Senegalese sole is an economically important flatfish species in aquaculture and an attractive model to decipher the molecular mechanisms governing the severe transformations occurring during metamorphosis, where retinoic acid seems to play a key role in tissue remodeling. In this study, a robust sole transcriptome was envisaged by reducing the number of assembled libraries (27 out of 111 available), fine-tuning a new automated and reproducible set of workflows for de novo assembling based on several assemblers, and removing low confidence transcripts after mapping onto a sole female genome draft. From a total of 96 resulting assemblies, two "raw" transcriptomes, one containing only Illumina reads and another with Illumina and GS-FLX reads, were selected to provide SOLSEv5.0, the most informative transcriptome with low redundancy and devoid of most single-exon transcripts. It included both Illumina and GS-FLX reads and consisted of 51,348 transcripts of which 22,684 code for 17,429 different proteins described in databases, where 9527 were predicted as complete proteins. SOLSEv5.0 was used as reference for the study of retinoic acid (RA) signalling in sole larvae using drug treatments (DEAB, a RA synthesis blocker, and TTNPB, a RA-receptor agonist) for 24 and 48 h. Differential expression and functional interpretation were facilitated by an updated version of DEGenes Hunter. Acute exposure of both drugs triggered an intense, specific and transient response at 24 h but with hardly observable differences after 48 h at least in the DEAB treatments. Activation of RA signalling by TTNPB specifically increased the expression of genes in pathways related to RA degradation, retinol storage, carotenoid metabolism, homeostatic response and visual cycle, and also modified the expression of transcripts related to morphogenesis and collagen fibril organisation. In contrast, DEAB mainly decreased genes related to retinal production, impairing phototransduction signalling in the retina. A total of 755 transcripts mainly related to lipid metabolism, lipid transport and lipid homeostasis were altered in response to both treatments, indicating non-specific drug responses associated with intestinal absorption. These results indicate that a new assembling and transcript sieving were both necessary to provide a reliable transcriptome to identify the many aspects of RA action during sole development that are of relevance for sole aquaculture.