Trans1 t1 clone vector

Total RNA was extracted from 3-day-old adult O. communa samples using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First strand cDNA synthesis was performed using 1.0 µg total RNA and the One-step Reagent Kit with gDNA Eraser (TransGen Biotech, Beijing, China). Subsequently, according to the sequence obtained from the previous transcriptome data (shown in the Supplementary Materials), a pair of specific primers were designed using Primer Premier 5 (PREMIER Biosoft International, Palo Alto, CA, USA; Table 1). They were used to amplify the MRLC-sqh sequence using the cDNA as a template. The amplification product was then purified using the Monarch Gel Extraction kit (NEB, Ipswitch, MA, USA) and cloned into a Trans1-T1 clone vector (TransGen Biotech) and sequenced [33 (link)].

Total RNA was extracted from the five female ovaries collected, using TRIzol Reagent (Invitrogen, United States) and following the manufacturer’s protocols. The first strand of complementary DNA (cDNA) was synthesized from 1 μg total RNA using a first-strand cDNA Synthesis Kit (Transgen Biotech, Beijing, China) according to the manufacturer’s protocols. The synthesized cDNAs were stored at −20°C until use. To identify the OcomCSP12 sequence, a pair of degenerate primers (in Table 1) were designed to amplify the nucleic acid sequence. The polymerase chain reaction (PCR) was performed under the following thermal program: 94°C 5 min, 35 cycles of 94°C for 30 s, 48°C for 30 s, and 72°C for 90 s, followed by one cycle at 72°C for 10 min. The PCR product was purified with a Monarch gel extraction kit (NEB, England) and cloned into a Trans1-T1 clone vector (Transgen Biotech, Beijing, China) and sequenced (Sangon Biotech, Shanghai, China).