 4°C for 4 hours; 16°C: 16°C for 4 hours; 34°C: 34°C for 12 hours; 36°C: 36°C for 4 hours; 20 g: 20 g/L NaCl salt content for 4 hours; 60 g: 60 g/L NaCl salt content for 4 hours; DS (dark stress): 18 hour dark period; DC (dark cycle): 12 h/12 h day/night cycle, sampled at midnight). Noon was selected as the reference condition for differential expression analyses. A total of 137 million small RNA reads resulted in the prediction of 219 miRNAs in 9 experimental treatments with the software miRDeep2, yielding a set of 21 smRNAs after further quality filtering. miRNA target gene prediction yielded 1,720 animal- and 19 plant-like miRNA binding sites via bowtie software in the set of 12,858 3'UTRs and 51,917 genes, respectively. Annotated miRNA targets were subsequently tested for GO category enrichment. A total of 302 million paired-end (PE) reads were assembled to a final gene set of 58,649 genes ≥ 250 bp with the Oases software. smRNA and mRNA expression over 9 experimental treatments was quantified with the DESeq software. Expression estimates of 21 smRNAs and 19,893 GO-annotated genes were assessed for correlation over 9 experimental treatments, and smRNAs-mRNA expression pairs displaying a correlation > 0.8 or < −0.8 (Spearman Rank) were tested for GO category enrichment.
Baumgarten et al. BMC Genomics 2013 14:704 doi:10.1186/1471-2164-14-704