ERC000011
ENA-CHECKLIST
2023-04-30T00:33:44Z
ENA-FIRST-PUBLIC
2023-04-30T00:33:44Z
ENA-LAST-UPDATE
SAMEA112945639
External Id
Centre of Marine Sciences (CCMAR) - Universidade do Algarve
INSDC center name
2023-04-30T00:33:44Z
INSDC first public
2023-04-30T00:33:44Z
INSDC last update
public
INSDC status
ERS14946219
SRA accession
E-MTAB-12842:NET_CTRL_9
Submitter Id
ArrayExpress
broker name
gilthead seabream
common name
Protocols: Throughout the experimental trials, fish were maintained in 500 L fiberglass tanks with a flow-through system with seawater (natural photoperiod, water temperature: 13.4 ± 2.2 °C, dissolved oxygen level: > 5 mg/L, and salinity: 34.7 ± 0.8 psu). Fish were fed once daily by hand (% body weight/day adjusted when necessary), with commercial feed (Standard Orange 6) from “AquaSoja, Sorgal, S.A” (Ovar, Portugal), according to the nutritional requirements of the species. By the end of the trials fish were randomly collected from each tank and immediately anesthetized using a lethal dose of tricaine methanesulfonate (MS-222; Merck KGaA, Darmstadt, Germany). Liver samples were collected, cut, immediately frozen in liquid nitrogen, and stored at -80 °C until further use. Total RNA was extracted from 70 mg of liver samples (n = 9) using 1 ml of TRI reagent®. After homogenizing the tissue with an autoclaved micropestle, homogenates were centrifuged at 12,000 × g for 10 min at 4°C and incubated at RT for 5 min. Phase separation was achieved with 200 μL of cold chloroform, followed by vortexing, incubation for 15 min at RT, and centrifugation at 12,000 × g for 30 min at 4 °C. RNA isolation was performed with 500 μL of cold isopropanol. For precipitation, samples were allowed to stand for 1 h at -20 °C followed by centrifugation at 12,000 × g for 15 min at 4 °C. Pellets were then washed twice with 1 ml 75% cold EtOH, centrifuged at 12,000 × g for 8 min at 4 °C, and dried for 5-10 min, on ice. The pellets were resuspended in RNase-free water in a ThermoMixer® C at 55 °C for 10 min at 500 rpm. RNA purification and DNase I treatment were performed using the Isolate II RNA Mini Kit (BIO-52073, Meridian BioScience®, Cincinnati, OH, USA). The yield and purity of extracted RNA were assessed using a NanoVue Plus spectrophotometer (GE Healthcare, Chicago, IL, USA). Total RNA quality and integrity were checked using a 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA), and all samples with an RNA integrity number (RIN) > 7 were considered for sequencing. RNA-seq libraries were prepared from 1 μg of total RNA using the Illumina TruSeq™ Stranded mRNA Library Prep Kit according to the manufacturer’s instructions (TruSeq Stranded mRNA Reference Guide # 1000000040498 v00).
description
adult
developmental stage
wild type genotype
genotype
27
individual
Sparus aurata
organism
Sparus aurata
organism
liver
organism part
Sparus aurata
scientific_name
NET_CTRL_9
title