title: Extract 1 (S1)
organism: Danio rerio
description: Protocols: The caudal fin of wild-type adult zebrafish aged 3-4 months was amputated 1-2 segments anterior to the bifurcation of the most peripheral branching lepidotrichia. After finectomy, 10 fish (n = 5) were placed at 33 ± 1 °C in 3 L-plastic containers at a density of 5 fish/L. Fish were exposed for 24 hours to the Skeletonema costatum ethanolic extract (SKLT) at 56 µg/mL or to ethanol (vehicle) at 0.1%, supplemented in fish water. Treatment was renewed daily. Moderate water dynamics and air-water exchanges were facilitated by bubbling fish tanks with an air pump. Water quality parameters were monitored daily and maintained stable for the duration of the experiment as follows: dissolved oxygen 7.0 ± 0.5 mg/L, pH 7.4 ± 0.2 and conductivity 680 ± 20 μS. Fish were sacrificed at 24 hours post-amputation (hpa) using lethal anesthesia of 0.6 mM tricaine methanesulfonate (MS-222, pH 7.0; Sigma-Aldrich), then 24 hpa- blastemas were dissected and pooled (10 blastemas/tube, 5 tubes/condition) for RNA extraction. Total RNA was extracted from pools of blastemas at 24 hpa (n = 5) using NZYol (NZYTech, Lisbon, Portugal) and quantified using a NanoDrop OneC spectrophotometer (Thermo Fisher Scientific, Waltham, USA). RNA integrity was confirmed using an Experion Automated Electrophoresis system (Bio-Rad, Hercules, USA). Only RNA with an RNA integrity number (RIN) higher than 7 was used. Bulk RNA sequencing was outsourced to STABVIDA Lda (Caparica, Portugal). DNA libraries were constructed using a Stranded mRNA Library Preparation kit (STABVIDA).