RNA-Seq of Cellana toreuma: foot
The radulae from limpet(N =60) were divided into three independent pools, each containing radular tissue from 20 individualtons. Each radula was divided into unmineralized and mineralized regions. The feet of limpet were dissected for use as control tissue. Total RNA was extracted from the radulae or feet of the chitons using an Animal Total RNA Rapid Extraction Kit (GENERAY, Shanghai, China) according to the turers protocol. The tissue was ground into powders in liquid nitrogen and then transferred to a 1.5 mL RNase-free EP tube. 1 mL of Trizol was added to immerse the sample, followed by vortex mixing for 30 s and incubation at 25 C for 5 min. Chloroform was then added and the mixture was vigorously vortexed for 30 s, then incubated at room temperature for 3 min before centrifugation (12,000 g, 5 min, 4 C). The aqueous phase containing the RNA was obtained. RNA quality was evaluated by measuring absorbance, with A260/A280 ratios of 1.82.1 considered to represent RNA of good quality. RNA integrity wasated using Agilent 2100 bioanalyzer, and samples with RNA integrity number of above seven were employed for subsequent analyses following storage at 80 C. RNA-seq library was constructed using Illumina TruseqTM RNA sample prep Kit, and RNA-seq was performed by Majorbio Co. (China). The raw paired end reads were trimmed and quality control was performed using the tool fastp with defaulteters. The clean data so obtained were employed for de novoscriptome assembly using Trinity. The quality of the assembly was enhanced by filtering all the assembled sequences using CD-HIT and TransRate, followed by assessment using benchmarking universal single-copy orthologs.