Galaxea astreata wound-healing time-course
Goal: study wound healing after injury in the stony coral Galaxea astreataMethod:Samples used in this study were taken from coral colonies approximately 5 cm in diameter, maintained under controlled conditions in an indoor FRP tank at the New Taipei City Marine Resource Restoration Center. For sample preparation, polyps were gently removed from these colonies using clippers. The wound surface area and polyp height were standardised. Subsequently, polyps were fixed onto 0.1 mm thick film sheets using instant adhesive and transferred into glass containers filled with 800 mL of artificial seawater. Each container held up to 30 polyps. The containers were maintained in an incubator under controlled conditions: a 12-hour light/12-hour dark photoperiod, light intensity of 100 uE m-2 s-1, temperature of 25C, and salinity of 35 ppt. Feeding and water changes were performed twice weekly.At designated time points, coenosarc tissues surrounding each polyp (excluding the polyp body itself) were carefully collected under a dissecting microscope. Tissues were immediately transferred into 500 uL of TRI reagent (Sigma-Aldrich T9424) and homogenized, then stored at -20C. Total RNA was extracted using TRI reagent according to the manufacturer's instructions. RNA libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit following the standard protocol. Briefly, mRNA was isolated from 1 ug of total RNA using oligo(dT)-attached magnetic beads and fragmented under elevated temperature. First-strand cDNA synthesis was performed using reverse transcriptase and random primers, followed by second-strand synthesis and 3' adenylation. Adaptors were ligated to the cDNA fragments, which were then PCR-amplified and purified using the AMPure XP system (Beckman Coulter). Library quality was evaluated using the Qsep400 System (Bioptic), and concentrations were measured with a Qubit 2.0 Fluorometer (Thermo Scientific). Sequencing was performed on an Illumina NovaSeq platform with 150 bp paired-end reads.