This protocol describes the production of a reference-quality de novo transcriptome assembly for the spiny mouse (Acomys cahirinus). These methods can be applied to other RNA-Seq datasets to generate high-quality transcriptome assemblies in other species. Validation and description of assembly output is described in 'De novo transcriptome assembly for the spiny mouse (Acomys cahirinus)' bioRxiv, p.076067.