AbstractWith advances in high‐throughput sequencing technologies, de novo transcriptome sequencing and assembly has become a cost‐effective method to obtain comprehensive genetic information of a species of interest, especially in nonmodel species with large genomes such as spiders. However, high‐quality RNA is essential for successful sequencing, and sample preservation conditions require careful consideration for the effective storage of field‐collected samples. To this end, we report a streamlined feasibility study of various storage conditions and their effects on de novo transcriptome assembly results. The storage parameters considered include temperatures ranging from room temperature to －80°C; preservatives, including ethanol, RNAlater, TRIzol and RNAlater‐ICE; and sample submersion states. As a result, intact RNA was extracted and assembly was successful when samples were preserved at low temperatures regardless of the type of preservative used. The assemblies as well as the gene expression profiles were shown to be robust to RNA degradation, when 30 million 150‐bp paired‐end reads are obtained. The parameters for sample storage, RNA extraction, library preparation, sequencing and in silico assembly considered in this work provide a guideline for the study of field‐collected samples of spiders.